Novel gold-based nanocrystals for medical treatments and electrochemical manufacturing processes therefor

NOVEL GOLD-BASED NANOCRYSTALS FOR MEDICAL TREATMENTS AND ELECTROCHEMICAL MANUFACTURING PROCESSES THEREFOR

This application includes 1) US Patent Application Serial Nos. 61/223,944, filed July 8, 2009; 2) US Patent Application Serial No. 61/226,153, filed July 16, 2009; 3) US Patent Application Serial No. 61/228,250, filed July 24, 2009; 4) US Patent Application Serial No. 61/235,574, filed Aug. 20, 2009; 5) US Patent Application Serial No. 61/249,804, filed Oct. 8, 2009; 6) US Patent Application Serial No. 61/263,648, filed on November 23, 2009; and 7) U.S. Patent Application Serial No. 61/294,690, filed on January 13, 2010, claims priority.

This application relates to novel gold nanocrystals and nanocrystal shape distributions having surfaces that are substantially free of organic or other impurities or films. Specifically, the surface is "cleaner" compared to the surface of gold nanoparticles produced using a chemical reduction process that requires organic reducing agents and/or surfactants to allow the gold nanoparticles to grow from gold ions in solution.

The present invention includes novel electrochemical fabrication apparatus and techniques for producing gold-based nanocrystals. The present invention relates to the treatment of diseases or conditions for which gold therapy is already known, more generally conditions from pathological cellular activation, such as inflammatory (including chronic inflammatory) conditions, autoimmune conditions, hypersensitivity reactions and/or cancerous diseases or conditions. or pharmaceutical compositions and uses thereof of gold nanocrystals or suspensions or colloids thereof for prophylaxis. In one embodiment, the condition is mediated by macrophage migration inhibiting factor (MIF).

<u>gold salt (</u><u>gold</u><u></u><u>salt</u><u>)</u>

Robert Koch gained fame for discovering the bacteriostatic action of gold cyanide against Mycobacterium tuberculosis. It has since been observed that tuberculosis patients often benefit from a reduction in certain inflammatory conditions upon injection of a given gold salt into the disease. The observed reduction in inflammation resulted in aurothiolate used by Forestier in 1927 as a treatment for rheumatoid arthritis (Panyala, 2009) (Abraham, 1997). Early gold-based products are usually injected intramuscularly or subcutaneously (later intraarterially), and in some cases are still used today and/or are still being used to treat rheumatoid arthritis.

Specifically, certain gold compounds have been known for many years to have anti-inflammatory activity. For example, (i) sodium gold thiomlate, marketed as Myocrisin and a related compound version, and marketed as Myochrisine and Myochrisis (also " referred to as "gold sodium thiomalte"); (ii) sodium gold thioglucose sold as Solganol (also referred to as "gold sodium thioglucose"); (iii) sodium gold thiosulfate, marketed as Sanocrysin and related compound versions, as Crisalbine, Aurothion and Sanocrysis; and (iv) sodium gold thiopropanosulfonate, marketed as Allocrysine, has been used for the treatment of rheumatoid arthritis (Sadler, 1976; Shaw, 1999; Eisler, p. 133, 2004). Only monovalent gold salts were known to show therapeutic efficacy for the treatment of rheumatoid arthritis. In 1961, the British Rheumatism Council confirmed that injectable gold salts showed efficacy, and that gold salts remained a widely used treatment method for progressive rheumatoid arthritis (Ueda, 1998).

Treatment with various gold salts has also been suggested or observed anecdotally to be effective in a variety of other diseases, including asthma, HIV, malaria and cancer. A substantial body of evidence exists for these diseases in both human and animal models, suggesting that gold may be a viable treatment option for these unmet medical needs (Dabrowiak, 2009).

<u>oral gold (</u><u>oral</u><u></u><u>gold</u><u>)</u>

Recently, Auranofin has been used in many parts of the world.^&amp;#174; or Ridaura^&amp;#174;An oral gold product, 2,3,4,6-tetra-acetyl I-thio BD-glucopyranosato-S-(triethyl-phosphine), marketed as [Ho &amp; Tiekink, 2005, Dabrowiak, 2009]. auranofin^&amp;#174;was approved for human use by the FDA until the mid-1980s; auranofin^&amp;#174;has the advantage of oral absorption, but was considered less effective than injectable gold thiolate (Sadler, 1976; Shaw 1999).

<u>Toxicity of gold salts and oral gold</u>

Historically, the use of all injectable and oral gold-based therapies has been limited due to toxicity, and in many places 30 to 50% of patients undergo various gold-based therapies due to undesirable or intolerable side effects. stopped. Side effects of many conventional gold therapies include rash or mucosal skin effects (eg, itching, dermatitis and stomatitis); blood changes (eg, thrombocytopenia); protein in the urine (proteinuria); inflammation of the mouth; a decrease in the number of circulating white blood cells; decreased platelet count; aplastic anemia due to organ damage; lung abnormalities; adverse immune responses such as eosinophilia, lymphadenopathy, hypergammaglobulinemia; extreme hypotension, angina pectoris, myocardial infarction, nephrotoxicity and nephrotic syndrome; hepatitis; colitis; and chrysiasis (pigmentation) of the cornea, lens and skin (Eisler, p. 133-134, 2004). The most common side effect of gold therapy is skin toxicity, accounting for up to 60% of all adverse reactions, particularly lichen lichen rash and non-specific dermatitis (Eisler, p.133-134, 2004). These side effects have been found to be related to the agents used (eg carrier molecules, oxidation status of gold in compounds, etc.) rather than gold itself (Ho &amp; Tiekink, 2005).

In 1978, Payne and Arena found auranofin in rats compared to injected gold controls.^&amp;#174;Subacute and chronic toxicity of several oral gold compounds including Sprague Dolly rats were dosed for 6 weeks, 6 months and 1 year. In follow-up trials, one year of investigation was repeated using sequential death and a modified dosing regimen.

The target organs identified by this experiment were the stomach and kidneys. The above changes consisted of superficial erosion of the mucosa extending to 1/3 of the thickness of the mucosal layer and to 5% of its surface area. These changes are dose-related and are associated with weight loss. Treatment lesions were also evident. In the kidneys of rats treated with SK&amp;F 36914 for 6 months, hypertrophy (giant cells) of cortical tubule epithelial cells was present. There was also a dose-related hypertrophy of the nucleus (giant cell nucleus) with evidence of polymorphism and multinucleated giant cells. Similar changes were observed in the 1-year trial, but other neocortical cell adenomas were also seen in dose-related events (0/38, 3/39, 6/37 and 8/ for control, low, medium and high doses, respectively). 37). The repeated 1-year trial showed an unexpectedly high mortality rate. This is due in many cases to ileal lesions that have progressed to ulcers that appear to penetrate the intestinal wall. Presumably, death was due to acute infectious peritonitis. In the injected control group, sodium thiomalate was administered by intramuscular injection once a week for 1 year, and in the second experiment, it was administered once a week for 46 weeks and daily for 330 days thereafter. In the 1-year experiment, renal tubular cell giant cells were observed, and renal cell adenomas appeared in 1/16 of females but not males. In the 21-month experiment, all surviving rats showed giant cells of neocortical tubular epithelium, and cystic tubules were often observed. Neoplastic adenomas were occasionally seen in 8/8 females and 3/7 males surviving 21 months with ascites (Payne &amp; Arena, 1978). Similar results were found in dogs (Payne &amp; Arena, The subacute and chronic toxicity of SK&amp;F 36914 and SK&amp;F D-39162 in dogs, 1978).

Szabo et al., 1978a reported that auranofin in pregnant rats and fetuses^&amp;#174;Efficacy of gold-containing compounds including Sodium thiomalate and auranofin, an oral gold compound for maternal and fetal toxicity and teratogenicity^&amp;#174;was studied. Oral gold was administered by intubation on the 6th to 15th days of pregnancy, and sodium thiomalate was administered by subcutaneous injection on the 6th to 15th days of pregnancy. This is the standard exposure period in this study, and this exposure is considered to correspond to the first trimester of human pregnancy. The fetuses were examined using standard procedures, and the size of the group was appropriate for the experiment. Maternal and fetal toxicity was evident, and fetuses of animals treated with sodium gold sodium thiomalate showed a pattern of dosing-related malformations. The dosing used resulted in a certain percentage of dam deaths and had a pronounced effect on body weight (including actual weight loss at the start of dosing) and reduced feed consumption. Anomalies include skeletal abnormalities, external malformations and degrees of hydrocephalus and eye disorders. SK&amp;F D-39162 did not affect feed intake or body weight gain, but was also associated with a decrease in fetal body weight compared to controls. The only major impairment found using SK&amp;F D-39162 treatment was edema. There is no evidence for the effect of sodium thiomalate on transplantation, resorption, fetal number or fetal body weight in sodium thiomalate-treated animals. These authors conclude that the effect on the fetus is indirect and is due to the accumulation of gold in the lysosomes of the visceral yolk sac epithelium and is due to the inhibition of bioenzymes related to fetal nutrition. This hypothesis accounts for the teratogenicity of other compounds and is quite convincing (Szabo, Guerriero, &amp; Kang, The effects of gold containing compounds on pregnant rats and their fetuses, 1978).

Szabo et al., 1978b reported the effect of gold-containing compounds on pregnant rabbits and fetuses. In this experiment, pregnant rabbits were dosed on the 6th to 18th days of pregnancy. Sodium thiomalate was administered by subcutaneous injection, and the oral compound was administered by intubation. Both routes of administration resulted in maternal death, and abortions were observed in live animals. A dose-related decrease in maternal feed consumption resulting in substantial weight loss was observed at higher doses of both injectable and oral gold. In addition, effects on offspring size, number of resorptions and mean fetal weight are evident. Fetal abnormalities and malformations are also mainly observed in the abdomen (abdominal rupture and umbilical hernia), and the incidence of abnormalities affecting the brain, heart, lungs and skeleton is low. The authors concluded that the incidence of celiac disease over all of their historical controls was indicative of a specific reduction in rabbits for the effect of gold (Szabo, DiFebbo, &amp; Phelan, 1978).

Based on these studies, oral administration of relatively high doses of gold-containing compounds is associated with a dose-related pathogenesis of gastric mucosal erosion, with significant ileocecal lesions (including ulcers) leading to death in a large number of animals in longer trials. ) is related to Examination of the presented data suggests that gastric lesions are typical of distinct direct local effects on the mucosa. The neocortical tubular epithelium is another target target, perhaps due to the formation of high local concentrations during the enrichment of urine. Cortical tubular epithelial lesions progress from giant cells to adenoma formation in a significant number of animals. Although it is a benign tumor, it cannot be ignored from a risk assessment point of view. However, although lesions of the rodent kidney are characterized as being relatively common, especially in males, these studies have shown that they are relatively more affected in females than males.

Gastric lesions occur after administration of relatively large amounts of gold solution. In addition, important toxic agents in these experiments are ionic gold (e.g., Au(III) or Au³⁺) suggests that This type of lesion is also produced by a number of NSAID agents used in the treatment of various forms of arthritis and is generally considered to be controllable despite undesirable side effects. Therefore, the absence of such side effects is an advantage over conventional gold-based therapies.

In 1996, Cheriathundam and Alvares reported on liver and kidney markers and metallotionine levels in Sprague Dawley rats and three mouse strains (Swiss-Webster, C3H/Hej and DBA/2J). Sodium thiolate and auranophine^&amp;#174;was evaluated. In rats, sodium gold thiolate increased 7-fold in hepatic metallotionine levels, whereas in mice, strain metallotionine levels increased 2-fold in Swiss-Webster and approximately 5-fold in inbred. Sodium gold thiolate showed minimal change in metallotionine levels in the mouse strain. The liver marker serum ALAT was not altered by sodium gold thiolate in any species or strain tested. BUN, an indicator of renal function, was elevated 3-fold in rats, but not in any mouse strain. These data are consistent with the observation that sodium gold thiolate is nephrotoxic in rats and humans, but it is interesting that there is no evidence of nephrotoxicity in mice (Cheriathundam &amp; Alvares, 1996).

2 Observations of embryotoxicity and fetal disorders after treatment in pregnant animals of the species suggest that many, if not all, of the forms already in use represent developmental risk. This is similar to a number of other current RA therapies, for example, where methotrexate should give a label warning regarding its potential adverse effects on the fetus.

Several possible pharmacological actions contributing to both clinical efficacy and adverse reactions have been identified for oral gold. For example, Waltz et al., Auranophin^&amp;#174;It was found to inhibit carrageenan-induced edema in rats in a dose-related manner at concentrations of 40, 20 and 10 mg/kg with serum gold levels of 10 μg/ml and maximal inhibition of 86% at the maximal dose. auranofin^&amp;#174;The two basic ligands of triethylphosphine oxide and 2,3,4,6-tetra-o-acetyl-1-thio-β-D glucopyranose do not show any significant biological activity and Gold sodium malate, gold thioglucose and thiomalic acid did not significantly affect rat paw edema. auranofin^&amp;#174;was shown to significantly inhibit adjuvant arthritis, whereas the ligand had no effect. auranofin^&amp;#174;inhibits antibodies that are dependent on complete lysis. auranofin^&amp;#174;has been shown to inhibit the release of lysosomal enzymes such as β-glucuronidase and lysozyme from stimulated polymorphisms. auranofin^&amp;#174;is an effective inhibitor of antibodies dependent on cellular cytotoxicity exhibited by polymorphisms from rats with adjuvant arthritis. auranofin^&amp;#174;Silver is much more effective than sodium thiomalate as an inhibitor of peroxide formation. In the immunophagocytosis assay, sodium gold thiomalate caused a marked inhibition of auranophine.^&amp;#174; It does not show inhibitory activity at 40 times the concentration (Walz, DiMartino, Intocca, &amp; Flanagan, 1983).

Waltz et al. also auranofin^&amp;#174;It was indicated to be more effective than sodium thiomalate as an inhibitor of subcutaneous migration, chemotaxis and phagocytosis by peripheral blood monocytes. Lipsky et al., auranofins such as sodium thiomalate^&amp;#174;It was found that the direct inhibition of mononuclear phagocytes inhibited lymphocyte proliferation in vitro. However, auranofin^&amp;#174;also has an inhibitory effect on lymphocyte activity, which was not observed with sodium thiomalate. Inhibition of monocytes is 10 to 20 times lower than the concentration of sodium thiomalate auranofin.^&amp;#174;(Walz, DiMartino, Intocca, &amp; Flanagan, 1983).

In general, patients suffering from active rheumatic disease have reduced doses for lymphoproliferation caused by mitogen-stimulated lymphoproliferative or mixed lymphocyte responses. Although patients initially treated with sodium thiomalate initially exhibited some inhibition of mitogen-stimulated lymphocyte proliferation, patients who ultimately responded to the drug showed normal lymphocyte responses in vitro. Conversely, auranofin^&amp;#174;Lymphocyte responses are significantly suppressed within a few weeks in patients treated with . So, auranofin^&amp;#174;Mostly, due to the major difference in the pharmacological properties of the oral compounds relative to the injectable gold-thiol compounds, they show a strong immunosuppressive effect in vitro to a lower degree than the injectable gold compounds (Dabrowiak, 2009).

The adverse reaction is auranorphine, in which about 30-50% of treated patients produce some form of toxicity.^&amp;#174;It is a major limiting factor for the use of oral gold compounds such as (Dabrowiak, 2009) (Kean &amp; Anastassiades, 1979) (Kean &amp; Kean, The Clinical Pharmacology of Gold, 2008).

Skin rash is the most common negative side effect, with a specific type of rash occurring in about 30% of patients. Most lesions occur on the hands, forearms, torso, and shins, but occasionally also on the face, with slight erythema accompanied by scaly patches 1-10 cm in size, similar to seborrheic rashes. Serious problems of skin rash in the form of coin eczema, total exfoliation, and intense itching have been rarely documented.

Mouth ulcers (painful and analgesic) similar to aphthous ulcers occurred in approximately 20% of patients treated with injectable gold therapy. The occurrence of stomatitis is an obvious contraindication to the continuation of gold therapy, since mouth ulcers are known to signal pemphigus-like blister skin lesions.

The significantly altered (0-40%) frequency of proteinuria in the trial was reported by Kean and Anastassiades, and seems likely to reflect the various definitions of what constitutes proteinuria. Microhematuria is responsible for intermittent oral gold treatment (Kean &amp; Anastassiades, 1979), although these trials have not been shown to be the cause of any long-term serious or permanent renal damage from gold therapy (Kean &amp; Anastassiades, 1979). .

Gold compound-induced thrombocytopenia occurs in two distinct types, the most common being associated with platelet surface IgG antibodies, and less common being secondary to myelosuppression. The genetic marker HLA DR3 may indicate an increased risk in patients who develop thrombocytopenia associated with platelet surface antibodies.

Idiopathic toxicity in the form of cholestatic jaundice or acute enterocolitis has also been associated with injectable gold compounds, particularly sodium gold thiomalate, but has not been reported for oral gold.

Deposition of elemental gold in the lens and cornea of the eye has been reported, but it does not appear to cause any specific damage to vision.

The specificity of oral gold therapy is that loose, soft stools are usually produced in the first months of therapy. Subsequent months of treatment may be associated with a low incidence of early dropout in patients prone to altered variant diarrhea. The formation of apparently watery diarrhea occurs in 2-5% of patients and appears to be dosing related.

In general, the incidence of adverse events is lower for oral gold than for injectable gold, but can still be significant.

A second major disadvantage of the use of available gold-based treatments is the very slow onset of efficacy. Patients often have to continue treatment with, for example, gold salts for 3-6 months before experiencing any significant benefit. The long wait for any observed benefit is a major impediment to patient compliance, thus adversely affecting efficacy in use.

Although knowledge about the pharmacokinetic profile of gold is mostly focused on the determination of elemental Au, not much is known about the structure of gold when it is present in various tissues or organs (e.g., its chemical or physical or crystalline structure). there is not

After oral ingestion, the oral gold complex is rapidly but incompletely absorbed. The gold-based portion of the injectable gold-based complex appears to be rapidly absorbed into the circulation following intramuscular injection. In the blood circulation, auranofin^&amp;#174;(or a ligand thereof) appears to bind primarily to albumin. Specifically, radiolabeled auranofin in human volunteers^&amp;#174;After oral administration of , about 25% of the administered dose was detected in serum, and a peak concentration of 6-9 μg/100 ml was achieved within 1 to 2 hours. The plasma half-life was on the order of 15 to 25 days with almost total elimination from the body after 55 to 80 days. Only about 1% of radiolabeled Au was detectable after 180 days, whereas less than 30% of gold from sodium gold thiomalate was detected at this time. Gold is widely distributed through the reticuloendothelial system of the liver, bone marrow, lymph nodes, spleen and also synovial membranes, particularly phagocytic cells. Deposition in the skin occurred and it was observed that there may be a quantitative correlation between the amount of gold in the dermis and the total dose of gold given. Electron-enriched deposits of gold have also been observed in renal tubular cells, with sulfhydryl-containing enzymes enriched at another site, but the presence of glomerular-associated gold was not common (Walz, DiMartino, Intocca, &amp; Flanagan). , 1983) (Dabrowiak, 2009).

<u>gold nanoparticles</u>

Other formulations of gold have existed and continue to be developed, most of which use gold nanoparticles produced by various chemical reduction techniques, some of which use submerged plasma arc treatment techniques, and most of them use various A stable or partially stable gold colloidal or gold nanoparticle suspension is produced.

<u>Colloidal Gold Nanoparticles by Chemical Reduction</u>

Michael Faraday produced the first colloidal gold suspension by a chemical reduction method around 1850 (Faraday, 1857). Faraday uses ethers (e.g. CH₃-CH₂-O-CH₂-CH₃) dispersed in phosphorus or carbon disulfide (i.e., CS₂) was used to chemically reduce an aqueous gold salt, chloroaurate (ie, gold(III) salt).

Today, most colloidal gold preparations are produced by reduction of chloric acid (tetrachlorohydrogen aurate) with a reducing agent such as sodium citrate to produce "Tyndall purple". Now, there are various "conventional" reduction chemistry methods used to form colloidal gold. Specifically, several types of synthetic pathways exist, each of which exhibits different characteristics in the end product (eg, colloidal gold nanoparticles) produced thereby. Note that in addition to the strength, amount and type of reducing agent used, the action of the stabilizing agent (eg, the compound used in the solution phase synthesis process) is important (Kimling, 2006).

Although Faraday introduced a colloidal gold solution, the homogeneous crystallization method of Turkevich and Friends (and variants thereof) is most commonly used today, typically for a range of particle sizes, mostly It produces spherical particles (Kimling, 2006). Specifically, the most common method uses a gold(III) complex such as tetrachloroaurate (or chloric acid) as a starting material, and an added chemical species reducing agent such as Na thiocyanate, white phosphorus, Na₃ Citrate &amp; Tannic acid, NaBH₄, citric acid, ethanol, ascorbic acid Na, Na₃ Citrate, hexadecylaniline, etc. are used to reduce gold in the gold complex to gold metal (ie, gold (0) or metallic gold) (Brown, 2008). However, another chemical reduction technique is AuP (Ph₃) as a chemical species reducing agent for sodium borohydride (Brown, 2008). Depending on the specific processing conditions used in these chemical reduction processes, the size of these most spheres, nanoparticles, formed range from about 1 nm to about 64 nm in diameter (Brown, 2008). In addition, the specific thermal citrate reduction method used by Kimling produced, in addition to spherical particles, a small fraction of triangular-shaped particles, with a triangular-shaped species of less than about 5% (Kimling 2006).

Further studies have focused on the shape control of colloidal metal nanoparticles. Biologists and biochemists have understood that "structure dictates action" with respect to protein action. Gold nanoparticles of different shapes also have different properties (eg, optical, catalytic, biological, etc.). Controlling nanoparticle shape provides an effective approach to optically tuning nanoparticles. All gold nanoparticles contain a face-centered cubic lattice, and when caused or permitted by particular processing conditions, gold nanoparticles can range from irregular ellipsoids with defective surfaces (e.g., steps) to polyhedra with relatively limited surface defects. A variety of crystalline shapes can be employed. Different crystalline morphologies are associated with different crystal faces (or sets of crystal faces). However, some of the most common gold nanoparticle morphologies do not consist of single domains, but of paired faces (Tao, 2008).

Yuan, et al. (Yuan, et al.) found that non-spherical gold nanoparticles were mixed with gold salts (i.e., HAuCl₄ or by providing seed crystals from the borohydride reduction of auric acid). The seed crystals are then added as a reducing agent and/or a surfactant (eg capping agent) as a species NH₂It is contacted with the same gold salt in solution with OH, CTAB and sodium citrate. Several different crystalline shapes are formed by this approach, including triangles, truncated triangles, hexagonal layers and pseudo pentagons. Yuan concluded that variations in processing using different chemical reduction techniques could affect the physical and chemical properties of the resulting particles. Researchers have pointed out that the choice of capping agent is a key factor in controlling the growth (and shape) of nanoparticles (Yuan, 2003).

The process described and used in Yuan's document is known as "heterogeneous nucleation", in which seed particles are produced in a separate synthetic step. Thus, this type of shape control can be regarded as an overgrowth process (Tao, 2008). Many chemical reduction techniques use a rather complex two-step heterogeneous nucleation and growth process. However, others use single step heterogeneous nucleation, so that the seed crystal is first nucleated, and nanoparticles are formed from the nucleated seed crystal. Typically, a series of chemical reactions occur simultaneously in homogeneous nucleation. The main objective in homogeneous nucleation is to balance the nucleation rate with the crystal growth rate and to control the grain size, since nucleation and growth are carried out by the same chemical process (Tao, 2008). .

Metal nanoparticle synthesis in solution(s) typically requires the use of surfactants (surfactants) and/or amphoteric polymers as stabilizers and/or capping agents. Surfactants and/or amphoteric polymers are known to play an important role in controlling the size, shape and stability of the dispersed particles (Sakai, 2008).

Some of the most common crystal morphologies observed in crystalline gold nanoparticles (eg in heterogeneous nucleation processes) do not consist of a single crystal or single domain, but often multiple crystal domains surrounded by paired faces. made of particles containing A rule decahedron (also referred to as a pentagonal bipyramid) is an equilibrium shape completely surrounded by triangular (III) faces, and can be judged to be five tetrahedra sharing a common edge along an axis of five times. Such structures are commonly observed for nanocrystalline particles synthesized by metal evaporation on solid substrates and seeded heterogeneous nucleation reduction chemical approaches (Tao, 2008). However, in the case of nanoparticles synthesized by Turkevich and Friends, it is difficult to observe because the decahedron acts as a preferred seed for the growth of nanowires and nanorods (Tao, 2008). Thus, various shapes can be achieved by controlling the processing conditions, along with the amount and type of surfactant and capping agent added and used during the reductive chemistry approach due to Turkevich and Friends.

In each colloidal gold composition produced by a reductive chemistry approach, a surface coating comprising one or more elements of a reducing agent and/or surfactant or capping agent is applied onto (or within) at least a portion of the suspended gold nanoparticles. ) is evident. The use of a reducing agent (ie, a reducing agent) typically helps to suspend the nanoparticles in a liquid (eg, water). However, reducing agent coatings or surface impurities are sometimes added to or even replaced by surfactant coatings or capping agents. Such reducing agent/surfactant coatings or films are seen as impurities located on and/or within the metallic nanoparticles, resulting in colloids or sols having substantially many of the properties of a protective coating or film than the gold nanoparticles themselves. can (Weiser, p. 42, 1933).

For example, surfactants and amphoteric polymers are highly involved in the formation of nanoparticles (and thus affect size and shape), as well as affect the nanoparticles themselves. The surface properties of nanoparticles are modified by reducing agent coatings and/or reducing agent molecular coatings (Sperling, 2008).

The absorption of hydrophobic tails, hydrophilic head groups and certain counter ions (at least in the case of the use of ionic surfactants) on the surface of the nucleated particles, as well as the formation of complexes of the formed particles with metal ions and surfactants and/or amphoteric polymers All can affect the shape of the nanoparticles, the surface of the nanoparticles, and/or can alter the function of the nanoparticles (Sakai, 2008).

Different surface chemistries or surface films (eg, thickness (eg, film) of reducing agent by-products and/or the presence of reducing agent by-product compositions) can result in different interactions of gold nanoparticles with, for example, various proteins within an organism. The biophysical binding force of nanoparticles to proteins (e.g., electrostatic, hydrophobic, hydrogen bonding, van der Waals) depends not only on the size, shape and composition of the nanoparticles, but also on the thickness and/or surface impurities or coating(s) on the nanoparticles. or has a tangible function. The Turkevich and Friends method (and variations thereof) for producing gold nanoparticles is the most widely understood and used chemical reduction method. The use of citric acid or sodium citrate results in citrate-based chemistry (ie, referred to as citrate stabilized) on the surface of gold nanoparticles (Lacerda, 2010).

Further, Daniel et al. (1) citrate reduction to produce a somewhat loose envelope of "[citrate-based] ligands attached to gold nanoparticles; (2) citrate (for size control) and both a modification of the citrate reduction method using a sexual surfactant; (3) the "Brust-Schiffrin" method to produce a thiol or thiolate ligand that "strongly binds gold"; (4) xanthate; Methods for producing sulfur-containing ligands including disulfides, dithiols, trithiols and resorcinarene tetrathiols and (5) phosphines, phosphine oxides, amines, carboxylates (which may replace citrate coatings); Key gold nanoparticle formation techniques were reviewed, including chemical synthesis and assembly methods, including other ligands for aryl isocyanides and iodides. The authors reiterated the explanation attributable to the gold nanoparticles formed: "The resulting physical properties are neither those of bulk metals nor those of molecular compounds, but these are the shape of the nanoparticles and the properties of the protective organic envelope, the interparticle distance, the particle size. is highly dependent on" (Daniel, 2004).

Organic ligands (e.g., citrate-based ligands or coatings or films) present on the gold nanoparticles help to stabilize the gold nanoparticles in the liquid and thus prevent the nanoparticles from binding to other nanoparticles, for example Prevents sedimentation and/or agglomeration from the suspension by gravity so that these organic ligands (e.g. organic envelopes) become impurities (i.e. to the gold nanoparticles underlying them), and interactions of gold nanoparticles with proteins in life systems contribute to Such coating(s) or film(s) can have strong biological effects (Lacerda, 2010).

Further, Wang et al. concluded that commonly used citrate-reduced gold nanoparticles interfered with the uptake of gold nanoparticles into colloidal solutions without reducing agents and stabilizers (Wang, 2007). ).

Similarly, Lacerda et al. (Lacerda, et al.) indicated that a better understanding of the biological effects of nanoparticles requires an understanding of the binding properties of the nanoparticles and the proteins in vivo to which they bind. Protein uptake (or protein corona) on nanoparticles can be altered depending on nanoparticle size and surface layer composition and thickness. Lacerda concluded that the protein layer "dressing" the nanoparticles modulates the tendency of the nanoparticles to agglomerate and strongly influences their interactions with biological materials (Lacerda, 2010).

<u>Cleaning of colloidal gold nanoparticles produced by chemical reduction technique</u>

In some cases, the reducing agent surface coating or film will remain as an impurity on the surface of the nanoparticles, but in other cases it is desired to be removed by a variety of rather complex and costly techniques. When the coating is removed, it is usually replaced by an alternative composition or coating that allows the nanoparticles to remain in suspension in suspension upon hydration. The effect of purity on the chemistry and properties of nanoparticles is often overlooked, but results show that the degree of purification can have a significant impact (Sweeney, 2006). These researchers report that the preparation itself is generally tedious, time consuming and wasteful procedures such as extensive solvent washing and sufficient purification of nanoparticles, including fractional crystallization, can be significant challenges. In the absence of such purification, the parameters of surface chemistry-related contaminants on the surface of chemically reduced nanoparticles affect the ability to understand/manage fundamental structure-action relationships (Sweeney, 2006).

Subsequent processing techniques may also require a set of washing steps, specific concentration or centrifugation steps and/or subsequent chemical reaction coating steps, all of which have desirable results and specific performance characteristics for nanoparticles and nanoparticle suspensions. (eg, stabilization by ligand exchange, efficacy, etc.) (Sperling, 2008). In other cases, strong stripping methods are used to form very clean nanoparticle surfaces (Panyala, 2009).

Thus, it was concluded that the development of gold nanoparticles in the management, treatment and/or prevention of diseases is hampered by the fact that conventional methods for the preparation of gold nanoparticles are by and based on chemical reduction processes. Specifically, in 1996 (Robyn Whyman reported that one of the major impediments to the progression of colloidal gold produced by various reduction chemistry techniques was any "relatively simple, reproducible and generally applicable possible synthetic procedures" (Whyman 1996). It is possible to produce colloidal gold (eg, alone or in suspension) and reductive coatings with a variety of different physical properties, respectively, both in living cells. Alternatively, there are many variations of the earlier reduction chemistry techniques taught by Faraday that, when used with cells, can result in different efficacy/toxicity profiles, none of which met Wyman's criteria. Thus, a relatively simple, reproducible, and generally applicable fabrication approach for producing gold nanocrystals has been favored. In addition, the ability to ensure that this manufacturing approach meets FDA cGMP requirements is even more important.

Other authors are beginning to recognize their inability to distinguish the completely detrimental physical/biological performance of nanoparticles formed from the chemical formation (ie, chemical reduction) methods used to make them. In this regard, a cleaning or cleaning process can be used to alter or clean the surface of nanoparticles produced by reduction chemistry, albeit somewhat complex, expensive, and non-environmental, wherein the elements of the chemical process are It remains on and can affect the surface of the nanoparticles (and thus can affect their function). In addition, the presence of certain compounds during the nanoparticle formation process will affect the morphology (ie, size and/or shape) of the nanoparticles being formed. Certain possible preferred morphologies known to exist in gold-based crystalline systems are not readily observed in many of the products produced by these reduction chemistry techniques.

<u>colloidal</u><u> Other techniques for the manufacture of gold</u>

Obtaining reducing agents and surfactants (e.g., no stabilizing, capping or reducing agents added to achieve reduction of the gold ionic species) has resulted in some detrimental consequences of reducing agent/surfactant coatings that exist from reducing chemistry approaches. It has become the target of some researchers who understand it as For example, a frequency of 950 kHz is applied to an aqueous solution of hydrogen tetrachloroaurate by using ultrasonic techniques. Spherical gold nanoparticles in the range of 20 to 60 nm are produced at temperatures above 50°C. Relatively larger triangular plates and some hexagonal spheres coexist when the mixture is treated below 50°C (Sakai, 2008).

HAuCl₄X-ray irradiation was developed to obtain stabilizer-free and reducing agent gold nanoparticles so as not to "confuse" the biocompatibility issue in biomedical applications. The authors use a "strong" X-ray beam to generate hydrogen-free radical electron donors to⁺It was considered to generate electrons necessary for the chemical reduction of (Wang, 2007).

Another older and more complex technique for minimizing or eliminating the need for reducing agents and/or minimizing undesirable oxidation products of reducing agents is ⁶⁰1.8×10 from Co source⁴ Use γ-irradiation with a dose rate of rad/h. In this case, Au(CN) as₂first generates hydrated electrons from the radiolysis of water and uses the hydrated electrons to reduce gold ions:

e_aq^- + Au(CN)₂ Au⁰ + 2CN^- (Henglein, 1998).

It is known that the surface of gold nanoparticles can be further treated by adding compound species such as polyethylene glycol (PEG) or other specific ligands. In this regard, PEG-coated gold nanoparticles are induced by various techniques for migrating to cancer or tumor sites, and then, for example, in the treatment of cancer, which is irradiated with infrared or electromagnetic waves to heat and decompose cancer cells. work was carried out (Panyala, 2009). Surface PEGylation is also known to increase the blood half-life of nanoparticles; Polysorbate-80 may improve the blood-brain-blocker transport of nanoparticles (Teixido &amp; Giralt, 2008).

<u>by submerged arc treatment </u><u>colloidal</u><u> gold</u>

Also, methods for preparing gold nanoparticles by submerged arc treatment methods are known in the art. This method was first pioneered by Bredig in the late 1800s. Breddick used direct current to create an underwater arc between two wires. Breddick used a current of 5-10 amps and a voltage of 30-110 volts. In some cases, Breddick also used 0.001N sodium hydroxide instead of pure water. Breddick saw this process as grinding a metal electrode. Bredick obtained hydrosols of gold in this way (Weiser, pp. 9-17, 45-46, 1933).

Svedberg later improved Breddick's method by using a high-frequency arc instead of a direct current arc. Svedberg pointed out that the arc allows the formation of a metallic gas, which subsequently condenses into particles with colloidal dimensions. Although there have been many controversies about the exact mechanism of this method, metal vaporization is considered to be important (Weiser, pp.9-17, 45-46, 1933).

The parameters important to Svedberg in controlling the electric grinding to form a colloidal solution are: a) grinding rate, b) ratio of precipitate to total metal dispersed, c) degree of decomposition of the medium, and d) current characteristics. The dependencies of (a)-(c) are mentioned. The amount of precipitate achieved by the Breddick and Svedberg methods ranged from about 30% to about 50% under various processing conditions (Kraemer, 1924).

A more recent study on palladium using the Bredic method was conducted by Mucalo et al. (Mucalo, et al.). These investigators tested whether the metal particles in Bredic's sol were "impure" due to impurities from the coexisting electrolyte decomposition of the electrolyte, and oxidized material was believed to be formed during arcing (Mucalo, 2001). These researchers used modern surface analysis techniques (ie, XPS or "X-ray photoelectron spectroscopy") to measure differences in surface speciation with respect to pH. At lower pH, a gray-black unstable material was produced. At higher pH the sol is more stable, but still fully aggregated within 1-2 weeks. Nanoparticles consist of irregularly shaped spheres. Although the materials produced at higher and lower pH are mostly metallic in nature, the surface properties of these labile colloids are different. Bredic sol at higher pH produces a thicker outer oxide layer on unstable nanoparticles (Mucalo, 2001).

The method of Bredick and Svedberg was later improved by other researchers to produce methods based on various underwater arcs. However, what each of these submerged arc treatment methods has in common is the production of rather irregularly shaped metallic spheres. In this regard, nanoparticles produced by the Breddick or Svedberg method have a non-specific, spherical shape that exhibits a metal-based vaporization followed by a rapid quenching method, and the nanoparticles are coated with varying amounts of different oxide-based materials. (coated and/or includes).

<u>Toxicity of colloidal gold nanoparticles</u>

A report on the toxicity of gold nanoparticles was performed by Johnston et al. [Johnston, et al.] and was reported in 2010. There are four routes of intravenous exposure summarized for both mice and rats and an intubation approach for rats. Regarding the four intravenous trials summarized, Johnston et al. found that, in quantitative order, the tissue sites of accumulation were liver-spleen in 3 out of 4 tests and liver-lung in 1 out of 4 tests (i.e., the highest gold Nanoparticle accumulation was liver). Specifically, the four intravenous tests reported by Johnston et al. are summarized below (Johnston, 2010).

Tissue distribution of metal particles after exposure through various routes (Johnston, et al., 2010).

<img file="KR20120052967A_D0001.tif" />

Johnston et al. were critical of the various uncertainties put into a number of reported toxicity studies, including that certain conclusions about toxicity as a function of unique particle size (made by other authors) were not accurate. Specifically, Johnstone et al. concluded that 1.4 nm gold nanoparticles were the most toxic gold nanoparticles among various nanoparticle sizes, including 1.2 nm diameter gold nanoparticles, Pan et al. (2007). reported to have fallen. While Pan et al believed that there was a difference in toxicity profile as a function of size, Johnston et al. reported that the 1.4 nm particles were produced by the researchers themselves and the 1.2 nm particles were obtained outside the company (thus, both suggesting that the surface characteristics of the nanoparticles are different). Johnston et al. concluded that "aggregated state or surface chemistry" is the reason for the different performance, both of which are "known to alter particle behavior and toxicity" (Johnston, 2010).

Johnston et al. also concluded that the experimental set-up influences the toxicity outcome and that the tissue distribution of gold nanoparticles in an organism is a function of the size, shape and surface chemistry of the nanoparticles, as well as the route of exposure. In addition, they observed that the liver appeared to be the major site of accumulation, and this result was speculated to be due to the presence of phagocytic cells in the liver. They also reported that nanoparticle uptake is probably a result of the degree and type of protein binding that occurs on the surface of nanoparticles (eg, protein corona), which is a function of the size, shape and surface coating of the nanoparticles. In particular, they reported the ability to internalize nanoparticles, for example by endocytosis of various cell types. This endocytosis mechanism appears to be a function of particle shape as well as particle surface features, such as protein uptake on its surface. In other words, biological uptake is a function of shape, size and charge, and is also highly serum dependent (Johnston, 2010).

<u>The efficacy of colloidal gold</u>

A study by Abraham and Himmel (reported in 1997) disclosed the use of colloidal gold in the treatment of 10 patients who had not previously responded to various other gold-based treatments. The colloidal gold used in this study was produced by a modification of the standard "citric acid method" using "several proprietary variations" of Maclagan and Friends. Auto-aggregation of gold particles was prevented by using maltodextrin (food grade) at a concentration of 2.5% (Abraham, 2008). The size of the resulting colloidal particles was reported to be less than 20 nm as confirmed by the method of passing the colloidal suspension through a 20 nm filter (ie manufactured by Whatman Anotop). Subsequent TEM studies led Abraham to conclude that 99% of the particles produced were less than 10 nm. Sodium benzoate was also added (Abraham, 2008).

The colloidal gold suspension yielded a concentration of 1,000 mg/L (ie, 1,000 pm). Dosage levels given to each patient ranged from 30 mgs/day to 60 mgs/day, with most dosages being 30 mgs/day for 24 weeks. These dosages were administered orally. Table 1 shows the sex, age and previous condition and/or treatment of the patient. In this paper, it was concluded that 9 out of 10 patients were "significantly improved by 24 weeks of intervention" (Abraham &amp; Himmel, 1997). Abraham also reported a decrease in the concentration of certain cytokines, including IL-6 and TNF (Abraham, 2008).

A study of collagen-induced arthritis in rats by Tsai by Tsai showed that nanoparticle gold was bound to the protein VEGF, and that this binding is the reason for the improved clinical performance of rats injected with colloidal gold intra-articularly. concluded. In this case, injected colloidal gold is produced by standard chemical reduction methods using gold chloroaurate reduced using sodium citrate. Sai et al. reported that gold nanoparticles are spheres with an approximate diameter of 13 nm as measured by transmission electron microscopy. The concentration of the intra-articular solution was 180 μg/ml (ie 180 ppm). Intra-articular injection was performed once on the 7th or 10th day after induction of CIA (Tsai, 2007).

Brown, et al. disclosed that a standard colloidal gold formulation (referred to as Tyndall Purple) is produced by a standard chemical reduction method, the so-called reduction of chloroauric acid with sodium citrate. The average particle size of the resulting gold nanoparticles was 27±3 nm. This colloidal gold was dispersed in isotonic sorbital and injected by parenteral and subcutaneous approaches into rats suffering from experimentally induced arthritis. The injected dose is 3.3 μg/kg concentration. Brown et al. also disclosed that when administered subcutaneously, colloidal gold was about 1,000 times more effective than the comparative sodium aurothiomalate. Brown et al. also disclosed that colloidal gold was ineffective when administered orally, and this ineffectiveness was due to coagulation of gold nanoparticles in the presence of gastric juice and sodium chloride (Brown, 2007).

Brown et al. reported an alternative preparation method for colloidal gold with various sizes and shapes (Brown, 2008). Brown et al. disclosed various properties related to "nano-gold hydrosols" in Table 2. The authors conclude that the experiments conducted by (and reported by) "suggest that gold nanoparticles (Au0)-based drugs may have a role in future clinical therapies targeting the modulation of phagocytic cells" ( Brown, 2008).

Documents cited through the "Background" section are set forth in detail below.

<img file="KR20120052967A_D0002.tif" />

<img file="KR20120052967A_D0003.tif" />

<img file="KR20120052967A_D0004.tif" />

<u>Summary of invention</u>

Novel gold nanocrystals are provided having a nanocrystalline surface that is substantially free (as defined herein) of organic or other impurities or films. Specifically, the surface is "clean" compared to those created using chemical reduction processes that require chemical reducing agents and/or surfactants to grow gold nanoparticles from gold ions in solution. The majority of grown gold nanocrystals have unique and identifiable surface properties, such as spatially extended low index crystal planes {111}, {110} and/or {100} and groups of such surfaces (and their counterparts). The resulting gold nanocrystalline suspension or colloid has a preferred pH range, such as 4.0 to 9.5, more typically 5.0 to 9.5, and a zeta potential value of at least -20 mV, more typically at least -40 mV, for that pH range; Even more typically -50 mV or higher.

The shapes and shape distributions of these gold nanocrystals produced by the preparation methods described below include triangular (eg, tetrahedron), pentagonal (eg, pentagonal bipyramid or decahedron), hexagonal (eg, hexagonal bipyramid, icosahedron, 8 tetrahedron), rhomboids (eg, octahedron, various elongated bipyramids, fused tetrahedra, sides of bipyramids), and "others". The shape distribution(s) of nanocrystals (e.g., grown by various embodiments described herein) comprising the aforementioned spatially extended low index crystal planes (which form the aforementioned shapes) and having a "clean" surface. is unique In addition, the proportion of tetrahedral and/or pentagonal bipyramids formed in the nanocrystalline suspension is also unique.

100 Any desired average size of sub-nm gold nanocrystals may be provided. The most preferred crystalline size range is that the average crystal size or "mode" (as measured and determined by certain techniques specifically disclosed herein and as reported as "TEM average diameter") is predominantly less than 100 nm; more typically less than 50 nm, more typically less than 30 nm, and in many preferred embodiments disclosed herein, the mode for the nanocrystal size distribution is less than 21 nm, even more preferably 8 to 18 nm .

Any concentration of gold nanoparticles can be provided by the present invention. For example, the concentration of these gold nanocrystals is in the range of a few ppm (eg, μg/ml or mg/l) to several hundred ppm, typically 2 to 200 ppm (eg, 2 μg/ml to 200 μg/ml), more often in the range of 2 to 50 ppm (eg, 2 μg/ml to 50 μg/ml), even more typically in the range of 5 to 20 ppm (eg, 5 μg/ml to 20 μg/ml).

A novel method is provided for providing these unique gold nanocrystals. Such methods involve the production of gold nanocrystals in water. In a preferred embodiment, water does not significantly bind to the formed nanocrystals, but includes an added "process enhancer" that promotes nucleation/crystal growth during the electrochemical-stimulated growth process. Process enhancers play an important role in processes that include providing charged ions in an electrochemical solution that allow crystals to grow. These novel electrochemical methods can be generated in batch, semi-continuous or continuous processes. This process produces controlled gold nanocrystalline concentration, controlled nanocrystal size and controlled nanocrystal size range, as well as controlled nanocrystal shape and controlled nanocrystal shape distribution. A novel fabrication assembly is provided to produce these gold nanocrystals.

Pharmaceutical compositions comprising effective amounts of these gold nanocrystals for treating a medical condition are also provided. The pharmaceutical composition may be, by way of non-limiting example, no more than 0.1 mg/kg/day or no more than 0.05 mg/kg/day or even more typically no more than 0.025 mg/kg/day, most typically no more than 0.001 mg/kg/day. A systemic dosage of

These gold nanocrystals have a substantially cleaner surface than conventionally available gold nanoparticles and may preferably include spatially extended low index crystal planes forming novel crystal shapes and/or crystal shape distributions so that the nanocrystals Crystals are more biologically active (and less toxic) than spherical-shaped nanoparticles as well as nanoparticles (or nanocrystals) containing surface contaminants resulting from conventional chemical reduction processes, such as chemical reducing agents and/or surfactants. less) was found. Therefore, medical treatment can be performed with lower doses of gold.

Pharmaceutical compositions suitable for systemic or topical use, including oral, intravenous, subcutaneous, intraarterial, buccal, inhalational, aerosol, propellant or other suitable liquid as further described herein are provided.

Using these substantially surface-clean or surface-pure crystals, any gold therapy is known, including a wide range of inflammatory and autoimmune diseases, as well as certain infectious diseases (eg, HIV, AIDS malaria and Chagas disease) and cancer. disease can be cured. Descriptions of many of their uses are set forth in the background of the invention.

Now, it has been surprisingly discovered as part of the present invention that gold nanocrystals inhibit phagocytic migration inhibitory factor ("MIF"). This is the first initiation of the activity of gold nanocrystals (or nanoparticles), and it has been found to date that it can provide a scientific basis for understanding the scope of medical uses for gold nanocrystals. In addition, gold nanocrystals are effective against other diseases mediated by macrophage migration inhibitory factors. It was also found that these gold nanocrystals inhibit IL-6 but not IL-10. For example, MIF and/or IL-6 appears in a variety of pathological and/or biological signaling pathways, and these findings suggest that novel gold nanocrystals may activate pathological cellular activation, such as inflammatory (including chronic inflammatory) conditions, autoimmunity. It has been found to be effective in the treatment or prevention of conditions, hypersensitivity reactions and/or diseases or conditions resulting from cancerous diseases or conditions.

In addition, by practicing the electrochemical preparation method of the present invention, the gold "coating" can be achieved with other metals (or other non-metallic species, such as SiO2).₂) or alternatively these gold-based metal nanocrystals can be alloyed or combined with other metals in the liquid so that the gold-based nanocrystals can be coated by other metals. In this case, the gold-based composite or alloy may be produced in a colloid or suspension. Additionally, certain composites comprising both gold and other metals may also be formed.

Still further, the gold-based metal nanocrystal suspensions or colloids of the present invention can be mixed or combined with other metal-based solutions or colloids to form novel solutions or colloidal mixtures (e.g., in this case distinct metal species can be identified ).

<u>Brief description of the drawing</u>

1A, 1B and 1C show cross-sectional views of a passive electrode assembly according to the present invention.

2a and 2b show a cross-sectional view of an automatic electrode adjustment assembly according to the present invention;

3a to 3d show four alternative electrode adjustment configurations for electrode 1 and electrode 5 adjusted by means of an automatic device 20 .

4a to 4d show four alternative electrode configurations for the manually controlled electrode 1 and electrode 5 .

5A-5E show five different representative embodiments of configurations for electrode 1 .

FIG. 6 shows a cross-sectional view of a plasma generated using one specific configuration of the electrode 1 corresponding to FIG. 5E .

7A and 7B show cross-sectional views of two electrode assemblies that can be used.

8a to 8d show schematic views of four different electrode assemblies arranged in a plane parallel to the flow direction F;

9a to 9d show schematic views of four different electrode assemblies aligned in a plane perpendicular to the flow direction F;

10A-10E show various cross-sectional views of various trough members 30 .

11A-11H show schematic views of various trough members 30 , FIGS. 11C and 11D show atmospheric control device 35 and FIG. 11D show support device 34 .

12A and 12B show various atmospheric control devices 35 for locally regulating the atmosphere around the electrode set(s) 1 and/or 5 .

13 shows an atmospheric control device 38 for regulating the atmosphere around substantially the entire trough member 30 .

FIG. 14 shows a cross-sectional view of a set of control devices 20 positioned on a trough member 30 through which liquid 3 flows into a storage vessel 41 .

15A and 15B show various angles θ for the trough member 30.₁ and θ₂shows a cross-sectional view of

16a , 16b and 16c show schematic views of various control devices 20 of the electrode assembly 1 and/or 5 disposed on top of the trough member 30 .

16D, 16E, and 16F show AC transformer electrical wiring diagrams for use with various embodiments of the present invention.

Fig. 16G shows a schematic diagram of a transformer 60, and Figs. 16H and 16I show two sinusoidal in-phase and above schematics, respectively.

16J, 16K and 16L each show schematic views of eight electrical wiring diagrams using eight electrode sets, respectively.

Fig. 17a shows schematic diagrams of gold wires 5a and 5b used in the trough section 30b of Fig. 22a in connection with Examples 8, 9 and 10;

Fig. 17b shows a schematic diagram of gold wires 5a and 5b used in the trough section 30b of Fig. 21a in connection with Examples 5, 6 and 7;

17C shows the electrode configuration used to create sample GB-118 in Example 16.

17D-17F show the apparatus 20 for producing samples GB-139, GB-141 and GB-144 in Example 16 and used in Examples 1-4 for suspensions GT032, GT031, GT019 and GT033. .

17G, 17H, 17I and 7K show wiring diagrams used to control the apparatus 20 used in Examples 1-4 and 16. FIG.

17J and 17L show wiring diagrams used in the power supply 20 .

17M-17N show an alternative design for device 20 . The device 20 in FIG. 17N is used in Example 18.

18A and 18B show a primary trough member 30a, wherein one or more plasmas 4 are generated. The discharge of the primary trough member 30a flows to the secondary trough member 30b as shown in Figs. 19A and 19B.

19A and 19B show two different types of trough members in which wires are arranged using one transformer (Example 8-10) and using two transformers (Example 5-7). A schematic diagram of (30a and 30b) is shown.

Figures 20a-20h are alternatives to the arrangement shown in Figures 19a and 19b (with different electrode 5 wire arrangement and/or different numbers of electrodes), wherein the trough members 30a' and 30b' are adjacent have.

21A-21G illustrate various trough members 30b in conjunction with FIGS. 20A-20H and various embodiments.

22A and 22B show the trough member 30b in relation to FIGS. 19A, 19B and 20 and various embodiments.

23A-23D show various schematic and perspective views of an alternative trough embodiment used in Example 19.

Figure 24a shows a schematic diagram of an apparatus used in a batch process, in which in a first step a plasma 4 is generated to condition a fluid 3 .

24B and 24C are diagrams of an apparatus used in a batch method using wires 5a and 5b to produce nanocrystals in suspension (eg, colloid) as discussed in the Examples and in connection with the apparatus shown in FIG. 24A. A schematic diagram is shown.

25A shows a representative TEM micrograph of gold nanocrystals from dried suspension GD-007 produced according to Example 5. FIG.

25B shows a histogram of particle size distribution from TEM measurements of nanocrystals of suspension GD-007 produced according to Example 5;

25C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 5;

26A shows a representative TEM micrograph of gold nanocrystals from dried solution GD-016 produced according to Example 6.

26B shows the particle size distribution from TEM measurements for the nanocrystals produced according to Example 6.

26C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 6. FIG.

27A shows a representative TEM micrograph of gold nanocrystals from dried solution GD-015 produced according to Example 7.

27B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 7.

27C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 7. FIG.

28A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-018 produced according to Example 8. FIG.

28B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 8.

28C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 8. FIG.

29A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-019 produced according to Example 9. FIG.

29B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 9;

29C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 9. FIG.

30A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-020 produced according to Example 10.

30B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 10.

30C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 10. FIG.

31A shows a representative TEM micrograph of gold nanocrystals from dried solution 1AC-202-7 produced according to Example 11. FIG.

31B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 11. FIG.

31C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 11. FIG.

32A shows a representative TEM micrograph of gold nanocrystals from the dried solution GT-033 produced according to Example 4.

32B shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 4. FIG.

33A shows a representative TEM micrograph of gold nanocrystals from dried solution 1AC-261 produced according to Example 12.

33B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 12.

34A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-154 (20 Hz sine wave) produced according to Example 13. FIG.

34B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 13. FIG.

35A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-157 (40 Hz sine wave) produced according to Example 13. FIG.

35B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-157.

36A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-159 (60 Hz sine wave) produced according to Example 13. FIG.

36B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-159.

37A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-161 (80 Hz sine wave) produced according to Example 13. FIG.

37B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-161.

38A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-173 (100 Hz sine wave) produced according to Example 13. FIG.

38B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-173.

39A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-156 (300 Hz sine wave) produced according to Example 13. FIG.

39B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-156.

40 shows a schematic diagram of the electrical apparatus used to produce nanocrystals in solutions GB-166, GB-165, GB-162, GB-163 and GB-164.

41 shows a schematic diagram of the electric waves used in solutions GB-166, GB-165 and GB-162.

42A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-166 (60 Hz sine wave) produced according to Example 14. FIG.

42B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-166.

43A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-165 (60 Hz square wave) produced according to Example 14. FIG.

43B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-165.

44A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-162 (60 Hz triangle wave) produced according to Example 14. FIG.

44B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-162.

45 shows a schematic diagram of the triangular-shaped electric wave form used to generate samples according to GB-163 and GB-164.

46A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-163 (maximum duty cycle triangle wave) produced according to Example 15. FIG.

46B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-163.

47A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-164 (minimum duty cycle triangular wave) produced according to Example 15. FIG.

47B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-164.

48A1 shows representative TEM micrographs of gold nanocrystals from dried suspension GB-134 produced according to Example 16. FIG.

48A2 shows representative TEM micrographs of gold nanocrystals from dried suspension GB-134 produced according to Example 16. FIG.

48B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 16. FIG.

48C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 16. FIG.

49A-61A show dried samples GB-098, GB-113, GB-118, GB-120, GB-123, GB-139, GB-141, GB-144, GB- Shown are representative TEM micrographs of each of 079, GB-089, GB-062, GB-076 and GB-077.

49B-61B show dried samples GB-098, GB-113, GB-118, GB-120, GB-123, GB-139, GB-141, GB-144, GB- The particle size distribution histograms from TEM measurements for nanocrystals corresponding to 079, GB-089, GB-062, GB-076 and GB-077, respectively, are shown.

49c to 61c show samples GB-098, GB-113, GB-118, GB-120, GB-123, GB-139, GB-141, GB-144, GB-079, Dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals corresponding to GB-089, GB-062, GB-076 and GB-077, respectively, are shown.

54D shows the current versus time of GB-139 produced by Example 16. FIG.

54D, 55D and 56D show the measured current (in amperes) versus process time for samples GB-139, GB-141 and GB-144 produced according to Example 16;

61D shows 14 suspensions/colloids produced according to Example 16 (eg, GB-098, GB-113 and GB-118) over an interrogation wavelength range of about 250 nm-750 nm; (GB-120 and GB-123); (GB-139); (GB-141 and GB-144); (GB-079, GB-089 and GB-062); and (GB-076 and GB-077) UV-Vis spectral patterns, respectively.

Figure 61E shows the UV-Vis spectral pattern of each of the 14 suspensions over the interrogation wavelength range of about 435 nm-635 nm.

62A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-151 produced according to Example 18. FIG.

62B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-151.

63A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-188 produced according to Example 18. FIG.

63B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-188.

64A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-175 produced according to Example 18. FIG.

64B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-175.

65A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-177 produced according to Example 18. FIG.

65B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-177.

FIG. 66A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-176 produced according to Example 18. FIG.

66B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-176.

67A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-189 produced according to Example 18. FIG.

67B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-189.

68A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-194 produced according to Example 18. FIG.

68B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-194.

69A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-195 produced according to Example 18. FIG.

69B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-195.

70A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-196 produced according to Example 18. FIG.

70B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-196.

71A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-198 produced according to Example 18. FIG.

71B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-198.

72A shows a representative TEM micrograph of gold nanocrystals from dried solution GB-199 produced according to Example 18. FIG.

72B shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example GB-199.

72C shows 11 suspensions/colloids (eg, GB-151, GB-188, GB-175, GB-177, GB) produced according to Example 18 over the interrogation wavelength range of about 250 nm-750 nm. -176, GB-189, GB-194, GB-195, GB-196, GB-198 and GB-199) respectively show the UV-Vis spectral patterns.

72D shows the UV-Vis spectral patterns for each of the 11 suspensions over the interrogation wavelength range of about 435 nm-635 nm.

73A1 and 73A2 show two representative TEM micrographs of sample Aurora-020.

73B shows a histogram of particle size distribution from TEM measurements for nanoparticles corresponding to the dried sample Aurora-020.

73C shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanoparticles corresponding to sample Aurora-020.

74A1, 74A2 to 80A1, 80A2 are two representative TEMs of dried samples GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013, respectively. The micrograph is shown.

74B-80B show particle size distributions from TEM measurements for each of the nanocrystals corresponding to dried samples GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013. Show the histogram.

74C-80C show dynamic light scattering data for each of the gold nanocrystals corresponding to samples GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013 (e.g., hydrodynamic radius).

81A shows a perspective view of a comparative Breddick-Arc apparatus used to produce representative/comparative gold nanoparticles.

81B shows a cross-sectional view of a comparative Bredic-Arc device used to produce representative/comparative gold nanoparticles.

82A shows a representative TEM micrograph of gold nanoparticles from dried solution ARCG-05 produced according to Example 21. FIG.

82B depicts a particle size distribution histogram from TEM measurements for nanoparticles produced by ARCG-05.

83A-90A show representative TEM micrographs of eight comparative commercial colloidal gold products discussed in Example 22.

83B-90B show particle size distribution histograms from TEM measurements for nanoparticles corresponding to the eight comparative commercial colloidal gold products discussed in Example 22.

FIG. 90C shows a suspension of eight commercially available gold nanoparticles discussed in FIG. 22A over the interrogation wavelength range of about 250 nm-750 nm (Utopia Gold, SNG911219, Nanoparts, Nanocomposix 15 nm, Nanocomposix 10). nm, harmonic gold and meso-gold) respectively, the UV-Vis spectral patterns are shown.

FIG. 90D shows the eight commercially available gold nanoparticle suspensions discussed in FIG. 22A (Utopia Gold, SNG911219, Nanoparts, Nanocomposix 15 nm, Nanocomposix 10) over the interrogation wavelength range of about 435 nm-635 nm. nm, harmonic gold and meso-gold) respectively, the UV-Vis spectral patterns are shown.

91 shows a graph showing the zeta potential.

92 shows a graph depicting conductivity.

93 shows dynamic light scattering data (eg, hydrodynamic radius) for nanocrystal suspension GD-006 produced according to Example 23a.

94A-94D show graphs of the amounts of four different cytokines produced by human PBMCs upon antagonism by LPS in the presence of different amounts of GB-079.

95 shows a control, two experimental mixtures (eg GT-033 and GD-007), in which the experimental results measured were compared with those from a conventional steroid model (eg not measured in this model). Results from a collagen-induced arthritis ("CIA") model in mice shown are shown.

96A-D show representative photomicrographs of mouse paw joint cross-sections at various stages of arthritis.

97A-97E show representative micrographs of mouse paw joint cross-sections at various stages of arthritis.

FIG. 98 is a graph showing results from an experimental autoimmune encephalitis ("EAE") model in Biozzi mice showing the proportion of symptomatic animals in water for control group 1 versus GB-056 treatment group 2. do.

99 depicts a graph showing results from an experimental autoimmune encephalitis ("EAE") model in Biochi mice showing mean clinical disease scores for water in Control 1 vs. GB-056 Treatment Group 2. FIG.

100A-100E show representative TEM micrographs of gold nanocrystals from dried solution GB-056 produced according to Example 17;

101A shows a histogram of particle size distribution from TEM measurements for gold nanocrystals produced according to Example 17. FIG.

FIG. 101B shows dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 17. FIG.

102A-D show representative TEM micrographs of the same gold nanocrystals from the dried solution GB-056 produced according to Example 17 after serving as a test compound for 24 hours in the EAE test of Example 26.

103a shows a histogram of particle size distribution from TEM measurements for gold nanocrystals produced according to Example 17 after serving as a test compound for 24 hours in the EAE test of Example 26. FIG.

103B depicts dynamic light scattering data (eg, hydrodynamic radius) for gold nanocrystals produced according to Example 17 after serving as a test compound for 24 hours in the EAE test of Example 26. FIG.

104A-104C show representative TEM micrographs of the same gold nanoparticles from the dried solution GB-056 produced by Example 17 after serving as a test compound for 24 hours in the EAE test of Example 26. .

105A shows a histogram of particle size distribution from TEM measurements for nanocrystals produced according to Example 17 after serving as a test compound for 24 hours in the EAE test of Example 26. FIG.

106 depicts the mean weight gain of all mice over the long-term experiment according to Example 27.

107 depicts the average amount of treatment and control fluid consumed for all mice over the long run according to Example 27.

108 depicts the mean weight gain of all mice over 35 days by Example 28.

109 depicts the average amount of treatment and control fluid consumed in all mice over 35 days according to Example 28.

Figure 110 shows the amount of gold present in the feces of mice according to Example 28.

111 shows the amount of gold present in the urine of mice according to Example 28.

Figure 112 shows the amount of gold present in the organs and blood of mice according to Example 28.

<u>DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS</u>

I. <u>Novel Gold Nanocrystals </u>

Novel gold nanocrystals having a nanocrystalline surface substantially free of organic or other impurities or films are provided. Specifically, the surface is "clean" compared to those produced using chemical reduction processes that require chemical reducing agents and/or surfactants to form gold nanoparticles from gold ions in solution. The novel gold nanocrystals are produced by the novel manufacturing procedure detailed herein. The novel manufacturing procedure avoids the conventional use of added chemical reducing agents and/or surfactants (eg organic compounds) or other agents that are usually entrained in or on the particles or coated on the surface of the chemically reduced particles. Exclusion or stripping or removal of reducing agents using undesirable processes that themselves affect the particles.

In a preferred embodiment, the method is a "process enhancer" or "processing enhancer" (usually an inorganic material or carbonate or the like) that does not significantly bind to the formed nanocrystals, but rather promotes nucleation/growth during the electrochemical-stimulated growth process. ) nucleation and growth of gold nanocrystals in water containing Process enhancers play an important role in methods that involve providing charged ions in an electrochemical solution that allow crystals to grow. Process enhancers critically remain in solution and/or do not form coatings (eg organic coatings) and/or do not adversely affect the formed nanocrystals or the formed suspension(s) and/or decompose, evaporate or the compound(s) that are not lost. A preferred process enhancer is sodium bicarbonate. Examples of other process enhancers include sodium carbonate, potassium bicarbonate, potassium carbonate, trisodium phosphate, disodium phosphate, monosodium phosphate, potassium phosphate or other salts of carbonic acid. Additional process enhancers include salts, including sodium or potassium, of bisulfites or sulfites. Still other process enhancers for producing gold nanocrystals for medical applications under certain conditions may be sodium or other salts including potassium, which are not substantially incorporated into or on the surface of the gold nanocrystals. , does not impart toxicity to nanocrystals or suspensions containing nanocrystals.

A preferred concentration range for a processing enhancer is typically 0.01 to 20 grams/gallon (0.0026 to 2.1730 mg/ml), more typically 0.1 to 7.5 grams/gallon (0.0264 to 1.9813 mg/ml), most typically 0.5 to 2.0 grams per gallon (0.13210 to 0.5283 mg/ml).

Because the grown gold nanocrystals have a "bare" or "clean" surface of gold metal (eg, in a zero oxidation state), the surface is either highly reactive or biocatalytic (and also highly bioavailable). ). The nanocrystals are substantially surrounded by a water jacket. This feature provides increased in vivo efficiency for nanoparticle surfaces containing organic matter present, for example, from reducing chemical processes. "Clean" surfaces may also reduce the toxicity of nanocrystals to nanoparticles comprising coated or "dressed" surfaces. This increased efficiency of "clean" gold nanocrystals can provide an increased therapeutic index at lower dosages to achieve a therapeutic effect. A comparative mouse model example (Example 25) herein compares a gold nanocrystal suspension of the present invention to auranofin, a commercially available, FDA-approved gold drug. This example shows that these novel gold nanocrystals are at least 5-fold more active than auranofin in a well-tolerated collagen-induced arthritis model with inflammation in rheumatoid arthritis in mice.

Specifically, a comparative mouse model (Example 25) compares the efficacious dosage level using the crystal suspension of the present invention with the efficacious dosage level using the commercially available, FDA-approved gold drug auranofin. Example 25 shows that these novel gold nanocrystals were at least 17-fold lower than the effective dosing level of auranofin in a well tolerated collagen-induced arthritis model of inflammation in mice, and included in the effective dosing level of auranofin. It shows that efficacy is achieved at dosing levels 5 times lower than the gold content. So, comparing the relative potency level of the novel gold nanocrystals with the gold content of the gold-based drugs auranofin and auranofin alone, the comparative potency of the novel gold nanocrystals is 5 times greater than that of the gold contained in auranofin and higher than that of auranofin. 17 times larger.

This efficacy benefit could be achieved at much lower levels of therapeutic efficacy (17-fold lower dose than auranorphine, 5-fold lower dose than the gold contained in auranorphine), or alternatively, potentially much greater efficacy. This means that it can be achieved at equivalent dosage levels.

There are other important advantages of the novel nanocrystals in two other dimensions: the relative rate of onset of benefits and the relative toxicity. With regard to both the relative toxicity observed and the relative rate of onset of the observed benefits, the novel gold nanocrystals in animal models differ significantly and significantly differ from auranofin, the only orally administered and FDA-approved gold pharmaceutical product in the prior art. surpass

In a preferred embodiment, the nanocrystals are not dried prior to use, but are instead used in the liquid or concentrate from which they are formed (eg to form a suspension) or a reconstituted concentrate thereof. Complete removal (eg, complete drying) of these crystals from their suspension may in certain cases affect the surface properties of the crystals (eg, partial oxidation may occur) and/or may, for example, remove the initially formed water jacket. It appears to be capable of altering it to affect its ability to rehydrate crystals. This suggests that it may be optimal to use sterile pharmaceutical grade water (eg USP) and the process enhancer described above in the manufacturing method.

The gold nanocrystals produced by the present invention can also be used in industrial applications where gold reactivity is important (eg, catalytic and/or electrochemical methods), but pharmaceutical grade products are not required. When produced for non-pharmaceutical use, gold nanocrystals can be produced in a variety of solvents and using a wider variety of process enhancers, depending on the application. According to the method of the present invention, gold nanocrystals have unique and identifiable surface features, such as spatially extended low index crystal planes {111}, {110} and/or {100} and groups of these planes (and their counterparts). can be grown in a way that provides of the shape of the gold nanocrystals produced by the method described herein.Non-limiting examples include triangular (eg, tetrahedron), pentagonal (eg, pentagonal bipyramid or decahedron), hexagonal (eg, hexagonal bipyramid, icosahedron, octahedron), rhombus (eg, octahedron, various elongated bipyramids, fused tetrahedrons, sides of bipyramids), and "others". The proportion of nanocrystals (eg, grown according to the various embodiments described herein) having a spatially extended low index crystal facet and having a "clean" surface described above is another novel feature of the present invention. In addition, the proportions of tetrahedra and/or pentagonal bipyramids formed in or present in the nanocrystalline suspension are also unique.

In a preferred embodiment, the proportion of the pentagonal bipyramid is at least about 5% or within the range of about 5% to 35%, more typically at least about 10% or within the range of about 10% to 35%, even more typically at least about 15% or in the range of about 15% to 35%, more typically at least about 25%, and in some cases at least about 30%.

In another preferred embodiment, the proportion of tetrahedra is at least 5% or within the range of about 5% to 35%, more typically at least about 10% or within the range of about 10% to 35%, even more typically at least about 15%. % or within the range of about 15% to 35%, more typically at least about 25% and in some cases at least about 30%.

Further, the combination of pentagonal bipyramid and tetrahedron is at least about 15% or within the range of about 15% to 50%, more typically at least about 20% or within the range of about 20% to 50%, even more typically at least about 30%. % or within the range of about 30% to 50%, more typically at least about 35% and in some cases at least about 45%.

Further, the combination of pentagonal bipyramid, tetrahedron, octahedron, and hexagon is at least about 50%, or within the range of about 50% to 85%, more typically at least about 60%, or within the range of about 60% to 85%. , even more typically at least about 70% or within the range of about 70% to 85%, more typically at least about 70%, and in some cases at least about 80%.

100 Any desired average size of sub-nm gold nanocrystals may be provided. The most preferred crystalline size ranges are those in which the average crystal size or "mode" (measured and determined by certain techniques detailed herein and reported as "TEM mean diameter") is predominantly less than 100 nm, more typically less than 50 nm. , more typically less than 30 nm, and in many of the preferred embodiments disclosed herein, the mode for the nanocrystal size distribution is less than 21 nm, and an even more preferred range is from 8 to 18 nm.

The resulting gold nanocrystalline suspension or colloid must or must be adjusted to obtain the desired pH range. For example, when produced using the sodium bicarbonate process enhancer in the amounts detailed herein, the pH range is typically 8 to 9, which can be adjusted as desired.

The nature and/or amount of surface charge (eg, positive or negative) on the formed nanoparticles or nanocrystals can significantly affect the behavior and/or effect of the nanoparticles/suspensions or colloids. For example, a protein corona, such as an albumin corona formed in vivo, can be affected by the surface charge or surface properties of the nanoparticles. This surface charge is commonly referred to as "zeta potential". It is known that the greater the zeta potential (positive or negative), the greater the stability of the nanoparticles in solution (eg, the more stable the suspension). The performance of the nanoparticle suspension can be controlled by controlling the nature and/or amount of surface charge of the formed nanoparticles or nanocrystals.

Zeta potential is known as a measure of the electrodynamic potential in colloidal systems and is also referred to as the surface charge on a particle. The zeta potential is the potential difference between the fluid in which the particles are dispersed and the stationary layer of the fluid. Zeta potential is often measured in millivolts (eg, mV). A zeta potential value of about 20-25 mV is any value chosen to determine whether the dispersed particles are stable in the dispersion medium. Thus, when referring to "zeta potential" herein, it should be understood that the referred to zeta potential is a quantification or description of the amount of electrical charge present in the bilayer.

The zeta potential is calculated from the electrophoretic mobility by the following Henry equation:

<img file="KR20120052967A_D0005.tif" />

In the above formula, z is the zeta potential, U_Eis the electrophoretic mobility, ε is the dielectric constant, η is the viscosity, and f(kα) is a function of Henry. For the Smoluchowski approximation, f(kα)=1.5.

The Smolukowski zeta potential ("ZP") for gold nanocrystals produced by the method of the present invention is typically at least -20 mV, more typically at least about -30 mV, even more typically at least about - 40 mV, even more typically at least about -50 mV.

II. <u>new</u><u> Uses of Gold Nanocrystals</u>

The gold nanocrystals of the present invention are known to be effective in gold therapy and can be used to treat any disease, including a wide range of inflammatory and autoimmune diseases, as well as certain infectious diseases and cancer. Descriptions of many of these uses will be presented in greater detail in the background of the invention above or below.

The subject to be treated may be a human or other animal, such as a mammal. Non-human subjects include, but are not limited to, primates, domestic animals (eg, sheep, cattle, horses, pigs, goats), domestic animals (eg, dogs, cats), birds and other animals (eg, mice, rats, guinea pigs) , rabbits).

Importantly, it is surprising that it was discovered as part of the present invention that gold nanoparticles (and particularly gold nanocrystals detailed herein) inhibit macrophage migration inhibitory factor ("MIF"). It has been found that this is the first disclosure of such activity of gold nanoparticles and can provide a scientific basis for understanding the scope of medicinal uses for gold compositions to this day. In addition, it provides a scientific basis for concluding that gold nanoparticles are effective against other diseases mediated by macrophage migration inhibitory factors. It was also found that these gold nanocrystals inhibit IL-6 but not IL-10. Because MIF and/or IL-6 appear in a variety of pathologies and/or biological signaling pathways, these findings suggest that novel gold nanocrystals arise from pathological cellular activation in diseases or conditions, such as inflammatory (including chronic inflammatory) It is determined that treatment of the condition, autoimmune condition, particular infection, hypersensitivity reaction, and/or cancerous disease or condition is effective for prophylaxis.

MIF is a macrophage-derived multifunctional cytokine that is initially important in many pro-inflammatory conditions. MIF was initially described as a product of activated T-lymphocytes that inhibit random migration of macrophages. While MIF was initially shown to activate macrophages at inflammatory sites, MIF was found to mediate various signaling agents in the immune system. MIF has been known to be expressed in diseases or conditions in humans and animals, including infections, inflammations, wounds, ischemia and/or malignancies. MIF appears to play an important role in cell proliferation, cell differentiation, angiogenesis and wound healing. MIF also appears to mediate glucocorticoid (steroid) activity against at least part of its anti-inflammatory effects.

As can be seen from Examples 25 and 26, the nanocrystalline compositions of the present invention are highly effective in animal models for CIA and EAE. The link between these two animal models (as well as human disease states) is the presence of MIF.

Recent experiments have shown that monoclonal antibody antagonism of MIF may be useful in the treatment of sepsis, certain types of cancer and delayed types of hypersensitivity. Sepsis appears to be induced by inflammation and an overreaction of the immune system. In certain infections, upon attack by microorganisms, the innate immune system responds first, thereby recruiting neutrophils, macrophages and natural killer cells ("NK cells"). Thus, cytokines (and MIFs) play an important role as mediators regulating the activation and differentiation of these cells. Finally, the innate immune system reacts with the adaptive immune system by these and other stimulatory molecules, where in addition to providing pathogen-specific protection, the adaptive immune system has the ability to construct immune memory.

MIF appears as a major mediator in sepsis as it stimulates the production of TNF, other pro-inflammatory cytokines and eicosanoids, and induces the expression of TLR-4, which recognizes LPS and inhibits activation of the innate immune response. seems to do MIF and glucocorticoids act as antagonists and are responsible, at least in part, for the modulation of the inflammatory response. MIF has an inhibitory effect on glucocorticoids that normally inhibit inflammation.

Therapeutic antagonism of MIF may provide a "steroid-sparing" effect or even be therapeutic in "steroid-resistant" disease. Unlike other pro-inflammatory molecules, such as certain cytokines, expression and/or release of MIF is coupled to (eg may be triggered by) glucocorticoids. MIF appears to be able to antagonize the effects of glucocorticoids. MIF plays a major role in the regulation of pro-inflammatory cytokines. This appears to be the case for macrophages that secrete TNF, IL-Iβ, IL-6 and IL-8. MIF also modulates IL-2 excretion. MIF also plays a role in regulating T cell proliferation. In vivo, MIF can be used in endotoxic shock and experimental arthritis [eg, collagen-induced arthritis or "CIA" models, such as those used herein in the Examples below, and colitis, multiple sclerosis (eg, Examples) 26), models of immune diseases and other inflammatory conditions, including atherosclerosis, glomerulonephritis, uveitis and certain cancers].

Additionally, MIF recently appears to be important in the regulation of leukocyte-endothelial interactions. White blood cells interact with vascular endothelial cells to obtain excretion from the vasculature into the tissue. The role of MIF in this process has been demonstrated to influence leukocyte-endothelial adhesion and migration. This process appears to be a substantial part for almost all inflammatory diseases and also for diseases less differentiated as inflammatory, including, for example, atherosclerosis.

MIF is also expressed in plants (hence "MIF" is also referred to as plant MIF) and, where appropriate, a gold nanocrystal suspension of the present invention (eg, aqueous gold-based metal nanocrystals and/or gold nanocrystals and other metal(s) ) and/or alloys of gold nanocrystals with other metal(s) and/or combination therapy approaches) can be used in plant/agricultural applications such as crop control.

MIF is a key cytokine in transforming the nature of the immune response. The immune response has two effector mechanisms. The Th1 immune response produces cytotoxic T cells that kill pathogens and damaged/dysfunctional cells. The Th2 response produces antibodies that promote phagocytosis and activate complement. The role of MIF in determining polarization in the immune system depends on other cytokines such as IL-10. IL-10 is an effective anti-inflammatory cytokine that blocks the action of MIF on Th1 cells and results in the generation of a Th2 response. In the absence of IL-10, MIF stimulates Th1 cells to produce a cytotoxic response. IL-10 is produced by monocytes and B cells in response to stimuli, whereas MIF is independently produced and stored, for example, in the pituitary gland and T cells. Therefore, MIF plays an important role in both T cytotoxic cell mediated diseases such as rheumatoid arthritis and Crohn's and antibody mediated diseases such as idiopathic thrombocytopenia.

Without wishing to be bound by any particular theory or explanation, when referring to "one or more signaling pathway(s)" herein, MIF or one or more proteins associated with MIF (eg, a receptor site, such as a CD74 receptor site) involved in the innate immune system (e.g., NK and phagocytic cells, complement proteins (e.g., C5a) and/or inflammatory pathways) and the adaptive immune system (e.g., T cell dependent cytotoxicity (Th1) and antibody (Th2) pathways) should be understood to mean For example, when MIF is involved in the Th1 signaling pathway that produces T cytotoxic cells, other proteins such as IL6, TNF and other cytokines are also involved.

When the Th1 signaling pathway is overactive, various diseases include, for example, rheumatic diseases, connective tissue diseases, vasculitis, inflammatory conditions, vascular diseases, eye diseases, lung diseases, cancer, kidney diseases, neurological diseases, complications of infectious diseases, allergies disease, bone disease, skin disease, type 1 diabetes, Crohn's disease, MS and gastrointestinal diseases. Thus, chronic disease conditions can be alleviated by reducing the amount of MIF action associated with this particular Th1 signaling pathway.

Conversely, without wishing to be bound by any particular theory or explanation, when the Th2 signaling pathway is overactive, various antibodies are produced, for example, hemolytic anemia, ITP (idiopathic thrombocytopenic purpura), neonatal hemolysis, etc. cause diseases, including Moreover, such hyperactivity of the Th2 signaling pathway can lead to underactivity of the Th1 pathway, leading to the growth of various parasites or cancers. For example, in the case of malaria, overproduction of one or more homologues of MIF results in the generation of an ineffective, null antibody response against the parasite (e.g., bacteria with various crystalline forms or homologues of MIF (or its equivalent); produced or presented by parasites, viruses, fungi, etc., wherein each may have a different responsiveness to, for example, "normal" human MIF and alter the host immune response to create at least a local environment of "immune privilege" What can be done is reasonable]. Thus, by reducing the amount of MIF action associated with this particular Th2 signaling pathway, other disease conditions may be alleviated.

Additionally, without wishing to be bound by any particular theory or explanation, MIF also plays a role in driving signaling pathways involved in innate immunity. Such pathways include activation of natural killer ("NK") cells, phagocytes and other non-specific pathogen cell types and certain proteins, such as complement proteins (eg, C5a). Excess MIF (and/or MIF homologues) or a similar effect thereof can lead to undesirable overexpression or overreaction in certain signaling pathways as seen in multiple organ failure as a result of sepsis. An example of this is systemic inflammatory response syndrome (SIRS). Thus, by reducing the amount of MIF activity associated with this particular signaling pathway, many inflammatory diseases can be alleviated.

Thus, when endogenous MIF is present (e.g., in excess under local environmental conditions), for example as determined by known bodily fluid measurement techniques such as ELISA, spectroscopy, etc., one or more innate or adaptive immune system signaling pathways are inflammatory/immune The component may be overexpressed, overactivated or overproduced. Human disease can occur, for example, when one or more forms of MIF present cause the production of an excess T cytotoxic response or an excess antibody response or an exaggerated NK/phagocytic cell response. For example, when too many T cytotoxic cells are expressed, a variety of chronic inflammatory conditions can occur. Similarly, when excess Th2 or innate responses are promoted by MIF, other diseases occur.

In addition, it is also known that malaria parasites and other parasites such as nematodes and filamentous worms and some cancers produce certain types of exogenous or unregulated MIF or MIF homologues. Again, without wishing to be bound by any particular theory or explanation, exogenous expression of MIF or a homologue thereof results in stimulation of the Th2 signaling pathway, and an immune response such that the specific activated signaling pathway is not detrimental to tumors or parasites, etc. It has been shown that this can be an attempt by a parasite or tumor (eg, an "intruder") to create a condition that is activated by MIF or its homologues.

In the context of malaria parasites, for example, parasites can present exogenous MIFs, resulting in the production of antibodies rather than T cytotoxic cells, thereby stimulating the Th2 signaling pathway. However, such antibodies usually do not damage the parasite. Therefore, the parasite appears to produce at least a local area of immune privilege. In this regard, if alternative pathways, such as the Th1 pathway or the natural killer (NK) cell pathway, are reactivated, damage may occur to the parasite (eg, the immune system may clear the parasite). However, when excess antibody or other immune/inflammatory products are produced, for example as a result of selective activation of the Th2 pathway, the excess antibody can cross-link to various cellular sites or activate other immune molecules. do. When such crosslinking or activation occurs, a significant inflammatory response can occur. While not wishing to be bound by any particular theory or explanation, this inflammatory response may precisely be a response that occurs in malaria-infected women who are pregnant and susceptible to severe malaria and malaria anemia. Pregnant women have been found to be particularly sensitive to this effect due to the immune effect of the placenta in promoting the Th2 response and in sequencing parasites in the immune-privileged zone.

Again, without wishing to be bound by any particular theory or explanation, cancer cells also explicitly express MIF in an attempt to at least partially modulate the immune response thereto and/or promote their own growth. In this regard, there also appears to be a desire to engineer cancer cells such that the immune system follows the Th2 signaling pathway, as opposed to the Th1 signaling pathway that damages or kills cancer cells. For example, by causing a local immune privilege to be generated, there is no (or little) a particular risk to cancer cells. Conversely, when MIF seeks to stimulate the Th1 signaling pathway, a cytokine cell/inflammatory response can occur, resulting in damage or death of cancer cells (eg, tumors can be eliminated naturally by the immune system).

Again, without wishing to be bound by any particular theory or explanation, children have an immature immune system, particularly innate and Th1 pathways. Immaturity in some children results in altered MIF metabolism. Thus, modulation of MIF in children appears to result in the prevention or amelioration of infectious or inflammatory diseases.

Thus, without wishing to be bound by any particular theory or explanation, the gold nanocrystal suspensions of the present invention may inhibit one or more signaling pathways (eg, Th1 signaling pathways, Th2 signaling pathways and/or innate immune pathways) alone. or other therapies that modulate signaling pathways. Thus, various immune turn-on and/or turn-off can be made by interacting with or modulating MIF (or MIF homologue) associated with one or more signaling pathway(s). can Thus, the response along with the Th1 and Th2 signaling pathways for the production of T cytotoxic cells or antibodies can be turned on and/or turned off. (eg, the Th1-Th2 switch can be modulated to carry out some of the immune pathways that are activated). Similarly, the innate immune system and the resulting inflammation can be turned on or turned off.

One With the knowledge that more than one signaling pathway can be turned on/off, very important therapeutic interventions can occur. For example, endpoints of various surrogates can be monitored or investigated for a variety of different diseases, including, for example, many cancers. For example, an antigen, "cancer-embryonic antigen" or "CEA" is a known surrogate marker for the amount of tumor or amount of tumor burden present in a variety of different cancers. For example, it is known that the higher the amount of CEA, the more tumors associated with ovarian cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, and the like. In this regard, the amount of cancer-embryonic antigen can be determined by, for example, drawing blood and testing for the presence of CEA by known techniques including ELISA and certain spectrophotometric techniques. In this regard, once blood is drawn and measurements are taken to determine the amount of CEA, the degree of treatment required (eg, dosing, duration, and/or amount) can be obtained by monitoring changes in the amount of CEA measured. have. For example, when 15 to 45 ml of 10 ppm product is taken 2-3 times a day, monitoring the amount of CEA may result in an increase or decrease in the dose depending on the desired result.

Likewise, prostate cancer has a known surrogate endpoint of "prostate-specific antigen" or "PSA". These surrogate endpoints can also be monitored by drawing blood and examining it by ELISA techniques.

In addition, various cancers, such as melanoma (eg, eye cancer, etc.) also express antigens for example against "GP100" and/or "Melan-A". These surrogate endpoints can also be determined by taking blood from a patient and measuring the amount of antigen present by a similar ELISA or spectrophotometric technique. In all such cases, the presence of the antigen can cause an increase/decrease in the amount of therapeutic treatment given.

"Table A" below lists a number of known "tumor markers" and associated cancers, as well as sites from which biological samples were taken to measure these markers.

<u>Table A</u>

<img file="KR20120052967A_D0006.tif" />

Additionally, various diseases of immune and inflammatory dysfunction, such as rheumatoid arthritis and Crohn's disease, can be assessed using inflammatory markers such as C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). These surrogate endpoints can also be determined by drawing blood from a patient and then measuring by visual ELISA or spectrophotometric techniques for the amount of marker present. In all such cases, changes in inflammatory/immune markers may result in an increase/decrease in the amount of therapeutic treatment given.

Additionally, various antibody-based diseases such as hemolytic anemia or rhesus disease can be monitored by the concentration of specific antibodies present. Such surrogate endpoints can also be determined by taking blood from a patient and measuring the amount of antibody present by a similar ELISA or spectrophotometric technique. In all cases, the presence of the antibody can result in an increase/decrease in the amount of therapeutic treatment given.

Inhibitors or modulators of MIF and/or one or more of the signaling pathway(s) of MIF may also be used in implantable devices, such as stents. Accordingly, in a further embodiment, the present invention provides

(i) a reservoir containing one or more compounds of metal-based compounds, including gold solutions or colloids and mixtures and alloys thereof; and

(ii) an implantable device, preferably a stent, comprising a means for discharging or eluting the inhibitor or modulator from the reservoir.

Therefore, according to the present invention, the nanocrystalline gold-based therapy of the present invention has desirable efficacy against various autoimmune diseases, tumors or chronic or acute inflammatory conditions or diseases, diseases, syndromes, conditions, tendencies or predispositions selected from the group comprising There are various signs of having:

Rheumatoid disease (including, but not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, Still's disease), spondyloarthropathies (including but not limited to, ankylosing spondylitis, reactive arthritis, Reiter's syndrome), arthropathy crystalline (non-limiting examples) , gout, pseudogout, calcium pyrophosphate deposition disease), Lyme disease, polymyalgia rheumatica;

connective tissue diseases (including, but not limited to, systemic lupus erythematosus, systemic sclerosis, scleroderma, polymyositis, dermatomyositis, Sjogren's syndrome);

vasculitis (including, but not limited to, polyarteritis nodosa, Wegener's granulomatosis, Chog-Strauss syndrome);

inflammatory conditions or tendencies, including as a result of trauma or ischemia;

sarcoidosis;

vascular diseases, including atherosclerotic plaque vascular disease and infarction, atherosclerosis and vascular occlusive disease (including but not limited to atherosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease) and vascular stent restenosis;

eye diseases, including uveitis, corneal disease, iritis, iridocyclitis and cataracts;

autoimmune diseases (including, but not limited to, diabetes, prostatitis, myasthenia gravis, sclerosing cholangitis, primary biliary cirrhosis);

lung diseases (including, but not limited to, diffuse interstitial lung disease, pneumoconiosis, fibrosing alveolitis, asthma, bronchitis, bronchiectasis obstructive pulmonary disease, chronic obstructive pulmonary disease, adult respiratory distress syndrome);

cancers that are primary or metastatic (including, but not limited to, prostate cancer, colon cancer, bladder cancer, kidney cancer, lymphoma, lung cancer, melanoma, multiple myeloma, breast cancer, gastric cancer, leukemia, cervical cancer and metastatic cancer);

kidney diseases including glomerulonephritis, interstitial nephritis;

diseases of the hypothalamic-pituitary-adrenal axis;

neurological diseases including multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease;

diseases characterized by altered angiogenesis (eg, diabetic retinopathy, rheumatoid arthritis, cancer) and endometriosis;

infectious diseases including, but not limited to, bacterial, parasitic or viral, including HIV, HBV, HCV, tuberculosis, malaria and worms (including FDA-designated neglected diseases in developing countries);

complications of infectious diseases including endotoxin (septic) shock, exotoxin (septic) shock, infectious (true sepsis) shock, complications of malaria (eg, cerebral malaria and anemia), infections and other complications of pelvic disease;

transplant rejection, graft-versus-host disease;

allergic diseases including allergies, atopic diseases, and allergic rhinitis;

bone diseases (eg, osteoporosis, Paget's disease);

skin diseases including psoriasis, eczema, atopic dermatitis, UV(B)-induced dermal cell activation (eg, sunburn, skin cancer);

diabetes and its complications;

pain, testicular dysfunction and wound healing;

Gastrointestinal diseases including, but not limited to, inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease), peptic ulcer disease, gastritis, esophagitis, liver disease (eg, but not limited to, sclerosis and hepatitis).

In one embodiment, the disease or condition is rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, psoriasis, eczema, uveitis, diabetes, glomerulonephritis, atherosclerotic vascular disease and infarction, asthma, chronic obstructive pulmonary disease, HIV, HBV, HCV, tuberculosis, malaria, insect and cancer(s).

III. <u>pharmaceutical composition</u>

Also provided are pharmaceutical compositions comprising an effective amount of gold nanocrystals for treating any medical condition described herein. In a preferred embodiment, the gold nanocrystals are administered as an orally delivered liquid, wherein the gold nanocrystals remain in the water of manufacture that can be concentrated or reconstituted, while the surface of the gold nanocrystals is a surface modified from the original state of manufacture. It is preferable not to dry to the point of having or completely drying.

Experiments have shown that the gold nanocrystals of the present invention contain substantially more gold than prior art gold-based materials, including both FDA-approved gold-based pharmaceutical products and non-FDA-approved gold colloids, due to their clean, highly active crystalline surface. appeared to be in a valid form. Because of this, it is expected to be able to use significantly reduced dosages of nanocrystals than the dosage levels required by conventional compositions, including the oral gold product auranofin.

For example, in the well-recognized mouse model of collagen-induced arthritis, the standard dosage is 40 mg/kg/day of auranofin, which is about 1 mg/mouse/day, and 0.30 mg gold/day of auranorphin contained in auranofin. it is gold It has been shown that this standard auranofin dosage level produces a response equivalent to that resulting from a dosage of about 0.06 mg/day of gold nanocrystals of the present invention (Example 25). So, in these experiments, the nanocrystals are calculated to be 17 times more numerous than auranofin and 5 times more effective than the gold species contained in auranofin.

The standard FDA-approved dosing level for auranofin in humans is 6 mg/day or 0.9 mg/kg/day. The gold contained in human dosage levels of auranofin is 1.74 mg or 0.025 mg/kg. For the relative potency of the novel gold nanocrystals compared to auranofin, as demonstrated in a live animal model, the approximate human dose level for the novel gold nanocrystals is equivalent to the relative potency of 17X the human dose level for auranofin. It can be calculated by dividing by the factor or dividing the human dose level by the relative potency factor of 5X. This results in an approximate human dosing level of 0.35 mg/day of new gold nanocrystals versus the required 6 mg/day for auranofin and 1.74 mg/day required for the gold contained in auranofin, 70 kg For humans, 0.35 mg/day would result in a dosage of 0.005 mg/kg/day.

It is normal to generate dosage levels to achieve sizes up to 10 times the predetermined range around the expected mg/kg dosage. In this case, if the approximate suggested base dosage is 0.348 mg/day, which is 1/17 of the base dosage of auranofin, or 0.005 mg/kg/day, this means that the effective dosage range for auranofin-type efficacy using the novel nanocrystals is It is suggested that greater efficacy can be achieved at dosage levels of 0.005 mg/kg/day, even at levels within the range of 0.01 mg/kg/day or 0.25 mg/kg/day.

It is important to realize that pharmaceutical products aim to achieve the minimum dosage necessary to achieve efficacy, thereby minimizing the potential for toxicity or complications. A novel orally administered product with significantly greater efficacy may achieve lower dosage levels than conventional products and/or may achieve substantially greater efficacy at equivalent dosage levels.

Furthermore, in animal experiments, the toxicity level of the novel nanocrystals was observed to be low even at the maximum dosage level, meaning that it is less toxic than a conventional product, such as auranofin, even at higher dosage levels.

In addition, it was observed that the therapeutic effect of the nanocrystals of the present invention appeared faster than auranofin because the onset of action was usually several weeks compared to several days in the case of the nanocrystals of the present invention (see Example 25). This is a major benefit in use, as it means that patients get remission earlier and are more likely to continue to adhere to the regimen and thus continue to benefit from the product.

It was further observed that the gold nanocrystals of the present invention had a better therapeutic index than auranofin due to the lower dosage and associated lower toxicity in achieving efficacy.

In addition, in order to have practical value as a pharmaceutical treatment, it is recognized that the product can be manufactured under high pharmaceutical grade manufacturing, sourcing and quality control standards as stipulated by the FDA as Good Manufacturing Practice (GMP). it is important to do

Conventional gold nanoparticles are produced by a variety of methods, most of which include a chemical reduction process. It has been shown that no conventional chemical reduction or other conventional methods for the preparation of GMP compliant gold nanoparticles exist, and given the nature of these methods, possibly GMP compliance is extremely challenging, It has been shown to require significant time, money and ingenious engineering skills. The method by which the novel gold nanocrystals of the present invention are produced is designed to be GMP compliant to achieve another major difference and advantage of the gold nanocrystals of the present invention.

A clinical trial is required to confirm a therapeutically effective dosage, and 0.05 mgs or more (or 0.1, 0.5, 1.0, 2.0 mg or more) to 10 mg or more with respect to the dosage (once, twice or multiple times a day) It is reasonable to conclude that dosing within the range is effective for treating any of the conditions described herein in humans. Given the low toxicity of these gold nanocrystals, for more problematic diseases, use at higher dosage levels including, but not limited to, 10 mgs or more, such as 20 mg or more per dose, is appropriate.

Any concentration of gold nanocrystals may be provided by the present invention. For example, the concentration of such gold nanocrystals may range from a few ppm (eg, μg/ml or mg/l) to several hundreds of ppm, but typically 2 to 200 ppm (eg, 2 μg/ml to 200 μg/l). ml), more often in the range of 2 to 50 ppm (eg, 2 μg/ml to 50 μg/ml). Typical convenient concentrations are in the range of about 5 to 20 μg/ml, more typically about 8 to 15 μg/ml.

Systemic or topical use, including oral, intravenous, subcutaneous, intraarterial, buccal, inhalation, aerosol, propellant or other suitable liquid and the like, as further described herein, including the specific gels or creams discussed in Example 23. A pharmaceutical composition suitable for

Alternatively, suitable dosages of the active ingredient may range from about 0.1 ng/kg body weight to about 1 g/kg body weight per dose. The dosage is usually in the range of 1 μg to 1 g per kg body weight per dose, such as in the range of 1 mg to 1 g kg body weight per dose. In one embodiment, the dosage is in the range of 1 mg to 500 mg/kg body weight per dose. In another embodiment, the dosage is in the range of 1 mg to 250 mg/kg body weight per dose. In another preferred embodiment, the dosage is from 1 mg/kg body weight to 100 mg/kg body weight per dose, such as up to 50 mg/kg body weight per dose. In yet another embodiment, the dosage is in the range of 1 μg to 1 mg per kg body weight per dose.

Appropriate dosages and dosing regimens can be determined by the attending physician or veterinarian, and include the desired level of inhibitory and/or modulating activity, the particular condition being treated, the severity of the condition, whether the dosing is prophylactic or therapeutic, as well as the individual subject. may depend on the overall age, health and weight of

For example, gold nanocrystals contained in an aqueous medium, colloid, suspension, foam, gel, paste, liquid, cream, etc. may be administered in a single dose or in a series of doses. For example, an aqueous medium comprising metallic nanocrystals may be administered alone in a colloidal formulation, which is acceptable to include active ingredient mixtures with other compositions and/or therapies. Additionally, various pharmaceutical compositions may be added to the active ingredient(s)/suspension(s)/colloid(s).

Thus, typically gold nanocrystal suspensions or colloids of the invention (eg, aqueous gold-based metals and/or mixtures of gold and other metal(s) and/or alloys and/or combination therapy of gold and other metal(s)) approach) is administered with a second-line treatment. More typically, the second therapeutic agent comprises a glucocorticoid.

In a further embodiment of the invention, a gold nanocrystal suspension or colloid of the invention (eg, an aqueous gold-based metal and/or a mixture of gold and other metal(s) and/or an alloy of gold and other metal(s) and /or a combination therapy approach) and a pharmaceutically acceptable carrier, diluent or excipient. The formulation of such compositions is known to those skilled in the art. The composition may include pharmaceutically acceptable excipients such as carriers, diluents or excipients. These include, as appropriate, all customary solvents, dispersants, fillers, solid earners, coatings, antifungal and/or antibacterial agents, dermal penetrants, ibuprofen, ketoprofen, surfactants, isotonic and absorbent agents, and the like. It is understood that the compositions of the present invention may also include other auxiliary physiologically active agents. In addition, various dietary supplements and homeopathic carriers may also be used. Specifically, the selection of such ingredients may be based, in part, on the known functionality or use of these ingredients to obtain additive or synergistic effects when combined with the active ingredients of the present invention.

The carrier must be pharmaceutically acceptable in terms of compatibility with the other ingredients in the gold nanocrystal suspension of the present invention, and must not be harmful (eg, toxic at a therapeutically active amount) to the subject. Compositions include those suitable for oral, rectal, inhalational, nasal, transdermal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy, homeopathy and/or dietary supplements. Such methods include the step of bringing into association a carrier consisting of the metallic nanocrystals or suspensions of the present invention and one or more accessory ingredients. In general, compositions are prepared by uniformly and intimately binding one or more active ingredients in solution/colloid under suitable non-reactive conditions that minimize or eliminate, to the extent possible, negative or adverse reactions.

Depending on the disease or condition to be treated, it may or may not be desirable for the gold nanocrystal suspension or colloid of the present invention to cross the blood/brain barrier.

Thus, the gold nanocrystal suspensions or colloids of the present invention have an appropriate size, suitable crystal face(s) and/or desired shape or shape distribution, etc. (as discussed herein) to help cross the blood/brain barrier. It can be manufactured to have

Suspensions of gold nanocrystals according to the invention suitable for oral administration are usually presented as stable solutions, colloids or partially stable suspensions in water. However, such gold nanocrystals may also contain discrete units in non-aqueous liquids, such as liquid capsules, sachets or even tablets (e.g., as long as such processing does not adversely affect the functionality of the pristine gold nanocrystal surface, drying the suspension or colloid to produce active ingredient gold-based nanocrystals, each containing a predetermined amount of, for example, gold nanocrystal active ingredient; as a powder or granules; as solutions, colloids or suspensions in aqueous liquids or as non-aqueous liquids; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In addition, the gold nanocrystal active ingredient may be combined into a lump, emollient or paste.

Tablets produced from the gold nanocrystal suspensions or colloids of the present invention (including, for example, aqueous gold-based nanocrystals and/or alloys of gold and other metal(s) and/or combination therapy approaches) and other substances or compounds of the present invention can be, for example, It can be produced by first drying the suspension or colloid, collecting the residual dried material, and compacting or molding the powder into suitable tablets or the like. For example, compressed tablets may contain, in a suitable device, the active ingredient nanocrystals, e.g., metallic nanocrystals, in free flowing form, e.g., optionally with binders (e.g., inert diluents, preservatives, disintegrants (e.g. sodium starch glycolate, Cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) can be produced by compacting into powder or granules mixed with surfactant or dispersing agent.Molded tablet, for example, wetted with an inert liquid diluent in a suitable device. It can be produced by molding or pressing a mixture of powdered compounds. Tablets may optionally be coated or scored and formulated to provide sustained or controlled release of the active ingredient, for example, using hydroxypropylmethyl cellulose in various proportions to provide the desired release profile. The tablet may optionally be provided with an enteric coating to provide release in the portion of the intestinal tract other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising a colloid or suspension comprising one or more active ingredient(s) gold nanocrystals in a flavored base such as sucrose and acacia or gum tragacanth; pastilles comprising the gold nanocrystal active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia gum; mouthwashes comprising the gold nanocrystal active ingredient in a suitable liquid carrier.

The gold nanocrystal suspensions or colloids of the present invention (including, for example, aqueous gold-based metals and/or mixtures of gold and other metal(s) and/or alloys of gold and other metal(s) and/or combination therapy approaches) Also, one or more components or colloids (eg gold nanocrystals) in solution may be administered intranasally or via inhalation, for example by means of a nebulizer, aerosol or nebulizer for inclusion in a mist or mist.

Compositions suitable for topical administration to the skin may comprise the gold nanocrystals of the present invention suspended in any suitable carrier or base, and may be in the form of lotions, gels, creams, pastes, ointments, and the like. Examples of suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol , carbopol and water. Transdermal devices, such as patches, may also be used to administer the compounds of the present invention.

Compositions for rectal administration may be presented as suppositories with a suitable carrier base comprising, for example, cocoa butter, gelatin, glycerin or polyethylene glycol.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations comprising, in addition to the active ingredient, carriers known to be suitable in the art.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection suspensions or colloids which may contain antioxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending and thickening agents. The compositions may be presented in single or multiple dose hermetically sealed containers, such as ampoules and vials, and stored in a lyophilized state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. can Extemporaneous injection solutions, colloids and suspensions can be prepared from sterile powders, granules and tablets of the type already described.

Preferred unit dosage compositions are those comprising a daily dose or unit, divided daily doses, or containing appropriate fractions of the active ingredient as described hereinabove.

In addition to the gold nanocrystal active ingredients specifically mentioned above, the composition of the present invention may contain other agents conventional in the art with respect to the type of composition in question, for example, those suitable for oral administration are binders, sweeteners, It is to be understood that additional agents such as thickening agents, flavoring agents, disintegrating agents, coating agents, preservatives, lubricants, time delay agents and/or location release agents may be included. Examples of suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Examples of suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Examples of suitable flavoring agents include peppermint oil, wintergreen oil, cherry, orange or raspberry flavored oils. Examples of suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or esters thereof, waxes, fatty alcohols, zein, shellac or gluten. Examples of suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulfite. Examples of suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Examples of suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Additionally, in practicing the electrochemical preparation methods of the present invention, these gold-based metal nanocrystals can have a gold "coating" of other metals (or other non-metallic species, such as SiO2).₂) may be alloyed or combined with other metals in the liquid to occur in the ) phase, or alternatively gold-based nanocrystals may be coated with other metals. In this case, the gold-based composite or alloy may be produced in a colloid or suspension. Certain composites comprising both additional gold and other metals may also be formed.

Additionally, the gold-based metal nanocrystal suspensions or colloids of the present invention can be mixed or combined with other metal-based solutions or colloids to form novel solution or colloidal mixtures (e.g., in which case distinct metal species are still identifiable do).

IV. <u>Method for preparing gold nanocrystals</u>

A novel method for producing such unique gold nanocrystals is provided. This method involves the production of gold nanocrystals in water. In a preferred embodiment, the water does not contain an added "process enhancer" that does not significantly bind to the formed nanocrystals, but promotes nucleation/crystal growth during the electrochemical-stimulated growth process. Process enhancers play an important role in processes that involve providing charged ions in an electrochemical solution to allow crystals to grow. This novel electrochemical process can be carried out as a batch, semi-continuous or continuous process. These processes result in controlled gold nanocrystalline concentration, controlled nanocrystal size and controlled nanocrystal size range, as well as controlled nanocrystal shape and controlled nanocrystal shape distribution. A novel fabrication assembly is provided to produce these gold nanocrystals.

In one preferred embodiment, the gold-based nanocrystal suspension or colloid is produced or grown by electrochemical techniques in a batch, semi-continuous or continuous process, wherein the amount, average particle size, crystal face(s) and/or particle The shape(s) and/or particle shape distribution is modulated and/or optimized to achieve high biological activity and low cellular/biological toxicity (eg, high therapeutic index). Preferred average crystal sizes include a variety of different ranges, but the most preferred ranges include those in which the average crystal size is primarily less than 100 nm, more typically less than 50 nm for many applications, and more typically varies, for example for oral use. Nanocrystal size distribution as determined by drying of the solution (as described in more detail herein) and building a particle size histogram from TEM measurements in many of the preferred embodiments disclosed herein. The mode for is less than 21 nm, and an even more preferred range is 8 to 18 nm. Additionally, the particles include crystals having a desired crystal shape and a desirable crystal shape distribution, and {111}, {110} and/or {100} facets that can produce better performance than gold spheres or randomly shaped particles. It is preferable to include a crystal plane that

In addition, in practice of the electrochemical preparation methods of the present invention, these gold-based metal nanocrystals are such that the gold "coating" is obtained from other metals (or other non-metallic species, such as SiO2).₂), or alternatively gold-based nanocrystals can be alloyed or combined with other metals in the liquid to be coated by the other metal. In such cases, the gold-based composite or alloy may be produced in a colloid or in suspension. Additionally, certain composites comprising both gold and other metals may also be formed.

Additionally, the gold-based metal nanocrystal suspensions or colloids of the present invention can be mixed or combined with other metal-based solutions or colloids to form novel solutions or colloidal mixtures (e.g., in such cases where distinct metal species may still be identified. can).

The process for the preparation of novel metallic nanocrystal suspensions or colloids according to the present invention generally comprises nanocrystals/liquid(s), solution(s), colloid(s) or suspension(s), micron-sized particles thereof, nano A novel process and novel apparatus for the continuous, semi-continuous and batchwise production of various components in liquids comprising crystals, ionic species and aqueous-based compositions. The resulting components and nanocrystals may be present in a variety of possible compositions, concentrations, sizes, crystal planes (e.g., , spatially extended low index crystal planes) and/or shapes. Liquid(s) used and created/transformed during processing may play an important role in the preparation and/or action of components (eg, nanocrystals) independently or synergistically with the liquid containing them. Particles (eg, nanocrystals) may be formed by, for example, one or more tunable plasmas (eg, generated by one or more AC and/or DC power sources) typically in communication with at least a portion of a surface of a liquid. to exist in a liquid (eg, water) (eg, to be created and/or to be present in a liquid (eg, to be conditioned)). However, active ingredient (eg nanocrystals) suspensions or colloids can likewise be achieved without the use of the plasma.

Metal-based electrodes having various composition(s) and/or unique configurations or alignments are preferred for use in the formation of the tunable plasma(s), although non-metal-based electrodes may also be used in at least part of the process. It is also preferred to use one or more subsequent and/or substantially simultaneous tunable electrochemical processing techniques. Metal-based electrodes of varying composition(s) and/or unique construction are preferred for use in electrochemical processing technique(s). Existing electric fields, magnetic fields, electromagnetic fields, electrochemistry, pH, zeta potential, chemical/crystal components, etc. can be positively affected by the tunable plasma(s) and/or tunable electrochemical processing technique(s) of the present invention. There are several possible variables. A plurality of tunable plasmas and/or tunable electrochemical techniques are in many embodiments preferred practice for producing a virtually limitless set of aqueous solutions, suspensions and/or colloids of the present invention, as well as numerous processing advantages of the present invention. It is desirable to practice the teachings of the embodiments to achieve many of the resulting novel nanocrystals and nanocrystal compositions.

In a continuous process embodiment of the present invention, at least one liquid, for example water, flows into, flows through and exits from at least one trough member, wherein the liquid comprises at least one controllable plasma and/or said one or more trough members. It is treated, conditioned, transformed and/or affected by more than one species of tunable electrochemical technique. The result of a continuous process may be novel constituents in liquids, novel and/or tunable sizes, hydrodynamic radii, concentrations, crystal sizes and micron-sized particles in the crystal size range, ionic constituents, nanocrystals (e.g., metallic nanocrystals). , crystal facets of the crystal shape, spatially extended low index crystal facets, crystal shape and distribution and composition, zeta potential, pH and/or properties, wherein the nanocrystal/liquid mixture is produced in an efficient and economical manner.

In a preferred embodiment, the method is not significantly bound to the nanocrystals formed, but rather a "process enhancer" or "processing enhancer" (usually an inorganic material) that promotes nucleation/growth during an electrochemical-stimulated growth process. Nucleation and growth of gold nanocrystals in water comprising Process enhancers play an important role in processes that involve providing charged ions in an electrochemical solution that allows crystals to grow. Process enhancers critically remain in solution and/or do not form coatings (eg organic coatings), do not adversely affect the formed nanocrystals or the formed suspension(s), and/or decompose, evaporate during the electrochemical process or even lost compound(s). A preferred process enhancer is sodium bicarbonate. Examples of other process enhancers include sodium carbonate, potassium bicarbonate, potassium carbonate, trisodium phosphate, disodium phosphate, monosodium phosphate, potassium phosphate or other salts such as carbonic acid. Additionally, the process enhancer may be a salt, including sodium or potassium, of bisulfite or sulfite. Other process enhancers for producing gold nanocrystals for medical applications under certain conditions can be other salts, including sodium or potassium, or any material that aids in the electrochemical growth process described herein, and can be any The material is not substantially incorporated into or onto the surface of the gold nanocrystals and does not impart toxicity to or to the suspension containing the nanocrystals.

Preferred concentration ranges for processing enhancers are typically 0.01 to 20 grams/gallon (0.0026 to 2.1730 mg/ml), more typically 0.1 to 7.5 grams/gallon (0.0264 to 1.9813 mg/ml), most typically 0.5 to 2.0 grams/gallon (0.13210 to 0.5283 mg/ml).

For example, certain processing enhancers can be broken down into positive ions (cations) and negative ions (anions). Anions and/or cations are propagated relative to the oppositely charged electrode or, depending on various factors, including liquid composition, concentration of ions, applied field, frequency of applied field, waveform of applied field, temperature, pH, zeta potential, etc. move towards it. When the ions are located at or near the electrode, the ions may participate in one or more reactions with the electrode(s) and/or other component(s) at or near the electrode(s). . Occasionally, ions may react with one or more substances in the electrode (eg, various metal chlorides (MCl, MCl) when NaCl is used as a processing enhancer.₂ etc) may be formed). Such reactions may be desirable in some cases or undesirable in other cases. Additionally, sometimes ions present in the solution between the electrodes do not react and produce products such as MCl, MCl.₂ etc., but may affect the material at (or near the electrode) to form metal nano-crystals that are "grown" from the material provided by the electrode. For example, certain metal ions inject liquid 3 from electrode 5 and, together (eg, nucleate) form components (eg, ions, nanocrystals, etc.) within liquid 3 .

In addition, it is important to select a process enhancer that does not confer toxicity to the gold nanocrystals or liquids that allow the crystals to have maximum pharmaceutically acceptable potential. For example, for certain applications, chlorides may be undesirable as they produce gold chloride salts, which may be toxic.

Additionally, depending on the particular product formed, drying, concentration and/or freeze drying may be used to remove at least some or substantially all of the liquid in suspension to produce, for example, partially or substantially completely dehydrated nanocrystals. can do. In the case of complete dehydration of a solution, suspension or colloid, the metallic species must be capable of rehydration by addition of a liquid (eg, having a composition similar to or different from that being removed). However, not all compositions/colloids of the present invention can be completely dehydrated without adversely affecting the properties of the compositions/colloids. For example, many nanocrystals formed in a liquid tend to agglomerate or stick together upon drying. Dehydration should be avoided unless this formation of clumps is reversible during the subsequent re-dehydration step.

In general, certain solutions, suspensions or colloids of gold produced by the present invention can be concentrated several times without destabilizing the composition. However, complete evaporation is difficult to achieve, for example due to agglomeration effects. In many of the embodiments disclosed herein, such agglomeration effects appear to initiate at an approximate volume of 30% of the initial or starting reference volume removed from the suspension or colloid. Further, after evaporating a certain amount of liquid, as characterized for example by FAAS, DLS and UV-Vis techniques, the amount of evaporated liquid is subsequently reconstituted or added back to achieve a very similar product. For example, a suspension of two 500 ml nanocrystalline colloidal gold prepared by a technique similar to the preparation of GB-139 (discussed in detail in the Examples section herein) is placed in a glass beaker each and until boiling. Heated on a hotplate. The suspension is evaporated to 300 ml and 200 ml, respectively, and subsequently reconstituted with the amount of liquid removed (e.g., water purified by deionization and reverse osmosis ("DI/RO") water in amounts of 200 and 300 ml, respectively). and later characterized. Further, in another case, two GB-139 suspensions were again evaporated to 300 ml and 200 ml and then characterized without rehydration. It has been found that this dehydration process has little or no adverse effect on nanocrystal size or nanocrystal shape (e.g., nanocrystal size range and nanocrystal shape distribution are not significantly altered upon dehydration of GB-139 colloids, or their initial dehydrate and rehydrate to gold concentrations or ppm levels).

One important embodiment of the present invention involves the generation of a tunable plasma, wherein the tunable plasma comprises one or more electrodes positioned adjacent (eg, over) at least a portion of the surface of the liquid and the surface of the liquid itself. is placed between at least a portion of The liquid is disposed in electrical communication with one or more second electrodes (or plurality of second electrodes) to cause a surface of the liquid to act as an electrode, thereby participating in the formation of a controllable plasma. This configuration has certain characteristics similar to the dielectric barrier discharge configuration, except that the surface of the liquid is the active electrode participating in this configuration.

Each tunable plasma utilized is caused by one or more electrically conductive electrodes disposed at any location in the liquid (eg, at least partially within the liquid) and one or more electrically conductive electrodes disposed on the surface of the liquid and one over the surface of the liquid. It may be positioned between the two or more electrodes. At least one power source (in a preferred embodiment, at least one source of current and voltage, such as a transformer or power source) has at least one electrode disposed over the surface of the liquid and at least one electrode in contact with the surface of the liquid (e.g. for example, located at least partially or substantially completely within the liquid). The electrode(s) may have any suitable composition and suitable physical configuration (eg, size and shape), which may be between the electrode(s) disposed on top of the surface of the liquid and at least a portion of the surface of the liquid itself. It results in the generation of the desired plasma.

The electrical power (eg, voltage and amperage) applied between the electrode(s) (eg, comprising a surface of a liquid that functions as one or more electrodes to form a plasma) is applied to both AC and DC sources and may be generated by any suitable source (eg, voltage from a transformer) including variations and combinations thereof. Generally, an electrode or electrode combination disposed therein (eg, at least partially below the surface of the liquid) participates in the creation of a plasma by supplying voltage and current to the liquid or solution. However, the tunable plasma is actually disposed between at least a portion of the electrode(s) disposed on top of the surface of the liquid (eg, at its tip or tip) and one or more portions or regions of the liquid surface itself. In this regard, the tunable plasma may be applied to the electrodes described above (ie, at least a portion of the surface of the liquid and the surface of the liquid) when a breakdown voltage of the gas or vapor is achieved or maintained between and/or around and/or between the electrode(s) and the surface of the liquid. electrodes disposed on top of its own part).

In one preferred embodiment of the present invention, the liquid comprises water (or water comprising a particular processing enhancer(s)) and a gas (ie a tunable plasma) between the surface of the water and the electrode(s) above the surface of the water. gases or atmospheres that participate in the formation of) include air. Air produced by the present invention has different compositions, concentrations, crystal size distributions and/or crystal shape distributions of constituents (e.g., nanocrystals) (e.g., a tunable plasma and/or different amounts of specific constituents in solution or suspension). may be a function of the water content in the air located above the surface of the liquid), as well as various different water content(s) or desired humidity, which may result in different processing times required to obtain specific concentrations of the various components in the liquid, etc. can be adjusted. A specific embodiment of the tunable plasma 4 is discussed in more detail in Examples 5-7.

The breakdown electric field at standard pressure and temperature for dry air is about 3 MV/m or about 30 kV/cm. Thus, for example, when the local electric field around a metal point exceeds about 30 kV/cm, a plasma can be created in the dry air. Equation 1 is the destructive electric field ("E_c") and the distance between the two electrodes ("d") in meters.

&lt;Equation 1&gt;

<img file="KR20120052967A_D0007.tif" />

Of course, the destructive electric field ("E_c") may change as a function of the properties and composition of the gas or vapor disposed between the electrodes. In this regard, in a preferred embodiment in which the water (or water comprising the processing enhancer) is a liquid, a significant amount of water vapor may be inherently present in the air between the "electrodes" (ie, between one or more electrodes disposed over the surface of the water and the surface of the water itself serving as an electrode for plasma formation), and such water vapor may be present between them. It should affect at least the destructive electric field required to create the plasma.In addition, a higher concentration of water vapor may be present locally in and around the plasma generated due to the controllable plasma interaction with the surface of the water. The amount of "humidity" present in and around the generated plasma can be controlled or regulated by various techniques described in more detail below. Likewise, at least a portion of the component present in any liquid forms a tunable plasma disposed between the surface of the liquid and electrode(s) disposed adjacent (eg, along the surface) of the liquid. can form. The tunable composition of the plasma as well as the physical properties of the plasma itself can have a significant impact on the liquid as well as the particular processing technique (described in more detail below).

The electric field strength generated at and near the electrode is typically maximum at the surface of the electrode and typically decreases with increasing distance therefrom. electrode(s) disposed over the surface of the liquid, when it involves generation of a controllable plasma between the surface of the liquid and one or more electrode(s) disposed adjacent (eg, over) the liquid; The portion of the volume of gas between at least a portion of the liquid surface itself may comprise a disruptive electric field sufficient to create a tunable plasma. These generated electric fields can affect, for example, the behavior of a tunable plasma, the behavior of a liquid, the behavior of components in the liquid, and the like.

In this regard, FIG. 1a shows, for example, a point source electrode 1 having a triangular cross-sectional shape disposed at a distance "x" on top of a surface 2 of a liquid 3 flowing in direction "F". ) is shown in one embodiment. The tunable plasma 4 is generated by the tip or tip 9 of the electrode 1 and the surface 2 of the liquid 3 when a suitable power source 10 is connected between the point source electrode 1 and the electrode 5. between which electrode 5 is in communication with liquid 3 (eg, at least partially under surface 2 of liquid 3 ).

The adjustable plasma region 4 produced in the embodiment shown in FIG. 1A may typically have a shape corresponding to a conical or ellipsoidal structure, at least in part of the process, and in some embodiments of the present invention substantially It is possible to maintain this shape (eg conical shape) in all processes. The volume, intensity, component (eg, composition), activity, precise location, etc., of the adjustable plasma(s) 4 may include, but are not limited to, the distance ("x"), the physical and / or chemical composition, shape of electrode 1 , power supply 10 (eg, DC, AC, rectified AC, applied polarity of DC and/or rectified AC, AC or DC, waveform, RF, etc.) , the power applied by the power source (e.g., an applied voltage that is typically 1,000 to 5,000 volts, more typically 1,000 to 1,500 volts, applied amperage, electron velocity, etc.), the electric field generated by the applied power source and/or or the frequency and/or magnitude or ambient of the magnetic field, an electric field, a magnetic or electromagnetic field, an acoustic field, a naturally occurring or supplied gas or atmosphere between and/or around the electrode 1 and the surface 2 of the liquid 3 ( For example, the composition of air, nitrogen, helium, oxygen, ozone, reducing atmosphere, etc.), the temperature, pressure, volume, flow rate of liquid 3 in direction ("F"), The spectral characteristics, the composition of the liquid 3, the conductivity of the liquid 3, the cross-sectional area (eg volume) of the liquid adjacent to and around the electrodes 1, 5 (eg, the liquid 3 is controlled possible amount of time (ie residence time) allowed to interact with plasma 4 and the strength of such interactions], atmospheric flow at or near surface 2 of liquid 3 (e.g. For example, air flow) may vary depending on a number of factors including the presence (eg, provided fan(s) or atmospheric movement means), etc. (discussed in more detail herein below).

The composition of the electrode(s) 1 involved in the generation of the tunable plasma(s) 4 of FIG. 1A is, in one preferred embodiment of the present invention, a metal-based composition (eg, a metal such as gold and and/or alloys or mixtures thereof, etc.), but electrodes 1 , 5 may be made of any suitable material suitable with the various embodiments of the invention (eg, processing parameters) disclosed herein. In this regard, generation of plasma 4 in air above surface 2 of liquid 3 (eg water) typically results in at least some ozone as well as a quantity of nitric oxide and other components (other components herein). will be described in more detail in this section). These resulting components can be controlled and can be beneficial or detrimental to the formation and/or performance of components (eg, nanocrystals) and/or nanoparticles/solutions in the liquid of the resulting nanocrystal suspension or colloid, as described herein. may need to be controlled by a variety of different techniques, described in more detail below. Additionally, the emission spectrum of each plasma 4 is also a function of a similar factor, as shown, for example, in Examples 5-7 (described in more detail herein below). As shown in FIG. 1A , the adjustable plasma 4 actually contacts the surface 2 of the liquid 3 . In an embodiment of the invention, the material (eg metal) from the electrode 1 may comprise a portion of the tunable plasma 4 (eg may be a portion of the emission spectrum of the plasma), For example, it may be "sputtered" onto and/or into the liquid 3 (eg water). Thus, when metal(s) are used as electrode(s) 1 , various components (such as those shown in Examples 5-7) can be formed in the electrical plasma, such that the elemental metal(s), of the processing liquid 3 (eg, water) including, but not limited to, metal ions, Lewis acids, Bronsted-Lowry acids, metal oxides, metal nitrides, metal hydrides, metal hydroxides and/or metal carbides, and the like. The particular component to be part of is dependent on a particular set of operating conditions associated with the tunable plasma 4 and/or subsequent electrochemical processing operations in the liquid 3 (eg, for at least a portion of the process and simultaneously/ may be involved in subsequent reactions). These components may be present temporarily in the processing liquid 3 or may be semi-permanent or permanent. If such a component is temporary or semi-permanent, the time of subsequent reaction (eg, electrochemical reaction) with the component formed may affect the resulting final product. If the ingredients are permanent, they should not adversely affect the desired performance of the active ingredient nanocrystals.

Also, for example, electric, magnetic and/or electromagnetic field strengths in and around liquid 3 and the volume of liquid 3 exposed to said field (described in more detail elsewhere herein), electrodes ( Depending on the physical and chemical makeup of (1, 5), atmospheric (naturally occurring or supplied), and liquid composition, greater or lesser amounts of electrode(s) material(s) [e.g., metal(s) ) or derivatives of metals] can be found in the liquid (3). In certain circumstances, the substance(s) [eg, metal(s) or metal(s) composite material(s)] or component found in liquid 3 or plasma 4 (permanently or temporarily) (eg Lewis acids, Bronsted-Lowry acids, etc.) can have very desirable effects, in which case relatively large amounts of these substances may be desirable, while in other cases in liquid 3 Certain substances (eg, by-products) found may have undesirable effects and therefore minimal amounts of such substances may be required for liquid-based end products. Thus, electrode compositions can play an important role in materials formed in accordance with embodiments disclosed herein. The interactions between these components of the present invention are described in greater detail hereinbelow.

In addition, the electrode(s) 1 , 5 may have different compositions and/or structures of the liquids described hereinbelow and/or similar chemical compositions (eg, the same chemistry as their primary components) to achieve certain effects. element) and/or mechanical configuration or completely different composition.

between electrode(s) 1 , 5 , or between electrode(s) 1 , 1 (indicated herein below) or between electrode(s) 5 , 5 (indicated herein below). The distance ("y") is one important embodiment of the present invention. In general, when working with a power source capable of generating plasma under operating conditions, the location of the minimum distance ("y") between the closest portions of the electrode(s) used in the present invention is determined by the electrode [e.g., electrode To prevent undesirable arcing or formation of unwanted corona or plasma occurring between (s)(1) and electrode(s)(5)] (if some type of electrical insulation is not provided between them) It must be greater than the distance ("x"). Features of the present invention relating to electrode design, electrode location, and electrode interactions between the various electrodes are described in greater detail hereinbelow.

The power applied through power source 10 may be any suitable power that produces the desired tunable plasma 4 under all process conditions of the present invention. In one preferred mode of the invention, alternating current from a set-up transformer is used. Preferred transformer(s) 60 (see, eg, FIGS. 16D-16L ) for use in the various embodiments disclosed herein are those made possible by the use of magnetic shunts in transformer 60 . Intentionally has poor output voltage regulation. These transformers 60 are known as neon sign transformers. This configuration limits the current to the electrode(s) 1/5. With a large change in the output load voltage, the transformer 60 maintains the output load current within a relatively narrow range.

Transformer 60 is rated for its secondary open circuit voltage and secondary short circuit current. An open circuit voltage OCV develops at the output terminal of transformer 60 only when no electrical connection exists. Likewise, a short-circuit current is drawn from the output terminals when a short is placed across these terminals (in this case the output voltage is zero). However, when a load is connected to these same terminals, the output voltage of transformer 60 must fall between zero and a rated OCV. In fact, when transformer 60 is properly loaded, the voltage will be approximately half the rated OCV.

Transformer 60 is known as a Balanced Mid-Point Referenced Design (eg, also known as Balanced Mid-Point Referenced Design). This is most common with medium to high voltage rated transformers and most 60 mA transformers. This is the only acceptable type of transformer in a "midpoint return wire" system. A "balanced" transformer 60 has one primary coil 601 and two secondary coils 603, one on each side of the primary coil 601 (generally schematically shown in Fig. 16G). same as bar). This transformer 60 may be implemented in a number of ways as two transformers. As with an unbalanced midpoint reference core and coil, one end of each secondary coil 603 is attached to the transformer enclosure followed by the core 602 , the other end of each secondary coil 603 . is attached to the output lead or terminal. So, with no connectors present, a 15,000 volt transformer without this type of load measured about 7,500 volts from each secondary terminal to the transformer enclosure, but about 15,000 volts between these two output terminals. The transformer 60 of this example is used to form the plasma 4 disclosed in the embodiments herein. However, it should be understood that other suitable transformers (or power sources) are included within the scope of the present invention. In addition, these transformers 60 are exclusively used in Embodiments 1-4 of the present specification. However, different AC transformers 50 and 50a (discussed herein) are used for electrodes 5/5' in most other embodiments disclosed herein.

In another preferred embodiment, a rectified AC source produces a positively charged electrode 1 and a surface 2 of negatively charged liquid 3 . In another preferred embodiment, the rectified AC source produces a negatively charged electrode (1) and a surface (2) of a positively charged liquid (3). Also, other power sources, such as RF power supplies, may be used with the present invention. In general, the combination of the electrode(s) components (1, 5), the physical size and shape of the electrode(s) (1, 5), the electrode manufacturing process, the mass of the electrodes (1 and/or 5), the liquid (3) ) the distance ("x") between the tip 9 of the electrode 1 on top of the surface 2 of the (if any) and/or flow direction ("F"), amount of liquid 3 provided, type of power source 10 , frequency and/or waveform of power output of power source 10 are all dependent on the surface ( 2) and contributes to the design and power requirements (eg, destructive electric fields) required to obtain a controlled or tunable plasma 4 between the electrode tip 9 .

With further reference to the configuration shown in Fig. 1A, the electrode holders 6a, 6b may be lowered and raised by any suitable means (thus, the electrodes may be lowered and raised). For example, the electrode holders 6a , 6b can be lowered and raised within and through the insulating member 8 (shown in cross-section). The mechanical embodiments shown herein include male/female threaded threads. The parts 6a, 6b can be covered, for example, by further electrical insulation 7a, 7b. Electrically insulated portions 7a, 7b may have undesirable currents, voltages, arcs that may occur when their respective interfaces interface with the electrode holders 6a, 6b (eg, attempting to adjust the height of the electrodes). It may be any suitable material that prevents generation and the like (eg, plastic, polycarbonate, poly(methyl methacrylate), polystyrene, acrylic, polyvinyl chloride (PVC), nylon, rubber, fiber material, etc.). Likewise, insulating member 8 can be made of any suitable material that prevents undesirable electrical events (eg, arcing, melting, etc.) from occurring, as well as any structurally and environmentally suitable for practicing the present invention. material can be made. Typical materials include structural plastics such as polycarbonate, plexiglass (poly(methyl methacrylate)], polystyrene, acrylic, and the like. Additional suitable materials for use with the present invention are described in greater detail herein.

1C shows another embodiment for raising and lowering the electrodes 1 , 5 . In this embodiment, the electrically insulating portion 7a, 7b of each electrode is held in place by the press-fit present between the friction mechanism 13a, 13b, 13c and the portion 7a, 7b. The friction mechanisms 13a, 13b, 13c may be made of, for example, elastic steel, flexible rubber, or the like, as long as sufficient contact is maintained between them.

A preferred technique for automatically raising and/or lowering the electrodes 1 , 5 is described below. Power supply 10 may be connected to electrodes 1 , 5 in any suitable electrical manner. For example, wires 11a, 11b may be connected to electrode holders 6a, 6b [and/or electrically insulating 7a, 7b)].

2a shows another schematic view of a preferred embodiment of the invention, wherein the control device 20 of the invention is connected to the electrodes 1 , 5 so that the control device 20 is connected to the surface 2 of the liquid 3 . The electrodes 1, 5 are raised and/or lowered remotely (eg, upon command from another device or component) relative to the . The control device 20 of the present invention is described in further detail hereinbelow. In one preferred embodiment of the invention, electrodes 1 , 5 can be lowered and controlled remotely, for example, a suitable controller or computer comprising a software program (described in more detail hereinbelow) 2a) can also be monitored and controlled. In this regard, FIG. 2b is a diagram except that a Taylor cone "T" is used for electrical connection between the electrode 5 and the surface of the liquid 3 (or effective surface 2'). An electrode configuration similar to that shown in 2a is shown. Accordingly, the embodiment shown in Figures 1A, 1B and 1C is to be considered as a manually controlled device for use with the technology of the present invention, whereas the embodiment shown in Figures 2A and 2B is subject to appropriate instruction. It should be contemplated to include an automatic device or assembly 20 capable of remotely raising and lowering electrodes 1, 5 in response. In addition, the preferred embodiment of the present invention of FIGS. 2A and 2B is also spaced apart from the surface 2 (described in more detail herein below) at the tip 9 of the electrode 1 (and of the electrode 5 ). computer monitoring and computer control of the distance ("x") of tip 9']; Alternatively, computer monitoring and/or control of the flow rate of electrode 5 to/through liquid 3 may be used (discussed in detail herein below). Accordingly, appropriate commands for raising and/or lowering electrodes 1 , 5 may come from a suitable control device and/or individual actuators, such as a controller or computer (not shown in FIG. 2A ).

3A largely corresponds to FIGS. 2A and 2B, but FIGS. 3B, 3C and 3D illustrate various alternative electrode configurations that may be used in connection with certain preferred embodiments of the present invention. Figure 3b shows an essentially mirror image electrode assembly from the electrode assembly shown in Figure 3a. In particular, as shown in FIG. 3b , with respect to a direction ("F") corresponding to the flow direction of the liquid 3 , the electrode 5 causes the fluid 3 to flow in the longitudinal direction "F". It is the first electrode in communication with and in contact with the plasma (4) generated at the electrode (1). FIG. 3c shows two electrodes 5a , 5b disposed in a fluid 3 . This particular electrode configuration corresponds to another preferred embodiment of the present invention. In particular, as will be described in greater detail herein, the electrode configuration shown in FIG. 3C may be used alone or in combination with the electrode configuration shown in FIGS. 3A and 3B, for example. Similarly, a fourth possible electrode configuration is shown in FIG. 3D . In this FIG. 3d , no electrode(s) 5 are shown, rather only electrodes 1a and 1b are shown. In this case, two adjustable plasmas 4a, 4b are present between the electrode tips 9a, 9b and the surface 2 of the liquid 3 . The distances "xa", "xb" are the distances ("xa", "xb"), respectively, between the electrodes 9a/9b and the surface 2 of the liquid 3 at which the plasma 4 can be formed. may be approximately the same or substantially different, as long as the maximum distance is not exceeded. As mentioned above, the electrode configuration shown in FIG. 3D may be used alone or in combination with one or more of the electrode configurations shown in FIGS. 3A, 3B and 3C. The desirability of using specific electrode configurations in combination with one another for the direction of fluid flow ("F") is described in greater detail hereinbelow.

Likewise, a set of manually controllable electrode configurations generally corresponding to FIG. 1A is shown in FIGS. 4A, 4B, 4C and 4D, all of which are shown in partial cross-section. Specifically, FIG. 4A corresponds to FIG. 1A. Moreover, FIG. 4B corresponds to the electrode configuration in which the electrode configuration is shown in FIG. 3B , FIG. 4C corresponds to FIG. 3C , and FIG. 4D corresponds to FIG. 3D . In essence, the passive electrode configuration shown in FIGS. 4A-4D functionally corresponds to the remotely adjustable (eg, remotely controlled by computer or controller means) electrode configuration shown in FIGS. 3A-3D . It is possible to form materials produced in accordance with certain aspects of the invention similar to these materials produced. The desirability of using various electrode configuration combinations is described in greater detail hereinbelow.

Figures 5a-5e show perspective views of various preferred electrode configurations for the electrode 1 shown in Figures 1-4 (as well as other figures and the embodiment described below). The electrode configurations shown in FIGS. 5A-5E are representative of a number of different configurations useful in various embodiments of the present invention. Criteria for selecting an appropriate electrode for electrode 1 are, but are not limited to, the following conditions: requirements for a very well defined tip or tip 9, composition, mechanical limitations, electrode 1 The ability to create a shape from the containing material, conditioning of the material containing the electrode 1 (eg, heat treatment or annealing), convenience, the components introduced into the plasma 4, the effect on the liquid 3, etc. include In this regard, a small mass of material comprising, for example, the electrode 1 shown in FIGS. ), generating operating temperatures at which the size and/or shape of the electrode(s) 1 may be adversely affected. In this regard, for example, the electrode 1 has a relatively small mass (eg the electrode(s) 1 is made of gold and has a weight of about 5 g or less) and a very fine tip as the tip 9 . Including, for example, a fine tip (for example, a thin wire or triangular-shaped piece of metal having a diameter of only a few millimeters and exposed to hundreds to thousands of volts) cannot function as the electrode 1 (for example, the electrode 1 ) may be improperly deformed or melted) is likely to lack some type of additional interaction (eg, internal cooling means such as a fan or external cooling means, etc.) under a certain set of conditions. Thus, the composition of the electrode(s) 1 (eg, the material comprising the electrode) may influence a possible suitable electrode physical shape due to, for example, melting point, pressure sensitivity, environmental response. For example, the local environment of the tunable plasma 4 may cause chemical, mechanical and/or electrochemical corrosion of the electrode(s)].

Moreover, it should be understood that, in an alternative preferred embodiment of the present invention, a well defined sharp tip is not always required for the tip 9 . In this regard, the electrode 1 shown in FIG. 5E comprises a rounded tip 9 . A partially rounded or arc-shaped electrode may also function as electrode 1, which is a controllable plasma ( It should be understood that 4) can be produced from rounded electrodes or electrodes with sharper or sharper features. During the practice of the inventive technique of the present invention, such a tunable plasma can be positioned or positioned along various points of the electrode 1 shown in FIG. 5E . In this regard, FIG. 6 shows the various points ("a to g") corresponding to the starting points 9 for the plasma 4a to 4g occurring between the electrode 1 and the surface 2 of the liquid 3 . is showing Accordingly, it should be understood that various sizes and shapes corresponding to the electrode 1 may be utilized in accordance with the teachings of the present invention. Also, each of the tips 9, 9' of the electrodes 1, 5 shown in the various figures herein may be shown as a relatively sharp tip or a relatively blunt tip. Unless specific aspects of these electrode tips are described in more conceptual detail, the actual shape of the electrode tip(s) 9 , 9 shown in the figures may not be very important.

FIG. 7A shows a cross-sectional perspective view of an electrode configuration corresponding to that shown in FIG. 2A (and FIG. 3A ) housed in a trough member 30 . This trough member 30 has a liquid 3 supplied to the trough member from the rear face identified as 31 in FIG. 7A, the flow direction "F" facing the reader from the ground and towards the cross-sectional area identified as 32 . The trough member 30 is shown herein as a single piece of single material, but is fitted together and secured (eg, glued, mechanically attached, for example, by any acceptable means for attaching the materials to each other). ) can be made of a plurality of materials. Further, the trough member 30 shown herein has a rectangular or square cross-sectional shape, but may include a variety of different and more desirable cross-sectional shapes (described in further detail herein below). Accordingly, the flow direction of the fluid 3 is towards the reader from the ground, and the liquid 3 is substantially mutually with respect to the longitudinal flow direction "F" of the fluid 3 in the trough member 30 in this embodiment. It flows past each of the electrodes 1 and 5 arranged in series. This causes the liquid 3 to first experience a controllable plasma interaction (eg, a conditioning reaction) with the controllable plasma 4 and then the conditioned fluid 3 interacts with the electrode 5 . Certain preferred aspects of these electrode/liquid interactions and electrode placement(s) or electrode locations within the trough member 30 are described in greater detail elsewhere herein.

Fig. 7b shows a cross-sectional perspective view of the electrode configuration shown in Fig. 2a (as well as Fig. 3a), however, these electrodes 1, 5 are relative to the electrodes 1, 5 shown in Figs. 2a and 3a. It is rotated 90 degrees on the ground. In this embodiment of the present invention, the liquid 3 comprises an adjustable plasma 4 generated between the electrode 1 and the surface 2 of the liquid 3 and the longitudinal flow direction of the trough member 30 ( It contacts the electrode 5 at substantially the same point along "F") (ie from the ground). The flow direction of the liquid 3 is longitudinal along the trough member 30 and is directed from the ground to the reader as in FIG. 7A . Various preferred aspects of this electrode configuration are described in further detail herein below.

Fig. 8a shows a cross-sectional perspective view of the same embodiment shown in Fig. 7a. In this embodiment, as in FIG. 7A , the fluid 3 first interacts with the tunable plasma 4 generated between the electrode 1 and the surface 2 of the liquid 3 . Thereafter, the plasma induced or conditioned fluid 3 , which has been altered (eg conditioned, modified or prepared) by the tunable plasma 4 , then communicates with the electrode 5 and , thus causing a variety of electrochemical reactions to occur, which include the chemical composition, pH, physical or crystalline structure of the fluid 3 [and semi-permanent or permanent constituents in the fluid 3], which are described in more detail herein; excited state(s), etc.}. An alternative embodiment is shown in FIG. 8B . This embodiment essentially corresponds in general construction to those embodiments shown in FIGS. 3B and 4B . In this embodiment, the fluid 3 first communicates with the electrode 5 , after which the fluid 3 is a controllable plasma 4 created between the electrode 1 and the surface 2 of the liquid 3 . communicate with In this embodiment, the fluid 3 may be pre-strained prior to interacting with the electrode 5 .

FIG. 8c shows a cross-sectional perspective view of two electrodes 5a, 5b (corresponding to the embodiment shown in FIGS. 3c and 4c ), wherein the longitudinal flow direction of the fluid 3 ("F") is in contact with the first electrode 5a in the fluid flow direction "F" and then the second electrode 5b.

Likewise, FIG. 8D is a cross-sectional perspective view and corresponds to the embodiment shown in FIGS. 3D and 4D . In this embodiment, the fluid 3 communicates with a first adjustable plasma 4a generated by the first electrode 1a and thereafter the surface 2 of the fluid 3 and the second electrode 1a in communication with a second adjustable plasma 4b created between 1b).

9A is a cross-sectional perspective view and corresponds to the electrode configuration shown in FIG. 7B (and generally the electrode configuration shown in FIGS. 3A and 4A but rotated 90 degrees thereto). All electrode configurations shown in FIGS. 9A-9D are arranged such that the electrode pairs shown are disposed at substantially the same longitudinal point along the trough member 30 as in FIG. 7B .

Likewise, FIG. 9B generally corresponds to the electrode configuration shown in FIGS. 3B and 4B , and is rotated 90 degrees relative to the configuration shown in FIG. 8B .

FIG. 9C shows an electrode configuration generally corresponding to FIGS. 3C and 4C , rotated 90 degrees relative to the electrode configuration shown in FIG. 8C .

9D shows an electrode configuration generally corresponding to FIGS. 3D and 4D , rotated 90 degrees relative to the electrode configuration shown in FIG. 8D .

The electrode configurations shown generally in FIGS. 7, 8 and 9 include the electrode orientation and position relative to the fluid flow direction ("F"), the cross-sectional shape and size of the trough member 30 and/or the trough member 30 . The amount of liquid 3 in the trough member 30 and/or the amount of liquid 3 in or around the electrodes 5a/5b and in the trough member 30, the thickness of the electrodes, the number of electrode pairs provided and the trough member 30 relative to each other. ), as well as the depth into the liquid 3 (i.e. the amount in contact with the liquid 3), the speed of movement of the electrode into/through the liquid 3 (in the case of an electrode, the surface profile or shape is maintained or control), different results (eg, different conditioning effects on fluid 3, different pH in fluid 3, different nanocrystal sizes) as a function of various characteristics including power applied to the electrode pairs. and size distributions, different nanocrystal shapes and nanocrystal shape distributions and/or amounts of components (e.g., nanocrystalline materials) found in fluid 3, different functions of fluid/nanocrystal combinations (e.g., different biological/biocatalytic effects), different zeta potentials, etc.]. Further, electrode composition, size, specific shape, number of different types of electrodes provided, applied voltage, amount of current applied and/or achieved in fluid 3, AC source (and AC source frequency and AC waveform shape, duty cycle) etc.), DC source, RF source (and RF soap frequency, duty cycle, etc.), electrode polarity, etc. are all liquid when liquid 3 contacts, interacts and/or flows across these electrodes 1 , 5 . (3) affect the properties of [and/or the nanocrystals formed or contained within the liquid (3)] and thus the final properties of the material produced therefrom (eg, the resulting nanocrystals and/or suspensions or colloids); can affect Additionally, the liquid containing trough member 30 comprises a plurality of electrode combinations shown in Figures 7, 8 and 9 in some preferred embodiments. The electrode assemblies may all be of the same construction or may be a combination of a variety of different electrode configurations (described in greater detail elsewhere herein). Moreover, the electrode configuration may be in sequential communication with fluid "F" or may be in simultaneous or parallel communication with fluid "F". Different exemplary and preferred electrode configurations are shown in the additional figures below and are further described herein below along with the different components resulting therefrom (eg, nanocrystals and solutions or nanocrystal suspensions or colloids resulting therefrom). described in detail.

FIG. 10A shows a cross-sectional view of the liquid-containing trough member 30 shown in FIGS. 7, 8 and 9 . This trough member 30 has a cross section corresponding to that of a rectangle or a square, and an electrode (not shown in Fig. 10A) can be suitably positioned therein.

Likewise, a number of additional alternative cross-sectional embodiments for the liquid containing trough member 30 are shown in FIGS. 10B, 10C, 10D and 10E. The distances ("S" and "S'") for the preferred embodiment shown in each of FIGS. 10A-10E are, for example, measured from about 0.25" to about 6" (about 0.6 cm to 15 cm). The distance "M" is in the range of about 0.25" to about 6" (about 0.6 cm to 15 cm). The distance "R" is in the range of about 1/2" to about 7" (about 1.2 cm to about 17.8 cm). These embodiments (as well as additional configurations, wherein representative alternative embodiments are within the bounds and scope of the present disclosure) may be utilized in combination with other embodiments of the present invention. It should be noted that the amount of liquid 3 contained in each of the liquid-containing trough members 30 is not only a function of the depth "d", but also a function of the actual cross-section. Briefly, the amount of fluid 3 present at and around the electrode(s) 1 , 5 controls not only the liquid 3 but also the electrochemical interaction(s) of the liquid 3 with the electrode 5 . It is possible to influence one or more effects of the plasma 4 . Additionally, the flow rate of liquid 3 in and around electrode(s) 1 and 5 may also affect many properties of the resulting colloid or nanocrystals formed in suspension. These effects include the controllable plasma 4 conditioning effect on the liquid 3 [eg, the interaction of plasma electric and magnetic fields, the interaction of the electrochemical radiation of the plasma, the various chemical species in the liquid (eg, Electrodes that can advantageously affect the resulting end product, such as the formation of Lewis acids, Bronsted-Lowry acids, changes in pH, changes in the temperature of the liquid (e.g., slower liquid flow, the size/shape of the nanocrystals formed, etc.) (which may produce a longer contact and/or higher liquid temperature or residence time with or around 1/5) as well as the interaction or concentration of the controllable plasma (4) with the liquid (3). Similarly, the effect of the multiple embodiments (eg, electrochemical interactions) of the electrode 5 on the liquid 3 is also at least in part the effect of the liquid juxtaposed to the electrode(s) 5 . is a positive function. All of these factors can affect the balance that exists between the growth and nucleation of nanocrystals growing in liquid 3 , so that, for example, control of particle size and size range and/or control of particle shape and shape range create

In addition, strong electric and magnetic field concentrations will also affect the interaction of the liquid 3 with the plasma 4 as well as the interaction of the liquid 3 with the electrode 5 . Some important embodiments of these important interactions are described in more detail herein below. Further, the trough member 30 may comprise more than one cross-sectional shape along its entire longitudinal length. The inclusion of a plurality of cross-sectional shapes along the longitudinal length of the trough member 30 may include, for example, a field produced by an embodiment of the invention described herein (described in more detail elsewhere herein) or It may result in a change in concentration or reaction effect. Further, the trough member 30 may not be linear or "I-shaped", but rather "Y-shaped" or "ψ-shaped", where each portion of "Y" (or "ψ") is have different (or similar) sets of cross-sectional shapes and/or dimensions and/or sets of reaction conditions occurring therein.

In addition, the initial temperature of the liquid 3 input into the trough member 30 may also affect various properties of the product made in accordance with the teachings herein. For example, the different temperatures of the liquid 3 may vary depending on the nanocrystal size(s) and nanocrystal shape(s), the concentrations or amounts of the various formed components (eg, temporary, semi-permanent or permanent components), pH, The zeta potential may be affected. Likewise, temperature control along at least a portion or substantially all of the trough member 30 may have a desirable effect. For example, by providing localized cooling, the final properties of the formed product (eg, nanocrystal size(s) and/or nanocrystal shape(s)) can be controlled. Preferred liquid 3 temperatures during their processing are from freezing to boiling, more typically from room temperature to boiling, still more typically from about 40° C. to 98° C., more typically from 50° C. to 98° C. This temperature may be controlled, for example, by conventional means for cooling located at or near various parts of the processing apparatus.

Additionally, certain processing enhancers may also be added to or mixed with the liquid(s) 3 . Processing enhancers include both solids and liquids (and in some cases gases). The processing enhancer(s) may provide certain processing advantages and/or desirable end product characteristics. Some portion of the processing enhancer(s) may include, for example, a desired seed crystal (eg, promoting a desirable seed crystal or involved in the creation of a nucleation site) and/or a crystal facet growth promoter in the electrochemical growth process of the present invention. It may function, affect, or be part of a /inhibitor, or simply act as a current or power regulator in the electrochemical process of the present invention. Such processing enhancers may also have a desirable effect on the current and/or voltage conditions between electrodes 1/5 and/or 5/5.

A preferred processing enhancer is sodium bicarbonate. Examples of other process enhancers are sodium carbonate, potassium bicarbonate, potassium carbonate, trisodium phosphate, disodium phosphate, monosodium phosphate, potassium phosphate or other salts of carbonic acid and the like. Additional process enhancers may be salts, including bisulfites or sulfites of sodium or potassium. Other process enhancers for producing gold nanocrystals for medical applications under certain conditions can be other salts, including sodium or potassium, or any material that aids in the electrochemical growth process described herein; No substance is substantially incorporated into or on the surface of the gold nanocrystals and does not impart toxicity to the nanocrystals or suspension comprising the nanocrystals. A processing enhancer may assist in one or more of the electrochemical reactions disclosed herein and/or may assist in achieving one or more desirable properties in a product formed in accordance with the teachings herein.

For example, certain processing enhancers can dissociate into positive ions (cations) and negative ions (anions). Anions and/or cations may be steered or moved against the oppositely charged electrode depending on the liquid composition, concentration of ions, applied field, frequency of the applied field, waveform of the applied field, temperature, pH, zeta potential, and the like. When the ions are located at or near such electrodes, they may participate in one or more reactions with the electrode(s) and/or other component(s) disposed at or near such electrode(s). Often ions can react with one or more substances at the electrode [e.g., when NaCl is used as a processing enhancer, various metal chlorides (MCl, MCl)₂ etc.) may be formed]. This reaction may be desirable in some cases or undesirable in other cases. Additionally, often ions present in the solution between the electrodes are MCl, MCl₂ It may not react to form products such as, etc., but rather affect the material within (or near the electrode) to form metal nano-crystals "grown" from the material provided by the electrode. For example, certain metal ions may enter the liquid 3 from the electrode 5 and gather together (eg, nucleate) and become components (eg, ions, nanoparticles, etc.) in the liquid 3 . ) can be formed.

In the case of gold, a variety of extended surface planes are available in which crystal growth can occur as long as impurities (eg organic impurities) do not inhibit or impede such growth. Gold is known to have a face-centered cubic (fcc) structure, where gold nanocrystals grown by the method of the present invention are not single crystals, but typically pair to produce a variety of desirable and highly reactive nanocrystalline shapes or shape distributions. do. For example, single crystal surfaces {111}, {100} and {110} are among the most frequently studied and well understood surfaces. The presence of certain species, such as ions (e.g., added or donated by electrode 5) during the electrochemical crystal nucleation growth process, may affect the presence or absence of one or more of these extended surfaces. eg, promoting nucleated and/or specifically formed nanocrystals or nanocrystal shape distribution). Certain ions (e.g., anions) under certain field conditions may have specific nanocrystal shapes to other shapes (e.g., more decahedral shapes to other shapes, such as tetrahedral, icosahedral, octahedral; or other crystalline shapes) It may help the presence of more {111} extended facets or surfaces for other crystalline surfaces, which may result in the presence of specific crystalline shape combination(s), etc.) for The face, crystal shape (e.g., hexagonal face, octahedron, tetrahedron and pentagonal bipyramid (i.e., decahedron)) and/or crystal size or the presence or absence of an extended crystal face comprising the face (e.g., relative By controlling the amount), the nanocrystal shape can be relatively controlled. Controlling the size and shape of nanocrystals (as well as the surface properties of nanocrystals) can modulate their action(s) in a variety of systems, including biological systems.

Specifically, the presence of specific nanocrystalline shapes (or shape distributions) comprising specific spatially extended low index crystal planes cause different responses (eg, different biocatalytic and/or biophysical responses) and/or or different biological signaling pathways may be made active/inactive in the absence of the shaped nanoparticles) and/or may cause different responses that selectively occur under substantially the same conditions. , a pentagonal bipyramid structure or one crystalline shape of a decahedron or a tetrahedron comprising {111} faces) allows a set of reactions to occur (e.g., bound to a particular protein or homologue and/or a protein or cyto Affecting specific biological signaling pathways of Kin), different crystal shapes (e.g., octahedrons comprising the same or different crystal faces, e.g., {111} or {100}) have different reaction endpoints (e.g., different biocatalysts) or signaling pathway effects). More dramatically, the lack of any extended crystal growth planes results in spherical nanoparticles (e.g., those produced by conventional homogeneous chemical reduction methods), significantly affecting the performance of the nanoparticles. eg for expanded facet nanocrystals). Such differences in performance may occur due to different surface plasmon resonances and/or the strength of these resonances. Thus, by controlling the amount (e.g., concentration), nanocrystal size, presence or absence of specific extended growth crystal planes, and/or nanocrystalline shape or shape distribution(s), a specific response (e.g., biological response and/or biological response) signaling pathways) can be preferably influenced and/or controlled. Such control may result in the prevention and/or treatment of a variety of different diseases or conditions in which certain biological responses and/or signaling pathways (discussed in the following sections herein) function.

Additionally, certain processing enhancers may also include substances that can act as charge carriers, but do not themselves become ions. Specifically, metallic particles formed in situ (e.g., heterogeneous or homogeneous nucleation/growth) or dosing by the electrochemical processing techniques disclosed herein may also be used as charge carriers, crystal nucleating agents and/or growth promoters. can act, which can lead to the formation of a variety of different crystalline shapes (eg, hexagonal faces, octahedrons, tetrahedra, pentagonal bipyramids (decahedrons), etc.). Once again, the presence of a particular particle crystal size, an extended crystal facet of said crystal and/or a shape or shape distribution is preferably dependent on a specific response (eg, binding to a specific protein or protein homologue and/or specific biological signaling pathway; eg by affecting inflammatory pathways or proteasome pathways). In addition, since the processing enhancers of the present invention are not intended for conventional organic-based molecules used in conventional reducing chemistry techniques, the lack of such chemical reducing agents (or added surfactants) is not intended to be the basis of the surface of the grown nanocrystals in the present invention. It means that it is very "clean" compared to nanoparticles formed by this conventional reduction chemistry approach. When the term "clean" is used in reference to a nanocrystal surface or the phrase "substantially free of organic impurities or films" (or similar phases), it means that the formed nanocrystals (1) alter the behavior of the nanocrystals and/ or (2) does not have a chemical component that adheres to or adheres to a surface forming a layer, surface or film that applies a significant portion (eg, at least 25% of the crystals or more typically at least 50% of the crystals) In a preferred embodiment, the nanocrystal surface is completely free of any organic contaminants that substantially alter its functionality. Additionally, it is to be understood that ancillary components that cause binding to the nanocrystals of the present invention and do not substantially or adversely affect the action of the nanocrystals of the present invention are still included within the scope of the present invention. An example of a nanocrystal completely free of organic impurities or films is given in Example 5 of the present invention.

The lack of added chemicals (e.g. organics) allows the growth of gold atoms to proceed to expanded crystal planes, resulting in novel crystalline shape distributions, which also affect the in vivo function of nanocrystals (e.g., e.g. affects the protein corona that forms around nanoparticles/nanocrystals in serum). For example, but without wishing to be bound by any particular theory or explanation, protein corona formation regulates the localization of nanoparticles/nanocrystals in vivo, as well as protein folding of proteins at or near the surface of nanoparticles/nanocrystals. can be adjusted. Such differences in performance may be due to factors including, but not limited to, surface charge, surface plasmon resonance, epitaxial effects F, surface bilayers, zones of influence, and others.

Additionally, once seed crystals are generated in the process and/or a set of expanded crystal facets begins to grow (eg, homogeneous nucleation) or seed crystals are provided separately (eg, heterogeneous nucleation), the formed The amount of time a particle (e.g., a metal atom) stays at or near one or more electrodes in an electrochemical process can result in an increase in the size of the nanocrystal as a function of time (e.g., a metal atom transforms into a metallic nanocrystal). They can be aggregated and, if not retarded by certain organic components in the liquid, they can grow into a variety of shapes and sizes). The amount of time the crystal nucleation/growth conditions are present can control the shape(s) and size(s) of the grown nanocrystals. Thus, residence time at/near the electrode, liquid flow rate(s), trough cross-sectional shape(s), etc. all contribute to the nanocrystal growth conditions as discussed herein.

In a preferred embodiment, the proportion of the pentagonal bipyramid is at least about 5% or within the range of about 5% to 35%, more typically at least about 10% or within the range of about 10% to 35%, even more typically at least about 15% or about within the range of 15% to 35%, more typically at least about 25% and in some cases at least about 30%.

In another preferred embodiment, the proportion of tetrahedra is at least 5% or within the range of about 5% to 35%, more typically at least about 10% or within the range of about 10% to 35%, even more typically at least about 15% or within the range of about 15% to 35%, more typically at least about 25% and in some cases at least about 30%.

Further, the combination of pentagonal bipyramid and tetrahedron is at least about 15% or in the range of about 15% to 50%, more typically in the range of at least about 20% or in the range of about 20% to 50%, still more typically in the range of at least about 30% or within the range of about 30% to 50%, more typically at least about 35% and in some cases at least about 45%.

Further, combinations of pentagonal bipyramids, tetrahedra, octahedrons and hexagons are at least about 50% or within the range of about 50% to 85%, more typically at least about 60% or within the range of about 60% to 85%, still more typical at least about 70% or within the range of about 70% to 85%, more typically at least about 70% and in some cases at least about 80%.

In many preferred embodiments of the present invention, more than one AC source is used. The rate of change from a "+" polarity on one electrode to a "-" polarity on the same electrode is known as hertz, Hz, frequency, or cycles per second. In the United States, the standard output frequency is 60 Hz, whereas in Europe it is mainly 50 Hz. As shown in the embodiments of the present invention, frequency may also affect the size and/or shape of crystals formed according to the electrochemical techniques disclosed herein. Preferred frequencies are from 5 to 1,000 Hz, more typically from 20 to 500 Hz, even more typically from 40 to 200 Hz, even more typically from 50 to 100 Hz. For example, and without wishing to be bound by any particular theory or explanation, a nucleated or growing crystal first grows crystals that participate in forming itself (or crystal(s), for example due to different attracting charges). After having an attractive force applied to a component, such as an ion or atom, there is a repulsive force (eg, due to a similar charge repulsion) applied to that component. Such factors also affect particle size and/or shape, as well as reducing agents or surfactants (e.g., those that must be added to participate in conventional reducing chemistry techniques) that result in the absence of the added chemical species on the nanocrystal surface. It plays a significant role in the nucleation and/or crystal growth of the new nanocrystals formed by allowing crystals to form without it. The lack of an organic-based coating on the surface of grown nanocrystals alters (and in some cases controls) their biological behavior.

In addition, the particular waveform used at a particular frequency will also affect the nanocrystal growth conditions and thus the nanocrystal size(s) and/or shape(s). The United States uses a standard AC frequency of 60 Hz, which also uses a standard waveform of a "sine" wave. As shown in the Examples of the present invention, changing the waveform from a sine wave to a square or triangular wave also affects the nanocrystal crystallization conditions and thus the resulting nanocrystal size(s) and shape(s). crazy Examples of preferred waveforms include sine waves, square waves, and triangle waves, but hybrid waveforms are also considered to be within the scope of the present invention.

Additionally, the voltage applied in the novel electrochemical techniques disclosed herein may also affect the nanocrystal size(s) and shape(s). A preferred voltage range is 20 to 2000 volts, a more preferred voltage range is 50 to 1,000 volts, and an even more preferred voltage range is 100 to 300 volts. In addition to voltage, the amperage range used with these voltages is typically 0.1 to 10 Amp, a more preferred amperage range is 0.1 to 5 Amp, and an even more preferred amperage range is 0.4 to 1 Amp.

Additionally, the "duty cycle" used for each waveform applied in the novel electrochemical techniques disclosed herein may also affect the nanocrystalline size(s) and shape(s). In this regard, while not wishing to be bound by any particular theory or explanation, the amount of time the electrode is positively biased can result in a first order set of reactions, and a different set of reactions can occur when the electrode is negatively biased. can By controlling the amount of time when the electrode is positively or negatively biased, the size(s) and/or shape(s) of the grown nanocrystals can be controlled. Additionally, the rate at which the electrode converts to + or - is also a function of the waveform shape and also affects the nanocrystal size(s) and/or shape(s).

Temperature also plays an important role. In some of the preferred embodiments disclosed herein, the boiling point temperature of the water is approached at least a portion of the processing vessel in which the gold nanocrystals are nucleated and grown. For example, the output water temperature in a continuous processing embodiment herein ranges from about 60°C to 99°C. However, as discussed herein, different temperature ranges are also desirable. Temperature can affect the final product (eg, the size and/or shape of the nanocrystals) as well as the amount of the final product (ie, the ppm level of the nanocrystals in suspension or colloid). For example, while it is possible to cool the liquid 3 in the trough member 30 by various known techniques (as disclosed in some of the embodiments below), many embodiments herein are 3) causing evaporation of part of the liquid 3 during its processing without cooling.

11A shows a perspective view of one embodiment of substantially all of the trough members 30 shown in FIG. 10B including an inlet portion or inlet end 31 and an outlet portion or outlet end 32 . Flow direction "F" described in other figures herein may enter at or near end 31 (eg, trough at or near inlet portion 31 ). corresponding to the liquid exiting the trough member 30 through the outlet end 32 ] using suitable means for delivering the fluid into the member 30 . FIG. 11B shows the trough member 30 of FIG. 11A comprising three control devices 20a , 20b and 20c removably attached to the trough member 30 . The interaction and operation of the control devices 20a , 20b and 20c comprising electrode 1 and/or electrode 5 are discussed later herein. However, in a preferred embodiment of the present invention, the control device 20 may be removably attached to the upper portion of the trough member 30 so that the control device 20 can be positioned at different positions along the trough member 30 . may affect certain processing parameters, the resulting component (e.g., the size and shape of the nanocrystals), the resulting component, as well as the reactivity of the resulting nanocrystal(s)/fluid(s). .

11C shows a perspective view of the atmospheric control device cover 35'. The atmospheric control device or cover 35' is attached to a plurality of control devices 20a, 20b and 20c controllably attached to the electrode(s) 1 and/or 5 . The cover 35' allows any adjustable plasma 4 formed between any electrode(s) 1 and the surface 2 of the liquid 3 to control voltage, current, current density, polarity, etc. (herein a substantial portion (eg, greater than 50%) of the longitudinal direction of the trough member 30 to be a function of the conditioned atmosphere (as also discussed in more detail herein) as well as) It seeks to provide the ability to control the atmosphere along and/or within.

11D shows additional support means 34 (eg an outer part thereof) for supporting (at least partially) the control device 20 (not shown in FIG. 11D ) as well as supporting the trough member 30 . The apparatus of FIG. 11c including a) is shown. Various details may vary with respect to, for example, the trough member 30, atmospheric control(s) (eg, cover 35') and external support means (eg, support means 34). It is to be understood that all of these are to be considered to be within the boundaries and scope of the present disclosure.

11E shows an alternative configuration for the trough member 30 . Specifically, the trough member 30 is shown in perspective and is "Y-shaped". Specifically, the trough member 30 includes upper portions 30a and 30b and lower portions 30o. Likewise, inlets 31a and 31b are provided with outlets 32 . Portion 30d corresponds to the point where portions 30a and 30b meet portion 30o.

11F shows the same "Y-shaped" trough member shown in FIG. 11E , except that portion 30d of FIG. 11E is presented as a more distinct mixing zone 30d. In this regard, for example, certain components prepared or produced in liquid 3 in one or all of portions 30a, 30b and/or 30c may preferably be mixed together at point 30d (or 30d'). can Such mixing may occur naturally at intersection 30d shown in FIG. 11E (eg, no specific or special zone 30d' may be required), or may be more specifically controlled in portion 30d'. can It should be understood that portion 30d' may have any effective shape, such as rectangular, circular, rectangular, etc., and may have the same or different depths relative to other portions of trough member 30 . In this regard, site 30d may be a mixing zone or subsequent reaction zone, or may be a zone to which processing enhancers may be added. Details of intersections 30d and 30d' are discussed later herein.

11G and 11H show a "ψ-shaped" trough member 30 . Specifically, a new part 30c is added. The other features of FIGS. 11G and 11H are similar to those shown in FIGS. 11E and 11F .

It should be understood that a variety of different shapes and/or cross sections may exist for the trough member 30 , any of which arise from various design and manufacturing considerations. For example, one or more components produced in portion(s) 30a, 30b, and/or 30c may be transient (eg, seed crystals or nucleation points) and/or may be semi-permanent (eg, in a colloid). grown nanocrystals present). The final product (final product) resulting from such mixing, when such component(s), for example produced in portion 30a, is preferably and controllably reacted with, for example, one or more components produced in portion 30d. nature of the product) can be a function of when the components formed in parts 30a and 30b are mixed together. Further, the temperature of the liquid entering zone 30d (or 30d') may be monitored/to maximize certain desirable processing conditions and/or desirable properties of the final product and/or minimize certain undesirable products. can be controlled. Additionally, processing enhancers may optionally be used in one or more of portions 30a, 30b, 30c, 30d (30d) and/or 30o (or at any selected point or portion in trough member 30).

Figure 12a shows the use of various localized gases to control and/or influence certain components in the plasma 4 which can be controlled between the electrode 1 and the surface 2 of the liquid 3, for example, as well as the electrode ( s) (5) a local atmospheric control device (35) acting as a means for regulating the local atmosphere around the electrode set (1 and/or 5) to effect a controllable electrochemical reaction at and/or around it (5) shows a perspective view of Through-holes 36 and 37 presented in the atmospheric control device 35 are provided to allow external communication within and through the device 35 . In particular, the hole or inlet 37 serves as an input connection for any gas species that is intended to be introduced into the interior of the device 35 . Holes 36 serve as communication ports for electrodes 1 and/or 5 which extend through the electrode being connected to a control device 20 located on top of device 35 , for example. The gas introduced through the inlet 37 may simply be provided at a positive pressure relative to the local external atmosphere, allowing this portion to sink at least partially below the surface 2 of the liquid 3 , for example. Deviation may be caused by any suitable means or route, including, but not limited to, bubbling around portions 39a and/or 39b of (35). Alternatively, secondary holes or outlets (not shown) may be provided in the atmospheric control device 35 . In general, portions 39a and 39b break the surface 2 of the liquid 3 such that the surface 2 effectively acts as part of a seal to form a local atmosphere around the electrode sets 1 and/or 5 . can do. When a given gas at positive pressure is introduced through the inlet port 37 , small bubbles may be generated, for example, through the portions 39a and/or 39b. Alternatively, the gas may be exhausted through a suitable outlet in the atmospheric control device 35 , such as in the trough hole 36 .

12B shows a perspective view of the first atmospheric control device 35a from the front of the trough member 30 housed in the support housing 34 . The secondary atmospheric control device 35b comprises and represents a control device 20 located on its surface. "F" represents the longitudinal flow of liquid through the trough member 30 . If necessary, locally controlled atmosphere(s) (e.g., substantially the same chemical composition, such as air or nitrogen, or substantially different chemical composition, such as helium and nitrogen), is achieved around different sets of electrodes 1 and/or 5 can be

13 shows a perspective view of an alternative atmospheric control device 38 in which the entire trough member 30 and support means 34 are accommodated within the atmospheric control device 38 . In this case, for example, the gas inlets 37, 37' may be provided together with the gas outlet(s) 37a, 37a'. Not only is the correct placement of the gas inlet(s) 37, 37' and gas outlet(s) 37a, 37a' on the atmospheric control device 38 important, but also the composition of the atmosphere contained therein. . In this regard, if the gas is heavier than air or lighter than air, the positions of the inlet and outlet may be changed accordingly. Specifics of these factors are discussed later herein.

14 shows a schematic diagram of a general apparatus used in accordance with the teachings of some preferred embodiments of the present invention. In particular, FIG. 14 shows a schematic side view of a trough member 30 comprising a liquid 3 therein. On the upper portion of the trough member 30, there are a plurality of control devices 20a to 20d detachably attached in this embodiment. Control devices 20a-20d can of course be permanently secured in place when practicing various embodiments of the present invention. Exact number of control devices 20 (and corresponding electrode(s) 1 and/or 5 as well as configuration(s) of these electrodes) and control devices 20 (and corresponding electrodes 1 and/or 5) The location or arrangement of the is a function of various preferred embodiments of the invention discussed in greater detail herein. However, in general, the dosing liquid 3 (eg water or purified water) is provided by a liquid conveying means 40 (eg water) for pumping the liquid 3 from the first end 31 to the trough member 30 . Gravity or liquid pumping means for pumping the liquid 3 , for example a liquid pump], for example a peristaltic pump 40 . Exactly how the liquid 3 is dispensed is discussed in more detail later in this specification. The liquid transport means 40 may comprise any means for moving the liquid 3 including, but not limited to, gravity feeding or hydrostatic means, pumping means, regulating or valve means, and the like. However, the liquid transport means 40 must be able to reliably and/or controllably introduce a known amount of liquid 3 into the trough member 30 . The amount of time the liquid 3 is accommodated in the trough member 30 (eg at or around one or more electrode(s) 1/5) also depends on the resulting product (eg, of the grown nanocrystals). size(s) and/or shape(s)).

Once the liquid 3 is provided to the trough member 30 , a means for continuously moving the liquid 3 in the trough member 30 may or may not be required. However, a simple means for continuously moving the liquid 3 is a slight angle [theta] relative to the support surface on which the trough member 30 is disposed (for example, to a low-viscosity fluid 3 such as water). and a trough member 30 disposed at an angle of 1° to less than a few° to each other. For example, as long as the viscosity of the liquid 3 is not too high (eg, any viscosity near the viscosity of water can be controlled by gravity flow once this fluid is contained or placed in the trough member 30 ) , only a difference in vertical height of less than 1 inch may be required between the inlet portion 31 and the outlet portion 32 spaced apart by about 6 feet (about 1.8 meters) relative to the support surface. In this regard, FIGS. 15A and 15B show two acceptable angles θ for the trough member 30 capable of processing a variety of viscosities, including low viscosity fluids such as water.₁, θ₂) are shown respectively. The need for a larger angle θ may be the result of processing a liquid 3 having a higher viscosity than water, a requirement to pass the liquid 3 through the trough member 30 at a higher speed, and the like. In addition, other phenomena, such as hydrostatic head pressure or the specific use of hydrostatic pressure, can also be used to achieve the desired fluid flow when the viscosity of the liquid 3 increases so that gravity alone is insufficient. Further, additional means for moving the liquid 3 along the trough member 30 may also be provided inside the trough member 30 . Means for moving these fluids include mechanical means such as paddles, fans, propellers, augers, etc., thermal means such as transducers, heaters (which may have additional processing advantages) and the like are also in accordance with the present invention. Preferred for use together.

14 also shows the storage tank or storage vessel 41 at the end 32 of the trough member 30 . This storage vessel 41 may be any acceptable vessel and/or pumping, for example made of one or more materials that do not negatively interact with the liquid 3 (or components contained therein) produced in the trough member 30 . may be a means. Acceptable materials include, but are not limited to, plastics such as high density polyethylene (HDPE), glass, metal(s) (such as certain grades of stainless steel), and the like. Moreover, although a storage tank 41 is shown in this embodiment, it is to be understood that the tank 41 comprises means for dispensing or directly bottling or packaging the processed fluid 3 to the trough member 30 . shall.

16a, 16b and 16c show perspective views of one preferred embodiment of the present invention. In these figures 16a, 16b and 16c, eight individual control devices 20a to 20h are shown in greater detail. Such a control device 20 may use, for example, one or more of the electrode configurations shown in FIGS. 8A, 8B, 8C and 8D. The precise positioning and operation of the control device 20 (and corresponding electrodes 1 and/or 5) is described in greater detail elsewhere herein. 16B includes the use of two air distribution or air handling devices (eg, fans 342a and 342b). Such a gas handling device may, for example, help remove the generated humid air around the electrodes 1/5. Specifically, in some cases a certain amount of humidity is desirable, but in other cases an excess of localized humidity may be undesirable. Similarly, FIG. 16C includes the use of two alternative air distribution or air handling devices 342c, 342d.

The electrode control device shown in general for example in FIGS. 2 , 3 , 14 and 16 is shown in more detail in FIGS. 17d , 17e , 17f , 17m and 17n . In particular, these Figures 17D, 17E, 17F, 17M and 17N show perspective views of various embodiments of the control device 20 of the present invention.

Reference is first made to FIGS. 17d , 17e and 17f in particular. In each of these three figures, a base 25 is provided, which base has an upper portion 25' and a base portion 25". The base 25 is made of, but is not limited to, structural plastic, resin , polyurethane, polypropylene, nylon, Teflon, polyvinyl, etc. A dividing wall 27 is provided between the two electrode conditioning assemblies. The silver may be made of a material similar to or different from the material comprising the base 25. Two servo-step motors 21a, 21b are fixed to the surface 25' of the base 25. Step motor 21a, 21b) is moved slightly (e.g., slightly less than 1 degree or 1 on a 360 degree basis) such that circumferential movement of the step motors 21a/21b results in a vertical rise or fall of the electrode 1 or 5 in communication therewith. may be any stepper motor capable of (slightly exceeded). In this regard, the first wheel-like component 23a is a drive wheel connected to the output shaft 231a of the drive motor 21a such that when the drive shaft 231a rotates a circumferential movement of the wheel 23a is created . Also, the slave wheels 24a are pressed against and towards the drive wheels 23a so that there is frictional contact therebetween. Drive wheel 23a and/or slave wheel 24a may include notches or grooves in their outer portions to assist in receiving electrodes 1 , 5 . The slave wheel 24a is urged toward the drive wheel 23a by a spring 285 disposed between the portions 241a and 261a attached to the slave wheel 24a. In particular, a coiled spring 285 may be positioned around the portion of the shaft 262a extending from the block 261a. The spring creates a suitable friction force between the drive wheel 23a and the slave wheel 24a so that the electrode assemblies 5a, 5b, 1a, 1b, etc.) It must have sufficient tension to move in the vertical direction. This rotation or circumferential movement of the drive wheel 23a results in a direct transmission of the vertical change of the electrodes 1 , 5 shown here. At least a part of the drive wheel 23a should be made of an electrically insulating material, whereas the slave wheel 24a may be made of an electrically conductive material or an electrically insulating material, but preferably made of an electrically insulating material.

The drive motors 21a/21b rotate a small rotation (eg, slightly less than 1°/360° or slightly greater than 1°/360°) such that a small rotational change of the drive shaft 231a is converted into a small vertical change of the electrode assembly. This may be any suitable drive motor possible. A preferred drive motor includes a drive motor manufactured by RMS Technologies Model 1MC17-S04 step motor which is a DC powered step motor. These step motors 21a/21b each include RS-232 connections 22a/22b that allow the step motor to be driven by a remote control device such as a computer or controller.

Portions 271 , 272 , 273 are the primary height adjustments for adjusting the height of the base member 25 with respect to the trough member 30 . Portions 271 , 272 , 273 may be made of the same, similar, or different material as base 25 . Portions 274a/274b, 275a/275b may also be made of the same, similar, or different material as base 25 . However, these parts must be electrically insulated in that they accommodate the various wire components involved in delivering voltage and current to the electrode assemblies 1a/1b, 5a/5b.

The electrode assembly specifically shown in FIG. 17D includes electrodes 5a and 5b (eg, corresponding to the electrode assembly shown in FIG. 3C ). However, this electrode assembly may include electrode(s) 1 alone, electrodes 1 and 5 , electrodes 5 and 1 , or electrode(s) 5 alone. In this regard, FIG. 17e shows an assembly in which two electrodes 1a/5a are provided instead of the two electrode(s) 5a/5b shown in FIG. 17d. All other elements shown in FIG. 17E are similar to those shown in FIG. 17D.

With respect to the size of the control device 20 shown in FIGS. 17D, 17E and 17F , the dimensions "L" and "W" determine the size of the step motors 21a/21b and the width of the trough member 30 . It can be of any dimension that accommodates it. In this regard, the dimension "L" shown in FIG. 17f needs to be sufficient so that the dimension "L" is at least as long as the trough member 30 and preferably slightly longer (eg 10-30%). . The dimension "W" shown in FIG. 17f should not be wide enough to accommodate the step motors 21a/21b and not to make unnecessarily poor use of the longitudinal space along the length of the trough member 30 . In one preferred embodiment of the present invention, dimension "L" is about 7 inches (about 19 millimeters) and dimension "W" is about 4 inches (about 10.5 millimeters). The thickness ("H") of the base member 25 is any thickness sufficient to provide structural, electrical, and mechanical rigidity to the base member 25 and is between about ¼ to 3/4 (about 6 mm to 19 mm). mm) or so. Although these dimensions are not critical, they provide an understanding of the general size of a particular component of one preferred embodiment of the present invention.

Additionally, in each of the embodiments of the present invention shown in FIGS. 17D, 17E, and 17F , the base member 25 (and components mounted thereon) includes a suitable cover 290 (shown first in FIG. 17F ). ) to create a local protective environment for all components attached to the base member 25 as well as being electrically insulated. This cover 290 may be made of any suitable material that provides adequate safety and operational flexibility. Exemplary materials include plastics similar to those used for the trough member 30 and/or other parts of the control device 20 and are preferably transparent. This cover member 290 may also be made of the same type of material used to create the base portion 25 . Cover 290 is also shown with two throw-holes 291 and 292 . Specifically, these throw-holes may be aligned with, for example, an excess portion of the electrode 5 , which may be connected to, for example, a spool of electrode wire (not shown in these figures).

17M and 17N show another configuration for the control device 20 . In this arrangement, the similarly numbered parts are substantially identical to the parts shown in Figures 17D, 17E and 17F. The main difference between the control device 20 shown in FIGS. 17M and 17N is that a similar master or drive-pulley 23a rather than the slave wheel 24a or 241 shown in the embodiment of FIGS. 17D, 17E and 17F is provided. ), a resilient electrical contact device 242 is provided as shown in FIG. 17M , and as 242A/242B in FIG. 17N . In this regard, portions 242 , 242a and 242b provide elastic tension between the wires 5a or 5b. Additionally, this control device design allows for an electrical connection between the power supply 50/60 and the electrode 1/5 to exist. Servo motor 21a operates as discussed above, but a single electrode (FIG. 17M) or two electrodes (FIG. 17N) is driven by a single servo drive motor 21a. Thus, a single drive motor 21a can replace two drive motors in the case of the embodiment shown in Fig. 17N. Additionally, providing an electrical connection between the wires 1/5 and the power source 50/60 provides all electrical connections on the top surface, i.e. the surface away from the liquid 3, providing certain design and production advantages. .

17d and 17e show a refractory material part 29 . Component 29 is made of a suitable refractory component including, for example, aluminum oxide or the like. The refractory component 29 may have transverse throw-holes that provide electrical connection to the electrode(s) 1 and/or 5 . Additionally, longitudinal throw-holes exist along the length of the refractory component 29 so that the electrode assembly 1/5 can extend therethrough.

17E shows a perspective view of a base portion of the control device 20 . In FIG. 17E as such, one electrode(s) 1a is shown as extended through a primary fire-resistant portion 29a, and one electrode(s) 5a extends through a secondary fire-resistant portion 29b. shown expanded.

Accordingly, each electrode assembly explicitly discussed herein, as well as what is referred to herein, may be used in combination with the preferred embodiments of the control device presented herein.

In order to operate the control device 20, two general processes must be carried out. The first process involves electrically actuating the electrode(s) 1 and/or 5 (eg, applying power from the desired power source 10 ), and the second general process implementation includes, for example, how many Determine whether power is applied to the electrode(s), and adjust the electrode 1/5 height in the reaction appropriately with this measurement (e.g. manually and/or automatically adjust the electrode 1/5 height control); or by adjusting the electrode height or moving the electrode into contact with the liquid 3 over time (eg the electrode(s) 5 trough progressively progressing through the liquid 3 ) or from contact with the liquid 3 . It involves simply moving. In the case of using the control device 20, an appropriate device communicates with the step motor 21 via RS-232 ports 22a and 22b. The components of the control device 20 as well as important embodiments of the electrode activation process are discussed herein.

A preferred embodiment of the present invention uses the automatic control device 20 shown in the various figures herein. For example, step motors 21a and 21b shown in FIGS. 17D-17F and 17M-17N can be used in FIGS. 17G-17J (eg, electrode set 1/5 or electrode set for generating plasma 4 ). in case of (5/5)] controlled by the electrical circuit diagrammed in each; Alternatively, in some embodiments herein the electrode set 5/5 is controlled by the electrical circuit diagrammed in FIGS. 17K and 17L, respectively.

In particular, in this embodiment, the electrical circuit of FIG. 17J is a voltage monitoring circuit. Specifically, the voltage output from each of the output legs of secondary coil 603 in transformer 60 is monitored across points ("PQ") and points "P-Q". Specifically, "R_LThe resistor indicated by " corresponds to the internal resistance of a multi-meter measuring device (not shown). The output voltage measured between the points ("PQ", "P'-Q'") is typically For many of the preferred embodiments shown in the examples range from about 200 volts to about 4,500 volts.However, higher and lower voltages can work with many of the embodiments disclosed herein. In 4, a preferred target voltage is determined for each electrode set 1 and/or 5 at each position along the trough member 30. Such preferred target voltages are for example shown in Figs. This is achieved as the actual applied voltage by using the circuit control shown in 17i. These Figure 17g refer to a set of relays controlled by a Welemann K8056 circuit assembly (with a Microchip PIC16F630-I/P). Specifically, a voltage is detected across the "PQ" or "P'-Q'" position, and this voltage is compared to a predetermined reference voltage (actually compared to a target voltage range). When the measured voltage crosses the point ("PQ") approaching, for example, the highest point of a predetermined voltage target range, for example, the Bellemann K8056 circuit assembly clocks the servo motor 21 (see specifically FIG. 17f ). direction to lower the electrode 5a towards and/or into the fluid 3 . In contrast, if the measured voltage across one of the points ("PQ" or "P-Q") approaches the lowest point of the target voltage, for example referring again to FIG. 17F , servo motor 21a is driven Rotating the wheel 23a to the counterclockwise position will thereby raise the electrode 5a relative to the fluid 3 .

Each set of electrodes of Embodiments 1 to 4 of the present invention has a set target voltage range. An acceptable range of dimensions or magnitudes varies by an amount from about 1% to about 10%-15% of the target voltage. Some embodiments of the invention are more sensitive to voltage changes, and these embodiments should typically have a smaller allowable voltage range, while other embodiments of the invention are less sensitive to voltage and typically have a larger allowable range. should have Thus, by using the circuit diagram shown in Fig. 17J, the actual voltage output from the secondary coil 603 of the transformer 60 is "R_L"(Across terminals "PQ" and "P'-Q'") and then compared to a predetermined voltage range. Servo motor 21 rotates by a predetermined amount in a clockwise or counterclockwise direction as required. Moreover, with particular reference to Figures 17G-17J, it is noted that the interrogation procedure occurs sequentially by determining the voltage of each electrode, adjusting the height (if necessary) and then advancing to the next electrode. In other words, each transformer 60 is electrically connected in the manner shown in Fig. 17j Each transformer 60 and its associated measurement points ("PQ", "P-Q") are connected to individual relays, for example point "PQ" corresponds to relay 501 in Fig. 17G, and point "P'-Q'" corresponds to relay 502 in Fig. 17G. Thus, two relays are required for each transformer 60 . Each relay 501 , 502 , etc. sequentially applies a first output voltage from the first leg of the secondary coil 603 and then a second output voltage from the second leg of the secondary coil 603 . Interrogation is performed, which interrogates a first output voltage from a second transformer 60b on a first leg of its secondary coil 603 and then on a second leg of its secondary coil 603 . persists on

Computer or logic control for the disclosed interrogation voltage regulation technique is accomplished by any conventional program or controller including, for example, standard Visual Basic programming steps used in a PC in a preferred embodiment. These programming steps include interrogating, reading, comparing, and sending appropriate actuation symbols to increase or decrease the voltage (e.g. raising or lowering the electrode relative to the surface 2 of the liquid 3). includes Such techniques will be understood by those skilled in the art.

Further, in another preferred embodiment of the invention used in Example 16 for the electrode set 5/5', the automatic control device 20 comprises the electrical circuits of FIGS. 17H, 17I, 17K and 17L. is controlled by In particular, the electrical circuit of FIG. 17L is a voltage monitoring circuit used to measure the current. In this case, the voltage and current are the same numerical value due to the choice of resistors (discussed later herein). Specifically, the voltage output from each transformer 50 is monitored at points "PQ" and points "P'-Q"'. Specifically, "R_LThe resistors denoted by " correspond to the internal resistance of a multimeter measuring device (not shown). The points "PQ" and "P'-, which are typical for some preferred embodiments presented in the following examples herein, are The output voltage measured between Q"' is in the range of about 0.05 volts to about 5 volts. However, higher and lower voltages can be operated with many of the embodiments disclosed herein. A preferred target voltage is a trough Measurements are made for each electrode set 5/5' at each location along the member 30b'. Such a preferred target voltage can be obtained using, for example, the circuit shown in Figures 17H, 17I, 17K and 171. This figure 17 refers to a set of relays controlled by a Bellemann K8056 circuit assembly (with a Microchip PIC16F630-I/P).

In particular, in the example 16 embodiment, the servomotor 21 is caused to rotate at a certain predetermined time in order to maintain the desired electrode 5 profile. The servomotor 21 reacts by rotating a predetermined amount clockwise. Specifically, the servomotor 21 is such that about 0.009 inches (0.229 mm) of the electrode 5 advances toward and into the female receiver portion o5 (shown in, for example, parts of FIGS. 20 and 21 ) into this portion. rotated enough. So, the electrode 5 progressively proceeds through the liquid 3 . In one preferred embodiment disclosed herein, this electrode 5 movement occurs about every 5.8 minutes. Thus, the vertical movement speed to the female receiver portion o5 of each electrode 5 is about 3/4 inch (about 1.9 cm) every 8 hours. Thus, a substantially constant electrode 5 shape or profile is maintained by its constant or gradual progression into and through liquid 3 . Additionally, once the traveling end of the electrode 5 has reached the longitudinal end of the female receiver portion o5 , the electrode 5 can be removed from the processing device. Alternatively, electrode collection means may be provided for collecting the "used" portion of the electrode.

Examples of the collecting means of such electrode(s) 5 include, but are not limited to, a winding or spooling device and an expanded portion o5, a wire clipping or cutting device, and the like. However, other rates of electrode movement are also within the scope of the present invention to achieve different current/voltage profiles (and thus various different nanocrystal size(s) and/or shape(s)).

Furthermore, referring to Figs. 17H, 17I, 17K and 17L, it should be noted that the interrogation procedure occurs sequentially by determining the voltage of each electrode, which is shown in Fig. 17L in the embodiment of Example 16. Since the resistors Ra and Rb are approximately 1 ohm, and thus V=I, they correspond to the amount of current. In other words, each transformer 50 is electrically connected in the manner shown in FIGS. 17H, 17I, 17K and 17L. Each transformer 50 and its associated measurement points ("PQ", "P-Q") are connected to a separate relay. For example, point "PQ" corresponds to relays 501, 501' in FIG. 17K, and point "P'-Q'" corresponds to relays 502, 502' in FIG. 17K. Thus, a relay is required for each electrode set 5/5. Each relay 501/501' and 502/502' etc. sequentially interrogates the output voltage from the transformer 50, followed by a second voltage, etc. from the same transformer 50.

The computer or logic circuitry for the disclosed electrode height adjustment technique is accomplished by any conventional program or controller including, for example, standard Visual Basic programming steps used in a PC in the preferred embodiment. This programming step includes reading and sending the appropriate actuation symbol to lower the electrode against the surface 2 of the liquid 3 . Such techniques will be understood by those skilled in the art.

<u>Justice</u>

With respect to the present invention, the following terms and expressions used in the detailed description and claims are meant to have the following meanings.

"Carbomer" as used herein in Example 23 refers to a type of synthetically derived crosslinked polyacrylic acid polymer that provides efficient rheological modification with improved self-wetting properties for ease of use. In general, the carbomer/solvent mixture is neutralized with a base such as triethanolamine or sodium hydroxide to achieve the desired thickening, suspension and emulsion stabilizing properties to fully open the polymer to produce a cream or gel.

As used herein, "substantially clean" when used to describe a nanocrystal surface, the nanocrystal substantially inhibits the action of the nanocrystal in one or more of the significant properties of the gold nanocrystal described in the Examples herein. It means that it has no chemical components attached or adhered to the surface in varying amounts. Alternatively, the gold nanocrystals do not have a layer, surface, or film covering a significant fraction (eg, at least 25% crystals or in other embodiments at least 50% crystals). It can also mean that the nanocrystal surface is bare and completely free of any organic contaminants that substantially alter its functionality on the gold crystal surface. It is to be understood that ancillary components that cause binding to the nanocrystals of the present invention and do not substantially or adversely affect the action of the nanocrystals of the present invention are still included within the scope of the present invention. These terms are to be understood as relative terms referring to the lack of conventional organic-based molecules (eg, those used in conventional reduction chemistry techniques) on the surface of the grown nanocrystals of the present invention.

As used herein, "diagnostic effective amount" means an amount sufficient to bind to MIF to enable detection of a MIF-compound complex to enable diagnosis of a disease or condition.

As used herein, an "effective amount" provides a given MIF cytokine inhibition or therapeutic or therapeutic activity, or disease/condition prophylaxis or MIF signaling pathway(s), e.g., when administered according to a given dosing regimen. means a specific amount of a solution or compound. Dosing may be effected at intervals of minutes, hours, days, weeks, months or years, or may be administered continuously over any one of these time periods.

As used herein, "immune privilege" refers to a region or region (eg, body) in the life system that is resistant to the presence of antigens that normally elicit a response from the immune system (eg, an inflammatory immune response).

The term "operably coating" a stent refers to a metal-based nanocrystal of the invention (eg, an aqueous gold-based metal and/or a mixture of gold and other metal(s) and and/or coating the stent to allow timely evacuation (including alloys of gold and other metal(s)).

As used herein, the term "processing-enhancer" or "processing-enhancer" or "process enhancer" refers to one or more substances (eg, solid, liquid and/or gas), typically formed of nano An inorganic material that does not significantly bind to crystals, but promotes nucleation/growth during an electrochemical-stimulated growth process. These materials play an important role in methods that involve providing charged ions in an electrochemical solution to allow crystals to grow. The process enhancer critically remains in solution and/or does not form a coating (in one embodiment an organic coating) and/or does not adversely affect the formed nanocrystals or the formed suspension(s), and/or electrically A compound(s) that is decomposed, evaporated, or lost during the chemical crystal growth process.

As used herein, the term "steroid-sparing" means providing a substance other than a steroid in a combination therapy that reduces the amount of steroid needed to be effective in treating/preventing a condition.

The phrase "trough member," as used herein, refers to a pipe, harp, present in a material or object, conduit, duct, chute, hose and/or spout, so long as it is compatible with the electrochemical process disclosed herein. It should be understood to mean various fluid handling devices including pipes, channels or grooves.

The following examples are illustrative of specific embodiments of the invention and should not be construed as limiting the scope of the invention as defined in the appended claims.

<u>Examples 1-4 </u>

gold<u> Preparation of nanoparticles/nanoparticle solutions GT032, GT031, GT019 and GT033</u>

In general, each of Examples 1-4 utilizes a particular embodiment of the invention in connection with the apparatus generally shown in FIGS. 16B, 16C and 16G. Certain differences in processing and apparatus will be apparent in the respective embodiments. The trough member 30 is made of plexiglass, all having a thickness of about 3 mm to 4 mm (about 1/8"). The support structure 34 is also about 1/4" thick (about 6 to about 1/8"). 7 mm thick) of plexiglass. The cross-sectional shape of the trough member 30 corresponded to the shape shown in FIG. 10B (ie, frustoconical "V"). The base ("R") of the truncated cone "V" measured about 0.5" (about 1 cm), and each side ("S", "S'") measured about 1.5" (about 3.75 cm) . The distance ("M") separating the side portions ("S", "S'") of the V-trough member 30 is about 2¼" to 2 5/16" (about 5.9 cm) (measured from side to side) ) was. The thickness of each part was also measured to be about 1/8" (about 3 mm) thick. The longitudinal length of the V-trough member 30 ("L_T") (see FIG. 11A ) measured about 6 feet (about 2 meters) long from point 31 to point 32. Vertical height from end 31 to end 32 of the trough member 30. The difference in s was about 1/4 to 1/2" (about 6 to 12.7 mm) (ie, less than 1°) over a 6 foot length (about 2 meters).

Purified water (described herein below) was used as input liquid 3 in Example 1. In Examples 2 to 4, a processing enhancer was added to the liquid 3 input into the trough member 30 . A specific amount of processing enhancer as well as a specific processing enhancer added were effective in these examples. However, other processing enhancer(s) and amount of processing enhancer should be considered as being within the boundaries and scope of the present disclosure, and these specific examples should not be considered as limiting the scope of the present disclosure. The depth "d" (see FIG. 10B ) of water 3 in V-shaped trough member 30 is from about 7/16" to about 1/2" (about 11 mm) at various points along trough member 30 . to about 13 mm). Depth "d" was controlled in part through the use of dam 80 (shown in FIGS. 15A and 15B ). Specifically, a dam 80 was provided near the end 32 and had a depth "d" (shown in FIG. 10B ) such that it was about 7/6" to 1/2" (about 11 to 13 mm) deep. ) to help create The height ("j") of the dam 80 was measured to be about 1/4" (about 6 mm), and the longitudinal length ("k") was measured to be about 1/2" (about 13 mm). The width (not shown) completely crosses the bottom dimension ("R") of the trough member 30 . Thus, the total volume of water 3 in V-trough member 30 is approximately 26 inches during its operation.³(about 430 ml).

The flow rate of the water 3 in the trough member 30 was about 90 ml/min. Due to some evaporation in the trough member 30, the outflow from the trough member 30 was slightly less, about 60-70 ml/min. This flow of water 3 to the trough member 30 is a Masterflex rated at 0.1 horsepower, 10 to 600 rpm.^&amp;#174; It was obtained by using the L/S pump drive (40). Masterplex^&amp;#174; The model number of pump 40 was 77300-40. The pump drive is a Masterflex known as Easy-Load model number 7518-10.^&amp;#174;also had a pump head manufactured by In general, the head of the pump 40 is known as a peristaltic head. Pump 40 and head are masterflex^&amp;#174; Controlled by LS digital modular drive. The model number for the digital modular drive is 77300-80. The correct setting for the digital modular drive was, for example, 90 ml/min. Tygon with 1/4" diameter (ie size 06419-25)^&amp;#174; Tubing was placed on the peristaltic head. Tubing is Masterflex^&amp;#174;was prepared by Saint Gobain. One end of the tubing was delivered to the first end 31 of the trough member 30 by a flow spreading means disposed therein. The flow diffusion means are intended to minimize any pulsing conditions created by the peristaltic pump 40 as well as obstructions and bubbles in the water 3 introduced into the trough member 30 . In this regard, a small reservoir served as a diffusion means and provided at a point perpendicular to the end 31 of the trough member 30 to provide a relatively stable flow to the end 31 of the V-shaped trough member 30 when the reservoir overflows. A flow of water 3 occurred.

16B and 16C , eight individual electrode sets (Set 1, Set 2, Set 3 to Set 8) were attached to eight individual control devices 20 . Each of Tables 1a to 1d below refers to each of the eight electrode sets by "set number". Also, in any set number, electrodes 1 and 5 similar to the electrode assemblies shown in FIGS. 3A and 3C were used. Each electrode of the eight electrode set was set to operate within a specific target voltage range. Actual target voltages are listed in each of Tables 1a to 1d. The distance ("cc") from the centerline of each set of electrodes to an adjacent set of electrodes (see Fig. 14) is also represented. In addition, the distance ("x") associated with any electrode(s) 1 used was also reported. For any electrode 5, no distance ("x") was reported. Other relevant distances are reported, for example, in Tables 1a-1d.

The power source for each electrode set was an AC transformer (60). Specifically, FIG. 16D shows an AC power supply 62 connected to a transformer 60 . In addition, a capacitor 61 is provided, for example, so that a loss factor in the circuit can be adjusted. The output of the transformer 60 is connected to the electrode(s) 1/5 via a control device 20 . A preferred transformer for use with the present invention is one that uses an alternating current flowing in the primary coil 601 to establish an alternating magnetic flux in the core 602 that facilitates guiding the flux.

When secondary coil 603 is positioned near primary coil 601 and core 602 , this flux will couple secondary coil 603 with primary coil 601 . This coupling of the secondary coil 603 induces a voltage across the secondary terminal. The magnitude of the voltage at the secondary terminal is directly related to the ratio of the number of turns of the secondary to the number of turns of the primary. A larger number of turns in the secondary coil 603 than in the primary coil 601 results in a voltage boost, and a smaller number of turns results in a voltage drop.

Preferred transformer(s) 60 for use in these examples have deliberately poor output voltage regulation enabled by the use of magnetic shunts in transformer 60 . These transformers 60 are known as neon sign transformers. This configuration limits the current flow to the electrode(s) 1/5. Due to the large change in the output load voltage, the transformer 60 maintains the output load current within a relatively narrow range.

Transformer 60 is rated for its secondary open circuit voltage and secondary short circuit current. An open circuit voltage OCV develops at the output terminal of transformer 60 only when no electrical connection is present. Likewise, short circuit current is drawn only from the output terminals when a short is placed across these terminals (in which case the output voltage is zero). However, when a load is connected across these same terminals, the output voltage of transformer 60 must drop to some fraction between zero and the rated OCV. In practice, if transformer 60 is properly loaded, the voltage will be approximately half the rated OCV.

Transformer 60 is known as a balanced midpoint referenced design (eg, also already known as balanced midpoint grounded). This is most commonly found in medium to higher voltage rated transformers and most 60 mA transformers. This is the only acceptable type of transformer in a "midpoint return wired" system. A "balanced" transformer 60 has one primary coil 601 with two secondary coils 603, one on each side of the primary coil 601 (shown schematically in FIG. 16G ). shown as ). This transformer 60 can be implemented in a number of ways as two transformers. As with the unbalanced midpoint referenced core and coil, one end of each secondary coil 603 is attached to the core 602 and then to the transformer enclosure, and the The other end is attached to the output lead or terminal. Thus, without the connector present, an unloaded 15,000 volt transformer of this type would measure about 7,500 volts from each secondary terminal into the transformer enclosure, but about 15,000 volts between the two output terminals.

In an alternating current (AC) circuit with a line power factor or 1 (or 100%), voltage and current each start at zero, rise to a maximum, fall to zero, progress to a negative maximum, and return to zero again. This completes one cycle of a typical sine wave. This happens 60 times per second in typical US application. Thus, this voltage or current has a certain "frequency" of power 60 times per second (or 60 hertz). The power factor is related to the position of the voltage waveform relative to the current waveform. When both waveforms pass zero together and their maximums sum together, they are in phase and the power factor is 1 or 100%. 16H shows two waveforms "V" (voltage) and "C" (current) that are in phase with each other and have a power factor of 1 or 100%, whereas FIG. 16I is out of phase with each other and with a power factor of about 60%. Shown are two waveforms "V" (voltage) and "C" (current) with a factor, neither waveform passing through zero at the same time. The waveforms are out of phase and their power factor is less than 100%.

The normal power factor of most such transformer 60 is magnetic shunt 604 and secondary coil 603 which effectively adds an inductor into the output of the circuit of transformer 60 to limit the current to electrode 1/5. ) is largely due to the effect of The power factor can be increased to a higher power factor by the use of capacitor(s) 61 disposed across the primary coil 601 of the transformer 60 to drive the input voltage and current waves further into phase. .

The unloaded voltage of any transformer 60 to be used in the present invention as well as its internal structure is important. Preferred unloaded transformers for use in the present invention include those that are about 9,000 volts, 10,000 volts, 12,000 volts and 15,000 volts. However, these specific unloaded volt transformer measurements should not be considered as limiting the range acceptable power supply as a further embodiment. Certain preferred transformers for use with the various embodiments of the invention disclosed herein are Francefomer, Catalog, operating at 120 volts in the primary, 60 Hz and 9,000 volts in the secondary, 60 mA. No. Made by 9060-PE.

16E and 16F show another embodiment of the present invention, wherein the output of the transformer 60 input into the electrode assembly 1/5 is rectified by a diode assembly 63 or 63'. The result is generally that AC waves are substantially similar to DC waves. In other words, an almost flat line DC output is produced (in practice, a few 120 Hz pulses can often be obtained). This particular assembly creates two further preferred embodiments of the present invention (eg with respect to electrode orientation). In this regard, a substantially positive terminal or output and a substantially negative terminal or output are produced from the diode assembly 63 . The opposite polarity is achieved by diode assembly 63'. These positive and negative outputs may be input to one of the electrode(s) 1 and/or 5 . Accordingly, electrode 1 may be substantially negative or substantially positive, and/or electrode 5 may be substantially negative and/or substantially positive.

Figure 16j shows eight separate transformer assemblies 60a - 60h, each of which is connected to a corresponding control device 20a - 20h, respectively. This set of transformer 60 and control device 20 is used in these Embodiments 1-4.

Fig. 16k shows eight individual transformers 60a' to 60h', each corresponding to the rectified transformer diagram shown in Fig. 16e. This transformer assembly also communicates with a set of control devices 20a - 20h and may be used as preferred embodiments of the present invention, although they have not been used in these examples.

FIG. 16L shows eight individual transformers 60a to 60h, each corresponding to the rectified transformer diagram shown in FIG. 16F. This transformer assembly also communicates with a set of control devices 20a - 20h and can be used as a preferred embodiment of the present invention, although they are not used in these examples.

Thus, each transformer assembly 60a-60h (and/or 60a'-60h', and/or 60a"-60h") can be the same transformer, or a combination of different transformers (as well as different polarities). have. Transformer, power factor, capacitor(s) 61, polarity, electrode design, electrode location, electrode composition, cross-sectional shape(s) of trough member 30, local or global electrode composition, atmospheric(s), local or the overall liquid 3 flow rate(s), the liquid 3 local component, the volume of liquid 3 locally subjected to various fields within the trough member 30, neighboring (eg upstream and downstream) The choice of electrode set, local field concentration, use and/or location and/or composition of any membrane 50, etc., are all produced in the liquid 3 prepared according to the various embodiments disclosed herein. Factors affecting the composition and/or volume of components, nanoparticles and nanoparticles/suspensions or colloids as well as processing conditions. Accordingly, a plethora of embodiments may be practiced in accordance with the detailed disclosure set forth herein.

The size and shape of each electrode 1 used were approximately the same. The shape of each electrode 1 was that of a right-angled triangle with a scale of about 14 mm×23 mm×27 mm. The thickness of each electrode 1 was about 1 mm. Each triangular electrode 1 also had at its base a hole through it, which allowed the tip formed by the 23 mm and 27 mm sides to be sharpened towards the surface 2 of the water 3 . . The material comprising each electrode 1 is 99.95% pure (ie 3N5) unless otherwise stated herein. When gold was used for each electrode 1, the weight of each electrode was about 9 grams.

The wire used to attach the triangular electrode 1 to the transformer 60 was a 99.95% (3N5) platinum wire having a diameter of about 1 mm in Examples 1 to 3.

The wire used for each electrode 5 also contained 99.95% pure (3N5) gold with a diameter of about 0.5 mm. All materials for the electrodes (1/5) were obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520.

The water 3 used in Example 1 (and used in Examples 2-4 in combination with a processing enhancer) as input to the trough member 30 is produced by a reverse osmosis process and a deionization process. Essentially, reverse osmosis (RO) is a pressure-driven membrane separation process that separates species that are dissolved and/or suspended substances from surface water. This is called "reverse" osmosis because pressure is applied to reverse the natural flow of osmosis (which seeks to balance the concentration of substances on both sides of the membrane). The applied pressure forces water to move through the membrane leaving contaminants on one side of the membrane and purified water on the other side. Reverse osmosis membranes use multiple thin layers or sheets bonded together and wound in a spiral shape around a plastic tube. (It is also known as a thin film composite or TFC membrane). In addition to the removal of dissolved species, the RO membrane also separates suspended material, including microorganisms, that may be present in the water. A mixed bed deionization filter was used after RO processing. Total dissolved solvent ("TDS") after both treatments was Accumet^&amp;#174; It was about 0.2 ppm as measured by an AR20 pH/conductivity meter.

These examples use gold electrodes for an 8 electrode set. In this regard, Tables 1a-d set forth the relevant operating parameters associated with each of the 16 electrodes in the 8 electrode sets used to prepare the gold-based nanocrystal/nanocrystal suspension.

&lt;Table 1a&gt;

<img file="KR20120052967A_D0008.tif" />

&lt;Table 1b&gt;

<img file="KR20120052967A_D0009.tif" />

&lt;Table 1c&gt;

<img file="KR20120052967A_D0010.tif" />

&lt;Table 1d&gt;

<img file="KR20120052967A_D0011.tif" />

Table 1a shows that the "1/5" electrode configuration was used for electrode set number 1 and electrode set number 4, and all other sets were in the 5/5 configuration, while Table 1b, Table 1c and Table 1d show the electrode set. No. 1 is the electrode set using only the 1/5 configuration, indicating that all other sets were in the 5/5 configuration.

Additionally, the following differences in manufacturing setup were also used.

Example 1: GT032: The input water 3 to the trough member 30 was cooled in the refrigerator unit until it reached a temperature of about 2° C. and then pumped into the trough member 30 .

Example 2: GT031: A processing enhancer was added to the input water 3 before the water 3 was input into the trough member 30 . Specifically, NaHCO₃About 0.145 grams/gallon (ie, about 38.3 mg/L) sodium bicarbonate ("soda") having a chemical formula of Soda was obtained from Alfa Aesar, soda had a formula weight of 84.01 and a density of about 2.159 g/cm 3 (ie, Stock No. 14707, lot D15T043).

Example 3: GT019: A processing enhancer was added to the input water 3 before the water 3 was input into the trough member 30 . Specifically, about 0.17 grams/gallon (ie, about 45 mg/L) sodium chloride ("salt") having the chemical formula of NaCl was added to and mixed with water 3 .

Example 4: GT033: A processing enhancer was added to the input water 3 before the water 3 was input into the trough member 30 . Specifically, NaHCO₃About 0.145 grams/gallon (ie, about 38.3 mg/L) sodium bicarbonate ("soda") having a chemical formula of Soda was obtained from Alpha Eisar, soda had a formula weight of 84.01 and a density of about 2.159 g/cm 3 (ie, stock number 14707, lot D15T043). A representative TEM micrograph of the dried solution GT033 is shown in FIG. 32A . 32B also shows the dynamic light scattering data (ie, hydrodynamic radius) of solution GT033.

The salt used in Example 3 was obtained from Fisher Scientific (Lot No. 080787), and the salt had a formula weight of 58.44 and actual analysis as follows.

<img file="KR20120052967A_D0012.tif" />

Table 1e below summarizes the physical property results for each of the three solutions GT032, GT031 and GT019. Although the full characterization of GT019 has not been completed, under the processing conditions described herein, both processing enhancers (ie, soda and salt) increase the measured ppm of gold in solutions GT031 and GT019 relative to GT032.

&lt;Table 1e&gt;

<img file="KR20120052967A_D0013.tif" />

<u>Example 5-7 </u>

gold<u> Preparation of Nanocrystals/Nanocrystal Suspensions GD-007, GD-016 and GD-015 </u>

In general, each of Embodiments 5-7 utilizes a particular embodiment of the present invention in connection with the apparatus shown generally in Figs. 17B, 18A, 19A and 21A. Certain differences in processing and apparatus will be apparent in each example. The trough members 30a and 30b were made of 1/8" (about 3 mm) thick plexiglass and 1/4" (about 6 mm) thick polycarbonate, respectively. The support structure 34 was also made of plexiglass that was about 1/4" thick (about 6-7 mm thick). The cross-sectional shape of the trough member 30a shown in Fig. 18A is the shape shown in Fig. 10B. (i.e., truncated cone "V"). The base ("R") of truncated cone "V" measured about 0.5" (about 1 cm), and each side portion ("S", "S'") ) measures about 1.5" (about 3.75 cm). The distance ("M") separating the side portions ("S", "S'") of the V-trough member 30 is about 2¼" to 2 5/16" (about 5.9 cm) (measured from side to side) ) was. The thickness of each part was also measured to be about 1/8" (about 3 mm) thick. The longitudinal length of the V-trough member 30 ("L_T") (see FIG. 11A ) measured about 3 feet (about 1 meter) from point 31 to point 32 .

Purified water (as described herein below) is about 0.396 g/L NaHCO₃was mixed with and used as the liquid 3 entered into the trough member 30a. NaHCO used₃Although effective, this amount should not be considered as limiting the boundaries and scope of the present invention, and other quantities are within the boundaries and scope of the present disclosure. The depth "d" (see FIG. 10B) of the water 3 in the V-shaped trough member 30a is from about 7/16" to about 1/2" (about 11 mm) at various points along the trough member 30a. to about 13 mm). Depth "d" was controlled in part through the use of dam 80 (shown in FIG. 18A ). Specifically, a dam 80 was provided near the end 32 and had a depth "d" (shown in FIG. 10B ) such that it was about 7/6" to 1/2" (about 11 to 13 mm) deep. ) to help create The height ("j") of the dam 80 was measured to be about 1/4" (about 6 mm), and the longitudinal length ("k") was measured to be about 1/2" (about 13 mm). The width (not shown) completely crosses the bottom dimension ("R") of the trough member 30a. Thus, the total volume of water 3 in V-trough member 30a is about 6.4 inches during its operation.³(about 105 ml).

The flow rate of water 3 in the trough member 30a was about 150 ml/min (note: there was minimal evaporation in the trough member 30a). This flow of water 3 to the trough member 30a is a masterflex rated at 0.1 horsepower, 10 to 600 rpm.^&amp;#174; It was obtained by using the L/S pump drive (40). Masterplex^&amp;#174; The model number of pump 40 was 77300-40. The pump drive is a Masterflex known as Easy-Road model number 7518-10.^&amp;#174;also had a pump head manufactured by In general, the head of the pump 40 is known as a peristaltic head. Pump 40 and head are masterflex^&amp;#174; Controlled by LS digital modular drive. The model number for the digital modular drive is 77300-80. The correct setting for the digital modular drive was, for example, 150 ml/min. Tygon with a diameter of 1/4" (ie size 06419-25)^&amp;#174; Tubing was placed on the peristaltic head. Tubing is Masterflex^&amp;#174;was prepared by Saint-Gobain for. One end of the tubing was delivered to the first end 31 of the trough member 30a by a flow spreading means disposed therein. The flow diffusion means are intended to minimize any pulsing conditions created by the peristaltic pump 40 as well as obstructions and bubbles in the water 3 introduced into the trough member 30a. In this regard, a small reservoir served as a diffusion means and was provided at a point perpendicular to the end 31 of the trough member 30a to provide a relatively stable flow to the end 31 of the V-shaped trough member 30a when the reservoir overflows. A flow of water 3 occurred.

Five electrode sets were used in Examples 5 to 7, and one set was a single electrode set 1a/5a disposed on the trough member 30a. Plasma 4 from electrode 1a in trough member 30a was produced with electrode 1a similar in shape to that shown in Fig. 5e and weighed about 9.2 grams. This electrode was 99.95% pure gold. The other electrode 5a included a right-angled triangular platinum plate of about 14 mm×23 mm×27 mm with a thickness of about 1 mm immersed in liquid 3' by about 9 mm. The AC transformer used to generate the plasma 4 is the transformer 60 shown in FIG. 16D and discussed herein. An AC transformer 50 (described below) was connected to another electrode set 5/5. All other relevant operating conditions are shown in Tables 2a, 2b and 2c.

The output of the processing enhanced conditioned water 3 is collected in a reservoir 41 and thereafter at substantially the same flow rate as the pump 40 (eg, minimal evaporation generated within the trough member 30a ). It is pumped into the second trough member 30b by another pump 40'. The second trough member 30b measured about 30 inches long by 1.5 inches wide by 5.75 inches high and contained about 2,500 ml of water 3" therein. Four electrode sets 5b, 5b' , 5e, 5e') each contained a 99.95% pure gold wire measuring about 0.5 mm in diameter and about 5 inches (about 12 cm) in length and was substantially straight. was immersed in water (3) to a depth of about 4.5 inches (about 11 cm).

19A and 21A, separate electrode sets (Set 2, Set 3, Set 4 and Set 5) were attached to two separate transformer devices 50, 50a, as shown in FIG. 19A. Specifically, the transformers 50 and 50a were electrically connected to each electrode set according to the wiring diagram shown in Fig. 19A. Each transformer device 50 , 50a was connected to a separate AC input line that was 120° out of phase with respect to each other. Transformers 50, 50a are electrically connected in such a way as not to overload a single electrical circuit, for example allowing an upstream circuit breaker to be disconnected [eg, when used under these conditions, a single transformer 50 /50a) can draw enough current to cause upstream electrical problems]. Each transformer 50/50a was a variable AC transformer consisting of a single coil/winding of wire. This winding acts as part of both the primary and secondary windings. An input voltage is applied across the fixed portion of the winding. The output voltage is taken between one end of the winding and the other connection along the winding. By exposing a portion of the winding and making the secondary connection using a sliding brush, a continuously varying ratio can be obtained. The ratio of output to input voltage is equal to the ratio of the number of turns of the winding to which they are connected. Specifically, each transformer was a Mastek TDGC2-5kVA, 10A voltage regulator, output 0-250V.

Each of Tables 2a to 2c contains processing information regarding each of the four electrode sets in the trough member 30b by "set number". Each electrode of the set of four electrodes in the trough member 30b was set to operate at a specific target voltage. An actual operating voltage of about 255 volts was applied across the electrode set, as listed in each of Tables 2a-2c. The distance ("cc") from the centerline of each set of electrodes to an adjacent set of electrodes (see Fig. 14) is also represented. In addition, the distance "x" associated with the electrode 1 used in the trough member 30a is also reported. At electrode 5, no distance ("x") was reported. Other relevant variables are reported, for example, in Tables 2a-2c.

All materials for the electrodes (1/5) were obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520.

Water (3) used in Examples 5 to 7 was produced by a reverse osmosis process and a deionization process, and NaHCO₃ It was mixed with the processing enhancer and fed together into the trough member 30a. Essentially, reverse osmosis (RO) is a pressure-driven membrane separation process that separates species that are dissolved and/or suspended substances from surface water. This is called "reverse" osmosis because pressure is applied to reverse the natural flow of osmosis (which seeks to balance the concentration of substances on both sides of the membrane). The applied pressure forces water through the membrane leaving contaminants on one side of the membrane and purified water on the other side. Reverse osmosis membranes use multiple thin layers or sheets bonded together and wound in a spiral shape around a plastic tube. (It is also known as a thin film composite or TFC membrane). In addition to the removal of dissolved species, the RO membrane also separates suspended material, including microorganisms, that may be present in the water. A mixed bed deionization filter was used after RO processing. Total dissolved solvent ("TDS") after both treatments is Accumet^&amp;#174; It was about 0.2 ppm as measured by an AR20 pH/conductivity meter.

&lt;Table 2a&gt;

<img file="KR20120052967A_D0014.tif" />

&lt;Table 2b&gt;

<img file="KR20120052967A_D0015.tif" />

&lt;Table 2c&gt;

<img file="KR20120052967A_D0016.tif" />

Representative transmission electron microscopy (TEM) micrographs ( FIGS. 25A , 26A and 27A ) were taken on each of the dried suspensions prepared according to each of these Examples 5-7.

<u>transmission electron microscope </u>

Specifically, TEM samples were prepared using a carbon-stabilized Formvar-coated grid with a mesh size of 200. The grid was first pretreated by plasma treatment under vacuum. The grid was placed on a microscope slide aligned with a rectangular piece of filter paper and then placed in a Denton Vacuum apparatus with the necessary plasma generator accessories installed. The vacuum was maintained at 75 mTorr and the plasma was initiated and run for about 30 seconds. Upon completion, the system was vented and the grid removed. The grid was stable for 7 to 10 days depending on the humidity conditions, but in all cases it was used within 12 hours.

Approximately 1 μl of each inventive nanoparticle solution was placed on each grid and allowed to air dry at room temperature for 20-30 minutes, or until the droplets evaporate. Upon complete evaporation, the grid was placed on a holder plate until TEM analysis was performed.

A Philips/FEI Tecnai 12 transmission electron microscope was used to interrogate all prepared samples. The tool was run at an accelerating voltage of 100 keV. After alignment of the beams, the samples were examined at various maximum magnifications, including 630,000x. Images were acquired via an attached Olympus Megaview III side-mounted camera that transfers images directly to a PC equipped with iTEM and Technai user interface software that provides both control of the camera and TEM tools, respectively.

Within the iTEM software, it was possible to move randomly around the grid by adjusting the position of the crosshairs on the circular reference plane. By selecting and moving the crosshairs, you can navigate around the grid. Using this function, samples were analyzed in the four quadrants of the circular criterion to allow for an unbiased representation of the samples. Images were then analyzed with ImageJ 1.42 software. Another similar software program measuring the number of pixels across each particle for a known number of pixels in the space bar was used to streamline the particle counting process. Particles were measured using a scale bar on the image as a method to calibrate the software prior to measuring each individual particle. Once calibrated, particles were measured based on the following parameters: tetrahedral particles were measured from the vertex to the base of the triangle. A pentagonal bipyramid measures from the vertex of the rhombus to the vertex of the rhombus or from the vertex of the pentagon to the base of the pentagon based on the grain orientation of the grid. The icosahedron is measured using the longest length between the two faces of a hexagonal particle. Particles having a spherical or irregular shape are measured along the longest axis. Data collected from each sample set was sent to Excel and histograms were generated using a simple histogram function with 50 bins of minimum 5 nm and maximum 50 nm.

Then, the data generated in Excel is sent to Prism (GraphPad) and substituted into one of two models, either normal distribution or lognormal distribution, each with a unique probability density function (PDF). Histogram data can be analyzed by non-linear substitution on the data that produces a distribution known as a normal distribution within the prism. In addition, log-normal substitution of the data was generated by performing log transformation on the non-linear data set to generate the data set, then substituting it into the non-linear model, and then transforming it by exponential transformation. Thereafter, the two models were visually compared with a histogram, and the model with better data input was selected. The particle diameters mentioned above and reported in the histogram figures and tables herein are the modes of the PDF defined as the maximum of the log-normal or normal PDF curves. These PDF curves are superimposed on all histogram values, where the modal values are given directly above and are cited in the literature as TEM mean diameters.

For example, Figs. 25b, 26b and 27b show the crystal size distributions measured from the TEM micrographs corresponding to the dried solutions GD-007, GD-016 and GD-015 corresponding to Examples 5, 6 and 7, respectively. is a histogram. Each value reported in these histograms corresponds to the discussion above.

25A, 26A and 27A are representative TEM micrographs corresponding to dried solutions GD-007, GD-016 and GD-015 corresponding to Examples 5, 6 and 7, respectively.

The results presented in FIGS. 25d and 25e were obtained using a Philips 420ST transmission electron microscope equipped with an energy dispersive X-ray spectroscopy detector (EDS). The microscope was located in the School of Electron Microbeam Analysis at Johns Hopkins University and was operated under the guidance of an experienced operator. Briefly, about 1 μl of the GD-007 nanocrystalline suspension was placed on a Formbar carbon-coated 200 square mesh nickel grid and allowed to air dry at room temperature for about 20-30 minutes or until the droplets evaporated. Upon complete evaporation, the grid was placed in a TEM sample holder and interrogated at an accelerating voltage of 120 keV. The microscope's EDS system consisted of an Oxford photoelectron detector, an Oxford XP3 pulse processor and a 4 pi multi-channel analyzer connected to a Macintosh computer. Particle composition was determined by energy dispersive X-ray spectroscopy, where a high-energy beam of electrons is directed to the surface of the nanocrystal to release electrons within the inner shell, causing the available sites for external electrons to "fall off", thus It emits characteristic X-rays. Then, X-rays were detected by a detector having a resolution of 173.00 eV.

25D shows one of the gold nanocrystals grown according to Example 5 (ie, GD-007). The nanocrystals were interrogated with an electron beam as discussed herein.

25E shows the energy dispersive X-ray pattern of interrogation beam points of nanocrystals from solution GD-007. Since this measurement technique is accurate around monolayers of atoms, the absence of a pattern corresponding to the sodium peak indicates that no sodium-based monolayer is present on the surface of the crystal. Likewise, no significant carbonaceous peaks were observed, indicating the absence of any carbonaceous monolayers. Note the presence of an oxygen peak corresponding to the nickel grid below. Thus, these Figures 25D and 25E show that 1) no organic material is present on these molecules, and 2) the nanocrystals comprise a relatively clean surface without adverse molecules or coatings.

Additionally, dynamic light scattering techniques are also used to obtain an indication of the crystal size (eg, hydrodynamic radius) produced by the embodiments herein. 25c, 26c and 27c show graphical results of separate dynamic light scattering data sets.

<u>dynamic light scattering</u>

Specifically, dynamic light scattering (DLS) measurements were performed on a Viscotek 802 DLS instrument. In DLS, when laser light is struck by small particles and/or is organized in a water structure around small particles (less than a wavelength), the light scatters in all directions, resulting in time-dependent fluctuations in scattering intensity. The intensity variation is due to the Brownian motion of the scattering particle/water structure combination and includes information about the particle size distribution.

Instruments were allowed to warm up for at least 30 minutes prior to experimentation. Measurements were made using a 12 μl quartz cell. The following procedure was used.

One. First, 1 ml of DI water was added to the cell using a 1 ml micropipette, then water was poured from the cell into a waste beaker, and the remainder of the water was removed by shaking from the cell measurement cavity. This step was repeated at least two times to thoroughly rinse the cells.

2. 100 μl of sample was added to the cell using a 200 μl micropipette. After that, all liquid was removed from the cell with the same pipette using the same pipette tip and withdrawn into a waste beaker. 100 μl of sample was added again using the same tip.

3. Cells with samples were placed in a temperature controlled cell block of a Viscotek tool with the frozen side of the cell facing left. A new experiment in the Viscotech Omnisize software has been opened. Measurements were started 1 min after temperature equilibration and the laser power was attenuated to an appropriate value. Results were saved after all runs were completed.

4. The cell was taken from the tool and the sample was removed from the cell using the same pipette and tip once used in step 2.

5. Steps 2-4 were repeated at least twice for each sample.

6. For a new sample, a new pipette tip for a 200 μl pipette was taken to avoid contamination with the old sample, and steps 1-5 were repeated.

Data collection and processing was performed with Omnisize software version 3,0,0,291. The following parameters: Run Duration - 3 s, Experiment - 100, Solvent - Water, 0 mmol, Viscosity - 1 cP, Refractive Index - 1.333, Spike Tolerance - 20%, Baseline Drift - 15%, Target Attenuation - 300 kCounts, Block Temperature +40° C. was used for all experiments. After the data for each experiment was saved, the results were displayed on the "Results" page of the software. The particle size distribution (ie, hydrodynamic radius) was analyzed in the "Intensity distribution" graph. In this graph, any peak outside the range of 0.1 nm to 10 μm was considered an artifact. In particular, clean water (free of particles) produced no peaks within the range of 0.1 nm to 10 μm and broad peaks less than 0.1 nm. This peak was taken as the noise peak (noise flow) of the tool. Samples with very low concentrations or very small sizes of suspended nanocrystals or nanoparticles may exhibit measurable noise peaks in the "Intensity Distribution" graph. If the peaks within the range of 0.1 nm to 10 μm have a higher intensity than the noise peaks, then these peaks are considered real, otherwise the peaks can be problematic and represent artifacts of data processing.

Figure 25c shows graph data corresponding to a representative Viscotek output data set for Example 5 (ie, GD-007), and Fig. 26c shows a representative Viscotek output for Example 6 (ie, GD-016). The graph data corresponding to the data set is shown, and FIG. 27C shows the graph data corresponding to the representative Viscotec output data set for Example 7 (ie, GD-015). The number reported at the top of each peak in FIGS. 25C , 26C and 27C corresponds to the average hydrodynamic radius of the nanocrystals, light scatters around these nanocrystals, and is detected in each solution. A number of (e.g., hundreds of) data points were examined to give the number reported within each data set, as represented by an "s-shaped" curve (i.e., each curve was a series of collected data points). representing data points). The "% transmittance" reported in each data set corresponds to the intensity of the interrogation beam required to achieve dynamic light scattering data. Generally, but not always, very strong particles and/or particle/regular water structures are present when the reported "% transmittance" is less than 50%. Also, when "% transmittance" approaches 100%, often ions and/or very small particles (eg, pico-sized particles) are present, and the reported hydrodynamic radius is more ordered or structured than true solid particles. may contain water.

Dynamic light scattering assumes that the particles are not all spherical (but not these) but also measure the hydrodynamic radius (e.g., the effect of nanocrystals on water is also detected and reported in addition to the actual physical radius of the particle). ) algorithm, it should be noted that the dynamic light scattering particle size information is different from the TEM measured histogram. Thus, as in the other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in TEM histogram data and those reported in dynamic light scattering data.

<u>Atomic Absorption Spectroscopy</u>

AAS values were obtained from a Perkin Elmer AAnalyst 300 spectrometer system.

I) Principle

The technique of flame atomic absorption spectroscopy requires that a liquid sample be aspirated, atomized, and mixed with a combustible gas such as acetylene and air. The mixture is ignited in a flame whose temperature ranges from about 2,100°C to about 2,400°C. During combustion, atoms of the element of interest in the sample are reduced to free, unexcited ground-state atoms, which absorb light at the intrinsic wavelength. The intrinsic wavelength is element-specific and is accurate to 0.01 to 0.1 nm. To provide the elemental intrinsic wavelength, a light beam from a hollow cathode lamp (HCL) made of the element whose cathode is determined is passed through a flame. A photodetector detects a decrease in light intensity due to absorption by the analyte. A monochromator is used in front of the photodetector to reduce background ambient light and select a specific wavelength from the HCL required for detection. In addition, the deuterium arc lamp compensates for background absorbance generated by non-atomic species in the atomic cloud.

II) Sample preparation

10 mL of sample, 0.6 mL of 36% v/v hydrochloric acid and 0.15 mL of 50% v/v nitric acid were mixed together in a glass bottle and incubated in a 70° C. water bath for about 10 minutes. If the gold concentration is expected to exceed 10 ppm, the sample is diluted with DI water prior to addition of acid to induce a final gold concentration in the range of 1-10 ppm. For example, for a gold concentration of approximately 100 ppm, 0.5 ml of sample is diluted with 9.5 ml of DI water prior to addition of acid. Aliquoting was performed with an adjustable micropipette and the correct amount of sample, DI water and acid were measured by an Ohaus PA313 microbalance. The weight of the component is used to calibrate the measured concentration for dilution with DI water and acid. Each sample was prepared in triplicate and allowed to cool to room temperature before measurements were made after incubation in a water bath.

III) Tool Setup

The following settings were used for the Perkin Elmer A-Analyst 400 Spectrometer System.

a) Burner head: 10 cm single slotted, aligned in 3 axes according to manufacturing procedure to obtain maximum absorbency with 2 ppm Cu standard.

b) Atomizer: plastic with spacers in front of the impact bead.

c) gas flow<b>:</b> An oxidant (air) flow rate of about 12 l/min, a fuel (acetylene) flow rate of about 1.9 ml/min.

d) Lamp/monochromatizer: Au hollow cathode lamp, 10 mA operating current, 1.8/1.35 mm slit, 242.8 mm wavelength, background correction (deuterium lamp) on.

IV) Analytical procedure

a) Run the Au ramp and flame for approximately 30 minutes to warm up the system.

b) Calibrate the instrument with 1 ppm, 4 ppm and 10 ppm Au standards in a matrix of 3.7% v/v hydrochloric acid. 3.7% v/v hydrochloric acid is used as blank.

c) Verify the calibration scale by measuring a 4 ppm standard as a sample. The measured concentration should be between 3.88 ppm and 4.12 ppm. If outside this range, repeat step b).

d) The sample is measured three times. If the standard deviation between repetitions exceeds 5%, the measurement is repeated, otherwise proceed to the next sample.

e) Perform verification step c) after measuring at least 6 samples. If verification fails, steps b) and c) re-measure all samples measured after the last successful verification.

V) data analysis

The measured concentration values for each iteration were corrected for dilution with water and acid to calculate the actual sample concentration. The reported Au ppm values are the average of three corrected values for individual iterations.

<u>Plasma irradiance and characterization</u>

This embodiment provides a spectroscopic analysis of a tunable plasma 4 using a gold electrode 1, all of which are used in the embodiments herein. Three different spectrometers with high sensitivity were used to collect spectral information for the plasma 4 . Specifically, spectroscopic analysis was performed on several gold electrode plasmons. Different intensities of some species were observed, as well as species in plasma 4 . The presence/absence of such species can affect (eg, positively and negatively) products and processing parameters made in accordance with the teachings herein.

In this regard, FIG. 25F shows a schematic diagram of the experimental setup used to collect radiation spectroscopy information from the tunable plasma 4 used herein.

Specifically, an experimental setup for collecting plasma emission data (eg, irradiance) is shown in FIG. 25F . Generally, three spectrometers 520 , 521 , 522 receive emission spectral data via UV optical fiber 523 that transmits collimated spectral radiation collected by assembly 524 along path 527 . Assembly 524 can be positioned vertically to collect spectral radiation at different vertical positions within adjustable plasma 4 by moving assembly 524 to XZ stage 525 . Accordingly, the presence/absence and intensity of the plasma species can be determined as a function of interrogation in the plasma 4 . The outputs of the spectrometers 520 , 521 , 522 were analyzed by appropriate software installed on the computer 528 . All irradiance data is collected through holes 531 disposed approximately opposite to anti-reflective material 530 . The base of the hole 531 was located on the upper surface of the liquid 3 . More details of the device for collecting emitted radiation are as follows.

Assembly 524 included one UV collimator (LC-10U) with a refocusing assembly (LF-10U100) for the 170-2,400 nm range. Assembly 524 also included an SMA female connector manufactured by Multimode Fiber Optics, Inc. Each LC-10U and LF-10U100 had one UV fused silica lens associated with it. Adjustable focusing was provided by the LF-10U100 at about 100 mm from the cavity of the lens of the LF-10U100 also included in assembly 524 .

The collimator field of view at both ends of the adjustable plasma 4 was determined by a 455 μm fiber core diameter containing a solarization resistant UV optical fiber 523 (range 180-900 nm, manufactured by Mitsubishi). It was about 1.5 mm in diameter as a bar. UV optical fibers 523 were terminated at each end by an SMA male connector (sold by Ocean Optics, QP450-1-XSR).

UV collimator-fiber systems 523 and 524 provided sensitivities in the range of 180 to 900 nm for plasma irradiance emanating from horizontally oriented 1.5 mm diameter plasma cylinders at different locations within the adjustable plasma 4 .

The XZ stage 525 included two linear stages PT1 manufactured by Thorlabs Inc that maintained the UV collimator 524 and controlled the movement along the X and Z axes. Thus, it is possible to scan the adjustable plasma 4 horizontally and vertically respectively.

The emission of plasma radiation collected by the UV collimator-fiber system 523, 524 was measured by three fiber coupled spectrometers 520, 521 or 522 manufactured by StellarNet, Inc. [i.e., 180-295]. EPP2000-HR for gratings of nm, 2,400 g/mm, EPP2000-HR for gratings of 290-400 nm, 1800 g/mm and EPP2000-HR for gratings of 395-505 nm, 1,200 g/mm] . Each spectrometer 520, 521, 522 had a 7 μm entrance slit, 0.1 nm optical resolution, and a 2048 pixel CCD detector. The measured instrument spectral line extension is from 313.1 nm to 0.13 nm.

Spectral data acquisition was controlled by SpectraWiz software for Windows/XP manufactured by Stellanet. All three EPP2000-HR spectrometers (520, 521, 522) interfaced with one personal computer (528) equipped with four USB ports. The integration time and number of averaging over the various spectral ranges and plasma discharges were appropriately set to give the best possible signal-to-noise ratio for the unsaturated signal intensities. Typically, the spectral integration time was on the order of 1 second, and the number average spectra ranged from 1 to 10. All recorded spectra were acquired with an audited optimal background. The optical background was acquired before the start of acquisition of the corresponding set of measurements, each with the same data acquisition parameters.

Each UV fiber-spectrometer system (i.e., 523/520, 523/521 and 523/522) was equipped with an AvaLight-DH-CAL radiation calibrated light source manufactured by Avantes (not shown). Corrected. After calibration, all obtained spectral intensities were not only expressed in (absolute) units of spectral irradiance (mW/m 2 /nm), but also corrected for the non-linear response of the UV-fiber-spectrometer. The relative error of the Avarite-DH-CAL radiation corrected light source in the range of 200 to 1,100 nm is less than 10%.

Alignment of the field of view of the UV collimator assembly 524 to the tip 9 of the metal electrode 1 was performed prior to each set of measurements. The center of the UV collimator assembly 524 field of view is at the tip 9 by alignment of the two linear stages and by sending light to the center of each metal electrode 1 through the UV collimator-fiber system 523, 524. placed.

The XZ stage 525 is capable of vertically moving the assembly 524 so that analysis of the spectral emission at different vertical heights within the adjustable plasma 4 can be made while the assembly into a generally horizontal center of the adjustable plasma 4 . (524) is used to move. In this regard, the assemblies 524 are arranged at different heights, the first height of which is arranged as close as possible to the tip 9 of the electrode 1 , and then moved away from the tip 9 by a certain amount. became The emission spectroscopy of the plasma does not change as a function of interrogation location.

For example, FIGS. 25G-25J show radiation data associated with a gold (Au) electrode 1 used to form a tunable plasma 4 . Each of FIGS. 25G-25J described above shows emission data associated with three different vertical interrogation positions within the adjustable plasma 4 . The vertical position "0" (0 nm) corresponds to emission spectroscopy data collected immediately adjacent to the tip 9 of the electrode 1 , and the vertical position "1/40" (0.635 nm) corresponds to 0.635 from the tip 9 . mm and corresponds to emission spectroscopy data towards the surface of water (3). The vertical position "3/20" (3.81 mm) corresponds to the emission spectroscopy data towards the surface of the water 3 at a distance of 3.81 mm from the tip 9 .

Table 2d specifically shows each of the spectral lines identified in the tunable plasma 4 when the gold electrode 1 is used to generate the plasma 4 .

&lt;Table 2d&gt;

<img file="KR20120052967A_D0017.tif" />

<img file="KR20120052967A_D0018.tif" />

Various species related to the gold metal electrode 1 were identified in Table 2d. These species are for example gold from electrode 1 as well as NO, OH, N₂ common species including, etc. It is interesting that the presence and/or intensity (eg, amount) of some species is a function of position in the plasma which is adjustable. Thus, this suggests that various species may arise as a function of various processing conditions of the present invention (eg, power, location, composition, etc. of electrode 1 ).

<u>Examples 8-10 </u>

gold<u> Preparation of nanocrystals/nanocrystal suspensions GB-018, GB-019 and GB-020</u>

In general, Examples 8-10 each utilize a particular embodiment of the invention in connection with the apparatus generally shown in Figures 17A, 18A, 19B and 22A (e.g., tapered trough member 30b). ]. Certain differences in processing and apparatus will be apparent in the respective embodiments. The trough members 30a and 30b were made of 1/8" (about 3 mm) thick plexiglass and 1/4" (about 6 mm) thick polycarbonate, respectively. The support structure 34 was also made of plexiglass that was about 1/4" thick (about 6-7 mm thick). The cross-sectional shape of the trough member 30a shown in Fig. 18A is the shape shown in Fig. 10B. (i.e., truncated cone "V"). The base ("R") of truncated cone "V" measured about 0.5" (about 1 cm), and each side portion ("S", "S'") ) measures about 1.5" (about 3.75 cm). The distance ("M") separating the side portions ("S", "S'") of the V-shaped trough member 30a is about 2¼" to 2 5/16" (about 5.9 cm) (measured from side to side) ) was. The thickness of each part was also measured to be about 1/8" (about 3 mm) thick. The longitudinal length of the V-shaped trough member 30a ("L_T") (see FIG. 11A ) measured about 3 feet (about 1 meter) from point 31 to point 32 .

Purified water (discussed hereinbelow) in the range of about 0.396 g/L to 0.528 g/L NaHCO₃was mixed with and used as the liquid 3 entered into the trough member 30a. NaHCO in this range used₃Although effective, this amount should not be considered as limiting the boundaries and scope of the present invention. The depth "d" (see FIG. 10B) of the water 3 in the V-shaped trough member 30a is from about 7/16" to about 1/2" (about 11 mm) at various points along the trough member 30a. to about 13 mm). Depth "d" was controlled in part through the use of dam 80 (shown in FIG. 18A ). Specifically, a dam 80 was provided near the end 32 and had a depth "d" (shown in FIG. 10B ) such that it was about 7/6" to 1/2" (about 11 to 13 mm) deep. ) to help create The height ("j") of the dam 80 was measured to be about 1/4" (about 6 mm), and the longitudinal length ("k") was measured to be about 1/2" (about 13 mm). The width (not shown) completely crosses the bottom dimension ("R") of the trough member 30a. Thus, the total volume of water 3 in V-trough member 30a is about 6.4 inches during its operation.³(about 105 ml).

The flow rate of the water 3 in the trough member 30a ranged from about 150 ml/min to at least 280 ml/min. This flow of water 3 is a Masterflex rated at 0.1 horsepower, 10 to 600 rpm.^&amp;#174; It was obtained by using the L/S pump drive (40). Masterplex^&amp;#174; The model number of pump 40 was 77300-40. The pump drive is a Masterflex known as Easy-Road model number 7518-10.^&amp;#174;also had a pump head manufactured by In general, the head of the pump 40 is known as a peristaltic head. Pump 40 and head are masterflex^&amp;#174; Controlled by LS digital modular drive. The model number for the digital modular drive is 77300-80. The correct setting for the digital modular drive was, for example, 150 ml/min. Tygon with a diameter of 1/4" (ie size 06419-25)^&amp;#174; Tubing was placed on the peristaltic head. Tubing is Masterflex^&amp;#174;was prepared by Saint-Gobain for. One end of the tubing was delivered to the first end 31 of the trough member 30a by a flow spreading means disposed therein. The flow diffusion means are intended to minimize any pulsing conditions created by the peristaltic pump 40 as well as obstructions and bubbles in the water 3 introduced into the trough member 30a. In this regard, a small reservoir served as a diffusion means and was provided at a point perpendicular to the end 31 of the trough member 30a to provide a relatively stable flow to the end 31 of the V-shaped trough member 30a when the reservoir overflows. A flow of water 3 occurred.

Five electrode sets were used in Examples 8 to 10, and one set was a single electrode set 1a/5a disposed on the trough member 30a. Plasma 4 from electrode 1a in trough member 30a was produced with electrode 1 similar in shape to that shown in Fig. 5e and weighed about 9.2 grams. This electrode was 99.95% pure gold. The other electrode 5a included a right-angled triangular platinum plate of about 14 mm×23 mm×27 mm with a thickness of about 1 mm immersed in liquid 3' by about 9 mm. The AC transformer used to generate the plasma 4 is the transformer 60 shown in FIG. 16D and discussed herein. An AC transformer 50 (discussed herein) was connected to another electrode set 5/5. All other relevant operating conditions are shown in Tables 3a, 3b and 3c.

The processing-enhanced output of conditioned water 3' is collected in a reservoir 41 and thereafter removed at a flow rate substantially equal to that of the pump 40 (eg, minimal evaporation generated within the trough member 30a). 2 is pumped into the trough member 30b by another pump 40'. The second trough member 30b shown in FIG. 22A is tapered and measures about 3.75 inches high at its end 32, about 3.75 inches wide, and about 1 inch wide at its end 31, so that A tapered shape was formed. This trough member 30b contained therein about 1,450 ml of liquid 3 that was about 2.5 inches deep. Each of the four electrode sets 5b, 5b, 5e, 5e contained Example 8 and 99.95% pure gold wire measuring about 5 inches (about 13 cm) long and about 0.5 mm diameter at 9 and about 1.0 mm diameter in Example 10. In each of Examples 8-10, approximately 4.25 inches (about 11 cm) of wire was immersed in water 3" having a depth of about 2.5 inches (about 6 cm). Each electrode set 5a, 5a' , 5d, 5d) were shaped like "J" as shown in Fig. 17A The distance ("g") as shown in Fig. 17A was measured to be about 1 to 8 mm.

19B and 22A , four separate sets of electrodes (Set 2, Set 3, Set 4 and Set 5) were attached to a single transformer device 50 . Specifically, the transformer 50 was the same transformer used in Examples 5 to 7, but was electrically connected to each electrode set according to the wiring diagram shown in Fig. 19B. In contrast, this winding configuration was different from that used in Examples 5-7 described above, where only a single transformer 50 required the low amperage requirements of the trough member 30b design of the present invention [eg, less wire is in contact with liquid 3].

Each of Tables 3a to 3c contains processing information for each of the four electrode sets by "set number". Each electrode of the set of four electrodes in the trough 30b was set to operate at a specific target voltage. An actual operating voltage of about 255 volts was applied to the four electrode sets, as listed in each of Tables 3a-3c. The distance ("cc") from the centerline of each set of electrodes to an adjacent set of electrodes (see Fig. 14) is also represented. In addition, the distance "x" associated with the electrode 1 used in the trough member 30a is also reported. At electrode 5, no distance ("x") was reported. Other relevant variables are reported, for example, in each of Tables 3a to 3c.

All materials for the electrodes (1/5) were obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520.

Water (3) used in Examples 8 to 10 was produced by a reverse osmosis process and a deionization process, and NaHCO₃ It was mixed with the processing enhancer and fed together into the trough member 30a. Essentially, reverse osmosis (RO) is a pressure-driven membrane separation process that separates species that are dissolved and/or suspended substances from surface water. This is called "reverse" osmosis because pressure is applied to reverse the natural flow of osmosis (which seeks to balance the concentration of substances on both sides of the membrane). The applied pressure forces water through the membrane leaving contaminants on one side of the membrane and purified water on the other side. Reverse osmosis membranes use multiple thin layers or sheets bonded together and wound in a spiral shape around a plastic tube. (It is also known as a thin film composite or TFC membrane). In addition to the removal of dissolved species, the RO membrane also separates suspended material, including microorganisms, that may be present in the water. A mixed bed deionization filter was used after RO processing. Total dissolved solvent ("TDS") after both treatments is Accumet^&amp;#174; It was about 0.2 ppm as measured by an AR20 pH/conductivity meter.

&lt;Table 3a&gt;

<img file="KR20120052967A_D0019.tif" />

&lt;Table 3b&gt;

<img file="KR20120052967A_D0020.tif" />

&lt;Table 3c&gt;

<img file="KR20120052967A_D0021.tif" />

28A, 29A and 30A are representative TEM micrographs corresponding to dried suspensions GB-018, GB-019 and GB-020 showing gold crystals grown in Examples 8, 9 and 10, respectively.

Figures 28b, 29b and 30b show the particle size distributions measured from TEM micrographs (i.e., using the software described above in Examples 5-7) corresponding to the dried suspensions taken from Examples 8, 9 and 10, respectively. is a histogram.

28c, 29c and 30c show dynamic light scattering data (ie, hydrodynamic radius) of gold nanocrystal suspensions prepared in Examples 8, 9 and 10, respectively. Each of these figures shows a graphical result of a dynamic light scattering data set.

Dynamic light scattering assumes that the crystals are not all spherical (but not these) but also measure the hydrodynamic radius (e.g., the effect of a particle on water is also detected and reported in addition to the particle's actual physical radius). It should be noted that the dynamic light scattering particle size information is different from the TEM measured histogram because of the algorithm used. Thus, as in the other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in TEM histogram data and those reported in dynamic light scattering data.

<u>Example 11</u>

<u>Preparation of gold-based nanoparticles/nanoparticle solutions or colloids IAC-202-7 by batch process</u>

This embodiment uses a batch process according to the present invention. 24a shows the device used to condition the liquid 3 . Once conditioned, the liquid 3' is processed in the apparatus shown in FIG. 24B.

Table 4a shows the processing enhancer baking soda (i.e., NaHCO₃) represents the matrix changed from about 1 gram/gallon to 2 grams/gallon (i.e., from about 0.264 g/L to about 0.528 g/L), and residence times reflected in Table 4A in the device of FIG. 24A [i.e., processing enhancer The amount of time that water 3 with The applied voltage for each plasma 4 produced by electrode 1 was about 750 volts. This voltage was achieved by the transformer 60 discussed herein (ie, a balanced midpoint referenced design). A second and different transformer was electrically connected to the electrodes 5a / 5b shown in Fig. 24c. This transformer was a hy AC power source with a voltage range of 0 to 300 V, a frequency of 47 to 400 Hz and a maximum power rating of 1 kVA. The applied voltage for each identified implementation in Tables 4a and 4b was about 250 volts. The changed current as a function of time with minimum and maximum volts is reported in Table 4b. All other process variables were kept constant.

Thus, Table 4a shows that a number of variables (eg, process enhancers and predetermined residence times) affect both the amount or concentration of gold nanocrystals in water and the size distribution of the gold nanocrystals. In general, as the concentration of a processing enhancer increases from about 1 g/gallon (0.264 g/L) to about 2 g/gallon (0.528 g/L), the concentration (i.e., "ppm") increases with that of a given processing condition. It increases somewhat under the set. However, in some cases, the particle size distribution ("psd") increases undesirably so that the formed nanocrystals are no longer stable and they "precipitate" as a function of time (eg, an unstable suspension is prepared). These precipitation states are not instantaneous and thus suggest that suspensions of nanocrystals in water can be immediately processed into useful products such as, for example, gels or creams. This embodiment clearly demonstrates the various important effects of a number of processing variables that can be translated, at least in a direction, into the continuous process of the present invention discussed herein. These data are exemplary and should not be considered as limiting the scope and boundaries of the present invention. Moreover, these exemplary data should provide those skilled in the art with a good direction of operation to seek.

As a specific example, Table 4C shows that the first electrode set number 1 (ie, FIG. 24A ) is operated at a voltage of about 750 volts to form plasma 4 . This is similar to the other plasmas 4 discussed herein. However, electrode set number 2 (ie, FIG. 24C ) is powered by the hy-AC source described above.

&lt;Table 4a&gt;

<img file="KR20120052967A_D0022.tif" />

&lt;Table 4b&gt;

<img file="KR20120052967A_D0023.tif" />

&lt;Table 4c&gt;

<img file="KR20120052967A_D0024.tif" />

31A shows a representative TEM micrograph of a gold crystal dried from a solution prepared according to Example 11.

31B shows a histogram of particle size distribution based on TEM measurement of dried gold nanocrystals prepared according to Example 11. FIG.

31C shows schematic dynamic light scattering particle size data (ie, hydrodynamic radius) from Example 11. FIG. Specifically, a representative Viscotec data set is illustrated in this figure similar to that discussed herein.

Dynamic light scattering assumes that the particles are not all spherical (but not these) but also measure the hydrodynamic radius (e.g., the effect of nanocrystals on water is also detected and reported in addition to the actual physical radius of the nanocrystals). It should be noted that the dynamic light scattering particle size information is different from the TEM measured histogram because of the use of the TEM algorithm. Thus, as in the other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in TEM histogram data and those reported in dynamic light scattering data.

<u>Example 12</u>

<u>Preparation of gold-based nanoparticles/nanoparticle solutions or colloidal IAC-261 by batch process</u>

This embodiment uses a batch process according to the present invention. 24a shows the device used to condition the liquid 3 . Once conditioned, liquid 3' was treated with the apparatus shown in FIG. 24C.

Processing enhancer baking soda (e.g. NaHCO₃) is about 1.5 grams/gallon (eg, about 0.396 g/l). The amount of time the water 3 using the processing enhancer was exposed to the plasma 4 prior to subsequent processing in the apparatus shown in FIG. 24C was about 60 minutes.

The applied voltage for each plasma 4 produced by electrode 1 is about 750 volts. This voltage was achieved by the balanced midpoint referenced design discussed herein).

The secondary and different transformers were electrically connected to the electrodes 5a/5b shown in Fig. 24c. These transformers are from hy AC power sources with a voltage range of 0 to 300 V, a frequency range of 47 to 400 Hz and a maximum power rating of 1 kVA. The applied voltage was about 300 volts. The current changed as a function of time of a minimum amp of 0.390 and a maximum amp of 0.420 amps over a 60 min operating time. The diameter of the gold wire electrode is 1 mm.

The amount of gold nanoparticles produced in the suspension is about 13.7 ppm as determined by atomic absorption spectroscopy as discussed herein. The size and shape of the nanoparticles produced by this example are explicitly discussed in Table 12 herein.

33A shows a representative TEM micrograph of dried gold crystals from suspension IAC-261 produced according to Example 12.

33B shows a histogram of particle size distribution based on TEM measurement of dried gold nanoparticles produced according to Example 12.

<u>Example 13 </u>

gold<u> Preparation of Nanocrystals/Nanocrystal Suspensions GB-154-20Hz, GB-157-40Hz, GB-159-60Hz, GB-161-80Hz, GB-173-100Hz and GB-156-300Hz</u>

In general, this Example used the same manufacturing setup used for the manufacture of GB-134 in Example 16, and the details of the trough apparatus used were discussed in detail in the Examples. The main difference in the manufacture of the suspension or colloid in this embodiment is that different sinusoidal frequencies from the programmable AC power source are used as electrical inputs to the electrodes 5a/5b.

In particular, nanocrystal suspensions or colloids have been produced using sinusoidal AC frequencies as low as 20 Hz and as high as 300 Hz in accordance with the teachings herein. AC Power The 501AC is used for the Chroma 61604 programmable AC power. The applied voltage was 300 volts. The waveform is a sine wave at six different frequencies: 20, 40, 60, 80, 100 and 300 Hz. The applied current varies between 4.2 amps and 4.8 amps.

34A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-154, and FIG. 34B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-154.

35A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-157, and FIG. 35B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-157.

FIG. 36A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-159, and FIG. 36B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-159.

37A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-161, and FIG. 37B shows a particle size distribution histogram based on TEM measurements of dried gold nanocrystals from suspension GB-161.

38A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-173, and FIG. 38B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-173.

FIG. 39A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-156, and FIG. 39B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-156.

It is clear from this example that both the particle size "mode" and the particle size distribution increased with increasing frequency AC sine wave under the conditions of this example.

<u>Example 14 </u>

gold<u> Preparation of nanocrystals/nanocrystal suspensions (GB-166-sine, GB-165-square and GB-162-triangle)</u>

In general, this example used the same manufacturing setup used to create the GB-134 in Example 16, and briefly, the details of the trough apparatus used are discussed in detail in this example. The main difference in the preparation of suspensions or colloids in this example is that three different types of waveforms (eg sine, square and sensory waves) were generated by a BK Precision 4040 20 MHz function generator, 501FG. The waveform output was input to the Chroma 61604 programmable AC power supply, 501AC. The applied voltage for the sine wave ("SI") and square wave ("SQ") was 300 volts, and the applied voltage for the triangle-shaped waveform ("TR") was 250 volts. Each of these waveforms is shown in FIG. 41 . Specifically, GB-166 used a sine wave as an electrical input to the electrodes 5a/5b; GB-165 used a square wave; GB-162 used a triangular wave.

42A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-166, and FIG. 42B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-166.

43A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-165; 43B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-165.

44A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-162, and FIG. 44B shows a particle size distribution histogram based on TEM measurements of dried gold nanocrystals from suspension GB-162.

<u>Example 15 </u>

gold<u> Preparation of nanoparticles/nanoparticle suspensions (GB-163 and GB-164)</u>

In general, this example used the same manufacturing setup used to create the GB-134 in Example 16, and briefly, the details of the trough apparatus used are discussed in detail in this example. The main difference in the preparation of the suspension or colloid in this example is the two different duty cycles for the triangular waveform from the signal generator 501FG, and a programmable AC power supply 501AC (eg discussed in Example 14) was used. The applied voltage for each triangular waveform is 250 volts. Specifically, each of GB-166 and GB-164 used the triangular-shaped waveforms TR-1, TR-2 and TR-3 shown in Fig. 45 as electrical inputs to the electrodes 5a/5b. Waveform TR-2 is the maximum duty cycle and TR-3 is the minimum duty cycle.

46A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-163, and FIG. 46B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-163.

47A shows a representative TEM micrograph of dried gold nanocrystals from suspension GB-164, and FIG. 47B shows a histogram of particle size distribution based on TEM measurements of dried gold nanocrystals from suspension GB-164.

<u>Example 16 </u>

gold<u> nanocrystals/nanocrystal suspensions (GB-134); (GB-098, GB-113 and GB-118); (GB-120 and GB-123); (GB-139); (GB-141 and GB-144); (GB-079, GB-089 and GB-062); and (GB-076 and GB-077)</u>

In general, this Example 16 uses a particular embodiment of the invention in connection with the apparatus shown generally in FIGS. 20C-H, 21B-G and 22B. Additionally, Table 5 below summarizes the key processing parameters used in connection with FIGS. 20C-H, 21B-G and 22B. Table 5 also shows 1) the resulting "ppm" (ie, gold nanoparticle concentration), 2) the average of the three highest amplitude peaks shown in each of FIGS. 49C-61 (discussed herein below). coming from a single number, and 3) for the "hydrodynamic radius" taken from and also discloses a "TEM average particle diameter" the mode that corresponds to the particle size of the most frequently occurs as determined by the TEM histogram illustrated in FIG. 49b through 61b . These physical properties are performed as discussed herein.

&lt;Table 5&gt;

<img file="KR20120052967A_D0025.tif" />

<img file="KR20120052967A_D0026.tif" />

All of the trough members 30a', 30b' in the preceding figures were made of 1/8" (about 3 mm) thick plexiglass and 1/4" (about 6 mm) thick polycarbonate, respectively. Support structure 34 (not shown in many figures, but discussed herein) was also made of plexiglass that was about 1/4" thick (about 6-7 mm thick). Shown in FIGS. 19A and 19B . In contrast to the illustrated embodiment, each trough member 30a is integral with the trough member 30b' and is thus designated 30a' [eg, as in certain previous embodiments, separately, pumping means is not provided after the trough member 30a.] The cross-sectional shape of each trough member 30a' used in this embodiment corresponds to the shape shown in Fig. 10B (that is, a trapezoidal-shaped cross-section). Lim). The relevant dimensions of each trough member portion 30b' are listed in Table 15 as "M1" (ie, the inner width of the trough at the inlet portion of the trough member 30b'), "M2" (ie trough member 30b'). Inner width of the trough at the outlet part of], "L_T"[i.e., the transverse length or flow length of the trough member 30b'], "S" [i.e., the height of the trough member 30b'] and "d" [i.e., the liquid in the trough member 30b' ( 3"). In some embodiments, the distance "M" separating the side portions "S", "S" (see FIG. 10A ) of the trough member 30b was the same. In these cases, Table 15 shows only the value dimension for "M1" and the inlet for "M2" is expressed as "N/A". In other words, some trough members 30b' tapered along their longitudinal length, and in other cases the trough members 30b' were substantially straight along their longitudinal length. The thickness of each sidewall portion was also measured to be about 1/4" (about 6 mm) thick. Three different longitudinal lengths ("L"_T") is reported for the trough member 30b' (ie, 610 mm, 914 mm or 1,219 mm), but other lengths L_T) should be considered to be within the boundaries and scope of the trough member of the present invention.

Table 5 shows processing enhancers (NaHCO₃) is added to purified water (discussed herein) in an amount of about 0.4 mg/ml or 0.53 mg/ml. It is to be understood that other amounts of this processing enhancer also function within the scope and boundaries of the present invention. Purified water/NaHCO₃ The mixture was used as the liquid 3 fed into the trough member 30a'. The depth "d" of the liquid 3' in the trough member 30a' (ie, the plasma(s) 4 formed) is about 7/16 at various points along the trough member 30a'. " to about 1/2" (about 11 mm to about 13 mm). Depth "d" is controlled in part through the use of dam 80 (shown in FIGS. 18A and 18B ). Specifically, a dam 80 was provided near the output end 32 of the trough member 30a', and had a depth "d" (about 7/6" to 1/2" (about 11 to 13 mm) deep. (shown as "d" in Figure 10b). The height "j" of the dam 80 was measured to be about 1/4" (about 6 mm), and the longitudinal length "k" was measured to be about 1/2" (about 13 mm). The width (not shown) is completely transverse to the base dimension "R" of the trough member 30a'. Thus, during their operation, the total volume of liquid 3' in trough member 30a' is about 2.14 inches.³(about 35 ml) to about 0.80 in³(about 14.58 ml).

The flow rate of liquid 3' into trough member 30a' as well as trough member 30b' was about 150 mL/min for all but one of the formed samples (i.e., GB-144, which was about 110 mL/min). , the flow rate from trough member 30b' at point 32 was about 110 mL/min (ie, due to evaporation) for all samples except GB-144, which was about 62 mL/min. The amount of evaporation generated in the GB-144 is higher than the other samples because the residence time of the liquid 3" in the trough member 30b' is longer for the other samples prepared according to this embodiment. Flow rates should be considered to be within the scope and boundaries of the present invention.

This flow of liquid 3' is a Masterflex rated at 0.1 horsepower, 10 to 600 rpm.^&amp;#174; Obtained using the L/S pump drive (40). Masterplex^&amp;#174; The model number of pump 40 was 77300-40. The pump drive is a Masterflex known as Easy-Road model number 7518-10.^&amp;#174;also had a pump head manufactured by In general, the head of the pump 40 is known as a peristaltic head. Pump 40 and head are masterflex^&amp;#174; Controlled by LS digital modular drive. The model number for the digital modular drive is 77300-80. The exact setting of the digital modular drive was 150 ml/min for all samples except GB-144 which was for example 110 mm/min. Tygon with a diameter of 1/4" (ie size 06419-25)^&amp;#174; Tubing was placed on the peristaltic head. Tubing is Masterflex^&amp;#174;was prepared by Saint-Gobain for. One end of the tubing was delivered to the first end 31 of the trough member 30' by a flow spreading means disposed therein. The flow diffusion means are intended to minimize any pulsing conditions created by the peristaltic pump 40 as well as obstructions and bubbles in the water 3 introduced into the trough member 30'. In this regard, a small reservoir served as a diffusion means and was provided at a point perpendicular to the top of the end 31 of the trough member 30' to the end 31 of the V-shaped trough member 30a' when the reservoir overflowed. A relatively stable flow of liquid 3' occurred.

Table 5 shows that a single electrode set 1a/5a or two electrode sets 1a/5a are used in this embodiment 18. Plasma 4 was produced with an electrode 1 similar in shape to that shown in FIG. 5E and weighed about 9.2 grams. This electrode was 99.95% pure gold. The other electrode 5a included a right-angled triangular platinum plate of about 14 mm×23 mm×27 mm with a thickness of about 1 mm immersed in liquid 3' by about 9 mm. All other relevant operating conditions are shown in Table 15.

20C-20H, the output from the trough member 30a' was a conditioned liquid 3', and the conditioned liquid 3' into a second trough member 30b'. moved directly. The second trough member 30b shown in FIGS. 21B-21G and 22B had measurements as reported in Table 5 . This trough member 30b' contained from about 600 ml to about 1,100 ml of liquid 3" depending on the dimensions of the trough and the depth "d"" of liquid 3" therein. Table 5 shows FIG. 20c 20H, 21B-21G, and 22B, various different electrode configurations are shown, for example, the previous examples herein have four electrodes with one electrode set (1/5). (5/5) The use of the set was initiated. In this example, 8 or 9 electrode sets were used (eg, one 1/5 set with 7 or 8 5/5' sets, or 2 with 7 5/5' sets). 1/5 of a set). Each electrode set 5/5' contained 99.99% pure gold wire, about 0.5 mm diameter or 1.0 mm diameter, as reported in Table 5. The length of each wire electrode 5 in contact with the liquid 3" ("W" in Table 5)._L") measured from about 0.5 inches (about 13 mm) long to about 2.0 inches (about 51 mm) long. Two different electrode set configurations (5/5') were used. FIG. 21B , FIG. 21c, 21e, 21f, 21g and 22b show electrode sets 5/5' all oriented along a plane (i.e., organized in the form of lines along the direction of flow of liquid 3") . On the other hand, FIG. 21D shows the electrode set 5/5' rotated about 90° with respect to the electrode set 5/5' described above. Also, the embodiment shown in FIGS. 20A-20H shows that the electrode sets 1/5, 5/5' are all arranged along the same plane. However, the imaginary plane created between the electrodes in each electrode set 1/5 and/or 5/5' is either parallel to the flow direction of liquid 3" or perpendicular to the flow direction of liquid 3" or Or it should be understood that it may have an angle with respect to the flow direction of the liquid 3.

20C-20H, 21B-21G, and 22B, each individual electrode set 5/5' (eg, Set 2, Set 3-8, or Set 9) is shown herein. Electrically connected to the transformer devices 50 and 50a as shown. Specifically, the transformers 50 and 50a were electrically connected to each electrode set according to the wiring diagrams shown in Figs. 20C to 20H. Exact wiring varies between examples and reference should be made to FIGS. 20C-20G for specific electrical connection information. In most cases, each transformer device 50, 50a was connected to a separate AC input line that was 120[deg.] out of phase with respect to each other. Transformers 50, 50a are electrically connected in such a way as not to overload a single electrical circuit, eg with an upstream circuit breaker disconnected [eg, when used under these conditions, single transformer 50/50a ) can draw enough current to cause upstream electrical problems]. Each transformer 50/50a was a variable AC transformer consisting of a single coil/winding of wire. This winding acts as part of both the primary and secondary windings. An input voltage is applied across the fixed portion of the winding. The output voltage is taken between one end of the winding and the other connection along the winding. By exposing a portion of the winding and making the secondary connection using a sliding brush, a continuously varying ratio can be obtained. The ratio of output to input voltage corresponds to the ratio of the number of turns of the winding to which they are connected. Specifically, each transformer was a Mastek TDGC2-5kVA, 10A voltage regulator, output 0-250V.

Table 5 refers to each electrode set by "set number" (eg, "set 1" through "set 9"). Each electrode in a 1/5 or 5/5 electrode set was set to operate over a specific voltage range. The voltages listed in Table 5 are the voltages used for each electrode set. The distance ("cc") from the centerline of each set of electrodes to an adjacent set of electrodes (see Fig. 14) is also represented. Additionally, the distance ("x") associated with each electrode 1 used was also reported. At electrode 5, no distance ("x") was reported. Sample GB-118 had a slightly different electrode (5a/5b) configuration than the other examples herein. Specifically, each of the tips or ends 5t, 5t' of the electrodes 5a/5b was positioned closer to each other than the other portions of the electrodes 5a/5b. The distance ("dt") between the tips 5t, 5t' varied between about 7/16 inches (about 1.2 cm) and about 2 inches (about 5 cm). Other relevant variables are reported in Table 5.

All materials for the electrodes (1/5) were obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520. Implementation of GB-139, GB-141, GB-144, GB-076, GB-077, GB-079, GB-089, GB-098, GB-113, GB-118, GB-120 and GB-123 All materials for the electrodes (5/5) were obtained from Alpha Eysar at 26 Parkridge Road, Ward Hill, USA 01835. All materials for the electrodes (5/5) in Run GB-062 were obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520.

49A-61A show two representative TEM micrographs of each of the gold nanocrystals dried from each of the suspensions or colloids mentioned in Table 5 and formed according to Example 16. FIGS.

49B-61B show the measured size distributions of gold nanocrystals formed according to Example 16 and measured using the TEM tool/software described above in Examples 5-7 for each of the solutions or colloids mentioned in Table 5. is showing

49C-61C schematically depict a set of dynamic light scattering data measurements for nanocrystals (ie, hydrodynamic radius) prepared according to each of the suspensions or colloids mentioned in Table 5 and formed according to Example 16; Dynamic light scattering particle size information assumes that dynamic light scattering measures not only the particles are all spherical (not these), but also measures the hydrodynamic radius (e.g., the effect of a particle on water also depends on the actual physical radius of the particle). It should be noted that it is different from the TEM measured histogram because it uses the additionally detected and reported) algorithm. Thus, as in the other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in TEM histogram data and those reported in dynamic light scattering data.

Reference is now made to FIGS. 20C, 20H, 21E, 21F, and 20G, which are representative of the structures used to prepare samples GB-139, GB-141 and GB-144. The trough member 30b' used to make these samples is 1) 8 electrode sets 1/5, 5/5, for example, each electrode 1/5 in each electrode set 1/5. or 5/5) are all connected to a control device 20, 20a to 20g (ie, see Fig. 20h) that automatically adjusts the height of the 5/5, and 2) each electrode set 5 when required or required. The female receiver tubes o5a/o5a' to o5g/o5g' are connected to the base portion of the trough member 30b' so that the electrodes in /5) can be removably inserted into each female receiver tube o5. Therefore, it was different from the other trough members 30b' used in this Example 16. Each female receiver tube o5 was made of polycarbonate and had an inner diameter of about 1/8 inch (about 3.2 mm) and was held in place by a solvent adhesive to the base portion of the trough member 30b'. The holes in the base of the trough member 30b' allow the outer diameter of each tube o5 to be fixed therein such that one end of the tube o5 is equal to the surface of the base portion of the trough member 30b'. make it high The inner diameter of tube o5 effectively prevented any significant amount of liquid 3 from entering female receiver tube o5. However, some liquid flows into the interior of one or more female receiver tubes o5. The length or vertical height of each female receiver tube o5 used in this embodiment was about 6 inches (about 15.24 cm), although shorter or longer lengths are within the scope and boundaries of the present invention. Also, while the female receiver tubes o5 are hereinafter shown as straight, these tubes are J-shaped so that their openings spaced from the trough member 30b' can be above the top surface of the liquid 3" if desired. or curved in a U-shaped manner.

21E, 21F and 21G, each electrode 5/5' is first placed in contact with liquid 3" and enters the female receiver tube o5. After a certain amount of process time, Gold metal is removed from each wire electrode 5 , for example causing the electrode 5 to be thinned (ie, reduced in diameter) which alters the current density and/or rate at which the gold nanoparticles are formed. Thus, the electrode 5 moves towards the female receiver tube o5 allowing the fresh thicker electrode 5 to enter the liquid 3 at their upper surface portion. In essence, a corrosion profile or tapering effect is formed on the electrode 5 after a certain amount of processing time has elapsed (i.e., the portion of the wire near the surface of the liquid 3" is typically a female receiver tube o5). typically thicker than the portion in the vicinity], this wire electrode profile or tapering can, if desired, remain essentially constant throughout the manufacturing process, such that essentially the same at any point in time after the initial pre-equilibration during a manufacturing run. Allows the product to be manufactured, for example, so that the process is cGMP under current FDA guidelines and/or conforms to ISO 9000 as well.

The movement of the electrode 5 into the female receiver tube o5 can be achieved by monitoring various specific process variables (eg, current, amperage, nanocrystal concentration, optical density or color, conductivity, pH, etc.) that change as a function of time. may occur, or may be more convenient under the aggregate processing situation, but may be moved in a predetermined amount at various time intervals to produce a fixed movement speed. In this regard, FIGS. 54d , 55d and 56d show that the current is monitored/controlled as a function of time for each of the 16 electrodes used to prepare samples GB-139, GB-141 and GB-144, respectively, to the female receiver The vertical movement of the electrode 5 into the tube o5 is shown. Under these processing conditions, each electrode 5 was moved at a rate of about 3/4 inch (about 2.4 mm/hour) every 8 hours to maintain the current reported in Figures 54d, 55d and 56d. . 55D and 56D show a typical rise or pre-equilibrium state where the current starts at about 0.2-0.4 amps and increases to about 0.4-0.75 after about 20-30 minutes. Samples were collected only from equilibrium. Because the concentration of nanoparticles produced in liquid 3 increases as a function of time when equilibrium conditions are reached that can remain substantially constant through the remainder of processing due to the control process disclosed herein. A pre-equilibrium condition occurs.

Energy absorption spectra were obtained for the sample of Example 16 using UV-Vis spectroscopy. This information was obtained using a dual beam scanning monochromator system capable of scanning a wavelength range of 190 nm to 1,100 nm. A Jasco V-530 UV-Vis spectrometer was used to collect absorption spectroscopy. The tool was set up to support the measurement of low concentration liquid samples using one of a number of fused quartz sample holders or "cuvettes". A variety of cuvettes allow data to be collected in the 10 mm, 1 mm or 0.1 mm optical path of the sample. Data are acquired over a wavelength range using the following parameters: a 250-900 nm detector with a bandwidth of 2 nm, a data pitch of 0.5 nm, and a silicon photodiode with a water baseline background. Both deuterium (D2) and halogen (WI) scan rates of a 400 nm/mm source were used as the primary energy source. The optical path of these spectrometers was set up so that the energy beam could pass through the center of the sample cuvette. Sample preparation is not limited to filling and capping the cuvette and then physically placing the sample in a cuvette holder within a fully enclosed sample compartment. The optical absorption of energy by the material of interest was determined. Data output was measured and expressed as absorbance units (according to Beer-Lambert Law) versus wavelength.

Spectral patterns in the UV-visible range were obtained for each of the solutions/colloids produced in Example 16.

Specifically, Figure 61d shows 14 suspensions/colloids (GB-134) (GB-098, GB-113 and GB-118), (GB-120 and GB-123), respectively, within the wavelength range of about 250 nm-750 nm. ), (GB-139), (GB-141 and GB-141); (GB-079, GB-089 and GB-062); and (GB-076 and GB-077) UV-Vis spectral patterns.

Figure 61E shows the UV-Vis spectral pattern for each of the 14 suspensions/colloids over a wavelength range of about 435 nm to 635 nm.

In general, UV-Vis spectroscopy is a measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic enough to promote external electrons to higher energy levels. UV-Vis spectroscopy can be applied to molecules and inorganic ions or complexes in solution or suspension.

UV-Vis spectra can be used for sample identification but have a wide range of characteristics that can also be useful for quantitative measurements. The concentration of the analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert law.

<u>Example 17 </u>

gold<u> Preparation of nanocrystal/nanocrystal suspension GB-056</u>

In general, Embodiment 17 utilizes a particular embodiment of the present invention in connection with the apparatus shown generally in Figs. 17A, 18A, 20B and 22A. The trough members 30a (30a' and 30b) are made of 1/4" (about 6 mm) thick plexiglass and 1/8" (about 3 mm) thick polycarbonate, respectively. The support structure 34 was also made of plexiglass that was about 1/4" thick (about 6-7 mm thick). As shown in FIG. 20B , the trough member 30a is coupled to the trough member 30b'. integrated and indicated by the reference numeral 30a' (eg, no separate pumping means are provided after the trough member 30a as in the particular above example). The cross-sectional shape of 30a' corresponds to the shape shown in FIG. 10B (ie, truncated cone "V"). The base ("R") of the truncated cone "V" measured about 0.5" (about 1 cm), and each side ("S", "S'") measured about 1.5" (about 3.75 cm) . The distance ("M") separating the side portions ("S", "S'") of the V-shaped trough member 30a is about 2¼" to 2 5/16" (about 5.9 cm) (measured from side to side) ) was. The thickness of each sidewall portion was also measured to be about 1/8" (about 3 mm) thick. The longitudinal length of the V-trough member 30a' ("L")_T") (see FIG. 11A ) measured about 1 foot (about 30 cm) from point 31 to point 32 .

Purified water (discussed herein) contains NaHCO in the range of about 0.396 g/L.₃was mixed with and used as the liquid 3 entered into the trough member 30a'. The depth "d" (see FIG. 10B) of liquid 3' in V-shaped trough member 30a' can range from about 7/16" to about 1/2" (see FIG. 10B) at various points along trough member 30a'. about 11 mm to about 13 mm). Depth "d" was controlled in part through the use of dam 80 (shown in FIG. 18A ). Specifically, a dam 80 was provided near the end 32 and had a depth "d" (shown in FIG. 10B ) such that it was about 7/6" to 1/2" (about 11 to 13 mm) deep. ) to help create The height ("j") of the dam 80 was measured to be about 1/4" (about 6 mm), and the longitudinal length ("k") was measured to be about 1/2" (about 13 mm). The width (not shown) is completely transverse to the base dimension "R" of the trough member 30a'. Thus, the total volume of liquid 3' in V-trough member 30a' is about 2.14 inches during its operation.³(about 35 ml).

The flow rate of liquid 3' in trough member 30a' was about 150 ml/min, and the flow rate from trough member 30b' at point 32 was in the range of about 110 ml/min (due to evaporation). . This flow of liquid 3' is a Masterflex rated at 0.1 horsepower, 10 to 600 rpm.^&amp;#174; It was obtained by using the L/S pump drive (40). Masterplex^&amp;#174; The model number of pump 40 was 77300-40. The pump drive is a Masterflex known as Easy-Road model number 7518-10.^&amp;#174;also had a pump head manufactured by In general, the head of the pump 40 is known as a peristaltic head. Pump 40 and head are masterflex^&amp;#174; Controlled by LS digital modular drive. The model number for the digital modular drive is 77300-80. The correct setting for the digital modular drive was, for example, 150 ml/min. Tygon with a diameter of 1/4" (ie size 06419-25)^&amp;#174; Tubing was placed on the peristaltic head. Tubing is Masterflex^&amp;#174;was prepared by Saint-Gobain for. One end of the tubing was delivered to the first end 31 of the trough member 30' by a flow spreading means disposed therein. The flow diffusion means are intended to minimize any pulsing conditions created by the peristaltic pump 40 as well as obstructions and bubbles in the water 3 introduced into the trough member 30'. In this regard, a small reservoir served as a diffusion means and was provided at a point perpendicular to the top of the end 31 of the trough member 30' to the end 31 of the V-shaped trough member 30a' when the reservoir overflowed. A relatively stable flow of liquid 3' occurred.

A single electrode set (1a/5a) was used in Example 17. Plasma 4 was produced with an electrode 1 similar in shape to that shown in FIG. 5E and weighed about 9.2 grams. This electrode was 99.95% pure gold. The other electrode 5a included a right-angled triangular platinum plate of about 14 mm×23 mm×27 mm with a thickness of about 1 mm immersed in liquid 3' by about 9 mm. All other relevant operating conditions are shown in Table 10.

As shown in FIG. 20B , the output from the trough member 30' was the conditioned liquid 3', and the conditioned liquid 3' flowed directly into the second trough member 30b'. . The second trough member 30b shown in FIG. 22A measured about 3.75 inches high, about 3.75 inches wide at its end 32 and about 1 inch wide at its end 31 . This trough member 30b' contained therein about 1,450 ml of liquid 3" that was about 2.5 inches deep. In this embodiment, the four electrode sets 5a, 5a', 5d, 5d' Each contained 99.95% pure gold wire measuring about 0.5 mm in diameter. Each wire 5 measured about 5 inches (about 12 cm) long. The liquid 3" was about 4.25 inches ( It was about 2.5 inches (about 6 cm) deep with a j-shaped wire of about 11 cm) immersed therein. Each electrode set 5b, 5b', 5e, 5e' was shaped like "J" as shown in Fig. 17A. The distance ("g") shown in FIG. 17A was measured to be about 1 to 8 mm.

20B and 22A, four separate electrode sets (Set 2, Set 3, Set 4 and Set 5) were attached to two separate transformer devices 50, 50a as shown in FIG. 20B. Specifically, the transformers 50 and 50a were electrically connected to each electrode set according to the wiring diagram shown in Fig. 19A. Each transformer device 50 , 50a was connected to a separate AC input line that was 120° out of phase with respect to each other. Transformers 50 and 50a are electrically connected in such a way as not to overload a single electrical circuit, for example allowing an upstream circuit breaker to be disconnected (eg, single transformer 50/50a when used under these conditions). can draw enough current to cause upstream electrical problems]. Each transformer 50/50a was a variable AC transformer consisting of a single coil/winding of wire. This winding acts as part of both the primary and secondary windings. An input voltage is applied across the fixed portion of the winding. The output voltage is taken between one end of the winding and the other connection along the winding. A continuously varying ratio was obtained by exposing a portion of the winding and making the secondary connection using a sliding brush. The ratio of output to input voltage is equal to the ratio of the number of turns of the winding to which they are connected. Specifically, each transformer was a Mastek TDGC2-5kVA, 10A voltage regulator, output 0-250V.

Table 6 shows each of the four electrode sets by "set number". Each electrode of the four electrode sets was set to operate within a specific voltage range. The actual voltage listed in Table 10 was about 255 volts. The distance ("cc") from the centerline of each set of electrodes to an adjacent set of electrodes (see Fig. 14) is also represented. In addition, the distance ("x") associated with electrode 1 is also reported. At electrode 5, no distance ("x") was reported. Other relevant variables are reported, for example, in Table 6.

All materials for the electrodes (1/5) were obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520.

&lt;Table 6&gt;

<img file="KR20120052967A_D0027.tif" />

100A-100E show five representative TEM micrographs of dried gold nanocrystals from solution/colloid GB-056 formed according to Example 16;

101A shows the measured size distribution of dried gold nanocrystals from suspension/colloids measured by using the TEM tool/software described above in Examples 5-7.

101B schematically shows three sets of dynamic light scattering data measurements (ie, hydrodynamic radii) for nanocrystals prepared according to this Example 17. Dynamic light scattering assumes that the particles are not all spherical (but not these) but also measure the hydrodynamic radius (e.g., the effect of particles on water is also detected and reported in addition to the actual physical radius of the nanocrystals). ) algorithm, it should be noted that the dynamic light scattering particle size information is different from the TEM measured histogram. Thus, as in the other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in TEM histogram data and those reported in dynamic light scattering data.

102A-D show additional representative TEM micrographs of the same suspension/colloid GB-056 produced according to Example 17, however, this suspension/colloid was administered to mice via a bottle of water in treatment group B, as discussed in Example 26. was exposed to It should be noted that this representative TEM nanocrystal image is from the dried solution GB-056, so certain drying conditions may affect the image. It is clear that some cluster formation of gold nanocrystals occurs, for example, during drying. However, FIG. 103A has a nanocrystal size distribution substantially similar to that shown in FIG. 101A. In this regard, the data shown in Figures 102 and 103 correspond to suspensions present in the drinking water bottles of mice for 24 hours between Days 2 and 3 of the Example 26 EAE experiment. The comparison of Figures 103b to 101b is important. In this regard, the dynamic light scattering data was altered. Specifically, the largest hydrodynamic radius shown in FIG. 101B is about 16.8 nm, while in FIG. 103B it is about 20.2 nm. It is clear that the dynamic light scattering data is recognized to be some type of cluster formation of nanocrystals in suspension shown by the dried suspension/gold nanocrystals TEM micrographs shown in FIGS. 102A-D.

Similarly, Figs. 104a to 104c; 105a; and FIG. 105C both correspond to the suspension/colloid GB-056 present in the drinking water bottle for 24 hours between Days 4 and 5 of the EAE experiment discussed in Example 26. Once again, it is clear that clumping of the nanocrystals has occurred.

101A, 103A, and 105A are all substantially similar for TEM measured nanocrystal sizes, where the dynamic light scattering radius (eg, hydrodynamic radius) of the nanocrystals is the smaller hydrodynamic radius reported in FIG. 101B . As shown in FIG. 103b for , it is clear that it is enlarged as shown in FIG. 105b.

These data suggest that exposure of the compositions of the invention disclosed herein to certain components, for example in mouse saliva, can cause the formation of clusters or clumps of nanocrystals suspended in liquid. Thus, prolonged exposure to certain proteins may have a "denaturing" effect on these inventive compositions. This "denaturing" effect is measurable and without wishing to be bound by any particular theory or explanation, such reactivity due to a very "clean" surface may support a desirable in vivo activity (eg, a particular protein-binding mechanism). It is very desirable to have

<u>Example 18</u>

gold<u> Nanocrystal/Nanocrystal Suspension (GB-151, GB-188, GB-175, GB-177, GB-176, GB-189, GB-194, GB-195, GB-196, GB-198 and GB-199 ) manufacturing</u>

In general, this embodiment utilizes certain embodiments of the present invention in connection with the apparatus shown generally in FIGS. 18A and 21D . A control device 20 (not shown in FIG. 21D ) was connected to electrodes 1/5 and 5/5, but the need to actuate the control device 20 due to the short runtime at each "run ID". there is no Accordingly, referring to FIGS. 3C and 9C , the ends 9' of the electrodes 5a and 5b are disposed in parallel with the base of the trough member 30b'. Additionally, Table 7 summarizes the key processing parameters used in connection with FIGS. 18A-21D . In addition, Table 7 shows that 1) the resulting "ppm" (ie, gold nanocrystal concentration), 2) the mode corresponding to the most commonly occurring crystal diameter, measured by the TEM histogram shown in FIGS. 62b-72b. "TEM average diameter" is disclosed. These physical properties are performed as discussed herein.

&lt;Table 7&gt;

<img file="KR20120052967A_D0028.tif" />

<img file="KR20120052967A_D0029.tif" />

All of the trough members 30a', 30b' in FIGS. 18A-21D described above are each made of 1/8" (about 3 mm) thick plexiglass and 1/4" (about 6 mm) thick polycarbonate, respectively. became The support structure 34 (not shown in the figure, but discussed herein) is also made of plexiglass that is about 1/4" thick (about 6-7 mm thick). In contrast to the embodiment, each trough member 30a is integral with the trough member 30b' and is thus denoted by reference number 30a' [eg, as in certain previous embodiments, no separate No pumping means are provided after the trough member 30a.] The cross-sectional shape of each trough member 30a' used in this embodiment corresponds to the shape shown in Fig. 10B (that is, a trapezoid-shaped cross-section). Lim). The relevant dimension of each trough member portion 30b' is shown in Table 7 as "M1" (i.e., the width of the trough at the outlet of the trough member 30b' equal to the inner width of the trough at the outlet portion of the trough member 30b'). inner width], "L_T"[i.e., the transverse length or flow length of the trough member 30b'], "S" [i.e., the height of the trough member 30b'] and "d" [i.e., the liquid in the trough member 30b' ( 3"). The thickness of each sidewall portion was also measured to be about 1/4" (about 6 mm) thick. Two different longitudinal lengths ("L"_T") is reported for trough member 30b' (ie, 762 mm or 914 mm), but other lengths L_T) should be considered to be within the boundaries and scope of the trough member of the present invention.

Table 7 shows processing enhancers (NaHCO₃) is added to purified water (discussed herein) in an amount of about 0.26 mg/ml or 0.53 mg/ml. It is to be understood that other amounts of this processing enhancer also function within the scope and boundaries of the present invention. Purified water/NaHCO₃ The mixture was used as the liquid 3 input into the trough member 30a'. The depth "d" of the liquid 3' in the trough member 30a' (ie, at which the plasma(s) 4 is formed) is about 7/16 at various points along the trough member 30a'. " to about 1/2" (about 11 mm to about 13 mm). Depth "d" is controlled in part through the use of dam 80 (shown in FIGS. 18A and 18B ). Specifically, a dam 80 was provided near the output end 32 of the trough member 30a', and had a depth "d" (about 7/6" to 1/2" (about 11 to 13 mm) deep. (shown as "d" in Figure 10b). The height "j" of the dam 80 was measured to be about 1/4" (about 6 mm), and the longitudinal length "k" was measured to be about 1/2" (about 13 mm). The width (not shown) is completely transverse to the base dimension "R" of the trough member 30a'. Thus, during their operation, the total volume of liquid 3' in trough member 30a' is about 2.14 inches.³(about 35 ml) to about 0.89 in³(about 14.58 ml).

The flow rate of liquid 3' to trough member 30a' as well as to trough member 30b' is varied (as shown in Table 7), and the flow rate from trough member 30b' at point 32. is changed due to different flow input and evaporation. Other acceptable flow rates should be considered to be within the scope and boundaries of the present invention.

This flow of liquid 3' is a Masterflex rated at 0.1 horsepower, 10 to 600 rpm.^&amp;#174; obtained by using the L/S pump drive (40). Masterplex^&amp;#174; The model number of pump 40 was 77300-40. The pump drive is a Masterflex known as Easy-Road model number 7518-10.^&amp;#174;also had a pump head manufactured by In general, the head of the pump 40 is known as a peristaltic head. Pump 40 and head are masterflex^&amp;#174; Controlled by LS digital modular drive. The model number for the digital modular drive is 77300-80. The exact setting of the digital modular drive was 150 ml/min for all samples except GB-144 which was for example 110 ml/min. Tygon with a diameter of 1/4" (ie size 06419-25)^&amp;#174; Tubing was placed on the peristaltic head. Tubing is Masterflex^&amp;#174;was prepared by Saint-Gobain for. One end of the tubing was delivered to the first end 31 of the trough member 30' by a flow spreading means disposed therein. The flow diffusion means are intended to minimize any pulsing conditions created by the peristaltic pump 40 as well as obstructions and bubbles in the water 3 introduced into the trough member 30a'. In this regard, a small reservoir served as a diffusion means and was provided at a point perpendicular to the top of the end 31 of the trough member 30a' to the end 31 of the V-shaped trough member 30a' when the reservoir overflows. A relatively stable flow of liquid 3' occurred.

Table 7 shows that there is a single electrode set 1a/5a used in Example 18. Plasma 4 was produced with an electrode 1 similar in shape to that shown in FIG. 5E and weighed about 9.2 grams. This electrode was 99.95% pure gold. The other electrode 5a contained 99.95% about 1 mm gold immersed in liquid 3'. All other relevant operating conditions are shown in Table 7.

The output from the trough member 30a' is a conditioned liquid 3', which flows directly into the secondary trough member 30b'. The second trough member 30b' shown in FIG. 21D has the measurements as reported in Table 7. This trough member 30b' contains from about 260 ml of liquid 3" to about 980 ml depending on the dimensions of the trough and the depth "d"" of liquid 3". Electrode configuration is used.For example, the above example herein discloses the use of 4 sets of electrode 5/5 and 1 electrode set 1/5.In this embodiment, 8 electrode sets are used (eg one 1/5 set and 7 or 8 5/5' sets.) Each electrode set 5/5' measures about 0.5 mm in diameter or 1.0 mm in diameter as reported in Table 7. 99.99% pure gold wire. The length of each wire electrode (5) in contact with the liquid (3") ("W" in Table 7)._L(reported as ") measured from about 0.75 inches (about 19 mm) long to about 1 inch (about 25 mm) long. FIG. 21D shows electrode set 5/5' aligned as shown in FIG. 5C is shown.

Each electrode set 5a / 5b is connected to a Chroma 61604 programmed AC power source (not shown, as discussed herein). The applied voltage is reported in Table 7. Specifically, Table 7 gave each electrode set a "set number" (eg, "set 1" to "set 8"). Each electrode of the 1/5 or 5/5 electrode set was set to operate within a specific voltage range. The voltages presented in Table 7 are the voltages used for each electrode set. The distance "cc" (see FIG. 14 ) from the centerline of each electrode set to the neighboring set of electrodes was also reported. Additionally, the distance "x" (see eg FIG. 2A ) with respect to each electrode 1 used was also reported. Other relevant variables are reported in Table 7.

All materials for electrodes 1/5 were obtained from Hi-Rel Alloys, 23 Lewis Street, Port Erie, Ontario, L2A2P6, Ontario, Canada.

62A-72A show two representative TEM micrographs of each gold nanoparticle referred to in Table 7 and dried from each solution or colloid formed according to Example 18.

62B-72B show the measured size distributions of gold particles measured using the TEM instrument/software discussed in Examples 5-7 for the dried solution or colloids mentioned in Table 7 and formed according to Example 18; do.

Energy absorption spectra were obtained for the sample of Example 18 using UV-Vis spectroscopy. This information was obtained using a dual beam scanning monochromator system capable of scanning a wavelength range of 190 nm to 1,100 nm. A Jasco V-530 UV-Vis spectrometer was used to collect absorption spectroscopy. The tool was set up to support the measurement of low concentration liquid samples using one of a number of fused quartz sample holders or "cuvettes". A variety of cuvettes allow data to be collected in the 10 mm, 1 mm or 0.1 mm optical path of the sample. Data are acquired over a wavelength range using the following parameters: a 250-900 nm detector with a bandwidth of 2 nm, a data pitch of 0.5 nm, and a silicon photodiode with a water baseline background. Both deuterium (D2) and halogen (WI) scan rates of a 400 nm/mm source were used as the primary energy source. The optical path of these spectrometers was set up so that the energy beam could pass through the center of the sample cuvette. Sample preparation is not limited to filling and capping the cuvette and then physically placing the sample in a cuvette holder within a fully enclosed sample compartment. The optical absorption of energy by the material of interest was determined. Data output was measured and expressed as absorbance units (according to Beer-Lambert Law) versus wavelength.

Spectral patterns in the UV-visible range were obtained for each of the solutions/colloids produced in Example 18.

Specifically, FIG. 72C shows 11 suspensions/colloids (GB-151, GB-188, GB-175, GB-177, GB-176, GB-189, GB-194, UV-Vis spectral patterns of GB-195, GB-196, GB-198 and GB-199) are shown.

Figure 72D depicts the UV-Vis spectral pattern for each of the 11 suspensions/colloids over a wavelength range of about 435 nm to 635 nm.

In general, UV-Vis spectroscopy is a measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample. Ultraviolet and visible light have sufficient energy to promote external electrons to higher energy levels. UV-Vis spectroscopy can be applied to molecules and inorganic ions or complexes in solution.

UV-Vis spectra can be used for sample identification but have a wide range of characteristics that can also be useful for quantitative measurements. The concentration of the analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert law.

<u>Example 19 </u>

<u>gold</u><u> Nanoparticle/nanoparticle suspension or colloid Aurora-002, Aurora-004, Aurora-006, Aurora-007, Aurora-009, Aurora-011, Aurora-012, Aurora-013, Aurora-014, Aurora-016, Aurora- 017, Aurora-019, Aurora-020, Aurora-021, Aurora-022, Aurora-023, Aurora-024, Aurora-025, Aurora-026, Aurora-027, Aurora-028, Aurora-029 and Aurora-030 Produce</u>

In general, embodiment 19 uses a different trough member 30 and electrode 1/5 combination than any other embodiment disclosed herein. Specifically, this embodiment uses a first set of four electrodes 1 and a single electrode 5a in a trough member 30a' to generate a plurality of plasmas 4, the conditioned liquid 3 ') is created. The conditioned liquid 3' flows into and through the longitudinal trough member 30', and parallel disposed electrodes 5b/5b' extend substantially the entire longitudinal or flow length of the trough member 30b'. are placed along Reference is made in particular to FIGS. 23A, 23B, 23C and 23D , which show various schematic and perspective views of this embodiment of the present invention. Additionally, Table 8 below includes relevant processing variables related to this embodiment of the present invention.

&lt;Table 8&gt;

<img file="KR20120052967A_D0030.tif" />

<img file="KR20120052967A_D0031.tif" />

<img file="KR20120052967A_D0032.tif" />

Referring to Fig. 23a, two AC power sources 60, 60a are electrically connected as shown, and at the first trough member portion 30a' at the four corresponding electrodes 1a, 1b, 1c, 1d. Four separate plasmas 4a, 4b, 4c, 4d are generated. As shown in FIG. 23A , only a single electrode 5a is electrically connected to all four electrodes 1 . These power supplies 60 and 60a are the same power supplies reported in other embodiments herein. Two different amounts of processing enhancer NaHCO before four plasmas 4a-4d were conditioned as reported in Table 13.₃was added to liquid (3). The amount and type of processing enhancers reported should not be construed as limiting the present invention. The flow rates of liquid 3/3' to trough member 30a' as well as trough member 30b' are also reported in Table 8. The flow rate from trough member 30b' was approximately 5% to 50% lower due to liquid loss in evaporation, with higher evaporation at higher power input at electrodes 5b/5b'. Variable flow rates for liquid 3/3' may be used in accordance with the teachings herein.

Only one set of electrodes 5b/5b' was used in this particular example. These electrodes 5b/5b' are connected to an AC power supply 50 as described in other examples herein. The metal wire electrodes 5b/5b' used in this particular example were the same gold wire with dimensions as reported in Table 8 used in other examples reported herein. However, a gold wire electrode of a relatively long length (ie, for other examples herein) may have a longitudinal length (L) of the trough member 30b'._T) along the The wire lengths for electrodes 5b/5b' are reported in Table 8. Two different wire lengths were used, either 50 inches (127 cm) or 54 inches (137 cm). In addition, different transverse distances between the wires 5b/5b' have also been reported. Two separate lateral distances are reported herein, namely 0.063 inches (1.6 mm) and 0.125 inches (3.2 mm). A plurality of different lateral distances between the electrodes 5b/5b' as well as different electrode 5b/5b' lengths are available.

The wire electrodes 5b/5b' are connected to the devices Gb, Gb', T8, T8', Tb, Tb' near the input end 31 (see FIG. 23c ) and the corresponding devices Gb near the output end 32 . , Gb, Cb, Cb, Cbb, Cbb) are spatially disposed in the liquid 3 in the trough member 30b. Various devices are used to ensure that the electrodes 5b/5b are It should be understood that those reported herein, which allow for continuous placement along the trough member 30b', are exemplary. An important requirement for placing the electrodes 5b/5b' is those in contact with the liquid 3". including the ability to maintain a desired lateral separation between electrodes along the entire length of Specifically, the electrodes 5b/5b' are drawn through guide members Gb and Gb' made of polycarbonate near the input end 31 and of glass near the output end 32 . At each end of the trough member 30b', the members Gb and Gb' have limits Cbb, Cb'b' and the trough member 30b' near the output end 32 of the trough member 30b'. The position is adjusted by the limits (Cb, Cb') at the opposite ends. Electrical connections to the electrodes 5b/5b' were made at the output end 32 of the trough member 30b' near the top of the guide members Gb, Gb'. Tension springs Tb and Tb' are used to hold the taught electrode wires 5b/5b' to hold the electrodes in a fixed spaced relation to each other. In this regard, the electrodes 5b/5b' may be substantially parallel along their entire length, or they may be closer at one end of them relative to the other (eg, measure their entire length). produce different lateral distances). Controlling the lateral distance(s) between electrodes 5b/5b' affects current, current density concentration, voltage, and the like. Of course, other positioning means may occur to those skilled in the art, and the same is within the scope and scope of the present invention.

Table 8 shows the specific results including, for example, "hydrodynamic radius" (ie, hydrodynamic radius (reported in nanometers)) as well as various relevant processing conditions as well as process applied across electrodes 5b/5b. current is shown. Additionally, final ppm levels have also been reported for various process conditions with low values of about 0.5 ppm and high values of about 128 ppm.

73A shows two representative TEM micrographs of gold nanoparticles dried from solution or colloidal Aurora-020 reporting the 128 ppm concentration of gold measured the next day after synthesis. Within 2 weeks, the concentration of this sample was reduced to 107 ppm, and after another 5 weeks the concentration was reduced to 72 ppm.

73B shows the measured size distribution of gold nanoparticles measured by the TEM tool/software described above in Examples 5-7 corresponding to dried Aurora-020.

73C schematically shows a set of dynamic light scattering data measurements (ie, hydrodynamic radius) for nanocrystals prepared according to Aurora-020 mentioned in Table 8 and measured 7 weeks after synthesis. The main peak in the intensity distribution graph is approximately 23 nm. Dynamic light scattering measurements on a fresh Aurora-020 sample (not shown) produced a major peak at 31 nm. Dynamic light scattering assumes that the particles are not all spherical (but not) but also measure the hydrodynamic radius (e.g., the effect of a particle on water is also detected and reported in addition to the particle's true physical radius) It should be noted that the dynamic light scattering particle size information is different from the TEM measured histogram because of the algorithm used. Thus, as in the other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in the TEM histogram data and those reported in the dynamic light scattering data.

Thus, it is clear from this continuous processing method that various process variables can affect the final product.

<u>Example 20</u>

<u>Preparation of gold-based nanoparticles/nanoparticle suspensions or colloids GA-002, GA-003, GA-004, GA-005, GA-009, GA-011 and GA-013 by batch process</u>

This embodiment uses a batch process according to the present invention. 24A shows a device used to condition the liquid 3 in this embodiment. Once conditioned, liquid 3' was processed in the apparatus shown in FIG. 24C. The primary goal of this embodiment is to represent a variety of different processing enhancers (listed as "PE" in Table 9). Specifically, Table 9 shows the voltage used for each of the electrodes 1 and 5, the residence time for the liquid 3 exposed to the plasma 4 in the apparatus of FIG. 24A, and each of FIGS. 24A and 24C. The volume of liquid used, the voltage used to create the plasma 4 of FIG. 24A and the voltage used for the electrodes 5a/5b of FIG. 24C are described.

&lt;Table 9&gt;

<img file="KR20120052967A_D0033.tif" />

With respect to the reported processing enhancer (PE), different mg/ml amounts were used with similar conductivity for each solution [e.g., similar molar mass of cations also present in liquid (3/3')) ]. The electrode wire diameter used in each example was the same, about 1.0 mm, obtained from ESPI, 1050 Benson Way, Ashland, Oregon, USA 97520, as discussed herein.

The amount of electrode contacting liquid 3' of the device shown in FIG. 24C was the same in each case, ie 0.75 inches (19.05 mm).

Table 9 also shows the same processing enhancer, NaHCO₃The effect of transverse electrode separation (ie, distance "b" between substantially parallel electrodes 5a/5b shown in FIG. 24C ) on It is clear that the electrode currents and corresponding final liquid temperatures were less for closer electrode placements (ie, smaller "b" values).

A voltage source 60 (discussed herein) was used to generate the plasma 4 shown in FIG. 24A . A voltage source 50 (discussed herein) was used to generate the voltage and current between the electrodes 5a/5b shown in FIG. 24C.

Table 9 also reports the measured hydrodynamic radii (i.e., the "hydrodynamic radius" taken from the average of the three highest amplitude peaks shown in each of FIGS. 74C-80C and shown in FIGS. 74B-80B. A single number for "TEM mean diameter" corresponding to the mean measured gold nanocrystal size calculated from the TEM bar graph with

74A1 , 74A2 to 80A1 , 80A2 show two representative TEM micrographs, respectively, of gold nanocrystals dried from each of the solutions or colloids mentioned in Table 9 formed in accordance with this example.

74b-80b show the measured particle size distribution of gold nanocrystals measured by using the TEM tool/software described above in Examples 5-7 for each of the suspensions or colloids mentioned in Table 9 formed in accordance with this Example. is showing

74C-80C graphically depict dynamic light scattering data (ie, hydrodynamic radius) for nanocrystals prepared according to each of the suspensions or colloids mentioned in Table 9 formed according to this example. Dynamic light scattering assumes that the particles are not all spherical (but not these) but also measure the hydrodynamic radius (e.g., the effect of a particle on water is also detected and reported in addition to the particle's actual physical radius) It should be noted that the dynamic light scattering particle size information is different from the TEM measured histogram because of the algorithm used. Thus, as in other examples included herein, it is not surprising that differences in reported particle sizes exist between those reported in TEM histogram data and those reported in dynamic light scattering data.

<u>Comparative Example 21 </u>

<u>Preparation of Gold-Based Nanoparticles/Nanoparticle Suspensions by the Breddick/Svedberg Process</u>

This example uses an AC plasma in water generated between two gold electrodes to prepare a gold nanoparticle suspension similar to the method by Breddick and Svedberg (discussed in Background).

Specifically, FIG. 81A shows a perspective view of a device designed to act like Svedberg's AC plasma device. 81b shows a cross-sectional view of the same device. In each of these figures, gold electrodes el and e2 each having a diameter of 1 mm are immersed in water 3 . About 1 gallon of water (3) was contained in a glass container. The electrically insulating sleeve members s1 and s2 prevent an electric arc if undesired. Electrodes e1 and e2 are energized by the same transformer 60 discussed herein. The electrode (e1) was made to be close to the end of the electrode (e2) at the portion marked "Sh". The end "ea" of the electrode e1 was struck to make it almost flat. Then, the flat end ea was brought close to the end of the electrode e2 in the vicinity of the portion Sh. When the electrode end ea was close to the portion Sh, an underwater plasma 4w was generated. Once stabilized, the underwater plasma (4w) was allowed to run for about 2.5 hours, producing about 1 gallon of colloid. The results of the 2.5 hour run are shown in Figures 82A and 82B.

82A is a representative TEM micrograph of gold nanoparticles produced according to this example. 82B shows a histogram of particle size distribution from TEM measurements of gold nanoparticles produced according to this example. As is evident from the TEM micrograph, there are no nanocrystals similar to the present invention.

<u>Comparative Example 22a</u>

<u>Commercially available colloidal nanoparticle suspensions </u>

For comparison, eight commercially available colloidal gold solutions were obtained. Trade names and sources are shown in Table 10 below.

&lt;Table 10&gt;

<img file="KR20120052967A_D0034.tif" />

FIG. 90C shows a suspension of eight commercially available gold nanoparticles discussed in FIG. 22A (Utopia Gold, SNG91 1219, Nanoparts, Nanocomposix 15 nm, Nanocomposix 10) over the interrogation wavelength range of about 250 nm-750 nm. nm, harmonic gold and mesogold).

FIG. 90D shows the eight commercially available gold nanoparticle suspensions discussed in FIG. 22A (Utopia Gold, SNG911219, Nanoparts, Nanocomposix 15 nm, Nanocomposix 10) over the interrogation wavelength range of about 435 nm-635 nm. nm, harmonic gold and mesogold).

<u>Particle-size and particle-shape analysis</u>

Transmission electron microscopy (TEM) images were analyzed by visual observation using the software mentioned in Examples 5-7. Each particle/crystal was assigned to one group of five species by the two-dimensional projection shown in the micrograph. The five categories are triangles, pentagons, hexagons, and rhombuses. These categories correspond to the three-dimensional morphologies identified in the literature and conventional TEM experiments using inclined sample holders. The 2D/3D correspondence of the particle/crystal shape categories is presented in Table 11 below.

&lt;Table 11&gt;

<img file="KR20120052967A_D0035.tif" />

Certain nanocrystal morphologies can be taken in multiple two-dimensional projections. For example, a possible shape for an icosahedron, gold nanocrystals, can appear as irregular heptagons or spheres in TEM micrographs. Care is taken to identify hexagons, octagons, and other shapes in a two-dimensional view, but crucial information regarding the true shape of such nanocrystals cannot always be identified in a two-dimensional projection. Therefore, only the tetrahedral and pentagonal bipyramid (eg decahedron) categories can be absolutely identified. Hexagonal, rhombic and other categories are grouped together.

A pentagonal bipyramid nanocrystal can be projected as a rhombus when viewed from its plane. This is unlikely given the planar nature of the sample substrate and considering the very small number of rhombuses counted through the analysis. A counted decahedron by means of a pentagonal two-dimensional projection is identified as such from the previous group, and its count is regarded as a measure of advantage or a method of identifying crystals of the present invention from the art. Likewise, a triangle or tetrahedron can also be readily identified and can also be used for comparison.

Aggregation and aggregation of particles or nanocrystals occurs during colloids or as artifacts of the drying process required for TEM sample preparation/analysis. Dark agglomerates and larger aggregates (more than about 50 particles per nanocrystal) were not analyzed due to possible counting errors. The crystal/particle count and particle/crystal shape of the smaller aggregates and visually soluble aggregates were analyzed. Additionally, only well resolved images were used for this investigation.

In order to be very careful during the analysis of TEM micrographs of all suspensions or colloids produced by the present invention, any questionable crystals were assigned to the group marked "Other". The crystal in question may fall into a defined crystal category, but some uncertainty exists (eg, a small pentagon with one edge obscured by a neighboring particle). Conversely, when analyzing particles among commercially available colloids, any particle having a questionable shape was regarded as a "doubtful advantage" and was classified as "category hexagonal" despite the uncertainty of its actual crystal structure. assigned. Thus, the crystal/particle shape comparison is not biased with respect to possible differences between the commercially available colloids and nanocrystalline colloids produced by the present invention, and is made very carefully.

It is evident from Table 12 below that the presence of the corresponding nanocrystals in the shape of pentagonal bipyramids and/or tetrahedra is significantly different from commercially available colloids and ARCG-05. Moreover, these nanocrystals have a substantially "clean" surface as discussed, shown and defined herein.

&lt;Table 12&gt;

<img file="KR20120052967A_D0036.tif" />

<u>Example 22b </u>

<u>Zeta Potential Example </u>

The nature and/or amount (eg, positive or negative) of surface changes on the formed nanoparticles can also have a significant impact on the behavior and/or effect of the nanoparticles/suspensions or colloids. For example, protein corona can be affected by surface changes on nanoparticles. This surface change is commonly referred to as "zeta potential". In general, it is known that the greater the zeta potential (positive or negative), the greater the stability of the nanoparticles in solution (eg, the more stable the suspension). However, by controlling the nature and/or amount of surface charge of the formed nanoparticles, the performance of the nanoparticle solution in various systems can be controlled. A variety of different chemical compounds (both semi-permanent and temporary) Nanoparticles (and nanoparticle components), as well as different nanoparticles/solutions (e.g. liquids (3 ) (eg, water), including modifying its own structure) will be apparent to those skilled in the art. Thus, this example measures the zeta potential of commercially available colloidal gold suspensions, as well as some suspensions produced according to the present invention.

"Zeta potential" is known as a measurement of the electro-dynamic potential in a colloidal system. Zeta potential is also referred to as the surface charge on a particle. Zeta potential is also known as the potential difference between the fluid in which the particles are dispersed and the static layer of the fluid. Zeta potential is often measured in millivolts (ie, mV). A zeta potential value of approximately 25 mV is an arbitrary value chosen to determine whether stability exists between dispersed particles in a dispersion medium. Therefore, when referring to "zeta potential" in this specification, it should be understood that the referenced zeta potential is a description or quantification of the magnitude of the charge present in the bilayer.

The zeta potential is calculated from the electrophoretic mobility by the following Henry equation:

<img file="KR20120052967A_D0037.tif" />

where z is the zeta potential and U_Eis the electrophoretic mobility, ε is the dielectric constant, η is the viscosity, and f(kα) is a function of Henry. For the Smolukowski approximation, f(kα)=1.5.

Electrophoretic mobility was obtained by measuring the velocity of particles in an applied electric field using a laser Doppler velocimeter ("LDV"). In LDV, an incident laser beam is focused on a particle suspension inside a folded capillary cell, and light scattered from the particle is combined with a reference beam. This produces a fluctuating intensity signal whose rate of change is proportional to the speed of the particle, i.e., its electrophoretic mobility.

In this example, the zeta potential was measured using a zeta-sizer "Nano-ZS" manufactured by Malvern Instruments. For each measurement, 1 ml of sample was loaded into clean disposable zeta cells DTS1060C. A zeta-sizer was performed and the zeta potential was calculated using Dispersion Technology software, version 5.10. The following settings were used: dispersant: water, temperature: 25° C., viscosity: 0.8872 cP, refractive index: 1.330, dielectric constant: 78.5, approach model: Smoluchowski. One run of 100 replicates was performed for each sample.

91 shows the zeta potential of two colloidal nanocrystal solutions (GB-134 and GB-151) versus pH. The pH is changed by titration with a 1% by weight solution of acetic acid. Measurements were made on a Malvern Instruments Zeta-Sizer Nano-ZS90 in folded capillary cells DTS 1060 at 25°C. 20 and 50 sub runs per measurement were used at low and high pH, respectively.

92 shows conductivity measurements for the same colloidal solution tested for zeta potential. Conductivity measurements were taken simultaneously with a Malvern Instruments Zeta-Sizer NanoZS90 when measuring the zeta potential.

<u>Example 23a </u>

This Example 13a used a set of processing conditions similar to the set described in Examples 5-7. This embodiment used a device similar to that shown in FIGS. 17B, 18A, 19 and 21 . Table 8 describes the specific processing conditions of this embodiment showing differences between the sets of processing conditions described in Examples 5-7. The main differences in this embodiment include more processing enhancer added to liquid 3 and faster liquid 3 dosing flow.

&lt;Table 13&gt;

<img file="KR20120052967A_D0038.tif" />

93 shows a representative Viscotek output for a suspension produced according to Example 23a. The reported values correspond to the hydrodynamic radius of the nanocrystals in suspension.

<u>Example 23b</u>

This Example 23b used the suspension of Example 23a to produce a gel or cream product. Specifically, about 1,300 grams of the suspension prepared according to Example 13 was heated to about 60° C. over about 30 minutes. Suspension 1 liter Pyrex^&amp;#174; Heated on a metal hotplate in a beaker. About 9.5 grams of Carbopol^&amp;#174;(ETD 2020, Carbomer manufactured by Noveon, Inc., Cleveland, Ohio) was slowly added to the heated suspension with continuous stirring using a Squirrel rotating plastic paint mixer. This mixing was carried out for about 20 minutes until the large mass of Carbopol dissolved.

About 15 grams of high purity liquid lanolin (Now Personal Care, Bloomingdale, IL) was added to the suspension and mixed with the stirrer described above.

Then about 16 grams of high purity jojoba oil was added and mixed into the suspension. Approximately 16 grams of high-purity cocoa butter chunks (Soap Making and Beauty Supplies, North Vancouver, British Columbia, Canada) were separated into 500 mL Pyrex^&amp;#174; Heat in a beaker and place on a hot plate until the mass is liquid, after which liquid cocoa butter is added and mixed into the suspension described above.

About 16 grams of potassium hydroxide (18% solution) was added and the above ingredients were mixed together to make the suspension a gel. The entire suspension was continuously mixed with a plastic squirrel rotary mixer to form a cream or gel. During this final mixing for about 15 minutes an additional flavor of "tropical island" (2 ml) was added. A pink cream gel was formed.

<u>Example 23c</u>

In this Example 23c, the suspension prepared according to Example 7 was used. Specifically, in this example, a gel or cream product was prepared using the product of Example 7 (eg, GD-015). Specifically, about 650 grams of the solution produced according to Example 7 was heated to 60° C. for about 30 minutes. Suspension 1 liter Pyrex^&amp;#174; Heated on a metal hotplate in a beaker. About 9.6 grams of Carbopol^&amp;#174;(ETD 2020, Carbomer manufactured by Noveon, Inc., Cleveland, Ohio) was slowly added to the heated suspension with continuous stirring using a Squirrel rotating plastic paint mixer. This mixing was carried out for about 20 minutes until the large mass of Carbopol dissolved.

About 7 grams of high purity liquid lanolin (Now Personal Care, Bloomingdale, IL) was added to the solution and mixed with the stirrer described above.

Then about 8 grams of high purity jojoba oil was added and mixed into the suspension. Approximately 8 grams of high-purity cocoa butter chunks [Soof Making and Beauty Surprise, North Vancouver, British Columbia, Canada] were separated into 500 mL Pyrex^&amp;#174; Heat in a beaker and place on a hot plate until the mass is liquid, after which liquid cocoa butter is added and mixed into the suspension described above.

Advil^&amp;#174; About 45 grams of liquid (eg, liquid ibuprofen and potassium) contained in a liquid gel cap is added to the suspension and mixed thoroughly.

About 8 grams of potassium hydroxide (18% solution) was added and mixed together to make the suspension a gel. The entire solution was continuously mixed with a plastic squirrel rotary mixer to form a cream or gel. Additional flavoring (2 ml) of "tropical island" was added during this final mixing for about 15 minutes. A pink cream gel was formed.

<u>Example 23d</u>

In this Example 23d, a gel or cream product was prepared using a suspension corresponding to GB-139. Specifically, about 650 grams of the suspension was heated to 60° C. for about 30 minutes. Suspension 1 liter Pyrex^&amp;#174; Heated on a metal hotplate in a beaker. about 6 grams of carbopol^&amp;#174;(ULTREZ10, Carbomer manufactured by Noveon, Inc., Cleveland, Ohio) was slowly added to the heated suspension with continuous stirring using a Squirrel rotating plastic paint mixer. This mixing was done for about 20 minutes until the large mass of Carbopol dissolved.

About 7 grams of high purity liquid lanolin (Now Personal Care, Bloomingdale, IL) was added to the suspension and mixed with the stirrer described above.

Then about 8 grams of high purity jojoba oil was added and mixed into the suspension.

Approximately 8 grams of high-purity cocoa butter chunks [Soof Making and Beauty Surprise, North Vancouver, British Columbia, Canada] were separated into 500 mL Pyrex^&amp;#174; Heat in a beaker and place on a hot plate until the mass is liquid, after which liquid cocoa butter is added and mixed into the suspension described above.

About 8 grams of potassium hydroxide (18% solution) was added and the suspension was mixed together to make the suspension a gel. The entire suspension was continuously mixed with a plastic squirrel rotary mixer to form a cream or gel. A pink cream gel was formed.

<u>Example 23e</u>

In this example 23e, a gel or cream product was prepared using a suspension substantially corresponding to 1AC-261. Specifically, about 450 grams of the suspension was heated to 60° C. for about 30 minutes. Suspension 1 liter Pyrex^&amp;#174; Heated on a metal hotplate in a beaker. About 4.5 grams of Carbopol^&amp;#174;(ULTREZ10, Carbomer manufactured by Noveon, Inc., Cleveland, Ohio) was slowly added to the heated suspension with continuous stirring using a Squirrel rotating plastic paint mixer. This mixing was done for about 20 minutes until the large mass of Carbopol dissolved.

About 6.5 grams of potassium hydroxide (18% solution) was added and mixed with the above ingredients to make the suspension a gel. The entire suspension was continuously mixed with a plastic squirrel rotary mixer to form a cream or gel. A pink cream gel was formed.

<u>Example 24</u>

<u>In vitro experimentation of the effect of gold nanocrystalline formulation GB-079 on monocyte cytokine production</u>

<u>summary</u>

This in vitro example was designed to determine the effect of gold nanocrystalline suspension GB-079 on four different cytokines/chemokines. Specifically, in this example, human peripheral blood mononuclear cells ("hPBMCs") were each treated with four different concentrations or ppm levels of gold nanocrystalline suspension GB-079 (eg, a suspension produced according to the disclosure of this Example or colloids) in the presence or absence of bacterial lipopolysaccharide ("LPS") (as disclosed herein).

Lipopolysaccharides bind to TLR4, a receptor expressed on a number of different cells of the immune system, and this binding is usually a cytokine, usually in an NFkB-dependent (e.g., nuclear factor kB-dependent) manner, and/or activation of a series of cytokines. or known to initiate expression. About 5% CO at about 37°C₂ And after 24 hours of incubation conditions in a humidified atmosphere at about 95% relative humidity, the supernatant was removed and analyzed for the presence of a series of different cytokines/chemokines including MIF, TNFα, IL-6 and IL-10. It is expected that most of these cytokines (but not the only source of them) in the hPBMC population are monocytes. Cultivation in the absence of LPS indicates whether treatment induces the production of these cytokines/chemokines, whereas these cultures in the presence of LPS indicate whether such treatment can modulate the production of cytokines in response to inflammatory stimuli. indicates whether Cytokine analysis by Luminex^&amp;#174; The extracellular assay protocol was performed. The Luminex system uses antibody-coated microsomes that bind specifically to the cytokine being analyzed. When excited by laser light, microsomes that bind antigen are measured, which is a direct analysis of the amount of cytokines produced, the data is provided as raw data, and the absolute amount of each cytokine/chemokine is measured. did.

<u>Preparation of hPBMCs</u>

<u>Substances used to make cells</u>

<u>PBMC Isolation</u><u>Supplier catalog number</u>

Histopark 1077 Sigma H8889

RPMI 1640 x10 Sigma R1145

Endotoxin Free Water (EFW) Gibco 15230-170

50 ml Falcon Test Tube Corning 430829

Citrate ACD Sigma C3821-50 ml

AB Serum National Blood Service

Plastic 24 Well Flat Plate Costa/Corning 3524

LPS Sigma

<u>medium replenishment</u>

Penicillin/Streptomycin Sigma P0871

HEPES Sigma H0887

Glutamine Gibco 25030-024

Sodium Bicarbonate (7.5%) Gibco 25080

<u>device</u>

NucleoCounter (e.g. cell count and viability counter manufactured by Chemometec)

Benchtop centrifugation

tissue culture hood

<u>human blood collection</u>

Blood was drawn by syringe from healthy volunteers and placed in a 50 ml Falcon test tube. 3.3 ml of citric acid anticoagulant (ACD, Sigma) was added in a sterile manner to a 50 ml Falcon tube. Mix by inverting the test tube.

<u>Cell manufacturing method</u>

One. Mix together 10x RPMI + supplement (25 ml 10x RPMI + 2.5 ml Penstrep + 2.5 ml L-glutamine + 5 ml HEPES + 6.7 ml sodium bicarbonate solution (7.5%)) referred to herein as "culture medium" in a Falcon test tube. did.

2. Blood was resuspended in an equal volume of 1x RPMI 1640 (diluted from 10x RPMI in EFW - the resulting 200 ml [20 ml in 180 ml]) and mixed by inversion in a Falcon tube.

3. The Histopark was warmed back to room temperature (RT) and 20 ml was added to a 50 ml Falcon tube.

4. Histopark was lightly covered with 30 ml blood/medium and mixed.

5. Histopaque blood mixture samples were spun (without brakes) at room temperature for about 25 minutes in a benchtop centrifuge at 1,600 rpm.

6. PBMCs were separated in the interfacial layer between the medium and histopark, the cells were removed by aspiration into a 50 ml Falcon test tube, and 10 ml of culture medium was added thereto.

7. Cell samples were spun at 1,800 rpm for about 10 minutes at room temperature.

8. Cell samples were washed twice with 30 ml RPMI and resuspended in culture medium (RPMI, supplemented as described above = RPMI/serum free).

9. RPMI supplemented with 5% AB serum was generated during rotation.

10. Cell samples were resuspended in 2 ml RPMI + supplement + serum.

11. Cell counting was completed, and viability assessment was performed using a NucleoCounter (eg, cell viability counter).

12. The cells were again suspended in 1x RPMI to 2.5x10⁶ A final concentration of cells/ml was obtained.

13. 500 μl of cells were transferred to a 24-well plate.

14. 10x RPMI + Supplement (500 μl PenStrep, 500 μl L-Glutamine, 1 mL HEPES, 2.5 mL AB Serum) was prepared by mixing together in a Falcon tube to form a "test medium".

15. The GB-079 gold nanocrystal suspension of the present invention was added to the wells in a 24-well plate (900 μl total volume).

16. 100 μl 10x RPMI + supplement was added to each well of a Costa 24 well plate.

17. 24 well plates in a humidified incubator set at 37° C./5% CO₂left for 1 hour.

18. LPS was produced in 1x RPMI at 4x final concentration.

19. 500 μl of LPS was added to each well or 500 μl medium was added to wells that did not receive LPS so that the total well volume of material into each well was 2 ml.

20. Plate the plate with a humidified thermostat set at 37° C./5% CO₂was left for 24 hours.

21. 1,800 μl (3×600 μl aliquots) of the supernatant was removed for ELISA analysis and Luminex analysis.

22. The supernatant was heated to -80°C with Luminex.^&amp;#174; Stored until analysis in the system.

<u>Luminex</u><u>^&amp;#174;</u><u> analysis system</u>

The supernatant amount is Luminex approved as of January 11, 2010 at the address below.^&amp;#174; Analyzed by extracellular assay protocol.

<u>http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.1540.File.dat/Luminex%20Extracellular%20Protocol.pdf </u>

&lt;Table 14&gt;

<img file="KR20120052967A_D0039.tif" />

Cells were stimulated with LPS (high dose of 1 mg/ml and low dose of 10 ng/ml), then supernatants were collected 24 hours later and analyzed for the abundance of the four cytokines disclosed herein. Control wells contain cells and the test compound GB-079 of the present invention, but no LPS. Results obtained for each of the other cytokines/chemokines are shown in FIGS. 94A to 94D.

94A depicts the effect of GB-079 on IL-6 production by human peripheral blood mononuclear cells (hPBMC). It is evident from Figure 94a that IL-6 levels were reduced by GB-079 in LPS stimulated PBMCs. Some IL-6 production was also observed using the highest concentration of GB-079 in the absence of LPS stimulation at five different concentration levels.

94B depicts the effect of GB-079 on IL-10 production by hPBMCs. It is clear from Figure 94b that the observed levels of IL-10 were not affected by the addition of GB-079 at all concentration levels.

94C depicts the effect of GB-079 on MIF production by hPBMC. Specifically, FIG. 94C shows that levels of MIF were reduced in a dose dependent manner following LPS stimulation. This decrease was observed at dilution levels of 1:5 and 1:10, and the MIF level reverted to that of the control sample by the 1:20 concentration of GB-079.

Additionally, FIG. 94D shows that higher concentrations of GB-079 resulted in a higher increase in TNFα levels (using all tested doses of LPS) than the vehicle control stimulated samples. Some TNFα production was also observed with the highest dose of GB-079 in the absence of LPS stimulation.

<u>Example 25</u>

<u>Collagen-Induced Arthritis (CIA) Experiments in Mice</u>

<u>summary</u>

This example illustrates the efficacy of two inventive gold nanocrystalline compositions (eg, GT033 and GD-007) in a mouse CIA model. Specifically, male DBA/1 mice (12 weeks old) were administered 100 μg chicken type II collagen ("CII/CFA") emulsified with complete Freund's adjuvant on day 0 of the experiment by injection into the base of the tail. Swelling of the clinical joint was graded from Day 14 to 3 times weekly until completion on Day 42. These results are summarized in FIG. 95 . Treatment was carried out according to the following protocol. Blood was drawn on days 0 and 42. At termination, animals were exsanguinated, hind limbs removed, and ankle joints made for histopathological examination. Histopathological results are presented in Tables 6 and 7 below.

<u>methodology</u>

animal

Species: Mouse

Strain: DBA/1

Source: Harlan

Gender and Male: Male, 30

Age: About 12 weeks of age at the start of the experiment

Identification: Each mouse was given a unique identification number.

Animal husbandry: Upon receipt, all animals were examined for external signs of diseased animals and all unhealthy animals were excluded from further evaluation. Animals were housed in groups of 5 under specific pathogen free (spf) conditions in a thermostatically controlled monitoring room (22±4° C.) in the animal unit. Animals were allowed to equilibrate for at least 72 hours prior to use under standard animal rearing conditions. The health status of the animals was monitored during this period and the suitability of each animal for laboratory use was assessed prior to initiation of the experiment.

Breeding: Animals were housed in groups of 10 animals per cage in controlled rooms to ensure correct temperature, humidity and a 12 hour light/dark cycle during the duration of the experiment.

Diet: irradiated with light. Pellets and water are retained, acclimatized and allowed to use freely during the post-dose period.

<u>Compounds and Agents</u>

Chicken Collagen Type II (Sigma, C9301).

Incomplete Freund's Adjuvant ("IFA") (Sigma, FF5506)

Mycobacterium tuberculosis H37Ra (BD Bioscience, 231141)

Phosphate Buffered Saline ("PBS")

Test compound gold nanocrystal formulations GT033 and GD-007.

Vehicle: Water.

<u>Treatment group and dosage</u>

Control 1: First treatment "Group 2" and second treatment "Group 3" of 10 animals each per group.

Group 1: CII/CFA on day 0, receiving normal drinking water from days 0-42

Group 2: Day 0 CII/CFA, gold nanocrystal formulation (GT033; Example 4/Table 1d; 2.0 ppm gold as drinking water from days 0-42).

Group 3: CII/CFA on day 0, gold nanocrystal formulation as the only liquid for drinking from days 0-42 (GD-007; Example 5/Table 2a; gold ppm 14.8).

<u>protocol</u>

One. Upon arrival, the health of all animals was checked, and after passing a health test, each was numbered with a unique ear tag.

2. Animals were allowed to acclimatize for at least 72 hours.

3. Chicken type II collagen was produced to achieve a suspension at a concentration of about 16 mg/ml in 0.1 M acetic acid. After dissolution at 4° C. overnight, the solution was diluted with cold PBS to achieve a suspension with a concentration of about 8 mg/ml.

4. New mycobacteria were generated by finely pulverizing with a mortar and adding about 7 ml of IFA dropwise to produce an emulsion or suspension of CFA with a final concentration of about 5 mg/ml.

5. An injectable suspension of collagen in CFA (eg, "CII/CFA") was created using approximately equal volumes of each emulsion of chicken type II collagen and CFA.

6. On day 0, animals were injected with 50 μl of CII/CFA solution at the base of the tail.

7. Treatment with gold nanocrystal formulation GT033 (eg, group 2) and gold nanocrystal formulation GD-007 (eg, group 3) was given by the above schedule until day 42. Specifically, each water bottle containing normal drinking water, GT033 or GD-007 was watered every other day or once every 3 days as needed. The bottles were not specifically cleaned or specifically emptied during the 42-day trial.

8. The grade of the legs was measured 3 times per week from the 14th day until the end of the experiment. Each limb was graded by:

0=Normal.

1=Slight swelling of the entire joint or inflammation of individual toes.

2=Entire joint reddened and/or moderately swollen with inflamed more than one toe.

3=Severe joint inflammation and redness spreading to multiple toes.

4=Severe joint inflammation and redness spread to multiple toes; A clear sign of bone remodeling.

9. All animals were bled on days 0 and 42, and the recovered sera were stored for any analysis.

10. On day 42 animals were sacrificed, ankle joints removed, and placed in neutral-buffered formalin to prepare for histopathology.

11. These sections were processed, stained with hematoxylin and eosin stains ("H &amp; E"), and qualified (and blinded to experiments) tissues using semi-quantitative measurements of the extent of infiltration and damage. Graded by a pathologist.

95 graphically depicts the results of the limb grade CIA-test. Obviously the gold nanocrystal formulation GD-007 (group 3) with a measured gold concentration of about 14.8 ppm performed best, perhaps performing average (or better) with conventional steroid treatment, and the results are shown below. 95 (even though not actually measured). The gold nanocrystal formulation GT033 (Group 2) performed better than Control 1 at a concentration of about 2.0 ppm gold nanocrystals suspended in water.

Histopathology was performed on the left and right paws from 10 mice in group 1 (control) and group 3 (GD-007), respectively. Mice in group 2 were not subjected to histopathology.

Each pair of paws was assigned a pathology numerical code (eg, R0248-09 for 1 mouse in group 1) and coded left ("L") or right ("R") from each numbered animal.

<u>Histopathology/Methodology</u>:

· The skin was incised from the foot.

· Sections were generated by demineralization from the sectioned samples.

The demineralized samples were routinely treated, sectioned, and one H &amp; E-stained sections were made for investigation. This includes two halves of each specimen cut in half.

Each histopathological paw was graded as described below. Samples were graded in a blind manner without knowledge of experimental protocols or group identification.

· Multiple phalanges and tarsal joints were generally present in each section. The rating relates to the most severely affected of these joints in each case.

&lt;Table 15&gt;

score meter

In this case, three appearances of joint pathology were scored to contribute to a composite score (eg, maximum possible score=9). So, the higher the score, the greater the damage. Representative micrographs of joints corresponding to the above-mentioned grades 0-3 are shown in FIGS. 96A to 96D, respectively. Representative compilations of these grades 0-3 are shown in FIGS. 97A (eg, grade 0) through 97E (eg, grade 9).

&lt;Table 15&gt;

<img file="KR20120052967A_D0040.tif" />

&lt;Table 16&gt;

<img file="KR20120052967A_D0041.tif" />

&lt;Table 17&gt;

<img file="KR20120052967A_D0042.tif" />

As is common for this type of murine CIA model, one animal in treatment "Group 3", GD-007 (eg, R0266-09) was correlated with its right and left joints with respect to the presence/absence of arthritis. indicates the absence of a relationship. Similar discrepancies occurred in some control mice, as well as differences in the severity of arthritis between different joints in the same mouse (eg, R0250-09).

However, it is clear that the most severe pathology occurred in control group 1 (eg drinking water) and the least severe pathology occurred in the first "treatment group 2" (eg gold nanocrystal formulation GD-007).

One animal in treatment group 3 (eg R0267-09) had a broken bone, which is probably the highest score. Exclusion of this animal resulted in a mean score of 0.22. Additionally, histopathological data suggest that all 8 out of 10 mice did not develop an injury (eg, a total of 16 paws were examined). It is clear that the gold nanocrystal formulation GD-007 exerts a significant amount of effect in the CIA test.

It is clear that the gold nanocrystal formulation produced by the present invention significantly reduced the negatively induced arthritis effect in the CIA model compared to the control. Both reduction of excess IL-6 and/or reduction of excess MIF are known to reduce the negative effects of arthritic conditions. Thus, without wishing to be bound by any particular theory or explanation, the arthritic condition may be reduced by reducing MIF and/or one or more signaling pathways associated with MIF. The gold nanocrystalline formulation GD-007 showed significantly improved results compared to the control. These results, together with the results presented in the in vitro example and the EAE mouse model example, show that the gold nanocrystal composition of the present invention can alter MIF and/or MIF, as well as more signaling pathways related to IL-6. suggest

<u>Example: Dosage Comparison</u>

As indicated above, in the gold nanocrystal experiments, each mouse used GD-007 solution as the sole source of drinking fluid. To calculate the dosage of gold consumed by mice per day, the following formula was used:

<img file="KR20120052967A_D0043.tif" />

here,

The dosage is nanocrystalline gold (mg/kg/day) consumed by one mouse per day,

· Volume is the average amount (ml/day) of the average GB134 solution consumed by mice per day,

Concentration is the amount of nanocrystalline gold in GD-007 solution (mg/ml),

· Body weight is the weight (kg) of the mouse.

The following assumptions were used to calculate the nanocrystalline gold dosage:

·Volume = 4 ml

Concentration = 0.0148 mg/ml

· Weight = 0.025 kg.

These results indicated that the nanocrystalline gold dose was 2.4 mg/kg/day.

A comparison of gold content at dosages commonly used for the treatment of auranofin in a mouse model of type II collagen-induced arthritis is presented below. A typical auranofin dosage is 40 mg/kg/day [Agata et al., 2000]. The gold content in auranofin is 29%, so the gold dosage is about 12 mg/kg/day.

The only known human experiments using gold nanoparticles [Abraham et al. 1997, 2008], a dosage of 30 mg/day gold nanoparticles was used in patients weighing between 108 and 280 pounds. This corresponds to a dosage of about 0.24 to 0.61 mg/kg/day gold nanoparticles.

A comparison of the dose levels of the gold content in the novel gold nanocrystals used in these different efficacy experiments, gold in gold nanoparticles, and gold in auranofin is presented in Table 17a below, indicating that the novel gold nanocrystals of the present invention, aura Molecular form in norphin or as described in Abraham, et. al.], which is fundamentally different compared to conventional gold, performs very differently, and demonstrates a much higher potency.

&lt;Table 17a&gt;

<img file="KR20120052967A_D0044.tif" />

<u>Example 26 </u>

<u>Acute Rat Model of Experimental Autoimmune Encephalitis ("EAE")</u>

<u>summary</u>

This example illustrates the efficacy of the inventive gold nanocrystalline composition GB-056 in a mouse EAE model. Female Biochi mice 7-8 weeks old were inoculated flank with mouse spinal cord homogenate in CFA on day 0 of the experiment by injection at the base of the tail. Ten treatment group mice were orally administered gold nanoparticle suspension treatment GB-056 (eg, as discussed in Example 17) with only liquid drinking using a standard water bottle. Raw gold nanocrystalline formulation GB-056 was provided daily using a clean water bottle. Control mice were given normal tap water. Clinical scores on the EAE test were completed on a standard scoring system of 0-5.0 scores from Day 1 to completion on Day 28. These results are presented in Tables 9a and 9b as well as FIGS. 98 to 99 . Treatment was provided by the following protocol.

<u>methodology</u>

<u>animal</u>

Species: Mouse

Strain: Biochi

Source: Harlan

Gender and number: female, 20

Age: About 7-8 weeks of age at the start of the experiment

Identification: Each mouse was given a unique identification number.

Animal husbandry: Upon receipt, all animals were examined for external signs of diseased animals and all unhealthy animals were excluded from further evaluation. Animals were housed in groups of 5 under specific pathogen free (spf) conditions in a thermostatically controlled monitoring room (22±4° C.) in the animal unit. Animals were allowed to equilibrate for at least 72 hours prior to use under standard animal rearing conditions. The health status of the animals was monitored during this period and the suitability of each animal for laboratory use was assessed prior to initiation of the experiment.

Breeding: Animals were housed in groups of 10 animals per cage in controlled rooms to ensure correct temperature, humidity and a 12 hour light/dark cycle during the duration of the experiment.

Diet: irradiated with light. Pellets diet and water are retained, acclimatized and are allowed to be used ad libitum during the post-dose period.

<u>Compounds and Agents</u>

Mice and spinal cord homogenates ("MSCH") were generated in-house.

Incomplete Freund's Adjuvant ("IFA") (Sigma, FF5506)

Mycobacterium tuberculosis H37Ra (BD Biosciences, 231141)

Phosphate Buffered Saline ("PBS") in-house.

Test Compound Gold Nanocrystalline Suspension GB-056 (discussed herein)

vehicle: water

<u>Treatment group and dosage</u>

Control 1 and Treatment 2 each have 10 animals per group.

Group 1: On day 0 a mixture of MSCH/IFA/TB (see protocol below) was injected into the base of the tail of each mouse, and each received normal drinking water dispensed from a water bottle from day 0 to day 28.

Group 2: On day 0, a mixture of MSCH/CFA/TB was injected into the base of the tail of each mouse, each from day 0 to day 28 from a daily rinsed water bottle with fresh GB-056 as the only liquid for drinking each day. A dispensed gold nanocrystal formulation (GB-056) was provided.

<u>protocol</u>

Upon arrival, the health of all animals was checked, and after passing a health test, each was numbered with a unique ear tag.

One. Animals were allowed to acclimatize for at least 72 hours.

2. The spinal cord was reconstituted in PBS containing Mycobacterium tuberculosis H37RA. This produced 6.6 mg/ml of MSCH and 400 μg/ml of H37RA. An equal volume of Freund's incomplete adjuvant was added to this mixture to generate the final immunogen (3.3 mg/ml SCH and 200 μg/ml H37RA). These mixtures are not considered complete Freunds because the amount of tuberculosis is much lower.

3. On day 0, animals were injected with 50 μl of the solution discussed in step 3 at the base of the tail.

4. Treatment with the gold nanocrystal formulation GB-056 was given by the above schedule until Day 28. Unused GB-056 was provided daily (eg, replaced approximately every 24 hours).

5. Scores were scored daily from Day 1 to the end of the experiment. Each mouse was scored as follows:

0: normal

0.5: incompletely paralyzed tail

1.0: relaxed tail

1.5: slow and/or no upright reflex

2.0: Paralysis of one hind leg

2.5: Paralysis of one hindfoot and abnormal gait

3.0: Paralysis of both hind feet

3.5: Paralysis of both hind paws + incomplete paralysis of one forelimb

4.0: Paralysis of both hind paws + paralysis of one or both forelimbs

5.0: Moribund

6. On day 28 animals were sacrificed, brains and spinal cords removed, and placed in neutral buffered formalin in preparation for histopathology.

7. These sections were treated and stained with hematoxylin and eosin stains ("H &amp; E"). Tables 9a and 9b present the raw scores for each of the 20 mice in this EAE experiment.

&lt;Table 18a&gt;

<img file="KR20120052967A_D0045.tif" />

&lt;Table 18b&gt;

<img file="KR20120052967A_D0046.tif" />

98 graphically depicts the proportion of animals developing any sign of disease in each of Control 1 and Gold nanocrystal treatment group 2 (eg, GB-056). Control group 1 showed that 90% of the mice developed at least some symptoms, whereas only 40% of the mice in treatment group 2 developed some level of symptoms.

99 depicts the EAE score mean for each group. Notably, the onset of any signs was delayed by 2 days in group 2 treated with gold nanocrystals, and the total score for treatment group 2 was significantly lower than the average reported in control group 1. The gold nanocrystal formulation GB-056 with a measured gold concentration of about 12 ppm performed significantly better than Control 1 in this EAE test.

As is customary for this EAE model, one animal in treatment group 2 (eg, animal 4) died, whereas in control group 1 three animals died.

The most serious pathology occurred in control group 1 and the least pathology occurred in treatment group 2.

One animal that died in treatment group 2 (eg animal 4) gave this group a higher score. It is evident that the gold nanocrystal suspension GB-056 of the present invention has a significantly positive effect in this EAE test. While not wishing to be bound by any particular theory or explanation, the results of the murine CIA model and the results of this example in combination with an in vitro MIF cytokine assay indicate that MIF, and/or the MIF signaling pathway, is a gold nanocrystalline composition of the present invention. strongly suggest that it is favorably influenced by

<u>Example 27 </u>

<u>Prolonged Exposure of Gold Nanocrystal Suspension GD-013 in Mice</u>

The purpose of this example was to observe what negative toxicological effects occurred in mice when they freely drank the gold nanocrystal suspension GD-013 as the sole liquid source for an extended period of time.

A total of 25 female mice were used in this example, 5 mice were used in the control group, and 10 animals were used in each of the 2 treatment groups. The control group received normal water in a drinking bottle. The two treatment groups were given two different concentrations of GD-013 as the only drinking water. The first treatment group received a 50% GD-013 crystal suspension (the remaining 50% was purified DI/RO water), and the second treatment group received a 100% GD-013 crystal suspension. All groups were given to drink as much or as little as they wanted, and food was given freely as well. The body weight of each animal and the average amount of fluid consumed were recorded weekly. At week 23 of the experiment, 6 mice were sacrificed for cadaveric dissection and pathology (3 mice from each GD-013 crystal suspension treatment group). The remaining mice continued to consume the two treatment suspensions for 46 weeks.

<u>Substances and methods</u>:

In this type of exposure study, it is permissible to use only one sex female for toxicity testing. Data from other experiments show that there is generally no difference between sexes, but when one sex responds more strongly, it is usually female. Only males should be used if there is some form of evidence showing that male responses are stronger. In the absence of information indicating whether males were affected in this way, only females were used. The females used were non-partuming and non-pregnant adults. A Swiss Webster strain of randomized mice was used in this Example. These strains were chosen because they are widely used for general purpose and toxicology studies. It is also known not to have any fatal genetic deficiencies that do not potentially interfere with data collection.

&lt;Table 19&gt;

<img file="KR20120052967A_D0047.tif" />

<u>Dosage Preparation</u>

All treatment groups included in this experiment received a reference GD-013 nanocrystalline suspension in their water bottles. Mice were allowed to drink water ad libitum. The control group was given purified bottled water.

&lt;Table 20&gt;

<img file="KR20120052967A_D0048.tif" />

<u>housing and feeding</u>

All laboratory personnel participating in the field of mouse experiments wore personal protective clothing (eg, gloves, face masks and shoe covers). Mice were purchased from Harlan Laboratories. Upon acquisition, the mice were given permanent identification in the form of a tail tattoo (Harvard Apparatus Tattoo). The mice were then randomly assigned and housed in groups of 5 mice per cage. is large enough to have sufficient space for each 5 animals, and must not be so small as to impede the distinct observation of each animal. The mice are allowed to acclimatize to the laboratory environment for 1 week. Maintain at temperature and relative humidity between 30%-50% Artificial full-spectrum light was used (PureLite 60w, 120v bulbs).Using a timer, a 12-hour light cycle and 12-hour A dark cycle was achieved. Food was provided ad libitum (Purina Certified Rodent Diet 5002). Standard concove bedding was provided to the cage. The cage was changed to once a week. If a caged animal was found dead in the cage, the dead animal was replaced immediately upon removal.

<u>Procedures and observations</u>

After the acclimatization period, both treatment groups were started to receive the GD-013 nanocrystalline suspension marked on the water bottle. The control group continued to receive purified drinking water. On the first day of treatment, each mouse was weighed and the body weight recorded. At the start of each week, all mice were reweighed and all body weights were recorded. In addition, the approximate amount of water consumed and GD-013 crystal suspension was recorded weekly. Mice were observed during the experiment for any signs of abnormality or distress.

<u>weight gain</u>

At the start of the experiment, all mice had approximately the same body weight. Each week, each animal was weighed and the body weight recorded. The individual body weight of each animal in the group was averaged and graphically plotted in FIG. 106 to show the average weight gain of all groups during the experiment. A vertical line at week 23 is shown in FIG. 106, indicating the time at which the histopathology was performed.

<u>average daily consumption</u>

Each group consumed (1) water, (2) 50% GD-013 and (3) 100% GD-013 each week was measured. The amount of liquid, 50% purified water consumed during the preceding week was measured and calculated to obtain an approximate daily intake per animal for that week. Liquid consumption data for 46 weeks is shown in FIG. 107 .

<u>Results/Discussion</u>:

<u>weight gain</u>

Statistical analysis of the mean body weight of the groups was performed to determine if there were any differences in weight gain and/or loss between the groups. Each treatment group was compared to the control group, and the two treatment groups were also compared to each other. Overall, there was a statistically significant weight loss between the 100% GD-013 treatment group and the control group (P&lt;0.05). There was no statistically significant weight gain/loss between the two treatment groups or between the 50% GD-013 treatment group and the control group.

<u>Average consumption per week</u>

All three groups consumed what was considered a normal amount of liquid per day, so dehydration was not a problem. Again, a statistical analysis of the consumption values for each group was performed to determine whether there was a significant difference in consumption. The two treatment groups were compared with the control group, and the two treatment groups were compared with each other. The control group consumed significantly less than the two treatment groups (P&lt;0.05). There was no statistical difference between the amount consumed by the treatment group (P&gt;0.05). There were no observable differences in problems related to health, behavior, or dehydration.

<u>death rate</u>

Two deaths were reported in the experiment, one in each treatment group. The first death occurred at week 20 in the 50% GD-013 group. The second death occurred at week 22 in the 100% GD-013 group. Mice from the 50% GD-013 treatment group were always much smaller than the rest and did not gain body weight; The cause is unknown. Other mice did not show any signs of distress or poor health. No pathology was possible for these two mice.

<u>pathology</u>

Three mice from each treatment group were treated pathologically at week 23. Histopathological evaluation of organs of the heart, thymus, lung, liver, kidney, spleen, stomach, duodenum, jejunum, ileum, cecum, colon, bladder, ovary, striated muscle, hairy skin, bone marrow (femur/tibia), pituitary gland and brain was processed for Although some abnormalities have been reported according to pathological findings, it is considered as an accidental finding related to normal mutation and normal wasting among all individuals. The findings in the pathology report did not indicate any degree of toxicity to the target organ. The pathologist has no knowledge of what treatment the mice under investigation have received, nor does the pathologist have any knowledge of the treatment in control mice in order to rule out possible biases in the pathological findings.

All tissues mentioned above were examined in full, and only the spleen and liver showed minimal to slight variations in color. The only specific histopathological findings were reported in Table 21. The numbers "2-3," "2-5" and "4-7" in the 50% GD-013 line refer to 3 different mice, where indicated in "Remarks". Likewise, the histopathology "Vigo" of the spleen relates to three mice "3-3," "5-9" and "5-10", whereas in "Vigo" of the liver, only one mouse ( eg "5-10"). All full investigations are consistent with euthanasia and/or congestion from fat storage and are considered to be within the limits of normal. No overall lesions were reported.

&lt;Table 21&gt;

<img file="KR20120052967A_D0049.tif" />

<u>Example 28 </u>

<u>35-Day Intake and Distribution and Acute Toxicity Study</u>

35 The purpose of one experiment was to determine the uptake and distribution and rapid toxicity (if necessary) of two crystal suspensions (GB-134 and GB-151) and to compare the results with a commercially available MesoGold product. Thirteen mice were used in the experiment. Gold concentrations were measured in the urine and feces of test animals, as well as in certain vital organs and blood. Additionally, selection of organs from some individuals was investigated to determine histologically whether any abnormalities were present. In addition, all mice were allowed to drink until death for the experiment. Such a procedure was performed, for example, to confirm whether an accurate gold concentration in blood could be measured.

<u>Substances and methods</u>:

&lt;Table 22&gt;

<img file="KR20120052967A_D0050.tif" />

<u>dosing preparation</u>

All treatment groups included in this experiment were given a solution in a water bottle. Mice were allowed to drink freely. Each group received (1) MesoGold, (2) GB-134 or (3) GB-151 (all undiluted) in a beverage bottle.

&lt;Table 23&gt;

<img file="KR20120052967A_D0051.tif" />

<u>Procedures and observations</u>

Metabolic cage collection of urine and feces was initiated after animals received each treatment for 1 day. A total of 9 animals per week were housed in metabolic cages and their urine and feces were collected. Within the metabolic cage, individual mice were given a bottle of water with a liquid allocated for drinking. The amount of liquid consumed during 24 hours was also measured and recorded. Urine and stool samples were then collected and tested for Au concentration. The volume of excreted urine and the weight of collected feces were also measured and recorded.

At the end of the experiment, all 13 animals were sent to Taconic Laboratories (Rockville, MD) for full cadaver dissection and pathology reporting or organ and blood samples were collected for further analysis (herein of (discussed below). Microscopic examination was performed on the heart, lung, liver, spleen, kidney, brain, stomach, duodenum, jejunum, ileum, cecum, and colon. Further specific heart, lung (left and right), liver, spleen, kidney (left and right) and brain were collected and sent back to empty sterile glass vials for further concentration analysis.

<u>Procedures for the disintegration of stool and urine samples</u>

Specific methods have been developed to measure the amount of gold in feces and urine. PTFE sample cups and microwave digestion bombs were ordered from Fisher Scientific and obtained from Parr Instrument Company (www.parrinst.com). A 23 ml PTFE sample cup (Fischer catalog number 0102322A) and a Parr 4781 microwave digestion balm (Fischer catalog number 0473155) were used for digestion.

The microwave used is a Panasonic 1300 watt model number NN-SN667W, serial number 6B78090247.

<u>Pee</u>

1.5 grams of urine was weighed into a PTFE sample cup. If the urine exceeds this mass, prepare for another digestion. If the urine sample mass was less than 1.5 grams, an appropriate amount of DI water was added to bring the weight to about 1.5 grams. 0.24 ml of 50% v/v HNO₃was added to the sample cup followed by 0.48 ml of 36% v/v HCl. The sample cup was sealed and placed inside a microwave balm. The microwave chest was sealed and placed in the center of the microwave. The sample was irradiated until the Teflon indicator screw was raised 1 mm from the top of the chestnut. The duration of the night in the microwave ranges from 30 to 60 seconds, depending on the urine sample. The microwave extinguishing chestnut was removed from the microwave and allowed to cool for 20-30 minutes until the Teflon indicator screw was lowered into its original position. The sample cup was removed from the microwave digestion balm, and the liquid sample was transferred to a vial for testing.

<u>credit</u>(1 pellet sample):

A single fecal pellet was weighed into a PTFE sample cup. 5 ml of DI water was added to the sample cup. 0.8 ml of 50% v/v HNO₃was added to the sample cup, followed by addition of 1.6 ml of 36% v/v HCl. The sample cup was sealed and placed inside a microwave balm. The microwave chest was sealed and placed in the center of the microwave. The sample was irradiated until the Teflon indicator screw was raised 1 mm from the top of the chestnut. The duration of the night in the microwave ranges from 20 to 30 seconds depending on the urine sample. The microwave extinguishing chestnut was removed from the microwave and allowed to cool for 20-30 minutes until the Teflon indicator screw was lowered into its original position. The sample cup was removed from the microwave digestion balm and the liquid sample was transferred to a vial for testing.

<u>bulk stool sample</u>

About 0.300 grams of stool was weighed into a PTFE sample cup. 5 ml of DI water was added to the sample cup. 0.8 ml of 50% v/v HNO₃was added to the sample cup, followed by addition of 1.6 ml of 36% v/v HCl. The sample cup was sealed and placed inside a microwave balm. The microwave chest was sealed and placed in the center of the microwave. The sample was irradiated until the Teflon indicator screw was raised 1 mm from the top of the chestnut. The duration of the night in the microwave ranges from 20 to 40 seconds, depending on the bulk stool sample. The microwave extinguishing chestnut was removed from the microwave and allowed to cool for 20-30 minutes until the Teflon indicator screw was lowered into its original position. The sample cup was removed from the microwave digestion balm and the liquid sample was transferred to a vial for testing. Bulk stool samples may require multiple digestions to break down all of the stool present in the initial sample.

Note: If the sample did not appear to be fully degraded (ie, solids were still present or discolored on the side of the PTFE sample cup), a secondary degradation was performed. This is DI water, 50% v/v HNO specified for the appropriate sample.₃ and a second addition of 36% v/v HCl volume (see procedure above for exact volume). The samples were then microwaved again and allowed to cool for 20-30 minutes before being transferred to sample vials for testing.

*DI water = deionized water

*PTFE = polytetrafluoroethylene

Once all samples were resolved, they were analyzed using the atomic absorption spectroscopy techniques discussed herein.

35 The pathology findings for one experiment are presented in Table 24 below. All tissues were fully examined, and only the spleen and liver showed minimal to slight variations in color. All full investigations are consistent with euthanasia and/or congestion from fat storage and are considered to be within the limits of normal. No overall lesions were reported. Remarks relate to specific mice and are presented in Table 24 below. designation "M-3" relates to one mouse in the mesogold group, while "GB-134-7" relates to one mouse in the "GB-134" group; "G151-9" relates to one mouse in the "GB-151" group.

&lt;Table 24&gt;

<img file="KR20120052967A_D0052.tif" />

108 depicts no significant difference in weight gain found between any groups (overall P&gt;0.05).

109 shows that there is no significant difference in consumption of fluid found between any groups (overall P&gt;0.05).

Figure 110 shows that there is a significant difference in the amount of Au found in the feces between the mesogold group and between the GB-134 and GB-151 groups (P&lt;0.01). There was no significant difference between the GB-134 and GB-151 groups (P&gt;0.05). Table 25 shows the actual recorded results.

&lt;Table 25&gt;

<img file="KR20120052967A_D0053.tif" />

111 shows that there was no significant difference in the mean amount of gold found in urine between any groups (overall P&gt;0.05).

&lt;Table 26&gt;

<img file="KR20120052967A_D0054.tif" />

<u>Procedures for Measurement of Neutron Activation Assays in Tissue Samples and Blood</u>

Specific samples of heart, liver, spleen, kidney, brain and blood were analyzed for gold content. Specifically, a neutron activation assay was used. Instrumental neutron activation analysis (NAA) is particularly powerful for its sensitivity and ability to accurately measure multiple elements in a single sample. NAA does not require any chemical treatment or special chemical preparation of the sample, thus minimizing, for example, loss, potential for contamination and dissolution of any incomplete tissue sample.

The NAA method involves weighing a tissue sample in a polyethylene vial. An inert material was added to each vial to prevent evaporation losses. Each vial was uniquely identified by a neutron flux monitor and bar code attached to the bottom of each vial. These vials were stacked into 1-foot long bundles for irradiation with neutrons from the nuclear reactor. Bundles contain randomly selected duplicate samples, and gold standards (or gold at known concentrations) are inserted at random locations within the bundles.

All bundles were processed in a similar manner. The bundles were exposed to barite flux in a nuclear reactor. Specifically, the bundle was inserted into the core of the nuclear reactor for about 45 minutes. The bundle was rotated during irradiation so that no horizontal velocity variations were present. (Vertical velocity variation was monitored by individual genus monitors). This irradiation makes any gold present in the sample radioactive and begins to emit radiation in the form of transmitted gamma rays whose energy (or wavelength) is characteristic of gold (eg Au 198, 411.8 ke V).

After a decay period of about 6 days, the irradiated samples were loaded into the counting system. Specifically, each irradiated and partially decayed sample was placed adjacent to a gamma-ray spectrometer using a high-resolution coaxial germanium detector. Gamma rays were continuously irradiated from each sample (as long as gold was present), and the interaction of the emitted gamma rays with the detector produced discrete voltage waves of height proportional to the incident gamma ray energy. The specially developed multi-channel analyzer classifies the voltage wave from the detector according to its magnitude, and digitally composes the spectrum of gamma-ray energy versus intensity. The counting time is about 45 minutes per sample. By comparing the spectral peak positions and areas with library standards, gold was qualitatively and quantitatively identified. The results of the analysis are specified below.

Referring to Table 27 below, FIG. 112 shows a bar chart of mouse organ types and colloids orally consumed by the identified mice. The number at the end of each colloidal identification refers to a specific mouse. Specifically, organs from two mice GB-151-4 and GB-151-5 were examined. GB-151-4 means mouse number 4 that consumed GB-151. Organs from another mouse, mouse GB-134-3 (eg, mouse number 3 that consumed suspension GB-134) were similarly examined. An organ from another mouse, mouse, mouse number 2 (meso-2), consumed commercial colloidal gold. Although the sample size was relatively small, the difference was significant.

Gold was not detected in two brain samples, GB-151-6 and GB-134-3, with detection limits of 0.35 ppb and 0.25 ppb, respectively. Blood samples GB-151-5 and GB-134-3 were not analyzed because they were insufficient for analysis.

&lt;Table 27&gt;

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