Compositions and methods for targeted thermomodulation

[0001] The field of the invention are nanoparticles for use in cosmetic, diagnostic and / or therapeutic procedures.

Background of the invention [0002] Laser treatments within the skin are widely known and their use for therapeutic and cosmetic purposes is highly valued. Therapeutically, potential applications in laser skin therapy include laser ablation of cancer cells in cancer patients and laser ablation of damaged tissue in patients with burns. Cosmetic applications of laser skin therapy are much more numerous and include hair removal / reduction, dyschromia treatment, skin contraction after surgery such as liposuction, acne treatment, chemical or physical abrasion of unwanted skin patches, surgery including nose correction and face lifting and neck, as well as other aesthetic skin modeling treatments. Different variations of such treatments are disclosed in e.g. US 2006/078578 A1, WO 2006/122222 A2, US 2005/203495, US 2005/175649 A1, US 2010/057068 A1, WO 2005/092286 A2 and WO 2006/051542 A1.

Summary of the Invention [0003] Despite the promising use of laser therapy in skin treatment and cosmetology, current laser treatments have limited efficacy, requiring a significant number of repeated treatments and generating increasing costs. Suboptimal laser treatments also have limited specificity, leading to debilitating side effects such as non-specific skin damage, skin irritation and scarring.

[0004] Light-based hair removal systems have particularly low efficiency in removing fair hair (vellus, blond, gray, red hair). Numerous (even 6 or more) treatments are insufficient to achieve a therapeutic effect in patients with blond, gray or red hair, even using topical chromophores such as carbon. In addition to removing light hair with light, thermoablation technology also has

Unused potential in wound healing, tissue remodeling, vascular repair and acne treatment.

[0005] Acne vulgaris is the result of obstruction of the hair-sebaceous unit consisting of the hair shaft, hair follicle, sebaceous gland and the pendular muscle, which leads to the accumulation of skin sebum produced in the sebaceous gland and subsequent bacterial colonization of the hair follicle. Micro-blackheads resulting from the accumulation of skin sebum (sebum) progress to non-inflammatory skin pustules (blackheads / blackheads) or pustules that recruit inflammatory cells and lead to the formation of papules, nodules and cysts. The consequences of untreated acne vulgaris often include discoloration, scarring and disfigurement, as well as significant psychological burden. Therefore, acne treatments tend to reduce the buildup of sebum and microbes within the hair follicles and sebaceous gland.

[0006] Although methods involving the use of light and laser appear promising in the treatment of skin disorders, their effectiveness is still insufficient. Ultraviolet (UV) / blue light has been approved by the FDA only for mild to moderate acne because of its anti-inflammatory effect on skin cells (keratinocytes), potentially through the effects of porphyrin photosensitizing agents in the follicles. Exogenous porphyrin precursors such as 5-aminolevulinic acid (5-ALA), which have been developed for topical or oral administration, have been shown to accumulate in the sebaceous vesicles, absorb red photons and form reactive oxygen species that directly damage cell membranes and proteins . It has been shown that this procedure combining the use of porphyrins and high-intensity red light, termed "photodynamic therapy", leads to a 50% reduction in sebum and acne production for 20 weeks after irradiation. However, destruction of the skin's sebaceous gland structures requires high intensity energy (50-150 J / cm²), and the transdermal penetration of porphyrin leads to undesirable side effects, which include photosensitivity, pain, inflammation, hyper / hypopigmentation and

Permanent scarring.

[0007] In order for laser therapy to be fully useful in the treatment of human skin disorders, methods must be provided for the local induction of light-induced destruction within skin structures without damaging the surrounding tissues.

[0008] In this application, see attached claims, new compositions and methods useful for targeted thermomodulation of target cell populations and target tissues for use in cosmetic procedures and the treatment and prevention of chronic and acute diseases and disorders.

[0009] The present application describes compositions comprising a cosmetically acceptable carrier and numerous plasmonic nanoparticles in an amount effective to induce thermomodulation in the target tissue region that is locally contacted with the composition.

[0010] The composition contains plasmonic nanoparticles, which are activated by exposure to energy supplied from a non-linear excitation source - surface plasmon resonance - to the target tissue region. The present application describes compositions comprising at least one plasmonic nanoparticle which comprises a metal, metal composite, metal oxide, metal salt, electric conductor, electric superconductor, electric semiconductor, dielectric, quantum dot or composite from the combinations mentioned above. This application describes a composition in which a significant amount of plasmonic particles present in the composition contain geometric nanostructures. This application describes in which plasmon particles have any geometric shape currently known or to be created, which absorb light and generate plasmon resonance at the desired wavelength, including nanoslabs, solid nanoshells, hollow nanoshells, nanopails, nanoris, nanospheres , nanofibers, nanowires, nanopyramides, nanoprisms, nanoparticles or a combination of the above. Plasmon particles contain silver and / or gold and may additionally contain nickel, copper, titanium, silicon, gallium, palladium, platinum or chromium. [0011] In certain embodiments in the present application

A composition is provided comprising a cosmetically acceptable carrier that contains an additive, a dye, an emulsifier, a fragrance, a humectant, a polymerizable monomer, stabilizer, solvent or surfactant. In one embodiment, the present application provides a composition in which the surfactant is selected from the group consisting of sodium lauryl sulfate, sodium dodecyl sulfate, ammonium lauryl sulfate, sodium sulfate vinegar-1 / deceth-1, lipids, proteins, peptides or derivatives thereof. In one embodiment, the present application provides a composition in which the surfactant is present in an amount of from about 0.1 to about 10.0% by weight of the carrier. In yet another embodiment, the solvent is selected from the group consisting of water, propylene glycol, alcohol, hydrocarbon, chloroform, acid, base, acetone, diethyl ether, dimethyl sulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran, dichloromethane and ethyl acetate. In one embodiment, the composition comprises plasmon particles having an optical density of at least 1 OD for one or more maximum resonance wavelengths.

[0012] Plasmon particles comprise a hydrophilic or aliphatic coating, wherein the coating is substantially non-adsorbing to the skin of a mammal, and the coating comprises polyethylene glycol, silica, silica oxide, polyvinylpyrrolidone, polystyrene, protein or peptide. In yet another embodiment, thermomodulation includes damage, ablation, lysis, denaturation, deactivation, activation, induction of inflammation, activation of heat shock proteins, disruption of cell signaling, or disruption of the cell microenvironment in the target tissue region. In addition, in certain embodiments, the target tissue area includes the sebaceous gland, sebaceous gland element, sebocyte, sebocyte element, tallow or funnel follicle. In further embodiments, the target tissue area is a protrusion, bulb, stem cell, stem cell niche, skin papilla, cortex, epidermis, hair sheath, core, pyloric muscle, Huxley's layer or Henle's layer.

[0013] The present application describes methods for carrying out targeted

Tissue ablation comprising the steps of i) topically applying to the skin of a patient a composition according to claim 1; ii) providing penetration agents for redistributing plasmonic particles from the skin surface to a skin tissue component; and iii) irradiating the skin surface with light. In further or additional embodiments, a method is provided in which the light source is excited mercury, xenon, deuterium or metal halide, phosphorescence, incandescence, luminescence, light emitting diode or sunlight. Still in further or additional embodiments, a method is provided wherein the penetration agent includes high frequency ultrasound, low frequency ultrasound, massage, iontophoresis, high pressure air flow, high pressure liquid flow, vacuum, pretreatment with fractional photothermolysis or dermabrasion, or their combination. In yet further embodiments, a method is provided wherein the irradiation comprises light with a wavelength from about 200 nm to about 10,000 nm, a fluence from about 1 to about 100 joules / cm², pulse width from about 1 femtosecond to about 1 second, and repetition frequency from about 1 Hz to about 1 THz.

[0014] The present application also describes a composition comprising a cosmetically acceptable carrier, an effective amount of sodium dodecyl sulfate and numerous plasmonic nanoparticles in an amount effective to cause thermal damage in the target tissue region with which the composition is locally contacted, wherein the optical density of the nanoparticles is at least 1 FROM for a resonance wavelength of about 810 nanometers or 1064 nanometers, the plasmon particles having a silica coating from about 5 to about 35 nanometers, wherein the acceptable carrier contains water and propylene glycol.

[0015] Also described is a hair laser ablation or acne treatment system comprising a composition and a plasmonic energy source suitable for use on human skin.

Brief description of the figures [0016]

EP 2 608 762 B1

Figure 1 illustrates diagrams showing certain embodiments of the use of preparations for removing hair and treating acne. Presented (A) for hair removal, the preparation of plasmonic nanoparticles (black) is 1. applied topically to human skin, 2. delivered deeply to the hair follicle and washed away from the skin surface, 3. irradiated with a clinical laser with a wavelength corresponding to the absorption peak of the plasmon particle, and 4. removed from the hair follicle with a damaged hair follicle; and (B) for the treatment of acne, the preparation of plasmonic nanoparticles (black) is 1. applied topically to human skin, 2. delivered to the sebaceous gland and washed away from the skin surface, 3. irradiated with a clinical laser with a wavelength corresponding to the absorption peak of the plasmon particle, and 4 . removed from the destination, where the accumulated sebum and the ability to produce sebum in the sebaceous gland are destroyed.

Figure 2 shows the temperature profile of some embodiments of the plasmonic nanoparticle preparations (SL-001, triangles) provided in this application compared to the exemplary clinical dyes currently used: carbon balm (circles), Meladine aerosol formulation (diamonds), and indocyanine green (squares) ), after exposure to 55 millisecond laser pulses 1064 nm, 20 J / cm². SL-001 and dyes were evenly diluted 1: 1000 from clinical concentration (SL001 1000 OD, carbon 20-200 mg / ml, Meladine 1 mg / ml, ICG 5 mg / ml). n = 3, error standard deviation (SD) from the mean.

Figure 3 shows the penetration of fluorescently-labeled nanoparticles deep into the hair follicles as determined using pig skin explants and confocal imaging of certain embodiments of the subject matter described in this application. Presented (A) a cross-sectional diagram of treated pig skin subjected to angular imaging in two 60 μm serial planes: "plane 1" (representing the funnel of a bellows) and "plane 2" (representing a deeply embedded bellows); (B) representative confocal images show nanoparticles with red fluorescence (548 nm) found in part

Surface and deep bellows, but not in the dermis below;

and (C) red fluorescence nanoparticles retained in the deep part of the bellows (approximately 400 μm) at high magnification. The green color is due to tissue autofluorescence.

Figure 4 shows the penetration of plasmon nanoparticles deep into the hair follicles, as determined using pig skin explants and dark field imaging. Presented (A) a diagram of treated pig skin, cut into sections and imaged horizontally relative to the bellows; (B) light blue plasmonic particles are visible over 1.2 mm deep and differ from (C) untreated pig skin (negative control) in which no dyes can be seen.

Figure 5 showing observations of human skin treated only with laser (left forearm) or plasmonic particles and laser (right forearm) shows nonspecific and specific photothermal damage. (A, B) In the upper panel, human skin was only irradiated with laser pulses of 810 nm (30 J / cm², 30 ms, 2 transitions) (A) or previously treated with a preparation of uncoated resonance plasmon nanoparticles at 830 nm in 20% propylene glycol (B). The plasmonic nanoparticle preparation was used together with a 3-minute massage, and the skin surface was washed 3 times with water and ethanol alternately before laser irradiation. Thirty minutes after laser irradiation, non-specific clinical burns were observed on the B surface compared to A, caused by significant photothermal heating of residual uncoated particles on the skin surface (C, D). In the lower panel, human skin was only irradiated with laser pulses of 1064 nm (40 J / w) cm², 55 ms, 3 passes) (C) or previously a preparation with silica-coated resonance plasmonic nanoparticles for 1020 nm in 20% propylene glycol (D). The plasmonic nanoparticle preparation was used together with a 3-minute massage, and the skin surface was washed 3 times with water and ethanol alternately before laser irradiation. Thirty minutes after laser irradiation, there were no signs of skin burns or erythema in Part D or C,

EP 2 608 762 B1 as the silica coated particles could be sufficiently washed off the skin surface. The enlarged photo of the D area shows specific photothermal damage (perimollicular erythema and edema) in the target region treated with nanoparticles.

Figure 6 is a photograph showing nanoparticles targeted photothermal damage to live human skin treated with a plasmonic nanoparticle preparation and a clinical laser. Live human skin was exposed to a 1020 nm resonant preparation coated with silica (200 nm diameter) plasmon nanoparticles in 20% propylene glycol and a 3-minute massage. The procedure was repeated three times and the skin surface was washed 3 times with water and ethanol alternately to remove residual particles. The treated skin was irradiated with laser pulses at a wavelength of 1064 nm (40

J / cm², 55 ms, 3 passes). After laser irradiation, clinical signs of erythema and peripillary edema were observed in the hair follicle area to which the nanoparticles were directed, but these symptoms did not occur in surrounding and untreated nanoparticle tissues.

Figure 7 shows the delivery of a plasmonic nanoparticle preparation to the sebaceous gland in human skin. (A) Confocal microscopic image of biopsy and cross-section of human skin, immunostained for collagen type IV basal membrane (blue) and nerve marker PGP 9.5 (green) shows microanatomy of the hair follicle (HF) and sebaceous gland (SG). Silica nanoparticles are colored red (200 nm). (B) Schematic image and dark field microscopy image of the excised human skin treated with the preparation of plasmonic nanoparticles, and then cut into sections and imaged in a horizontal plane relative to the bellows. Light blue plasmonic particles are visible up to a depth of 400 μm and inside the human sebaceous gland.

Figure 8 shows cosmetic preparations of plasmonic nanoparticles containing surfactants for targeted treatment of sebaceous glands. Silica-coated nanoparticles (diameter 200 nm, OD 100), prepared in 20% propylene glycol with the addition of surfactants, sodium dodecyl sulfate (SDS) or lauryl sulfate

Sodium (SLES) was applied to human skin with massage and ultrasound, and then the skin was cut into sections in a horizontal plane for dark field microscopic observation. (A) Plasmon particle preparations in 1% SDS / 20% PG penetrated into the sebaceous gland to a depth of 400 μm as in Fig. 7. (B) Plasmon particle preparations in 1% SLES / 20% PG penetrated into the sebaceous gland to a depth of 600 um . In the inserted frame a piece of skin without visible particles is shown (40 μm scale strip). The outline of the sebaceous gland is schematically marked.

Figure 9 is a picture showing a comparison of the effects of massage and ultrasound on the orientation of nanoparticles to human hair follicles and sebaceous glands. Silica-coated nanoparticles (diameter 200 nm, OD 100) were prepared in 1% SDS / 20% propylene glycol and applied to human skin along with massage or ultrasound. Images in the dark cross-sectional area in the horizontal plane taken at low (20x) and high (50x) magnification show (A) little or no accumulation of plasmon particles in the hair follicle funnel after the massage alone compared with (B) the expansion of the hair follicle funnel and significant accumulation plasmonic particles after ultrasound only.

Figure 10 shows an embodiment of the cosmetic preparations of plasmonic nanoparticles for targeted therapy of sebaceous glands. Plasmon nanoparticles of various shapes and coatings were prepared in 1% SDS / 20% propylene glycol and applied to human skin with massage and ultrasound, then the skin was cut into sections in a horizontal plane for dark field microscopic observation. (A) Polyethylene glycol (PEG) coated nanowires (gold, size 15 x 30 nm) were observed inside the funnel of the hair follicle to a depth of 200 μm (white arrow). (B) A lower concentration (10 OD) of silica-coated nanoslabs (silver, diameter 200 nm) was observed at a depth of up to 600 μm in the hair follicle and sebaceous gland (hollow arrow). In the inserted frame, sections of the skin without visible particles are shown (100 pm scale bar).

EP 2 608 762 B1

Detailed description of the invention [0017] The biology of physiological and pathophysiological tissue growth and remodeling, as well as changes in cell morphology are more complex than is commonly known and include interactions within networks of biological compounds, physical forces and cell types.

[0018] The object of the invention described in the present application is to provide compositions, methods and systems for non-invasive and minimally invasive treatment of skin and underlying tissues or other available tissue spaces using nanoparticles. Treatment includes hair removal and removal or reduction of acne. This application also describes hair growth and regrowth, skin rejuvenation or reconstruction, wrinkle reduction, pores reduction, ablation of cellulite and other skin lipid deposits, removal of warts and fungi, shallowing or removal of scars, including hypertrophic scars and keloids, incorrect pigmentation (e.g. red wine stains), removal of tattoos and skin imperfections (e.g. texture, color, color, elasticity, hydration). Other therapeutic or prophylactic methods described in this application include, but are not limited to, treating excessive sweating, lack of sweat, Frey syndrome (sweating syndrome), Horner syndrome and Ross syndrome, actinic keratosis, keratosis folliculitis, dermatitis, albinism, dandruff, psoriasis, lichen planus, eczema, alopecia, psoriasis, malignant or benign skin tumors.

[0019] Unless otherwise explained, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described in this application may be used in the practice or testing of the present disclosure, appropriate methods and materials are provided in the present application. Materials, methods and examples are illustrative only and are not limiting. Other features of the disclosure are derived from the following detailed description and claims.

[0020] As used herein, the terms "administer" or

"Administration" means providing or causing delivery of material to a subject, e.g., by topical, subcutaneous, intradermal, enteral, parenteral, rectal, intranasal, intravenous, intramuscular, intraperitoneal or other routes.

[0021] A "carrier suitable for administration" to a subject is any material that is physiologically compatible with the local or [other] route of administration to the desired vertebrate individual. Carriers may contain solid, dry materials for the formulation; or the carrier may contain liquid or gel materials for the preparation of liquid or gel forms. The specific type of carrier and final formulation depend in part on the chosen route (s) of administration and the type of product.

[0022] A "comparable amount" is an amount that is measurably similar to a given reference or standard.

[0023] The "ingredients" of the formulation include any products or compounds related thereto or contained therein.

[0024] An "effective dose", "effective amount" or "therapeutic amount" is an amount sufficient to produce the desired pharmacological, cosmetic or therapeutic effect, and thus leading to the effective prevention or treatment of a disease or disorder or providing benefits to a vertebrate subject.

[0025] A "therapeutic effect" or "therapeutically desirable effect" refers to a change in a treated domain or region that shows signs of being affected in a desired manner, e.g., cancer treatment causes the destruction of cancer cells or inhibits the growth of cancer cells, treatment of acne reduces the number of and / or exacerbation of pimples, hair removal leads to pronounced hair loss, or wrinkle reduction surgery results in the disappearance of wrinkles.

[0026] An "isolated" biological component (such as a nucleic acid molecule, protein or cell) has been substantially separated from or purified from other biological components within which the component was produced, including other proteins, lipids, carbohydrates and other components.

[0027] As used herein, the term "nanoparticle" generally refers to a particle wherein at least one of its dimensions is from about 0.1 nm to about 9000 nm.

[0028] The term "subject" or "patient" as used in this application means any species of vertebrate.

[0029] The term "substantially pure" or "substantially isolated" as used in this application is a compound that is substantially free of one or more other compounds.

[0030] "Target tissue" includes a region of an organism in which physical or chemical strength or change is desired. As described in this application, exemplary target tissues for acne treatment include sebaceous gland, while exemplary target tissues for hair removal include a sebaceous unit, a funnel, a hair follicle, or an epidermis without hair follicles. The "region" of the target tissue includes one or more tissue components. Exemplary regions of target tissue include stem cell niche, protrusion, sebaceous gland, skin papilla, cortex, epidermis, inner sheath of the hair, outer sheath of the hair, core, Huxley's layer, Henle's layer or the efferent muscle. The "domain" of the target tissue region includes the basement membrane, extracellular matrix, cell surface proteins, unbound proteins / analytes, GlycoMatrix, glycoproteins or lipid bilayer.

[0031] A compound that is "substantially free" of some additional content is substantially or completely free of this content.

[0032] A "plasmon nanoparticle" is a metallic size structure expressed in nanometers in which surface plasmons excited by light are found. These surface plasmons are surface electromagnetic waves that propagate in a direction parallel to the metal-dielectric tangent surface (e.g. metal / air or metal / water). [0033] "Light-absorbing nanometallic" includes a nanomaterial capable of exhibiting a quantum size effect.

[0034] As described in the present application, compositions are provided that contain plasmonic nanoparticles to induce selective

EP 2 608 762 B1 thermomodulation in target tissue.

Plasmon nanoparticles.

[0035] Such compositions contain from about 10⁹ up to about 10¹⁶ nanoparticles, e.g. 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶ nanoparticles. Preferably the compositions contain about 10¹¹ up to 10¹³ particles so that the number of particles placed in effective therapeutic volumes of 1 ml is from 10⁹up to 10¹¹. In certain embodiments where a higher concentration of nanoparticles is desired in the target region, the compositions comprise particle concentrations with optimal optical densities (ODs) of 10 OD 100 OD or optical densities above 1000 OD. In some embodiments, they correspond to concentrations of about 1- 10% w / w / nanoparticles or more.

[0036] Nanoparticles can be homogeneous or heterogeneous in size and other characteristics. The size of nanoparticles is generally about 0.1 nm to about 5000 nm with respect to at least one dimension. Some variation in the size of the nanoparticle population should be expected. For example, the variation may be less than 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 25%, 50%, 75%, 100%, 200% or more than 200%. In certain embodiments where optimal plasmon resonance is desired, particle sizes in the range from about 10 nm to about 100 nm are provided. Optionally, in embodiments where an increased penetration of nanoparticles into a targeted tissue area, such as a hair follicle, is desired, particle sizes in the range from about 100 nm to about 1000 nm are provided. Particle size modulation present in the composition is also a useful means to concentrate the composition in the target domain. In addition, as described in this application, nanoparticles having a size range from about 10 nm to about 100 nm can be used as a component of larger molecular structures, generally in the range of from about 100 nm to about 1000 nm. For example, a plasmonic nanoparticle may have a coated surface to increase its size, deposited on an acceptable carrier, or it may be crosslinked or combined with other particles or other materials to form a larger particle. IN

In certain embodiments in which at least one dimension of at least one nanoparticle in a solution of plasmonic nanoparticles is less than 50-100 nm, the surface of the nanoparticle may be coated with a matrix (e.g. silica) with a thickness of 10-100 nm or greater in to increase this dimension or particle to 50-100 nm or more. Increasing the dimension can increase the delivery of all nanoparticles to the target region (e.g. hair follicle) and limit delivery to the non-targeted region (e.g. dermis).

[0037] Important issues to consider when making nanoparticles include: 1) zeta potential (positive, negative or neutral) and the charge density of particles and resulting compositions; 2) hydrophilicity / hydrophobicity of particles and resulting compositions; 3) presence of an adsorption layer (e.g. particle slip plane) and 4) target cell adhesion properties. Nanoparticle surfaces can be functionalized by thioled groups with a negative, positive or neutral charge (e.g. carboxylic acid, amines, hydroxyls) in various ratios. In addition, a surface coating made with anions (e.g. acrylate, citrate and others), a coating with a surfactant (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1, lecithin and other surfactants, including cetyltrimethylammonium bromide (CTAB), lipids, peptides) or protein / peptide coatings (e.g. albumin, egg albumin , egg white, milk protein, protein from other foods, plants, animals, bacteria, yeast or proteins obtained by recombination techniques). Block copolymers are also useful. In addition, the usefulness of any other compound or material that adheres to light-absorbing surfaces of particles is known to stimulate or counteract specific molecular interactions and to increase the penetration of particles into pores or hair follicles. In some embodiments, the surface of the particles is not modified. The modulation of hydrophilicity compared to hydrophobicity is carried out by modifying the surface of the nanoparticles with

Chemicals known in the art, including silanes, isothiocyanates, short polymers (e.g., PEG) or functionalized hydrocarbons. Polymer chains (e.g. biopolymers such as proteins, polysaccharides, lipids and their hybrids; synthetic polymers such as polyethylene glycol, PLGA and others; and hybrids of biopolymers with synthetic polymers) with different lengths and packing densities are useful to distinguish the adsorption layer / particle slip plane .

[0038] Optical absorption. Preferred nanoparticles have an optical absorption of from about 10 nm to about 10,000 nm, e.g. 100-500 nm. In certain embodiments, nanoparticles exhibit optical absorption useful for excitation using standard laser devices or other light sources. For example, nanoparticles absorb wavelengths of about 755 nm (alexandrite lasers) in the range of about 800810 nm (diode lasers) or about 1064 nm (Nd: YAG lasers). Similarly, nanoparticles absorb intense pulsed light (IPL), e.g. in the range from about 500 nm to about 1200 nm.

[0039] Organization. The nanoparticles provided in this application may generally contain a collection of unorganized nanoparticles. The term "disorganized" means that the nanoparticles in such a set are not connected with each other by physical force or chemical bonds either directly (particle-particle) or indirectly by means of certain intermediaries (e.g. particle-cell-particle, particle-protein- particle, particle-analyte-particle). In other embodiments, the nanoparticle compositions are organized into ordered grids. In particular, such ordered grids may include any three dimensional grids. In some embodiments, only a portion of the nanoparticles are organized, e.g. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 86, 90, 95, 99% or more than 99% of nanoparticles are organized into an ordered grid. Nanoparticles are organized by van der Waals attraction, London forces, hydrogen bonding, dipole-dipole interaction or covalent bond or a combination thereof.

[0040] "Ordered grid" or "ordered grids" may take the form of

A macrostructure consisting of individual parts that can be structured or unstructured in the form of spheres, colloids, spheres, ovals, squares, rectangles, fibers, wires, rods, scales, thin films or a flat surface. In contrast to the "unstructured network", which basically has no macrostructure.

[0041] Geometrically developed nanostructures.

The nanoparticles provided in this application can be formed into any currently known shapes or to be created to absorb light and generate plasmon resonance at a maximum wavelength or composition from 200 nm to 10,000 nm. In non-limiting examples, nanoparticles have the shape of spheres, ovals, cylinders, squares, rectangles, rods, stars, tubes, pyramids, stars, prisms, triangles, branches, plates or consist of a flat surface. In non-limiting examples, plasmonic nanoparticles contain nanoslabs, solid nanoshells, hollow nanoshells, nanowires, nanorysis, nanospheres, nanofibers, nanowires, nanopyramides, nanoprisms, nanoparticles and a combination thereof. Plasmon particles contained in the composition contain a significant amount of geometrically developed nanostructures defined as 5, 10, 15, 25, 50, 75, 80, 85, 90, 95, 98, 99, 99.9 or more than 99.9% of particles.

[0042] Composition. The nanoparticle contains gold and / or silver. The nanoparticle described in this application is a metal, metallic composite (e.g. silver and silica, gold and silica), metal oxide (e.g. iron oxide, titanium oxide), metal salt (e.g. potassium oxalate, strontium chloride), intermetallic phase (e.g. titanium aluminum, alnico), electric conductor (e.g. copper, aluminum), electric superconductor (e.g. yttrium, barium and copper oxide, strontium, calcium and copper oxide), electric semiconductor (e.g. silicon, germanium), dielectric (e.g. silica, plastic) or quantum dot (e.g. zinc sulfide, cadmium, selenium). The materials described in this application are gold, silver, nickel, platinum, titanium, palladium, silicon, gallium. Optionally, the nanoparticle described in this application contains a metal and dielectric, metal and semiconductor or metal, semiconductor and dielectric composite.

EP 2 608 762 B1 [0043] Coating. The composition contains coated nanoparticles.

<td>Type material</td><td>properties</td><td>Sample materials</td>

<td>Material biorozpoznawa Default</td><td>A group with substrate or analyte affinity or avidity</td><td>Antibody, peptide, phage, DNA, RNA</td>

<td>Material bioactive</td><td>A group (e.g. protein, analyte) that processes and modulates the activity of an individual biological or cellular</td><td>Growth factor (e.g. VEGF), cytokine, cell surface receptors, receptor ligands, G protein, kinase / phosphatase</td>

<td>Material biological</td><td>Material derived from living matter</td><td>Albumin, egg albumin, egg protein, milk protein, protein derived from another food, plant, animal, bacterium, yeast or obtained using techniques recombination; peptides; enzymes, lipids, fatty acids, sugars</td>

<td>Material biocidal</td><td>A material capable of killing, destroying or damaging biological matter</td><td>Synthetic pesticides or natural or natural antimicrobials</td>

<td>materials Dielectric</td><td>Insulator that can be polarized by an electric field</td><td>Silicon, semiconductors doping</td>

<td>Material chemorozpozn awalny</td><td>A material capable of interacting with a group to form bonds, biological or chemical reactions</td><td>Receptor, receptor ligand, chemical molecule</td>

<td>Chemically active Material</td><td>Material that transforms the substance</td><td>Aldehyde, halogens, metals</td>

<td>Polymer / dendritic mayor</td><td>Long chain molecule (linear or branched, block or co-block)</td><td>PLGA, PEG, PEO, polystyrene, carboxylated styrene, rubbers, nylons, silicones, polysaccharides</td>

<td>Polymer</td><td>Surface particle</td><td>PH sensitive binding, binding</td>

EP 2 608 762 B1

<td>Type material</td><td>properties</td><td>Sample materials</td>

<td>sensitive to incentives environment</td><td>which changes under the influence of the environment (e.g. acid)</td><td>sensitive to light, heat-sensitive bond, enzyme-sensitive bond, hydrolytic bond</td>

<td>hydrogel</td><td>Polymer with high hydrophilicity and ability to "manage" water</td><td>Based on synthetic 2-hydroxyethyl methacrylate (HEMA), based on polyethylene glycol (PEG), PLGA, PEG-diacrylate; natural ionic gels, alginate, gelatin, hyaluronic acids, fibrin</td>

<td>Metal</td><td>A thin metal coating to improve resonance and / or functionalization</td><td>Gold, silver, nickel, platinum, titanium and palladium.</td>

<td>Semiconductors</td><td>A semiconductor layer or core that increases plasmon resonance</td><td>Silicon and gal</td>

<td>Polymer containing a fluorescent marker</td><td>Fluorophore crosslinked with a polymer coating or directly with the particle surface</td><td>Fluorescein, rhodamine, cy5, cy5.5, cy7, Alexa dyes, Bodipy dyes</td>

<td>Matrix</td><td>A matrix coating that increases the solubility of nanoparticles and / or reduces "stickiness" to biological structures</td><td>Silica, polyvinylpyrrolidone, polysulfone, polyacrylamide, polyethylene glycol, polystyrene, cellulose, carbopol.</td>

[0044] Biological molecules. The composition may contain a peptide, nucleic acid, protein or antibody. For example, a protein, antibody, peptide or nucleic acid that binds the hair follicle stem cell protein (e.g., keratin 15), protein, Glyco Matrix or cell surface or stem cell lipid, protein, peptide, GlycoMatrix extracellular matrix or basement membrane.

EP 2 608 762 B1 [0045] Charged groups. The coated nanoparticles may contain charged groups by which these charges mediate increased or reduced binding to components located inside or outside the hair follicle by electrostatic or chemical interactions.

<td>Group class</td><td>properties</td><td>Sample groups</td>

<td>Polar groups</td><td>Indifferent charge, but increases hydrophilicity in water</td><td>Hydroxyl groups, isothiocyanates</td>

<td>group nonpolar</td><td>Increases hydrophobicity and / or improves solubility</td><td>Hydrocarbons, compounds myristoylated silanes isothiocyanates</td>

<td>group charged</td><td>Functional modifications surface, which change the zeta potential, isoelectric point or pKa and affect the adsorption / binding with compounds with complementary charge</td><td>Amines, carboxylic acids, hydroxyls</td>

<td>Ionic groups</td><td>Single ion surface groups</td><td>Ammonium salts, chloride salts</td>

<td>group alkaline</td><td>Groups that give off hydrogen ions</td><td>Amides, hydroxides, metal oxides, fluorides</td>

<td>group acid</td><td>Groups that accept hydrogen ions</td><td>Carboxylic acids, sulfonic acids, mineral acids</td>

<td>group oxidising</td><td>Groups that oxidize</td><td>Manganese ions, reactive oxygen species</td>

<td>group hydrophobic</td><td>Groups that increase the solubility in the non-aqueous solution and / or increase the adsorption on the skin in the hair follicle</td><td>Hydrocarbons, compounds myristoylated, silanes</td>

<td>group hydrophilic</td><td>Groups that like water and prevent adsorption</td><td>PEG, PEO, PLGA</td>

<td>Indifferent groups</td><td>Groups that bind the target</td><td>Antibodies, peptides, proteins</td>

EP 2 608 762 B1

<td>Group class</td><td>properties</td><td>Sample groups</td>

<td></td><td>cell, structure or protein of interest interests</td><td></td>

<td>group antagonistic e</td><td>Groups that block binding to the target of interest</td><td>Antibodies, peptides, proteins</td>

<td>group reactive</td><td>Groups that respond with biological or non-biological ingredients causing a change in structure on target or</td><td>aldehydes</td>

Description of target tissues.

[0046] Topical and dermatological applications. Target tissues for topical and dermatological use include the skin surface, epidermis and dermis. Diseases or conditions suitable for treatment with topical and dermatological applications include acne and hair removal. Warts, fungal infections, psoriasis, scar removal, hair growth, reduction of hypertrophic or keloid scars, skin imperfections (e.g. textures, colors, colors, elasticity, hydration) and malignant and benign skin tumors.

[0047] The term "acne" as used herein includes acne vulgaris and other forms of acne and associated skin conditions, including spring acne, concentrated acne, cosmetic acne, fulminant acne, acne prone acne, mechanical acne, acne pox, acne necrotic chlorine acne, drug-induced acne, acne pruritus, halogen acne, facial milf lupoid (sclerosis), acne caused by hair pomade, acne caused by tar and tropical acne.

[0048] Subcutaneous applications. Target tissues for subcutaneous uses described herein include adipose tissue and connective tissue below the common coating. The diseases or conditions described in this application suitable for treatment by subcutaneous uses include

EP 2 608 762 B1 wrinkles and tattoos. Other described uses include skin rejuvenation and / or restoration, removal or reduction of stretch marks and fat ablation.

[0049] According to the invention, the specific region of the target tissue is a hair follicle, sebaceous gland, merocrine sweat gland, apocrine sweat gland or paranasal muscle within which the specific target domain is located. For example, the target is the convex region of the hair follicle. Because in one embodiment, nanoparticles are useful in thermoablating hair follicle stem cells to remove hair, regions containing hair follicle stem cells are of particular interest. Thus, the target tissue region may include stem cell niche, bulge, sebaceous gland, dermal papilla, cortex, epidermis, inner sheath of the hair, outer sheath of the hair, core, Huxley's layer, Henle's layer or posterior muscle. Each of these regions may contain cells, stem cells, basement membrane, extracellular matrix, growth factors, analytes or other biological components that mediate hair follicle renewal. Disruption or destruction of these ingredients would cause a therapeutic effect, e.g., slowing or stopping processes that mediate hair regrowth, preventing sebum from the sebaceous gland, damaging or stopping the development of cancer cells, and reducing wrinkle formation. Targets can also be structures located in close proximity to the desired ablation target, especially if they are able to conduct heat efficiently.

[0050] Location domains. Compositions containing nanoparticles that are preferably localized in the domain of the target tissue region of a mammal subject to which the composition is administered are described.

[0051] Targeting groups. Nanoparticles can be designed to bind selectively to the target tissue domain. For example, nanoparticles are operably linked to a domain via a biological group to effectively target nanoparticles to the target tissue domain. Preferably the group comprises a stem cell component, progenitor cell, extracellular matrix component, basement membrane component,

The hair stem component, the hair follicle epithelium component, or the epidermal component without the hair follicle. Biological groups include proteins such as cell surface receptors, glycoproteins or extracellular matrix proteins, as well as carbohydrates, analytes or nucleic acids (DNA, RNA) and membrane components (lipid bilayer components, microsomes). [0052] Delocation domains. The nanoparticles present in the composition are preferably delocalized from the domain of the target tissue region. Delocation domains include specific areas of the tissue in which nanoparticles do not substantially aggregate or, if appropriate, are more effectively removed from the domain. In preferred embodiments, the relocation domain is an epidermis without a hair follicle, dermis, a hair follicle component (e.g. hair stem cell, stem cell niche, bulge, sebaceous gland, skin papilla, cortex, epidermis, inner hair sheath, outer sheath of the hair, core, Huxley's layer, Henle's layer, or paranasal muscle), funnel follicle, sebaceous gland, component of sebaceous gland , sebocyte, sebocyte component or tallow.

[0053] Energy sources. Nonlinear plasmon resonance excitation sources are described herein, which include various light sources or optical sources. Examples of light sources include a laser (ion laser, semiconductor laser, Q-switched laser, free-running laser or fiber laser), light emitting diode, lamp, sun, fluorescent light source or electroluminescent light source. Typically, the energy source may emit radiation at a wavelength of from about 100, 200, 300, 400, 500, 1000, 2000, 5000 nm to about 10,000 nm or more. A non-linear source of plasmon resonance excitation is capable of emitting electromagnetic radiation, ultrasound, heat energy, electricity, magnetic energy or electrostatic energy. For example, energy is radiation with an intensity of about 0.00005 mW / cm² up to about 1000 TW / cm². Optimal intensity is selected to induce high heat gradients from plasmon nanoparticles in areas from about 10 microns to hundreds of microns in surrounding tissue, with minimal residual effect on heated tissues with no particles in the radius

About 100 microns or more from the nanoparticle. In certain embodiments, the difference in heat gradient between the target tissue region and regions of other tissues (e.g., skin) is greater than 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 50 times, 100 - times or more than 100 times.

[0054] Energy can be regulated by monitoring thermal heat gradients on the skin surface using a thermal / infrared camera. As demonstrated in this application, the methods and systems of the present disclosure provide better efficacy if a surface plasmon is generated on nanoparticles by radiation. Typically, the plasmon is produced in a single-photon method or, optionally, a two-photon method, a multi-photon method, a stepwise method or an up-conversion method.

[0055] Delivery of radiation. Physical means for supplying energy from a non-linear source of plasmon resonance excitation to the target tissue area include fiber, waveguide, contact tip, or a combination thereof. [0056] Optical sources include a continuous optical (CW) optical source or pulsed optical source, which may be a polarized (or optionally unpolarized) single wavelength optical source capable of emitting radiation at a frequency from about 200 nm to about 10,000 nm. Optionally, the optical source is a polarized (or optionally unpolarized) optical source with multiple wavelengths capable of emitting radiation at a frequency from about 200 nm to about 10,000 nm. The pulsed optical source is generally capable of emitting pulsed radiation at a frequency of about 1 Hz to about 1 THz. A pulsed optical source is generally capable of producing a pulse lasting less than a millisecond, microsecond, nanosecond, picosecond or femtosecond. The optical source can be connected to a skin surface cooling device to reduce the heating of particles or structures on the skin surface and to focus heating on components in the hair follicles or tissue structures located in deeper layers.

EP 2 608 762 B1 [0057] Compositions containing nanoparticles. In order to provide optimal penetration through the skin into the target tissue, in some embodiments, plasmon nanoparticles are prepared in various compositions. Preferably the nanoparticles are prepared in compositions containing 1-10% v / v. surfactant (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1). Surfactants break down and emulsify sebum or other hydrophobic liquids, allowing better targeting of hydrophilic nanoparticles to the hair follicle, funnel, sebaceous gland or other skin regions. Surfactants also reduce the free energy needed to deliver hydrophilic nanoparticles to small hydrophobic gaps, such as the space between the hair shaft and hair follicle or to the sebaceous gland. Compositions containing nanoparticles may also contain emulsions at various concentrations (1-20% w / v) in aqueous solutions, silicone / oil solvents, propylene glycol or creams (e.g. containing alcohols, oils, paraffins, colloidal silicas). In other embodiments, the formulation includes a non-degradable polymer, e.g. synthetic poly (lactide-co-glycolide) copolymer, porous nylon copolymer of lauryl lactam and caprolactam, hydroxyethyl cellulose, polyelectrolyte monolayers or, optionally, in natural hydrogels such as hyaluronic acid, gelatin and others. In further embodiments, a PLGA hydrogel, PEG acrylate, is included in the formulation. Optionally, a matrix component such as silica, polystyrene or polyethylene glycol is provided in the formulation. Other preparations contain ingredients for surfactants, lipid bilayer, liposome or microsome. The nanoparticle can be a larger micron-sized particle.

[0058] Effective doses. As described in this application, an effective dose of compositions comprising nanoparticles contains a certain amount of particles required, in some aspects, to produce an effective heat gradient in a target tissue region so that a portion of the target tissue region is subjected to thermal energy from excited

Nanoparticles. The "minimum effective dose" is the smallest number or lowest concentration of nanoparticles in the composition that is effective in achieving the desired biological, physical and / or therapeutic effect (s). Preferably, the optical density of plasmonic nanoparticles is 10 OD - 1000 OD for one or more wavelengths corresponding to maximum resonance.

[0059] Cosmetically acceptable carriers. Cosmetic or pharmaceutical compositions with numerous plasmonic nanoparticles and a cosmetically or pharmaceutically acceptable carrier are provided. In general, the carrier and composition must be suitable for topical application to the skin of a mammalian subject such that plasmonic nanoparticles are present in an amount effective to selectively thermomodulate the skin component. Preferably, the nanoparticles are prepared with a carrier containing 1-10% v / v. surfactants (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1) to break the epidermal skin barrier, emulsify sebum, increase the mixing of hydrophilic nanoparticles with hydrophobic solutions, and reduce entropic particle delivery for hydrophobic skin regions (e.g. between the hair shaft and the surrounding sheath of the hair follicle). In some embodiments, the carrier comprises a polar or non-polar solvent. Examples of suitable solvents include alcohols (e.g. n-butanol, isopropanol, n-propanol, ethanol, methanol), hydrocarbons (e.g. pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane), chloroform, diethyl ether , water, water with propylene glycol, acids (e.g. acetic acid, formic acid), bases, acetone, isooctane, dimethyl sulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran, dichloromethane, ethyl acetate, tetramethylammonium hydroxide, isopropanol and others. In other embodiments, a stabilizing agent is provided, such as antioxidants, to prevent undesired oxidation of materials, sequestrants forming chelate complexes and inactivating metal ion traces that would otherwise act as catalysts, emulsifiers, ionic or nonionic surfactants, cholesterol or phospholipids. for stabilizing the emulsion (e.g.

Egg yolk lecithin, sodium stearoyl lactate, sodium bis- (2-ethylhexylsulfosuccinate (AOT)), ultraviolet stabilizers, protective materials, especially plastics, against the harmful effects of ultraviolet radiation. In further embodiments, the composition with the cosmetically acceptable carrier is prepared in such a way that the nanoparticles are substantially suspended.

[0060] Other ingredients including emulsion, polymer, hydrogel, matrix, lipid bilayer, liposome or microsome are also optionally included. In addition, a detectable dye (e.g. pigment), fragrance, wetting agent and / or skin protection agent may be included. In some examples, the viscosity of the formulation is more, less or within 0.1-1000, measured in millipascals-seconds (mPa • s).

[0061] The amounts of nanoparticles per milliliter in the composition may be subject to modification in order to obtain a specific binding and may range from 10⁹ up to 10¹⁸ particles, however, usually from 10¹¹ it's 10¹³ nanoparticles per milliliter. In certain embodiments where a higher concentration of nanoparticles in the target region is desired, the compositions comprise particle concentrations with optical densities of 10 OD - 1000 OD or optical densities above 1000 OD. In some embodiments, they correspond to nanoparticle concentrations of about 0.1-10 % w / w or more.

[0062] Before using nanoparticle formulations, skin and hair follicles can be pre-prepared to increase delivery of nanoparticles to the target region. In some embodiments, the hair stems are cut or removed using shaving, wax, cyanoacrylate surface peels, calcium thioglycolate, or other techniques to remove the hair stem and / or hair follicles and create a hollow space in which nanoparticles can accumulate. Openings of active or inactive hair follicles can be blocked by plugs formed by corneocytes and / or other materials (e.g. skin impurities, soot, hydrocarbons, cosmetics). In some embodiments, the initial exfoliation of the surface, including mechanical exfoliation (e.g., "salt" sea salt

(Glow "or microdermabrasion) and chemical peeling (e.g. enzymes, alpha hydroxy acids or beta-hydroxy acids) removes plugs from the holes in the hair follicles to increase the targeting of nanoparticle preparations to target areas in the hair follicle.

[0063] The present application describes nanoparticle preparations prepared for application with a sponge applicator, fabric applicator, direct contact with a hand or hand in a glove, spray, aerosol, vacuum suction, high pressure air flow or high pressure liquid flow, roller , brush, flat surface, semi-flat surface, wax, ultrasound or other sonic forces, mechanical vibrations, manipulation of the hair shaft (including pulling, massaging), physical strength, thermal manipulation and other treatments. Nanoparticle preparation treatments can be performed alone, in combination, sequentially or in repetitions 1-24 times. Plasmon nanoparticles capable of selective localization in the first skin component are described herein, where physical massage or pressure, ultrasound or heat increase the selective location of the nanoparticles in the first component. In addition, nanoparticles can be selectively removed from skin components other than the first component, such removal being carried out using acetone, alcohol, water, air, skin peeling, chemical peeling, waxing or plasmonic compound reduction. In addition, nanoparticles have a coating layer that increases the solubility of nanoparticles in the carrier and / or reduces "stickiness" and accumulation in non-target areas. The object described in this application contains nanoparticles in which at least part of the outer surface of the nanoparticle is modified to contain a layer of polymer, polar monomer, nonpolar monomer, biological compound, metal (e.g., a thin metal layer, metal composite, metal oxide or metal salt ), dielectric or semiconductor. Optionally, the modification of the outer surface is polar, non-polar, charged, ionic, basic, acidic, reactive, hydrophobic, hydrophilic, agonist or antagonistic. If at least one dimension of at least one nanoparticle found in solution

Plasmonic nanoparticles are below 50-100 nm, the surface of the nanoparticles can be coated with a matrix (e.g. silica) 10-100 nm thick or coarser to increase the particle size to 50-100 nm or more. Increasing the dimension can increase the delivery of all nanoparticles to the target region (e.g., hair follicle) and limit delivery to the non-target area (e.g., dermis).

Penetration Agents [0064] The compositions of the present disclosure are administered topically. In this application, agents are provided for redistributing plasmonic particles from the skin surface to a skin tissue component, including a hair follicle, hair follicle component, hair follicle funnel, sebaceous gland or sebaceous gland component using high frequency ultrasound, low frequency ultrasound, massage, iontophoresis, high pressure air flow, high pressure liquid flow, vacuum, pretreatment with fractional photothermolysis or dermabrasion or combinations thereof. For example, the compositions can be administered using a sponge applicator, fabric applicator, spray, aerosol, vacuum suction, high pressure air flow or high pressure liquid flow, direct hand contact, ultrasound or other sonic forces, mechanical vibration, bellows manipulation hair (including pulling, massaging), physical strength, thermal manipulation and other treatments. Nanoparticle preparation treatments can be performed alone, in combination, sequentially or in repetitions 1-24 times.

Cosmetic and therapeutic applications of plasmonic nanoparticles.

[0065] In general, the applicant has created systems and methods for cosmetic and therapeutic treatments for skin conditions, diseases and disorders using treatment methods based on the use of nanoparticles.

Acne Treatment [0066] Acne is caused by a combination of diet, hormonal imbalance, bacterial infection (Propionibacterium acnes), predisposition

Genetic and other factors. The nanoparticle-based methods and systems described in this application for the treatment of acne are able to focus focally to the causal regions of the dermis, sebaceous gland and hair follicle, and therefore show advantages over existing techniques known in the art, including chemical treatment (peroxides , hormones, antibiotics, retinoids and anti-inflammatory compounds), dermabrasion, phototherapy (lasers, blue and red light treatment or photodynamic treatment) or surgery.

[0067] In particular, laser-based techniques are becoming an increasingly popular type of acne treatment, but the significant limitation is the lack of selective absorption properties for natural dyes (e.g. fat) for specific wavelengths of light such that selective heating of a single cell, structure, or tissue component, in particular in the sebaceous glands, bellows funnel and hair follicle regions, is not achievable without heating adjacent non-target tissues. The nanoparticles described here provide a much greater photothermal conversion than natural dyes, enabling the focusing of laser energy on specific cells, structures or tissue components in the sebaceous gland, bell funnel or hair follicle areas for selective photothermal damage.

[0068] The use of materials and techniques described in this application may provide acne treatment with a longer shelf life than existing methods. In certain embodiments, tuned, selective ablation of the sebaceous gland or funnel of the hair follicle is achieved as described in this application. In particular, plasmonic nanoparticles are specifically located in regions of the hair follicles inside or near the sebaceous gland or hair follicle funnel.

[0069] Plasmonic nanoparticles exhibit strong absorption at wavelengths emitted by standard laser hair removal devices (e.g. 755 nm, 810 nm, 1064 nm) relative to surrounding epidermal tissue. Thus, irradiation of targeted plasmonic nanoparticles with laser light causes heat radiation from the particles to the adjacent sebum,

EP 2 608 762 B1 of the sebaceous gland, funnel of the hair follicle and other factors causing acne.

Hair removal.

[0070] The nanoparticle-based methods and systems described in the present application for skin treatment have advantages over existing techniques known in the art, including laser techniques, chemical techniques, electrolysis, electromagnetic wave techniques and mechanical techniques (e.g. waxing, tweezers) ). Such techniques do not provide permanent hair removal for many entities. In particular, these techniques are not effective for subjects with light or medium colored hair that have side effects, including pain and no beneficial cosmetic effect, including hair removal. Laser techniques are common in various applications, but the significant limitation is the lack of selective absorption properties of natural dyes (e.g. melanin) for specific wavelengths of light such that selective heating of a single cell, structure or tissue component, in particular in the sebaceous glands, bellows funnel and hair follicle regions, is achieved without heating adjacent non-target tissues. The nanoparticles described in this application provide significantly more photothermal conversion than natural dyes, enabling the focusing of laser energy on specific cells, structures or tissue components to cause photothermal damage.

[0071] In the present application, more permanent reduction or removal of all hair types is provided compared to hair removal treatments known in the art. In certain embodiments, tuned selective ablation of the hair shaft and damage to the stem cells in the bulging region is provided as described herein. In particular, plasmonic nanoparticles are specifically localized in areas of the hair follicles within or near the bulging region, the domain rich in hair follicle stem cells. In addition, plasmonic nanoparticles are located in a very close approx. 50-75% of the hair shaft structure.

[0072] Plasmonic nanoparticles exhibit strong absorption at wavelengths emitted by standard laser hair removal devices (e.g. 755 nm, 810 nm, 1064 nm) relative to surrounding epidermal tissue. Thus, irradiation of targeted plasmon nanoparticles with laser light induces heat emission from the particles to adjacent stem cells (or in some cases the architecture of the hair shaft itself), leading to cell death and disruption of the normal regeneration pathway.

Benign and Malignant Skin Tumors [0073] Laser therapies used to prevent and treat benign, malignant, melanoma and non-melanoma skin tumors focus mainly on photodynamic therapeutic methods in which photosensitive porphyrins are applied to the skin to locate laser light to produce reactive oxygen species and destruction of cancer cells with toxic radicals. For example, 5-ALA in combination with laser therapy has been approved by the FDA for the treatment of non-melanoma skin cancers such as actinic keratosis, and is used out of registration to treat largely disseminated, inoperable or recurrent basal cell carcinomas (BCCs). However, this procedure causes photosensitivity, burning, peeling, scarring, hypo- and hyperpigmentation, and other side effects due to the non-specific percutaneous absorption of porphyrin molecules. The nanoparticles described in this application provide much more photothermal conversion than natural pigments and dyes, enabling focusing of laser energy on specific cells, structures or tissue components for selective thermomodulation.

[0074] The use of the materials and techniques described in this application may provide cancer therapies with greater sophistication and durability than existing methods. In this application, tuned selective ablation of specific target cells is described. In particular, plasmon nanoparticles specifically localized in areas of the hair follicles, where stem cells of the bulging of the hair follicle giving rise to cancer are described in this application

Basal cell nodules and other cancers. Plasmonic nanoparticles can also be delivered to other target cells that cause cancer to develop, for example, the intercellular epithelium in which cells are found that develop superficial basal cell carcinomas.

[0075] Plasmon nanoparticles exhibit strong absorption at wavelengths emitted by standard laser hair removal devices (e.g., 755 nm, 810 nm, 1064 nm) relative to surrounding epidermal tissue. Thus, irradiation of targeted plasmon nanoparticles with laser light induces heat emission from the particles to adjacent keratinocytes, melanocytes, a follicle bulging stem cell, a cancer cell, or a cancer precursor cell, leading to cell death or inhibiting cell growth to prevent and treat cancer. [0076] Subcutaneous applications. Target tissues for subcutaneous uses described herein include adipose tissue and connective tissue below the common coating. Diseases or conditions suitable for treatment with the subcutaneous uses described in this application include wrinkles and tattoos. Other described uses include skin rejuvenation and / or restoration, removal or reduction of stretch marks and fat ablation.

[0077] Vascular applications. Target tissues for vascular applications described herein include arteries, arterioles, capillaries, veins and venules. Diseases or conditions suitable for treatment with the vascular applications described herein include telangiectasias, leaky valves, and vasoconstriction. In particular, venous abnormalities are responsible for a significant proportion of vascular diseases or cosmetic conditions. People who have venous abnormalities such as telangiectasias or inefficient venous valves experience pain, itching or undesirable aesthetics. [0078] In addition, the present application describes several indications where the ablation of other vessels, including arteries, arterioles or capillaries, could provide therapeutic or cosmetic benefits, including: 1) ablation of vessels supplying adipose tissue and / or fat cells, 2 ) ablation of tumor supply vessels / cancer cells, 3) ablation of birthmarks

EP 2 608 762 B1 (port wine stains, hemangiomas, stork pinching), and 4) any other indication in which vessel ablation causes tissue damage and apoptosis or necrosis of cells supplied by these vessels, bringing therapeutic benefits or cosmetic. Described herein are methods of using compositions described herein for the selective damage of a vein component (s) using plasmonic nanoparticles distributed in the blood in a focal or diffuse manner. Plasmon nanoparticles can be combined with a pharmaceutically acceptable carrier as described above and introduced into the body by intravenous injection. Nanoparticles are dispersed in the blood and localized in specific vascular tissues. The nanoparticles can then be activated by laser or light-based systems known in the art for the treatment of skin conditions, e.g., hair removal or telangiectasia ablation. Alternatively, a fiber optic fiber laser can be used under control with or without imaging techniques to ablate the vessel or blood components in larger vessels. Devices with dual functions can be used for both nanoparticle injection and light emission by the optical fiber. Activated nanoparticles warm up blood and adjacent tissues (vessels, vascular walls, endothelial cells, components located on or inside endothelial cells, components of the endothelial basement membrane, components supplying mesenchymal tissues, cells or cellular components around the vessel, blood cells, blood cell components, other blood components) for ablation temperatures (38-50 ° C or higher).

[0079] A composition comprising a pharmaceutically acceptable carrier and numerous plasmonic nanoparticles in an amount effective to induce thermomodulation of the target region of the vascular or endovascular tissue to which the composition is intravenously contacted is described in the present application. In addition, the plasmonic nanoparticle composition may contain microvascular targeting agents selected from the group consisting of antibodies directed against microvascular endothelial cells and ligands for microvascular endothelial cell receptors.

EP 2 608 762 B1

Also described is a method of thermoablating a target vascular tissue in a mammalian subject, comprising the steps of contacting the target region of the vascular tissue with a composition comprising multiple plasmonic nanoparticles and a pharmaceutically acceptable carrier in such conditions that an effective amount of plasmonic nanoparticles is located in the domain of the target vascular region; and exposing the target region of the vascular tissue to energy provided from a non-linear surface excitation source of plasmon resonance in an amount effective to cause thermoablation of the domain of the target vascular region.

[0080] Oral and nasal uses. Target tissues for the oral uses described herein include the oral cavity, nose, throat, larynx and trachea. Diseases or conditions suitable for treatment with the vascular applications described herein include oral cancer, polyps, throat cancer and Mounier-Kuhn syndrome.

[0081] Endoscopic applications. Target tissues for the endoscopic applications described in this application include stomach, small intestine, large intestine, rectum and anus. Diseases or conditions suitable for treatment with the endoscopic applications described herein include gastrointestinal cancer, ulcerative colitis, Crohn's disease, irritable bowel syndrome, celiac disease, short bowel syndrome or infectious diseases such as lambliosis, tropical sprue, tapeworm infection, celandine, enteritis, ulcers, Whipple's disease and dilatation of the colon.

[0082] Thermomodulation methods. Methods are provided for thermomodulating the target tissue region. A composition of nanoparticles containing numerous plasmonic nanoparticles under such conditions that an effective amount of plasmonic nanoparticles is located in the domain of the target tissue region; and exposing the target tissue region to energy provided from a non-linear surface plasmon resonance excitation source in an amount effective to cause thermomodulation of the target tissue region.

EP 2 608 762 B1 [0083] Removal of non-specific bound nanoparticles. Nanoparticles located on the surface of the skin can be removed by contacting the skin with acetone, alcohol, water, air, a cleansing agent or wax. Optionally, physical purification can be performed. Optionally, a reduction of the plasmonic compound can be carried out.

[0084] Amount of energy supplied. The skin is irradiated at a fluence of 1-60 joules per cm² at laser wavelengths of around e.g. 750 nm, 810 nm, 1064 nm or other wavelengths, especially in the infrared range. Different repetition frequencies from continuous to pulse operation are used, e.g. 1-10 Hz, 10-100 Hz, 100-1000 Hz. Although part of the energy is reflected, the advantage of the object described in this application is that a significant amount of energy is absorbed by the particles and less is absorbed by the skin. Nanoparticles are delivered to the hair follicle, hair follicle funnel or sebaceous gland in a concentration sufficient to absorb e.g. 1.1-100x more energy than other skin components of similar volume. This is achieved in some embodiments by obtaining a concentration of particles in the hair follicle with absorbance at a peak laser power of 1.1-100x relative to other skin components of similar volume.

[0085] In order to allow tunable destruction of target skin structures (e.g. sebaceous glands, hair follicle funnel, hair follicles), light-absorbing nanoparticles are used in combination with a laser or other excitation source of appropriate wavelength. The laser light can be used in a continuous or pulsed manner, it can be single or multiple pulses of light. The heating intensity and distance beyond which photothermal damage occurs are controlled by the intensity and duration of exposure to light. In some such embodiments, pulsed lasers are used to provide local thermal damage. In some such embodiments, pulses of varying duration are provided to limit areas of thermal damage to 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 30, 50, 75, 100, 200 , 300, 500, 1000 microns from particles. Impulses continue

At least femtoseconds, picoseconds, microseconds or milliseconds. In certain embodiments, the maximum tissue temperature induced by heating by nanoparticles is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or 500 degrees Celsius. In certain embodiments where pulse heating is used, the highest temperatures are achieved locally within the hair shaft, without increasing the temperature of the macroscopic tissue by more than 0.1, 0.5, 1, 2, 3, 4, 5, 7, 9, 12, 15 or 20 degrees Celsius. In some embodiments, short pulses (100 nanoseconds - 1000 microseconds) are used to create very large transient heat gradients within and around the target skin structure (e.g. sebaceous gland and / or hair follicle) from introduced particles to locate damage in close proximity to the location of the particles. In other embodiments, longer pulses (1-500 ms) are used to generate heat gradients distant from the target structure to locate thermal energy in stem cells in the bulging region or other components more than 100 μm from the localized particles. Fluences of 1-30 joules per cm² they are generally sufficient for thermoablation of hair follicles with high particle concentrations, and therefore higher absorbance compared to the skin (e.g. 1.1-100 times per skin volume absorbance). These fluencies are often lower than currently used (e.g. diode laser: 25-40 J / cm², alexandrite laser: 20 J / cm², Nd: YAG laser: 3060 J / cm²) and cause less damage to areas without hair follicles and potentially less pain.

[0086] Plasmon resonance systems. Plasmon resonance systems that contain a surface that contains multiple plasmon nanoparticles and a non-linear excitation source are described in this application. The surface is a component of the skin that is intended for cosmetic or therapeutic treatment (e.g., a bulging region for hair removal, a funnel or a sebaceous gland to prevent acne). Means for delivering plasmonic nanoparticles to the skin surface, such as applicator, spray, aerosol, vacuum suction, have also been described as a component of the system.

EP 2 608 762 B1 high pressure air flow or high pressure liquid flow. In addition, agents for localizing plasmonic nanoparticles in the skin component (e.g., hair follicle, bulging region, sebaceous gland, funnel) were provided. Useful surface delivery means include a high frequency ultrasound generating device, low frequency ultrasound, heat, massage, contact pressure, or a combination thereof.

[0087] Systems have also been described that contain means for removing nanoparticles found on a skin region devoid of follicles. Such removal agents include at least one of acetone, alcohol, water, air, chemical peeling, wax or a compound that reduces the plasmonic compound.

[0088] In addition, the systems of the present disclosure provide a non-linear excitation source that produces a continuous wave optical source or pulsed optical source. Alternatively, a non-linear excitation source has the ability to generate electromagnetic radiation, ultrasound, thermal energy, electricity, magnetic energy or electrostatic energy. Systems have been described in which a non-linear excitation source has the ability to irradiate nanoparticles with an intensity from about 0.00005 mW / cm² up to about 1000 TW / cm². In addition, the non-linear excitation source may function in a single-photon method, a two-photon method, a multi-photon method, a gradual method or an up-conversion method. The described systems may use a fiber, waveguide, contact tip, or a combination thereof.

[0089] In some embodiments, the system includes a monitoring device such as a temperature sensor or a heat energy detector. In other embodiments, the systems also include control means for modulating a non-linear excitation source (e.g., "feedback control device"). In a related embodiment, the system comprises means for detecting surface temperature or a target tissue adjacent to the surface, wherein the controlling agent modulates the intensity of the non-linear excitation source and / or duration.

Excitation. In such embodiments, the control means preferably modulate the intensity of the non-linear excitation source such that the first hair follicle component is selectively thermoablated relative to the second hair follicle component. In further embodiments, during irradiation, the skin is contacted with a cooling device to minimize heating of the nanoparticles or skin on the surface, while heating the nanoparticles that penetrated deeper into the bellows, skin or sebaceous gland to temperatures that cause selective ablation of adjacent tissues.

[0090] The skin is an exemplary target tissue. The skin preferably contains a hair follicle and / or a sebaceous gland in which a non-linear excitation source produces energy that warms the skin in an amount effective to induce thermomodulation of the hair follicle, funnel, sebaceous gland, or a component thereof, e.g. by heating to increase skin temperature to more than 37 ° C, e.g. 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, up to about 50 ° C or higher.

[0091] Preparation methods. Nanoparticle compositions can be made by:

a) forming a first mixture containing multiple nanoparticles and a first solvent;

b) exchanging the first solvent for a second solvent to form a second mixture;

c) combining the second mixture with a cosmetically or pharmaceutically acceptable carrier;

thus creating a composition of nanoparticles.

[0092] The exchange step may optionally be carried out using liquid chromatography, a solvent exchange system, centrifugation, precipitation or dialysis. Typically, nanoparticles have a modified surface through a controlled reduction step or an oxidation step. Such surface modification may include a coating step such as adsorption of the monomer, polymer or biological unit on the surface of the nanoparticle. Typically, the coating step involves contacting the nanoparticles with

Oxidizing environment. In addition, the coating step may include polymerizing the monomer to form a polymer coating.

[0093] The methods described in the present application may also include the steps of dissolving nanoparticles in a non-polar solvent followed by mixing the dissolved nanoparticles in a polar solvent so that the nanoparticles are encapsulated in an emulsion. In addition, surfactants (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1) in concentrations of 0.1-10% to break the epidermal barrier of the skin, emulsify sebum and allow better mixing of hydrophilic nanoparticles in aqueous solutions. In addition, concentration of nanoparticles can be used, e.g. by centrifugation or lyophilization. In addition, nanoparticles can be subjected to pre-heating or irradiation. An optional step of coupling a biological entity or multiple biological entities to nanoparticles is also described. Such a coupling step may involve attaching biological units via a thiol, amino or carboxy bond to nanoparticles.

[0094] Diseases and disorders. The present invention can be applied to human (or animal) skin to reduce acne or reduce hair growth. Treatment for wrinkles and other changes associated with photoaging or chronological aging (generally referred to as skin rejuvenation), treatment of diseases, including skin diseases, rosacea reduction, folliculitis, chin follicle inflammation, or proliferative or papulomatous disorders such as psoriasis, stimulation of hair growth, reduction of cellit, warts, hypopigmentation such as port-wine stains (PWS: nevus flammeus), congenital birthmarks, excessive sweating, varicose veins, pigmentation disorders, tattoos, vitiligo, melanoderma, scars, stretch marks, fungal infections, bacterial infections, inflammatory skin disorders, disorders musculoskeletal system (e.g. tendonitis or arthritis), healing of surgical wounds, treatment of burns to improve healing and / or reduction of scarring, improvement of blood circulation in the skin

EP 2 608 762 B1 etc.

[0095] The present disclosure may also be useful in improving wound healing, including but not limited to chronic skin ulcers, diabetic ulcers, thermal burn wounds, ulcers or viral disorders, periodontal disease and other dental diseases. The present disclosure is also useful in enhancing the effects of devices that cause damage or wound in the process of cosmetic surgical procedures, including non-ablative techniques for thermal wound formation to treat wrinkles, scars, stretch marks and other skin disorders. In such circumstances, it may be beneficial to use traditional non-ablative thermal treatments in combination with the methods described in the present disclosure. Combination with microabrasion or surface abrasion, dermabrasion or enzymatic or chemical skin peeling, or topical cosmetic applications with or without the use of nanoparticles to improve treatment has also been described, as removal of the stratum corneum (and possibly additional epithelial layers) may be beneficial for some treatment regimens. The methods of the present disclosure have particular utility in the treatment of acne and hair removal. Also described are methods used to stimulate hair / hair follicle growth, reduce / prevent malignant and benign skin tumors, and skin rejuvenation.

[0096] The dermatologically therapeutic methods described herein can be created using irradiation of nanoparticles alone, irradiation of nanoparticles in combination with nano- or microparticles, or irradiation of nanoparticles with a composition comprising nano- or microparticles and one or more therapeutic agents. Such nanoparticle irradiation can be produced by any known nanoparticle generator, preferably a focused nanoparticle generator capable of generating and irradiating focused nanoparticle waves.

Examples

EP 2 608 762 B1

Example 1. Preparation of plasmon nanoparticles for thermomodulation.

[0097] Plasmonic nanoparticles, including nanowires, hollow nanoshells, silicon nanoshells, nanoslabs, nanorysis, nanowires, nanopyramides, nanoprisms, nanoplates and other configurations described in this application and known in the art configurations are produced in the size range from 1 to 1000 nm in such conditions so that the surface properties facilitate deep penetration into the hair follicle. The surface properties can be changed in one or many (2, 3 or 4) different dimensions to increase the concentration of nanoparticles in the target tissue domain. The penetration of 10-200 μm hair follicles can be maximized by using the nanoparticles described in this application. In the present disclosure, nanoparticles of from about 10 to about 100 nm are produced and are preferably organized or prepared for multiparticulates with a size in the range of 100 to 300 nm or more. Optionally, a single-particle structure (e.g., silica) is formed to increase the particle size to 100-300 nm or larger.

[0098] Plasmon nanoparticles with modified surface. An example of the production of plasmon nanoparticles with modified surface is presented below. Plasmon nanoparticles were synthesized with a stable cetyltrimethylammonium bromide (CTAB) coating and concentrated from an optical density of 10 OD to 100, 200, 300, 400, or 500 OD using 1 to 3 centrifugation cycles at 16,000 rpm, with decantation supernatant. Optionally, CTAB coated nanoparticles were concentrated and resuspended in 250 Amol / L 5-kDa methyl polyethylene glycol (PEG) thiol to produce PEG coated nanoparticles. To verify that the PEG polymer is fully reduced, spectrophotometry was performed to measure the thiol activity of the thiol-5,5-dithiobis (2-nitrobenzoic acid) polymer relative to a DTT gradient. The solution of methyl-PEG-thiol coated nanoparticles and CTAB was stirred at room temperature for 1 hour and then dialyzed against 5 kDa MWCO in 4 L of distilled water for 24 hours. Dialyzed samples were passed through 100-kDa filters to remove excess polymer.

EP 2 608 762 B1

Calculation of the number of PEG polymers per particle was performed using surface modification of nanoparticles with the aminoPEG-thiol polymer and calculation of the number of amines using the SPDP test. In the case of test preparations, solutions with an optical density of 100 OD of CTAB coated plasmonic nanoparticles in distilled water and solutions with an optical density of 100 OD of PEG coated plasmonic nanoparticles in distilled water, ethanol, DMSO or mineral oil were prepared. Plasmon nanoparticles with silica coatings were prepared by reacting nanoparticles with silicas such as tetraethylorthosilicate (TEOS), sodium silicate, aminopropyltriethoxysilane (APTS) etc. to a thickness of 5-50 nm or more. Control preparations containing only the carrier did not contain nanoparticles.

[0099] Embedded nanoparticles. Nanoparticles are embedded (or encapsulated) in materials, which allows different sizes to be made to adjust the size. Particles 100-2000 nm have been shown to penetrate the hair follicle without penetrating the dermis. The nanoparticles are encapsulated in silica, a synthetic poly (lactide-co-glycolide) copolymer, a porous nylon copolymer of lauryl lactam and caprolactam, hydroxyethyl cellulose, polyelectrolyte monolayers or, optionally, in natural hydrogels such as hyaluronic acid, without significantly changing plasmon properties. Nanoparticles are deposited in 100-2000 nm materials without creating a covalent connection or crosslinking of amines, carboxyls or other groups on the surface of the nanoparticle with the polymer structure. The surface area of ​​1002000 nm can be modified to obtain optimal zeta potential, hydrophilicity / hydrophobicity and / or adsorption layer using the techniques described in this application. In addition, the low to high polymer ratio can be modified to increase the concentration and depth of penetration of deposited plasmonic nanoparticles. Preferably, the nanoparticle ratio is more than about 1. Example 2. Preparation of thermoablative plasmon nanoparticles for topical administration.

[0100] Nanoparticles are prepared as in Example 1 using a suitable solvent (eg water, ethanol, dimethyl sulfoxide). The mixture containing numerous nanoparticles in water is concentrated to about 100500 OD and the solvent is replaced with a new one using liquid chromatography, solvent exchange system, centrifugation, precipitation or dialysis. The solvent may contain alcohol (e.g. n-butanol, isopropanol, n-propanol, ethanol, methanol), hydrocarbon (e.g. pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane), chloroform, diethyl ether, water, acid (e.g. acetic acid, formic acid), base, acetone, dimethyl sulfoxide, dimethylformamide, acetonitrile, tetrahydrofuran, dichloromethane or ethyl acetate. The new solvent combines with a cosmetically or pharmaceutically acceptable carrier, thereby forming a nanoparticle composition. Generally, the particles and carrier will form an emulsion.

[0101] Plasmonic nanoparticle preparations that increase or accelerate the penetration of nanoparticles into the hair follicles are provided. In some embodiments, nano- and microemulsions facilitate separation in lipid-rich skin compartments, such as the hair follicle. In certain embodiments, nanoparticles are prepared in compositions containing 0.5-2% v / v. surfactants to enable disruption of the epidermal skin barrier, emulsification of sebum and better mixing of hydrophilic nanoparticles in hydrophobic solutions or directing them to hydrophobic skin spaces (e.g. between the hair shaft and the surrounding follicle). Nanoparticle preparations are also supplied in various concentrations (1-20% w / v) in aqueous solutions, silicone / oil solvents, propylene glycol or creams (e.g. containing alcohols, oils, paraffins, colloidal silicas). In some embodiments, the light-absorbing nanoparticles are used in solutions with adjusted pH, temperature, osmolytic concentration, viscosity, volatility and other properties to improve the penetration of light-absorbing nanoparticles into the hair follicles.

[0102] Formulations are made to maximize nanoparticle stability (degree of aggregation in solution), nanoparticle concentration, and

The absorbance of nanoparticles (the degree of laser-induced heating at different concentrations).

[0103] When plasmonic nanoparticle formulations are illuminated with a clinical laser with a wavelength corresponding to the wavelength of maximum particle absorption, the formulation heats up to thermoablation temperatures faster and to a greater extent than conventional clinical absorption dyes. Figure 2 compares the temperature profile of plasmon particles (maximum absorption wavelength 1020 nm) with a fluid containing conventional clinical carbon dyes, Meladine spray and indocyanine green after exposure to 1064 nm laser pulses, 20 J / cm², 55 ms. The increase in temperature due to 1064 nm pulsed laser light was more than 2.5-fold greater for the plasmonic solution compared to conventional clinical dyes used at the same dilution (1: 1000 dilution from clinical concentration, with clinical concentrations of: 20-200 mg / ml for carbon, 1mg / ml for Meladine, 5 mg / ml for indocyanine green.

Example 3. Use of plasmonic nanoparticles for thermomodulation of hair.

[0104] For people with blond, red, gray or light hair, hair removal using existing light-based techniques is not appropriate. The present application provides methods for using the compositions described in the present application for the selective removal or reduction of untreated blond, red, gray or light hair. Plasmonic nanoparticles produced and prepared as described above are introduced into the target tissue region, generally the skin region, and activated by means of laser hair removal systems known in the art to achieve effective hair removal.

[0105] To achieve maximum penetration depth and concentration of plasmon nanoparticles in the hair follicle and / or near the sebaceous gland components, including the sebaceous duct, sebum, epithelial sebaceous junction and / or near the bulging area, including stem cells, niche stem cells, epithelial junction

In the bulging region and / or near the bulging of the bellows, an optimal particle size of 30-800 nm containing one or more plasmon nanoparticles has been developed. Nanoparticles encapsulating plasmon nanoparticles can be prepared from any number of polymers or matrices. In some embodiments, the formulation comprises a non-degradable polymer, e.g. synthetic poly (lactide-co-glycolide) copolymer, porous nylon copolymer of lauryl lactam and caprolactam, hydroxyethyl cellulose, polyelectrolyte monolayers or, optionally, in natural hydrogels such as hyaluronic acid, gelatin and others. In further embodiments, a PLGA hydrogel, PEG-acrylate, is included in the formulation. Preferably, a matrix component such as silica, polystyrene or polyethylene glycol is provided in the formulation to improve particle stability and allow easy removal from the skin surface after application and targeting of the bellows. Other formulations contain a surfactant component (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1), lipid bilayer, liposome or microsome. Plasmonic nanoparticles, including nanowires, nanoscores, nanospheres, nanoslabs or nanoris can be encapsulated in a polymer or lipid nanoparticle or matrix, or deposited on the surface of the particles. Alternatively, nanoparticles of 100-250 nm, 250-500 nm, 800 nm-1500 nm or more than 1500 nm can be used.

[0106] In some embodiments, pre-mechanical or chemical exfoliation of the skin is used to remove hair follicles and "open" the follicles to provide particles. In addition, hair can be removed by shaving or waxing to create an empty space in the hair follicle to fill with particles. The use of physical or thermal force strengthens or accelerates the penetration of light-absorbing nanoparticles and their conjugates into the hair follicles, in part by causing the hair follicle to expand before applying the nanoparticles. For example, to increase the penetration of light-absorbing nanoparticles into the hair follicles, sonic forces, mechanical vibrations, manipulations of the hair shaft (including pulling), force are used

Physical manipulation, thermal manipulation and other treatments. Treatments using the preparation of nanoparticles are performed alone, in combination, sequentially or in repetitions 1-24 times.

[0107] An applicator is used to uniformly apply nanoparticles to the bellows. The applicator may be a sponge, a fabric, direct contact with a finger, a tube, a syringe, a suction device, an aerosol, a spray or other means known in the art. In one example, 1 ml of a 100 O OD plasmonic nanoparticle preparation with a maximum resonance at 810 nm is applied to about 200 cm² the surface of the skin of an adult human with a syringe. For uniform distribution of the solution on the skin surface and deep into the hair follicles, fabric is used. To introduce particles deep into the hair follicles, a mechanical vibrator is used for 2 minutes with or without ultrasound (1 MHz, 5 minutes). The particles penetrate to a depth of 50-75% of the full thickness of the hair shaft in concentrations sufficient to warm the skin in a 100 μm radius at incremental temperatures 5-20 times higher than those generated in similar volumes of adjacent skin irradiated with a diode laser (810 nm). Acetone, ethanol or a cleansing agent can be used to remove any particles from the skin surface that have not accumulated in the bellows and to reduce or prevent heating of the skin without hair follicles.

[0108] Nanoparticle preparations are tested ex vivo on animal samples, ex vivo on human skin samples, and in vivo on human skin, and the following are evaluated: 1) the depth of penetration of nanoparticles into the hair follicles; 2) achieved particle concentration; 3) the degree of heating achieved at the supplied nanoparticle concentrations; and 4) efficiency of photothermal destruction, including temporary and permanent hair removal, 5) removal of nanoparticles after surgery. To assess the depth of penetration of nanoparticles, observation of surface functionalized plasmon nanoparticles by fluorescence microscopy after preparation of histological sections or hair follicle biopsies is performed

EP 2 608 762 B1 (removal of the hair shaft). Alternatively, direct observation of plasmonic nanoparticles using dark field microscopy after preparation of histological sections or hair follicle biopsies can be performed. To assess the concentration of nanoparticles at various depths along the hair follicle, the cut-out skin samples are separated by tape-stripping or heat-based techniques, the samples are dissolved for analysis in full volume of metal concentration by ICP-MS (inductively coupled plasma mass spectrometry) ). The macroscopic degree of warming is assessed using infrared thermography of skin samples and evaluation of skin sections exposed to laser radiation for thermal damage markers. Finally, the effectiveness of photothermal damage at the site of nanoparticle accumulation can be measured by analyzing the histological changes of cells at the target site, including the hair stem in the follicle, the inner hair sheath, the outer hair sheath and the bulging region containing the niche of the stem cells in which the stem cells are found participation in the growth of new hair. Since the bulging region is most often located halfway (about 50% down the length) of the hair shaft, permanent hair removal is sufficiently achieved by the accumulation of plasmonic nanoparticles to this depth. In some situations, the delivery of nanoparticles can also cause a heat gradient to be emitted further down the hair shaft. Animal studies are useful to demonstrate the effectiveness of non-dye hair removal by comparing thermal profiles, thermoablation of the hair shaft and thermal damage to bulging stem cells in treated hairless rodents, albino rodents and dark-colored rodents. Efficacy on live human skin is measured by measuring the number of hairs after observation after 3 and 12 months. Selected patients receive a biopsy at observation period 2, 4 and 6 to determine if nanoparticles have been removed from the skin without being deposited in the dermis.

[0109] Penetration of fluorescently labeled nanoparticles into bellows

In the case of hair follicles determined using pig skin explants and confocal imaging. A 25 mg / ml aqueous solution of silicon dioxide-coated nanoparticles (200 nm diameter) was contacted with freshly thawed pig skin, after which excess nanoparticle suspension was removed and a 3-minute manual massage was applied. The explant was cut into sections and subjected to confocal imaging. As shown in Figure 3A, sections of the explant were imaged at an angle to the hair follicles in 60 μm planes; Plane 1 represents the bellows funnel, while Plane 2 represents the further regions of the hair follicle. Figure 3B shows representative confocal images, which show that red nanoparticles (absorbance 548 nm) are visible in both surface and deep hair follicles, but are not detectable in the dermis layers below the follicles. Figure 3C shows high magnification imaging of red nanoparticles located and retained in deeply embedded hair follicles (approximately 400 μm). Green indicates tissue autofluorescence (488 nm).

[0110] Penetration of plasmonic nanoparticles into hair follicles determined using pig skin and dark field imaging. A suspension of plasmonic nanoparticles with an optical density of 100 OD (diameter 200 nm) was contacted with freshly thawed pig skin, after which excess nanoparticle suspension was removed and a 3-minute manual massage was applied. The procedure was repeated 3 times and the residue on the surface was removed with several (3-5) alternating applications of water and ethanol. The skin was cut, fixed, cut into sections along a horizontal plane and subjected to dark field imaging. As shown in Figure 4A, skin samples were cut into sections and imaged horizontally to the hair follicle at various depths. In skin images, plasmonic nanoparticles were visible as light blue spots at depths up to 1.2 mm in the spaces of the porcine hair follicles (Figure 4B). Control samples without plasmon nanoparticles were clearly differentiated (Figure 4C). ICPMS was also carried out on skin sections to assess the concentration of nanoparticles at various depths along the hair follicle.

[0111] Penetration of nanoparticles into hair follicles in hairless, albino and dark haired rodents. White- haired Swiss Webster mice (8 weeks old) (n = 3) were anesthetized by injecting an anesthetic ketamine / xylazine solution, followed by washing and drying the skin and dorsal hair. Before administration of the preparation with a permanent marker, three 10 cm x 10 cm areas were determined on each mouse and subjected to hair removal using 1) electric shaver, 2) Nair depilation reagent or 3) applying a warm wax / resin mixture and picking with a patch. Each mouse was treated with a pipette up to 3 nanoparticle preparations in 4 replicates with a 5 µL spot size per designated area of ​​the skin (up to 12 spots per area or 36 spots per mouse). The exact location of the spots was determined with a pen before pipetting. Duplicate spots on the left side of the back were massaged into the skin for 5 minutes, while duplicate spots on the right side of the back were applied without massage. Thirty minutes after application, the mice were sacrificed by carbon dioxide asphyxiation and cervical dislocation, after which the skin was carefully dissected and scraped along spot sizes. Skin biopsies were fixed in 10% paraformaldehyde, embedded in paraffin and cut in a transverse microtome into 5 μm thick sections. Slides with sections embedded in paraffin were deparaffinized and stained with hematoxylin and eosin (H&amp;E) or left unstained and subjected to dark field microscopic observation. The use of H&amp;E staining, light microscopy and / or dark field microscopy allows the visualization of over 50 follicles per preparation, while skin sections are assessed for visible, macroscopic accumulation of nanoparticles in the hair follicle, along the hair shaft, at the presumed niche of stem cells in region bulging and at the depth of the hair bulb. For serial histological sections, a staining kit using silver and sodium thiosulfate can be used to increase the signal of plasmonic nanoparticles by precipitation of silver metal. photomicrographs

Phase contrast and dark field are made and used to record the depth of penetration into the hair follicles for each nanoparticle preparation and method of application. ICP-MS is also carried out on skin sections to assess nanoparticle concentrations at various depths along the hair follicle.

[0112] Assessment of photothermal damage at the site of nanoparticle accumulation. The treated areas of pig, human or mouse skin were irradiated with a laser wavelength corresponding to the maximum absorption wavelength of the particles (e.g. YAG 1064 nm laser for 1029 nm plasmon particles) using clinical parameters (exposure 1s, 30-50 J / cm² and pulse width 10-50 ms). To determine photothermal damage to target skin structures, such as the hair follicle and bulging stem cells, 10 days after irradiation, patients were injected with lidocaine to anesthetize the treated areas and carefully cut the skin, which was then cut into sections along the spot size limits. Fresh human skin biopsies or explant human and animal skin samples were fixed in 10% paraformaldehyde, embedded in paraffin and cut in a transverse direction into 5 μm thick sections or fixed in Zamboni solution with 2% picric acid and cryosected on a sledge freeze microtome. Slides with paraffin sections were deparaffinized and stained with hematoxylin and eosin (H&amp;E). Histological sections were examined at various depths for markers of thermal damage and inflammation. Hematoxylin and eosin (H&amp;E) are used for imaging the skin and hair follicles microanatomy, as well as determining hair stem degeneration, sebaceous gland atrophy, and cell vacuolization (indicating cell damage). To assess keratinocyte damage, tetrazole chloride blue (NBTC) is used, a lactate dehydrogenase dye that is lost due to thermal damage to the cells. Cell damage in the hair follicles of skin samples treated with plasmonic nanoparticles and laser was evaluated and compared with samples treated only with laser. Alive, treated

Areas of human skin were also observed for a period of 2 weeks to 3 months after application of plasmonic nanoparticles and laser exposure, or during multiple treatments involving the use of plasmonic nanoparticles and laser, and then compared against the digital photo taken before the first treatment and relative to negative control, i.e. the laser treatments themselves. Clinical observations of hair removal as well as erythema, edema, discomfort, irritation or scarring determine the degree of non-specific thermal damage.

[0113] Effect of plasmonic particle coating on photothermal delivery and heating specificity. Preferably, a matrix component such as silica, polystyrene or polyethylene glycol is provided in the formulation to improve particle stability and allow easy removal from the skin surface after application and targeting of hair follicles. Acetone, ethanol or a cleansing agent can be used to remove any particles from the skin surface that have not accumulated in the bellows and to reduce or prevent heating of the skin without hair follicles. In Figure 5, live human skin was treated with uncoated plasmon particles compared to silica-coated plasmon particles before laser irradiation, and compared with controls without the application of nanoparticles (only laser). In both groups, preliminary skin preparation was used, including razor shaving and microdermabrasion on both forearms (15 seconds, medium setting) to remove bellows plugs and "open" the bellows to deliver particles. Human forearm skin was irradiated with only 810 nm (30 J / cm) laser pulses alone², 30 ms, 2 transitions) (Figure 5A) or after treatment with 830 nm resonant formulation uncoated plasmon nanoparticles in 20% propylene glycol (Figure 5B). The plasmonic nanoparticle preparation was applied together with a 3-minute massage and repeated 3 times, and the skin surface was cleaned using 3 alternating applications of water and ethanol prior to laser irradiation. Thirty minutes after laser irradiation, non-specific clinical burns were observed due to significant heating of the remaining uncoated

Particles on the surface of the skin (Figure 5B). Living human skin was also irradiated with only laser pulses at a wavelength of 1064 nm (40 J / cm², 55 ms, 3 transitions) (Figure 5C) or after treatment with 1020 nm resonant formulation with silica coated plasmonic nanoparticles in 20% propylene glycol (Figure 5D). The plasmonic nanoparticle preparation was applied together with a 3-minute massage and the procedure was repeated 3 times and the skin surface was cleaned using 3 alternating applications of water and ethanol before laser irradiation. Thirty minutes after laser irradiation, no signs of skin burns or erythema were observed, since the silica-coated particles could be sufficiently removed from the skin surface (Figure 5D). Magnified photo of the skin area treated with silica and laser coated particles shows specific photothermal damage (erythema and perimenal edema) in the place where nanoparticles were directed, with no damage to surrounding or untreated tissue particles (Figure 6). Example 4. The use of plasmonic nanoparticles for the treatment of acne.

[0114] The present application provides methods of using the compositions described herein in the treatment of acne vulgaris and other types of acne and acne-like skin conditions, simultaneously with selective targeting of sebaceous vesicles, in particular sebaceous glands and / or hair follicles. Plasmon nanoparticles produced and prepared as described above were introduced into the target tissue region, generally the skin region, and activated by laser-based systems known in the art to achieve effective hair removal.

[0115] To achieve maximum penetration depths and concentrations of plasmonic nanoparticles in the hair follicle and / or nearby components of the sebaceous gland, including the sebaceous duct, sebum, epithelial junction of the sebaceous gland and / or near the bulging region, including in stem cells , stem cell niche, combined

An optimal particle size of 100-800 nm containing one or more plasmonic nanoparticles has been developed for the epithelial region of the bulge and / or near the hair bulb. Nanoparticles encapsulating plasmon nanoparticles can be prepared from any number of polymers or matrices. In some embodiments, the formulation comprises a non-degradable polymer, e.g. synthetic poly (lactide-co-glycolide) copolymer, porous nylon copolymer of lauryl lactam and caprolactam, hydroxyethyl cellulose, polyelectrolyte monolayers or, optionally, in natural hydrogels such as hyaluronic acid, gelatin and others. In further embodiments, a PLGA hydrogel, PEG-acrylate, is included in the formulation. Preferably, a matrix component such as silica, polystyrene or polyethylene glycol is provided in the formulation to improve particle stability and allow easy removal from the skin surface after application and targeting of the bellows. Preferably the formulations contain surfactants (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1), components of the lipid bilayer, liposome or microsome. Surfactants break the epidermal skin barrier, emulsify sebum, improve the mixing of hydrophilic nanoparticles with hydrophobic solutions, and reduce entropy barriers to delivering hydrophilic particles to hydrophobic skin regions (e.g., between the hair shaft and the surrounding sheath). Plasmonic nanoparticles, including nanowires, nanoscores, nanospheres or nanoris, can be encapsulated in a polymer nanoparticle or matrix or deposited on the surface of the particle. Alternatively, nanoparticles of 100-250 nm, 250-500 nm, 800 nm-1500 nm or more than 1500 nm can be used.

[0116] The application of physical or thermal force strengthens or accelerates the penetration of light-absorbing nanoparticles and their conjugates into the hair follicles and / or sebaceous glands, in part by causing the hair follicle to expand prior to application of the nanoparticles. For example, to increase the penetration of light-absorbing nanoparticles into the hair follicles and / or sebaceous glands, sonic forces, mechanical vibrations, manipulations of the hair shaft (including pulling), force

Physical manipulation, thermal manipulation and other treatments. Treatments using the preparation of nanoparticles are performed alone, in combination, sequentially or in repetitions 1-24 times.

[0117] Prior to the application of plasmonic nanoparticles, a step of preliminary removal of excess sebum from the skin surface can be carried out using chemical and / or mechanical means. In some embodiments, pre-mechanical or chemical peeling of the skin is used to remove the hair follicle plugs and "open" the follicles to provide particles. In addition, hair can be removed by shaving or waxing to create an empty space in the hair follicle to fill with particles.

[0118] An applicator is used to uniformly apply nanoparticles to the bellows. The applicator may be a sponge, a fabric, direct contact with a finger, a tube, a syringe, a suction device, an aerosol, a spray or other means known in the art. In one example, 1 ml of a 100 OD plasmonic nanoparticle preparation with a maximum resonance of 810 nm is applied to about 200 cm² the surface of the skin of an adult human with a syringe. For uniform distribution of the solution on the skin surface and deep into the hair follicles, fabric is used. To introduce particles deep into the hair follicles, a mechanical vibrator is used for 2 minutes with or without ultrasound (1 MHz, 5 minutes). The particles penetrate to a depth of about 50% of the full length of the hair shaft at concentrations sufficient to warm the skin in a 100 μm radius at incremental temperatures 5-20 times higher than those generated in similar volumes of adjacent skin irradiated with a diode laser (810 nm). Acetone, ethanol or a cleansing agent can be used to remove any particles from the skin surface that have not accumulated in the bellows and to reduce or prevent heating of the skin without hair follicles.

[0119] Delivery of plasmonic nanoparticles to the sebaceous gland determined using human skin abdominoplasty and dark field imaging. The human sebaceous gland is located in the sebaceous unit

EP 2 608 762 B1 consisting of hair, hair follicle, pendular muscle and sebaceous gland. In Figure 7A, human skin biopsy was immunostained with antibodies directed against type IV collagen (basal membrane marker, blue) and PGP 9.5 (neural marker, green) to visualize representative microanatomy of the sebaceous hair units, including the hair follicle (HF), the sebaceous gland ( SG) and pertaining muscle. To deliver nanoparticles to the hair follicle and sebaceous gland, the skin was first pre-shaved to remove growing hair, microdermabrasion (15 seconds, medium setting) to remove hair follicle and corneocyte plugs and chemical depilation to 'open' hair follicles for delivery particles. A suspension with an optical density of 100 OD containing plasmonic nanoparticles (diameter 200 nm) prepared in 1% sodium dodecyl sulfate (SDS) and 20% propylene glycol (PG) was contacted with cut human skin after abdominoplasty, after which excess nanoparticle suspension was removed and manual massage was applied three times, followed by ultrasound ( 1 MHz, 5 minutes). The procedure was repeated three times and the skin surface was washed 3-5 times with water and ethanol alternately to remove residual particles from the surface. The skin was cut, fixed, cut into sections along a horizontal plane and subjected to dark field imaging. Dark field imaging assessment of horizontal skin sections showed that plasmon nanoparticle compositions with a cosmetically acceptable carrier 1% SDS / 20% PG used together with massage and ultrasound can be delivered to a depth of 400-600 μm to the human hair follicle and surface to the sebaceous gland (Figure 7B).

[0120] Cosmetic preparations for delivery to the hair follicles and sebaceous glands in human skin. Preferably the formulations contain surfactants (e.g. sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, sodium sulfate octech-1 / deceth-1), components of the lipid bilayer, liposome or microsome. Surfactants break the epidermal skin barrier and emulsify sebum, allowing better mixing of hydrophilic nanoparticles in hydrophobic solutions. In order to

For improving local viscosity and maintaining physiological pH, moisture retaining substances such as propylene glycol are used. In order to demonstrate the effectiveness and mechanism of sample cosmetics for delivery to human sebaceous glands, the skin was first subjected to pre-shave to remove growing hair, microdermabrasion (15 seconds, medium setting) to remove hair follicle and corneocyte plugs and chemical depilation to "open" hair follicles to deliver particles. Two separate 100 OD suspensions were prepared containing plasmonic nanoparticles (diameter 200 nm) in 1% sodium dodecyl sulfate and 20% propylene glycol (SDS / PG) or in 1% sodium lauryl sulfate and 20% propylene glycol (SLES / PG). The preparations were contacted with two separate samples of human skin from abdominoplasty and a 3-minute massage followed by ultrasound (1 MHz, 5 minutes) to introduce particles deep into the hair follicles. The procedure was repeated three times and the skin surface was washed 3-5 times with water and ethanol alternately to remove residual particles. The skin was cut, fixed, cut into sections along a horizontal plane and subjected to dark field imaging to evaluate particle delivery. Dark field imaging assessment of horizontal skin sections showed that plasmon nanoparticle compositions with a cosmetically acceptable 1% SLES / 20% carrier used together with massage and ultrasound can be delivered to a depth of 400-600 μm to the human hair follicle and surface to the sebaceous gland ( Figure 8B).

[0121] Comparison of the effects of massage and ultrasound on the delivery of nanoparticles to human hair follicles and the sebaceous gland. In order to increase the penetration of light-absorbing nanoparticles into the hair follicles and / or sebaceous glands, sonic forces, mechanical vibrations, manipulations of the hair shaft (including pulling), physical strength, thermal manipulations and other treatments are used. Mechanical massage improves the penetration of the bellows through the "pumping" mechanisms of the hair shaft, while ultrasound increases the transdermal delivery of drugs through

Temporary disruption of the lipid bilayer of the skin, formation of vesicles and formation of liquid micro-streams. To characterize the effect of ultrasound massage, the skin was pre-shaved to remove hair, microdermabrasion (15 seconds, medium setting) to remove plugs in hair follicles and corneocytes, and chemical depilation to 'open' hair follicles to deliver particles. A suspension of 100 OD containing plasmonic nanoparticles (diameter 200 nm) in 1% sodium dodecyl sulfate (SDS) and 20% propylene glycol (PG) were contacted with three separate samples of human skin from abdominoplasty. In order to introduce particles deep into the hair follicles, three treated human skin samples were treated with a 3-minute massage alone, with ultrasound alone (1 MHz, 5 minutes) or massage, followed by ultrasound. No particles were used for the fourth skin sample. The procedure was repeated three times and the skin surface was washed 3-5 times with water and ethanol alternately to remove residual particles. The skin was cut, fixed, cut into sections along a horizontal plane and subjected to dark field imaging to evaluate particle delivery. Dark field imaging of horizontal skin sections showed that plasmon nanoparticle compositions with a cosmetically acceptable 1% SLES / 20% carrier used with ultrasound provide more plasmon nanoparticles in the funnel compared to massage, although both mechanisms facilitate delivery (Figure 9).

[0122] Additional preparations of plasmonic nanoparticles for delivery to hair follicles in human skin. In certain embodiments, plasmonic nanoparticles comprise nanoscale, nanoshells, nanospheres, or nanorys, or plasmonic nanoparticles encapsulated in a polymer nanoparticle or matrix or deposited on the surface of the particle. Preferably, a matrix component, such as silica, polystyrene or polyethylene glycol, is provided in the formulation to improve particle stability and to allow easy removal from the skin surface after application and targeting of hair follicles. To demonstrate the preparation of additional shapes and concentrations

For plasmonic nanoparticles for delivery to the follicle, funnel and sebaceous gland, the skin was first pre-shaved to remove growing hair, microdermabrasion (15 seconds, medium setting) to remove plugs in hair follicles and corneocytes and chemical depilation for "Opening" the hair follicles to deliver particles. Separate suspensions with 10 OD optical density were prepared containing silica-coated nanoparticles, 30 OD optical density suspensions containing polyethylene glycol-coated plasmonic nanowires and fluorescent silica particles prepared in 1% sodium dodecyl sulfate and 20% propylene glycol. The preparations were contacted with three separate samples of human skin from abdominoplasty and a 3-minute massage followed by ultrasound (1 MHz, 5 minutes) to introduce the particles deep into the bellows. The procedure was repeated three times and the skin surface was washed 3-5 times with water and ethanol alternately to remove residual particles. The skin was cut, fixed, cut into sections along a horizontal plane and subjected to dark field imaging to evaluate particle delivery. Assessment by horizontal field imaging of horizontal skin sections showed that compositions of nanoparticles coated with polyethylene glycol (PEG) (gold, dimensions 15 x 30 nm) in a cosmetically acceptable carrier, used together with massage and ultrasound were observed in the hair follicle funnel at a depth of 200 μm (Fig. 10 A). The compositions of plasmonic nanoparticles (silica coated nanoplates) at lower concentration (10 OD) were visible at a depth of 400-600 μm in the hair follicle and sebaceous gland (empty arrow), although at a lower concentration than particles in a similar cosmetic carrier at a concentration of 100 OD ( Fig. 10B).

[0123] Assessment of photothermal damage to the sebaceous glands and target skin structures. Nanoparticle preparations were tested ex vivo using animal skin samples, ex vivo using human skin samples, and in vivo using human skin as described in Example 3. The efficiency of photothermal destruction at the site of nanoparticle accumulation can be measured by measuring thermal damage within sebocytes and

EP 2 608 762 B1 reduced sebum production in treated sebaceous glands. To measure photothermal damage, the skin was pre-shaved to remove growing hair, microdermabrasion (15 seconds, medium setting) to remove plugs in hair follicles and corneocytes, and chemical depilation to "open" hair follicles to deliver particles. The skin was contacted with resonance suspension (100 OD) at a wavelength of 810 nm plasmonic nanoparticles (diameter 200 nm) and subjected to a 3-minute massage and ultrasound (1 MHz, 5 minutes) to introduce the particles into the bellows. The procedure was repeated three times and the skin surface was washed 3-5 times with water and ethanol alternately to remove residual particles. Samples of the treated human skin were irradiated with a 810 nm wavelength laser (5 pulses 40 J / cm², 30 ms) and compared to human skin exposed only to laser. Human skin was biopsied, fixed in Zaboni's solution with 2% picric acid and cryosected on a sledge freezing microtome. Slides with sections in paraffin were deparaffinized and stained with hematoxylin and eosin (H&amp;E). Histological sections were examined at various depths for markers of thermal damage and inflammation. Hematoxylin and eosin (H&amp;E) are used to image microanatomy of the skin and hair follicles, and to determine hair stem degeneration, sebaceous gland atrophy, and cell vacuolization (indicating cell damage). To assess keratinocyte damage compared to sebocytes, tetrazole chloride blue (NBTC), a lactate dehydrogenase dye, is used which is lost due to thermal damage to the cells. Oil-Red-O intracellular dye can be used to determine the lipid and tallow oil content of the treated samples. The rate of sebum secretion is measured on the skin in vivo after 1-3 months of observation, using tallow absorbent tapes to show functional changes in sebum flow. The removal and prevention of acne lesions is measured based on the results reported by patients and the counting of acne lesions after 1-3 months of observation.

EP 2 608 762 B1

An example of a preparation containing thermoablative plasmonic nanoparticles for vascular ablation.

[0124] In this example, preparations are prepared for maximizing the stability of nanoparticles (degrees of aggregation in solution), the concentration of nanoparticles, and the absorbance of nanoparticles (the degree of laser-induced warming at various concentrations) after injection into the bloodstream. Nanoparticles are prepared as in Example 1, using a suitable solvent. The mixture containing many nanoparticles in water is concentrated to about 100-500 OD at maximum absorbance and exchange for a new solvent using liquid chromatography, solvent exchange system, centrifugation, precipitation or dialysis. Typical exchange solvent is NaCl 0.15 mol / L, phosphate buffer Na 0.1 mol / L (pH 7.2).

An example of the use of plasmonic nanoparticles for thermoablation of the vessel component (s) and microvessels.

[0125] In this example, compositions containing nanoparticles were typically administered intravascularly. After such administration of plasmonic nanoparticles, a laser adapted to the maximum plasmon resonance of the particles (e.g. 755 nm, 810 nm or 1064 nm) was used to heat the nanoparticles and surrounding tissue. Pulse widths of 10-100ns, 100ns-1ms, 1-10ms, 10-100ms, 100-1000ms or continuous wave irradiation were used to achieve thermal heat gradients and local heating around a 20-200nm particle or particle. 200nm ^ m, 2-20μm, 20200μm, 200μm -2mm. Thermal gradients of 20-200 nm were obtained from individual particles. Over one-millimeter thermal gradients are achieved by depositing the combined heat of many particles in veins with diameters of several hundred microns or more. Irradiation was used from 1 pulse to many pulses in a few seconds to minutes. To reduce pain and prevent thermal damage elsewhere, a cooling device for the epidermis layer was used. In order to achieve irradiation of vessels at specific depths from 0 to 10 mm, while avoiding heating of non-target vessels, the position of the laser was modified,

Fluence, wavelength, angle of incidence and radiation characteristics. Alternatively, a laser or light was used using a fiber optical fiber via a catheter to heat the particles in larger veins.

[0126] Typically, the tissue side is irradiated with a 2 W / cm laser beam², wavelength 810 nm, diameter 1 cm after injection of PEG coated nanowires with maximum plasmon resonance at 810 nm. Thermographic imaging is used to assess the tissue surface temperature immediately after irradiation.

[0127] Assessment of thermal damage to a component (s) of vessels, microvessels or capillaries. Thirty minutes after application, target vessels and surrounding supportive tissue (e.g. skin) were removed. The biopsies were fixed in 10% paraformaldehyde, embedded in paraffin and cut on a microtome in transverse direction into 5-μm thick sections. Slides with sections in paraffin were deparaffinized, stained with hematoxylin and eosin (H&amp;E) or stained with silver. The use of H&amp;E staining and light microscopy allows the visualization of one or more vessels, microvessels and capillaries. The visible thermal damages of the vascular structures are assessed. In addition, vascular staining (e.g. with CD21 dye) is performed to accurately identify vascular structures in tissue samples.

[0128] To the knowledge of the skilled person, the subject described in the present application may be implemented in other specific forms without departing from its nature or essential features. The above embodiments should therefore in all respects be considered illustrative rather than restrictive of the invention described herein.

EP 2 608 762 B1