Lipophilic drug delivery vehicle and methods of use thereof

Lipophilic drug delivery vehicles and methods of use

This application is about compositions and methods for delivering biologically active agents. Specifically, this application is related to bioactive agent delivery particles, which include a lipid-binding polypeptide, a lipid bilayer, and a bioactive agent.

<b>Reference before and after related applications</b>

This application is requesting the rights and interests of the U.S. Provisional Patent Application 60/447,508 filed on February 14, 2003 and 60/508,035 filed on October 1, 2003. The disclosures of the two cases are incorporated in their full text. This article is for reference.

<b>Statement on research or development sponsored by the federal government</b>

Part of the present invention was completed during the research work supported by grant case number HL65159 from the National Institutes of Health. The government may have certain rights in this invention.

Biologically active substances such as therapeutic agents, vaccine immunogens and nutrients are often not administered in pure form, but must be incorporated into biocompatible formulations, which will enhance the solubility of biologically active substances and package them in an appropriate form In order to achieve the most suitable beneficial effects, while minimizing undesirable side effects. The effective delivery of bioactive agents is often hindered by the short clearance time of the agent in the body, inefficient targeting of the site of action, or the nature of the bioactive agent itself, such as poor solubility or hydrophobicity in aqueous media. As a result, many formulation strategies have been developed to improve delivery, including controlled release formulations, emulsions, and liposome formulations.

The liposome medicine delivery system has been described. Liposomes are completely enclosed spherical lipid bilayer membranes containing trapped water volume. This lipid bilayer consists of two lipid monolayers, composed of lipids with a hydrophobic tail region and a hydrophilic head region. The structure of the film bilayer is such that the hydrophobic non-polar tail of the lipid molecule faces the center of the bilayer, while the hydrophilic head faces the water phase on both the exterior and interior of the liposome. The water-hydrophilic core region of liposomes may contain dissolved biologically active substances.

The delivery of pharmaceutically acceptable hydrophobic substances is often particularly problematic because it is insoluble or difficult to dissolve in an aqueous environment. For hydrophobic compounds used as medicines, direct injection is impossible or highly problematic, causing some dangerous conditions such as hemolysis, phlebitis, allergies, organ failure and/or death. There is a need for improved formulations for hydrophobic biologically active substances that will promote stability in aqueous environments and allow such substances to be effectively delivered to the desired site of action.

The present invention provides compositions and methods for delivering bioactive agents to individuals.

In one aspect, the present invention provides bioactive agent delivery particles comprising a lipid-binding polypeptide, an internal lipid bilayer containing a hydrophobic region, and a bioactive agent associated with the hydrophobic region of the lipid bilayer. Bioactive agent delivery particles generally do not contain a hydrophilic or aqueous core.

The bioactive agent delivery particle contains one or more bioactive agents, the package includes at least one hydrophobic region, and is incorporated into or associated with the hydrophobic interior of the lipid bilayer. The hydrophobic region of the bioactive agent is generally associated with the hydrophobic surface inside the lipid bilayer, such as a fatty acid chain. In a specific embodiment, the bioactive agent is amphotericin B (AmB). In another specific embodiment, the bioactive agent is camptothecin.

The particles are typically dish-shaped, having a diameter in the range of about 7 to about 29 nanometers.

The bioactive agent delivery particles contain lipids that can form a bilayer, such as phospholipids. In some embodiments, the bioactive agent delivery particle contains two lipids that can form a bilayer and those that cannot form a bilayer. In some embodiments, the lipid bilayer of the bioactive agent delivery particle comprises phospholipids. In a specific embodiment, the phospholipids incorporated into the delivery particles include dimyristylphospholipid choline (DMPC) and dimyristylphospholipid glycerol (DMPG). In a specific embodiment, the lipid bilayer comprises DMPC and DMPG at a molar ratio of 7:3.

In a preferred embodiment, the lipid-binding polypeptide is apolipoprotein. The main interaction between lipid-binding polypeptides, such as apolipoprotein molecules, and lipid bilayers is generally the residues on the hydrophobic surface of the amphiphilic structure such as the alpha-helical structure of the lipid-binding polypeptide, and the outer surrounding particles The hydrophobic interaction between the lipid fatty acid base chains on the surface. The particles of the invention may comprise exchangeable and/or non-exchangeable apolipoproteins. In a specific embodiment, the lipid-binding polypeptide is apolipoprotein AI.

In some embodiments, particles are provided, which comprise lipid-binding polypeptide molecules, such as apolipoprotein molecules, which have been modified to increase the stability of the particles. In a specific embodiment, the modification system includes the introduction of cysteine residues to form intramolecular and/or intermolecular disulfide bonds.

In another specific embodiment, a particle is provided, which comprises a chimeric lipid-binding polypeptide molecule, such as a chimeric apolipoprotein molecule, having one or more bound functional partial groups, such as one or more Target partial groups and/or one or more partial groups with the desired biological activity, such as antimicrobial activity, which can increase the activity of the biologically active agent incorporated into the delivery particle, or produce Synergistic.

In another aspect, a pharmaceutical composition is provided, which comprises bioactive agent delivery particles in a pharmaceutically acceptable carrier. A method for administering a biologically active agent to an individual is also provided, which comprises administering to the individual a pharmaceutical composition containing biologically active agent delivery particles in a pharmaceutically acceptable carrier. In some embodiments, the therapeutically effective amount of the biologically active agent is administered in a pharmaceutically acceptable carrier. In some embodiments, the administration system is parenteral, such as intravenous, intramuscular, transmucosal or intrathecal. In other embodiments, the particles are administered as an aerosol. In some embodiments, the bioactive agent is formulated for controlled release. In a specific embodiment, a method of treating fungal infections in individuals is provided, which comprises administering an antifungal agent, such as AmB, which has been incorporated into the bioactive agent delivery particles of the present invention, often in a therapeutically effective amount , In a pharmaceutically acceptable carrier. In another embodiment, there is provided a method of treating tumors in individuals, which includes administering an antitumor agent that has been incorporated into the bioactive agent delivery particles of the present invention, such as camptothecin, which is often used therapeutically. The effective amount is in a pharmaceutically acceptable carrier. In a specific embodiment, the bioactive agent delivery particle comprises a lipid-binding polypeptide having a group that is linked to the target portion of the vascular intestinal peptide, and the tumor is a breast tumor.

In yet another aspect, a method of formulating the above-mentioned bioactive agent delivery particles is provided. In a specific embodiment, the formulation method includes contacting a bilayer-forming lipid mixture with a biologically active agent to form a lipid vesicle-biologically active agent mixture, and bringing the lipid vesicle-biologically active agent mixture into contact with the lipid Binding polypeptide contacts. In another embodiment, the formulation method includes forming a dispersion of lipid vesicles containing a pre-formed bilayer, and adding a bioactive agent that has been dissolved in a suitable solvent. For this procedure, suitable solvents used to facilitate the dissolution of the bioactive agent include solvents with polar or hydrophilic properties, which can facilitate the dissolution of the bioactive agent to be incorporated into the delivery particles of the present invention. Examples of suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO) and dimethylformamide (DMF). In the vesicle/bioactive agent mixture, the lipid-binding polypeptide is added, followed by incubation, sonication, or both. In a specific embodiment, the biologically active agent incorporated into the delivery particles by any of the above methods is amphotericin B. In a specific embodiment, amphotericin B is dissolved in DMSO. In another specific embodiment, the bioactive agent is camptothecin. In a specific embodiment, the camptothecin system is dissolved in DMSO.

The present invention includes biologically active agent delivery particles prepared according to any of the above methods, and pharmaceutical compositions comprising particles prepared according to any of the above methods and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a kit comprising any of the above-mentioned biologically active agent delivery particles or pharmaceutical compositions, or delivery particles made by any of the above-mentioned methods, and/or reagents for formulating these particles, and/or Instructions for the method of personal administration of bioactive agents.

<b>Detailed description of the invention</b>

The present invention provides compositions and methods for delivering bioactive agents to individuals. The delivery vehicle is provided in the form of a biologically active agent incorporated into particles, which includes a lipid-binding polypeptide and a lipid bilayer. The inside of the particle contains the hydrophobic region of the lipid bilayer, which contains the hydrophobic part of the lipid molecule, such as the fatty acid base chain of the lipid. Surrounded by lipid hydrophilic surface. The hydrophobic nature of the particles of the present invention allows the incorporation of hydrophobic molecules, for example, by sandwiching between lipid molecules in the bilayer, or by sequestering into the hydrophobic region between the leaflets of the bilayer. The bioactive agent containing at least one hydrophobic region can be incorporated into the hydrophobic interior of the particle. As used herein, "incorporating" a biologically active agent into the hydrophobic region of the lipid bilayer means to solubilize in the hydrophobic region or hydrophobic portion of the lipid molecule of the bilayer, or to associate with it, for example, to form The fatty acyl chain of the lipid of the bilayer, or it is sandwiched in the fatty acyl chain.

These particles are roughly dish-shaped, and when measured by natural pore-limited gradient gel electrophoresis, they have a diameter in the range of about 7 to 29 nanometers when compared with a standard of known Stokes diameter. Blanche et al. (1981)<i>Biochim. Biophys .Acta</i>665(3):408-19. In some embodiments, the particles are stabilized in a solution, and can be lyophilized for long-term storage, and then reconstituted in an aqueous solution. The lipid-binding polypeptide component defines the boundary of the disc-shaped bilayer and provides structure and stability to the particles.

Chimeric lipid-binding polypeptide molecules (such as apolipoprotein molecules) are also provided, and can be used to incorporate various other functional properties into the delivery particles of the present invention.

The particles can be administered to individuals to deliver bioactive agents to individuals.

<b><i>Bioactive agent delivery particles</i></b>

The present invention provides "particles" (also referred to herein as "delivery particles" or "biologically active agent delivery particles"), which comprise one or more types of lipid-binding polypeptides, and one or more types of lipids that can form bilayers. Double layer, and one or more biologically active agents. In some embodiments, the delivery particles also include one or more types of lipids that cannot form a bilayer. Compositions containing these particles are also provided. In a specific embodiment, a pharmaceutical composition is provided, which includes delivery particles and a pharmaceutically acceptable carrier.

The particles contain hydrophobic regions (for example, composed of lipid fatty acid chains). The particles of the present invention typically do not contain a hydrophilic or aqueous core. The particles are roughly dish-shaped, with a flat, dish-like, or approximately round lipid bilayer, which is circumscribed by the amphiphilic α-helical structure and/or β-sheet of the lipid-binding polypeptide, which is connected to the double layer surrounding the dish Hydrophobic surface association. An illustrative example of the dish-shaped bioactive agent delivery particles of the present invention is outlined in<b>Image 6</b>middle.

Typically, the diameter of the dish-shaped transport particles is about 7 to about 29 nanometers, often about 10 to about 25 nanometers, and often about 15 to about 20 nanometers. "Diameter" refers to the diameter of one of the approximately round surfaces of the dish.

<b><i>Lipid-binding polypeptide</i></b>

As used herein, "lipid-binding polypeptide" refers to any synthetic or naturally-occurring peptide or protein, which forms a stable interaction with the lipid surface and can perform functions to stabilize the lipid bilayer of the particles of the present invention. The particles may contain one or more types of lipid-binding polypeptides, which means that the lipid-binding polypeptides in a single particle may be the same, or may be composed of two or more different polypeptide sequences. Lipid-binding polypeptides circumscribe the surrounding particles.

In some embodiments, lipid-binding polypeptides that can be used to deliver particles according to the present invention include proteins with amino acid sequences of naturally occurring proteins or fragments, natural variants, isoforms, analogs, or chimeric forms. , Proteins with non-naturally produced sequences, and lipid binding properties consistent with known apolipoproteins, and can be purified from natural sources, recombinantly produced or synthetically produced proteins or peptides of any length. Analogs of naturally occurring proteins can be used. Lipid-binding polypeptides may contain one or more amino acid analogs, such as D-amino acids, peptidomimetic structures, in which the peptide bond is replaced by a structure that is more resistant to metabolic degradation, or individual amino acids have similar structures Replacement.

In a preferred embodiment, the lipid-binding polypeptide is apolipoprotein. Any apolipoprotein or fragment or analog thereof capable of being associated with the phospholipid bilayer to form the dish-shaped bioactive agent delivery particles of the present invention can be used. The particles may contain exchangeable, non-exchangeable, or a mixture of exchangeable and non-exchangeable apolipoprotein molecules.

Apolipoproteins generally have a common structural body of type A amphiphilic α-helix (Segrest et al. (1994)<i>Adv. Protein Chem.</i> 45:303-369) and/or β-sheet body. Apolipoprotein generally contains a high content of α-helical secondary structure and has the ability to bind to hydrophobic surfaces. The characteristic feature of these proteins is their ability to interact with certain lipid bilayer vesicles and transform them into disc-shaped complexes (for review, see Narayanaswami and Ryan (2000)<i>Biochimicaet Biophysica Acta</i> 1483:15-36). When in contact with lipids, the protein system undergoes conformational changes, modifying its structure to conform to lipid interactions.

Generally speaking, the main interaction between the apolipoprotein and the lipid bilayer in the particle is through the residues on the hydrophobic surface of the amphiphilic α-helical structure of the apolipoprotein molecule and the lipid surrounding the disc-shaped bilayer. Hydrophobic interaction between acyl chains. The apolipoprotein molecules amphiphilic α-helical structure consists of a hydrophobic surface, which is in contact with the fatty acid chain of the lipid bilayer around the particle, and a hydrophilic surface. When the particle is suspended in an aqueous medium, it faces the outside of the particle and interacts with it. Exposure to aqueous environments. In some embodiments, the apolipoprotein may comprise an amphoteric β-sheet structure, in which the hydrophobic residues of the β-sheet interact with the hydrophobic surface of the lipid around the dish.

Bioactive agent delivery particles, each particle often contains about 1 to about 10 molecules of one or more types of apolipoproteins. The amount of the amphoteric α-helical structure contributed by the apolipoprotein in the particle is generally sufficient to cover the hydrophobic surface of the lipid molecules located at the edge of the disc-shaped lipid bilayer (that is, around the particle) otherwise exposed. In a specific embodiment, the apolipoprotein is human apolipoprotein AI (ApoA-I), and the lipid bilayer includes palmitoyl oleyl phospholipid choline, and one particle contains 2 ApoA-I molecules, The ratio is about 80 molecules of phospholipid to about 1 molecule of ApoA-I.

Examples of apolipoproteins that can be used to form the delivery particles of the present invention include, but are not limited to, apolipoprotein AI (ApoA-I), apolipoprotein E (ApoE) and apolipoporphyrin III (ApoIII), apolipoprotein A- IV (ApoA-IV), Apolipoprotein AV (ApoA-V), Apolipoprotein CI (ApoC-I), Apolipoprotein C-II (ApoC-II), Apolipoprotein C-III (ApoC-III) , Apolipoprotein D (ApoD), Apolipoprotein A-II (ApoA-II), Apolipoprotein B-100 (ApoB-100), Apolipoprotein J (ApoJ), Apolipoprotein H (ApoH), or Fragments, natural variants, heterogeneous recombinants, analogs or chimeric forms thereof. In some specific embodiments, the apolipoprotein is human ApoA-I. In other specific embodiments, the apolipoprotein is the C-terminal or N-terminal functional part of apolipoprotein E3 or its isoforms. In some specific embodiments, the apolipoprotein contains functional partial groups, which have been connected either synthetically or recombinantly, such as targeting partial groups or biologically active partial groups, which are not Inherent to apolipoprotein (see, for example<b>Figure 7</b>)。

In some embodiments, exchangeable apolipoproteins are used. The "exchangeable apolipoprotein" can be replaced by another protein or peptide with lipid binding affinity from the preformed dish-shaped particle of the present invention without destroying the integrity of the particle. Exchangeable apolipoproteins include synthetic or natural peptides or proteins capable of forming stable binding interactions with lipids. More than twelve unique exchangeable apolipoproteins have been identified in both vertebrates and invertebrates (see, for example, Narayanaswami and Ryan, ibid.).

In some embodiments, non-exchangeable apolipoprotein is used. As used herein, "non-exchangeable apolipoprotein" means that it will form a stable interaction with the lipid surface, and can work to stabilize the phospholipid bilayer of the particles of the present invention, but cannot be removed from the surface of the particles without being damaged The protein or peptide of the inherent structure of the particle.

<b><i>Bioactive agent</i></b>

The delivery particles contain one or more biologically active agents. As used herein, "biologically active agent" refers to any compound or composition that has biological activity, including therapeutic or diagnostic activity. The biologically active agent may be a medicament, drug, compound, or composition that can be used for medical treatment, diagnosis, or prevention.

The bioactive agent incorporated into the delivery particle as described herein generally contains at least one hydrophobic (e.g., lipophilic) region capable of associating with or integrating into the hydrophobic portion of the lipid bilayer. In some embodiments, at least a portion of the bioactive agent is sandwiched between lipid molecules inside the delivery particle. Examples of bioactive agents that can be incorporated into the delivery particles according to the present invention include, but are not limited to, antibacterial or antimicrobial (e.g., antibacterial, antifungal, and antiviral) agents, antimetabolites, antitumor agents, steroids, peptides, Proteins, such as cell receptor proteins, enzymes, endocrines and neurotransmitters, radioactive markers, such as radioisotope and radioisotope-labeled compounds, fluorescent compounds, anesthetics, bioactive lipids, anticancer agents, anti-inflammatory agents, nutrients, Antigens, pesticides, insecticides, herbicides or photosensitizers used in photodynamic therapy. In a specific embodiment, the bioactive agent is the antifungal agent Amphotericin B (AmB). In other specific embodiments, the bioactive agent is camptothecin, all-trans retinoic acid, annamycin, nystatin, paclitaxel, docetaxel or The hydroxylizarin. The bioactive agent containing at least one hydrophobic region is known in the art, and includes but not limited to ibuprofen, diazepam<img file="TW201106988A_D0001.tif" />, Griseofulvin, cyclosporine, cortisone, proleukulin, etoposide, taxane, α-tocol, vitamin E, vitamin A and lipopolysaccharide. See, for example, Kagkadis et al. (1996)<i>PDA J Pharm Sci Tech</i> 50(5):317-323; Dardel(1976)<i>Anaesth Scand</i> 20:221-24; Sweetana and Akers (1996)<i>PDA J Pharm Sci Tech</i> 50(5):330-342; US Patent 6,458,373.

In some embodiments, the bioactive agent incorporated into the delivery particles of the invention is a non-polypeptide. In some embodiments, the bioactive agent and the delivery particles containing the bioactive agent are substantially non-immunogenic when administered to the individual.

<b><i>Lipid bilayer</i></b>

The particles of the present invention comprise a lipid bilayer, a substantially circular surface with a dish-shaped object, a polar head group facing away from the inside of the particle, and the inside of the particle (that is, the space between the circular surfaces) contains a formable The hydrophobic fatty acid base chain of the lipid of the bilayer, and other lipid components, if present. The lipid molecules at the edge of the bilayer (the surface around the bioactive agent delivery particle), as discussed above, are the lipid-binding polypeptides that contact the particle. The particles may comprise one or more types of lipids that can form bilayers, or a mixture of one or more types of lipids that can form bilayers and one or more types of lipids that cannot form bilayers. As used herein, "lipid" refers to a substance of biological or synthetic origin, which is soluble or partially soluble in organic solvents, or when present in an aqueous phase, which partitions into a hydrophobic environment.

Any lipid capable of forming a bilayer that can associate with a lipid-binding polypeptide to form a disc-shaped structure can be used in accordance with the present invention. The "lipid that can form a bilayer" as used herein refers to a lipid capable of forming a lipid bilayer with a hydrophobic inner and a hydrophilic outer. Lipids that can form a bilayer include, but are not limited to, phospholipids, sphingolipids, glycolipids, alkyl phospholipids, ether lipids, and plasmalogens. One type that can form a bilayer lipid or a mixture of two or more types can be used. In some embodiments, the lipid bilayer comprises phospholipids. Examples of suitable phospholipids include, but are not limited to, dimyristyl phospholipid choline (DMPC), dimyristyl phospholipid glycerol (DMPG), palmitoyl oleyl phospholipid choline (POPC), dipalmitoyl Phospholipid choline (DPPC), dipalmitoyl phospholipid serine (DPPS), cardiolipin, dipalmitoyl phospholipid glycerol (DPPG), distearyl phospholipid glycerol (DSPG), egg yolk phospholipid Choline, soy phosphatidylcholine, phosphatidylinositol, phosphatidic acid, sphingomyelin and cationic phospholipids. Other suitable examples of lipids capable of forming bilayers include cationic lipids and glycolipids. In a specific embodiment, the particles comprise a phospholipid bilayer of DMPC and DMPG, often in a molar ratio of about 7:3. In another embodiment, the particles comprise a phospholipid bilayer of POPC. In some embodiments, a mixture of lipids that can form a bilayer can be used, at least about 1:50, 1:20, 1:10, 1:5, 1:2, 1:1 or 7:3. One of Mo Erbi.

The particles may also contain lipids that cannot form bilayer lipids. Such lipids include but are not limited to cholesterol, cardiolipin, phospholipid ethanolamine (this lipid can form a bilayer under certain conditions), oxysterols, plant sterols, ergosterol, sitosterol, cationic lipids , Cerebrosides, sphingosine, ceramide, diacylglycerol, monoacylglycerol, triacylglycerol, ganglioside, ether lipid, alkyl phospholipid, plasmalogen, prostaglandin and hemolysis Phospholipids. In some embodiments, the lipid used to prepare the delivery particles may contain one or more functional moieties, such as target moieties, biologically active agents, or for purification or detection. mark.

<b><i>Chimeric lipid-binding polypeptide</i></b>

The present invention provides chimeric lipid-binding polypeptides, which can be used to prepare the aforementioned delivery particles. The chimeric lipid-binding polypeptide may contain one or more linked "functional partial groups", such as one or more target partial groups, partial groups with the desired biological activity, and affinity for purification Mark and/or reporter molecules for feature identification or positioning research. The linked moiety group with biological activity may have an activity capable of increasing the biological activity of the biologically active agent incorporated into the delivery particle and/or synergistically with it. For example, some of the groups with biological activity may have antimicrobial (for example, antifungal, antibacterial, antiprotozoal, bacteriostatic, fungicidal, or antiviral) activity. In a specific embodiment, when the lipid-binding polypeptide is incorporated into the bioactive agent delivery particle, the linked functional portion of the chimeric lipid-binding polypeptide is not in contact with the hydrophobic surface of the lipid bilayer. In another specific embodiment, when the lipid-binding polypeptide is incorporated into the bioactive agent delivery particle, the functional moiety is connected to the hydrophobic surface of the lipid bilayer. In some embodiments, the functional part of the chimeric lipid-binding polypeptide may be inherent to the natural protein. In some embodiments, the chimeric lipid-binding polypeptide contains ligands or sequences that are recognized by cell surface receptors or capable of interacting with them.

In some embodiments, the chimeric lipid-binding polypeptide is a chimeric apolipoprotein. In a specific embodiment, the chimeric apolipoprotein contains a targeting moiety, which is not inherent to natural apolipoproteins such as yeast α-pairing factor peptide, folic acid, transferrin, or cholesferric lactate. In another embodiment, the chimeric apolipoprotein contains a partial group with the desired biological activity, which will increase the activity of the biologically active agent incorporated into the delivery particle and/or synergize it with it , Such as histatin-5, magnin peptide, bruscellin, protectin, colistin, N-terminal biliary iron lactate, echinocandin, hepcidin , Bactericin (bactenicin) or cyclosporine. In a specific embodiment, the chimeric lipid-binding polypeptide may comprise a functional part of the group inherent in apolipoprotein. An example of the intrinsic functional part of apolipoprotein is the intrinsic targeting part formed by the amino acid 130-150 of human apolipoprotein E, which contains the low-density lipoprotein receptor The region of receptor binding identified by members of the ethnic group. Other examples of the intrinsic functional part of apolipoprotein include the ApoB-100 region, which interacts with the low-density lipoprotein receptor, and the ApoA-I region, which interacts with the scavenger receptor type B1. In other specific embodiments, the functional part of the group can be added synthetically or recombinantly to produce a chimeric lipid-binding polypeptide.

As used herein, "chimeric" refers to two or more molecules that can exist individually and join together to form a single molecule with all the required functionality of its constituent molecules. The constituent molecules of the chimeric molecule can be joined synthetically by chemical conjugation, or in the case where the constituent molecules are all polypeptides or their analogs, the polynucleotides encoding the polypeptides can be fused together in a recombinant manner, resulting in A single continuous polypeptide is represented. Such chimeric molecules are called fusion proteins. A "fusion protein" is a chimeric molecule in which the constituent molecules are all polypeptides and are connected (fused) to each other, so that the chimeric molecules form a continuous single chain. The various components can be directly connected to each other or can be coupled via one or more linking agents.

As used herein, the "linking agent" or "spacer" of the chimeric molecule refers to any molecule that connects or joins the constituent molecules of the chimeric molecule. A variety of linker molecules are commercially available, for example from Pierce Chemical Company (Rockford Illinois). Suitable linking agents are known to those skilled in the art, and include but are not limited to linear or branched chain carbon linking agents, heterocyclic carbon linking agents or peptide linking agents. In the case where the chimeric molecule is a fusion protein, the linking agent may be a peptide that joins the protein containing the fusion protein. Although spacers generally do not have specific biological activities other than binding proteins or preserving certain minimum distances or other spatial relationships between them, the amino acid composition of peptide spacers can be selected to affect certain properties of the molecule, such as Folding, net charge, or hydrophobicity.

In some embodiments, chimeric lipid-binding polypeptides, such as chimeric apolipoproteins, are made by chemically conjugated lipid-binding polypeptide molecules with functional moieties to be linked. The way of chemically conjugated molecules is known to those who are familiar with this art. This method varies according to the structure of the part of the group to be connected, but it can be easily determined by a person familiar with the art.

Polypeptides typically contain a variety of functional groups, such as carboxylic acid (-COOH), free amine group (-NH₂) Or sulfhydryl (-SH) groups, which can be used to react with appropriate functional groups on the functional partial groups or on the linking agent to bind partial groups to it. The functional part of the group can be attached to the N-terminus, C-terminus of the apolipoprotein molecule or to the functional group on the internal residue (meaning the residue at the middle position between the N- and C-terminus) . Alternatively, the apolipoprotein and/or part of the groups to be labeled can be derivatized to expose or connect other reactive functional groups.

In some embodiments, the lipid-binding polypeptide fusion protein containing a functional part of the polypeptide is synthesized using a recombinant expression system. Typically, this system involves the establishment of a nucleic acid (eg DNA) sequence that encodes a lipid-binding polypeptide and a functional moiety, so that when expressed, the two polypeptides will be structurally under the control of the promoter Place the DNA, express the protein in the host cell, and isolate the expressed protein.

The sequence of the lipid-binding polypeptide and the sequence encoding the functional part of the group as described herein can be asexually propagated or amplified by in vitro methods, such as polymerase chain reaction (PCR), ligase chain reaction ( LCR), transcription-based amplification system (TAS) or self-sustaining sequential replication system (SSR). A great variety of asexual reproduction and in vitro amplification operation methods are well known to technicians. A technique sufficient to instruct the technician to go through the in vitro amplification method, an example of which can be found in Mullis et al., (1987) U.S. Patent 4,683,202;<i>Guidelines for PCR Proposal, Method and Application</i>(Innis et al.) University Publishing Company, San Diego, CA (1990) (Innis); Arnheim &amp; Levinson (October 1, 1990)<i>C&amp;EN</i> 36-47；<i>NIH Research Journal</i>(1991) 3:81-94; (Kwoh et al. (1989)<i>Proc. Natl. Acad Sci. USA</i> 86:1173; Guatelli et al. (1990)<i>Proc. Mad. Acad. Sci. USA</i> 87, 1874; Lomell et al. (1989)<i>J. Clin. Chem.,</i>35:1826; Landegren et al. (1988)<i>Science</i> 241:1077-1080; VanBrunt (1990)<i>Biotechnology,</i>8:291-294; Wu and Wallac (1989)<i>Gene,</i>4:560; and Barringer et al. (1990)<i>Gene,</i>89:117。

In addition, the DNA encoding the sequence of the fusion protein can be synthesized synthetically using methods familiar to those skilled in the art, including, for example, direct chemical synthesis, by some methods, such as Narang et al. (1979) <i>Meth. Enzymol.</i> 68: 90-99 Phosphate Triester Method, Brown et al. (1979) <i>Meth. Enzymol.</i> 68: 109-151 Phosphodiester Method, Beaucage et al. (1981) <i>Tetra. Lett.,</i>22:1859-1862 diethyl phosphamate method, or the solid support method of US Patent 4,458,066.

The nucleic acid encoding the chimeric lipid-binding polypeptide fusion polypeptide can be incorporated into a recombinant expression vector in a form suitable for expression in host cells. The "expression vector" used herein is a nucleic acid, which can be transcribed and translated into a polypeptide when introduced into a suitable host cell. This vector may also contain regulatory sequences, such as promoters, enhancers, or other performance control elements (such as polyadenylation messages). This adjustment sequence is known to those who are familiar with the art (see, for example, Goeddel (1990)<i>Gene expression technology: Meth. Enzymol.</i> 185, University Press, San Diego, CA; Berger and Kimmel,<i>Technical guidelines for molecular asexual reproduction, enzymatic methods</i>152 University Publishing Company, San Diego, CA; Sambrook et al. (1989)<i>Molecular Asexual Reproduction-Laboratory Manual (2nd Edition) Volumes 1-3</i>, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, etc.).

In some embodiments, the recombinant expression vector used to produce the chimeric lipid-binding polypeptide is a plasmid or an adhesive plastid. In other specific embodiments, the expression vector is a virus or a part thereof, which allows the expression of the nucleic acid introduced into the viral nucleic acid. For example, replication-deficient reverse transcriptase viruses, adenoviruses, and viruses associated with glands can be used. Expression vectors can be derived from phages, including all DNA and RNA phages (such as adhesive plastids), or viral vectors derived from all eukaryotic viruses, such as baculovirus and reverse transcriptase virus, adenovirus and adenovirus. Related viruses, herpes virus, vaccinia virus, and all single-stranded, double-stranded and partially double-stranded DNA viruses, all positive and negative-stranded RNA viruses, and replication-deficient reverse transcriptase viruses. Another example of an expression vector is the yeast artificial chromosome (YAC), which contains one centromere and two telomeres, allowing YAC to replicate into small linear chromosomes. Another example is bacterial artificial chromosomes (BAC).

The chimeric lipid-binding polypeptide fusion protein of the present invention can be expressed in host cells. The term "host cell" as used herein refers to any cell or cell line in which the recombinant expression vector used to produce the chimeric apolipoprotein fusion protein can be transfected for expression as described above. A host cell includes the progeny of a single host cell, and this progeny may not necessarily be identical (in morphology or in the overall genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. Host cells include cells transfected or transformed with expression vectors as described above in vivo. Suitable host cells include, but are not limited to, bacterial cells (e.g. Escherichia coli), fungal cells (e.g. Saccharomyces cerevisiae), invertebrate cells (e.g. insect cells, such as SF9 cells) and vertebrate cells, including mammalian cells.

The expression vector encoding the chimeric lipid-binding polypeptide fusion protein can be transfected into host cells using standard techniques. "Transfection" or "conversion" refers to the insertion of an exogenous polynucleotide into a host cell. The exogenous polynucleotide can be maintained as an unintegrated vector, such as a plasmid, or it can be integrated into the host cell genome. Examples of transfection techniques include, but are not limited to, calcium phosphate co-precipitation, DEAE-dextran mediated transfection, lipidation, electroporation, and microinjection. For suitable methods for transfecting host cells, see Sambrook et al. (1989)<i>Molecular asexual reproduction: a laboratory manual</i>, 2nd edition, ColdSpringHarbor Laboratory Press, and other laboratory textbooks. Nucleic acids can also be transferred into cells via a delivery mechanism suitable for introducing nucleic acids into cells in vivo, such as via retroviral vectors (see, for example, Ferry et al. (1991)<i>Proc. Natl. Acad Sci.,USA</i> 88:8377-8381; and Kay et al. (1992)<i>Human gene therapy</i>3:641-647), adenovirus vectors (see, for example, Rosenfeld (1992)<i>Cell</i> 68:143-155; with Herz and Gerard (1993)<i>Proc. Natl. Acad. Sci.,USA</i> 90:2812-2816), DNA uptake mediated by the recipient (see, for example, Wu and Wu (1988)<i>J. Biol. Chem.</i> 263:14621; Wilson et al. (1992) <i>J. Biol. Chem.</i> 267:963-967; and US Patent 5,166,320), direct injection of DNA (see, for example, Acsadi et al. (1991)<i>Nature</i> 332:815-818; and Wolff et al. (1990)<i>Science,</i>247:1465-1468) or particle impact (list of organisms) (see, for example, Cheng et al. (1993)<i>Proc. Natl. Acad. Sci.,USA</i> 90:4455-4459; and Zelenin et al. (1993)<i>FEBSLett.</i> 315:29-32)。

Once expressed, the chimeric lipid-binding polypeptide can be purified according to standard procedures in the art, including but not limited to affinity purification, ammonium sulfate precipitation, ion exchange chromatography, or gel electrophoresis.

In some embodiments, the chimeric lipid-binding polypeptide can be produced using a cell free expression system or via solid-state peptide synthesis.

<b><i>Modified lipid-binding polypeptide</i></b>

In some embodiments of the present invention, a lipid-binding polypeptide that has been modified is provided, so that when the polypeptide is incorporated into the bioactive agent delivery particle as described above, the modification will increase the stability of the particle , Or give aiming ability. In some embodiments, the modification allows the lipid-binding polypeptide of the particle to stabilize the dish-shaped structure or configuration of the particle. In a specific embodiment, the modification system includes the introduction of cysteine residues into the apolipoprotein molecule to allow the formation of intramolecular or intermolecular disulfide bonds, for example, mutations caused by position priming. In another embodiment, a chemical cross-linking agent is used to form the intermolecular link between the apolipoprotein molecules to enhance the stability of the particles. The intermolecular cross-linkage prevents or reduces the dissociation of the apolipoprotein molecule from the particle, and/or prevents the replacement of the apolipoprotein molecule in the body of the individual to which the particle is administered.

In other embodiments, the lipid-binding polypeptide is modified by either chemical derivatization of one or more amino acid residues or mutations caused by location induction to confer targeting ability to cell surface receptors or Recognized by it.

<b><i>Delivery system for delivering biologically active agents to individuals</i></b>

The present invention provides a delivery system for delivering a biologically active agent to an individual, which comprises the biologically active agent delivery particles as described above, and a carrier, optionally a pharmaceutically acceptable carrier. In some embodiments, the delivery system contains an effective amount of bioactive agent.

"Individual" as used herein refers to any prokaryotic organism or eukaryotic organism to which we want to deliver a biologically active agent. In some specific embodiments, the individual system is a prokaryote, such as bacteria. In other specific embodiments, the individual system is a eukaryote, such as fungi, plants, invertebrates (such as insects), or vertebrates. In some specific embodiments, the system is a vertebrate, such as humans, non-human primates, laboratory animals, such as mice or rats, pet animals, such as cats or dogs, or farm animals, such as horses, sheep, cows Or pigs, birds (meaning individual birds) or reptiles (meaning individual reptiles).

In some embodiments, the delivery particles are formulated in a suitable carrier for administration to an individual. As used herein, "carrier" refers to a relatively inert substance that helps the administration of the biologically active agent. For example, the carrier can impart dosage form or consistency to the composition, or can act as a diluent. "Pharmaceutically acceptable carrier" refers to a carrier that is biocompatible (meaning that it is not toxic to the host) and is suitable for a specific route of administration of a pharmacologically effective substance. Appropriate pharmaceutically acceptable carriers include, but are not limited to, stabilizers, wetting and emulsifying agents, salts with varying osmolarity, coating agents, buffers, and skin penetration enhancers. Examples of pharmaceutically acceptable carriers are described in<i>Remington's Medical Science</i>(Edited by AlfonsoR. Gennaro, 18th edition, 1990).

As used herein, "effective amount" refers to the amount of bioactive agent sufficient to achieve the desired result. "Therapeutically effective amount" or "therapeutic dose" refers to the amount of the bioactive agent sufficient to achieve favorable clinical results, such as reducing or alleviating the symptoms of diseases, reducing or reducing fungal or bacterial infections, etc.

In some embodiments, the delivery system is a pharmaceutical composition comprising bioactive agent delivery particles and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a bioactive agent delivery particle, which contains a non-polypeptide bioactive agent and a pharmaceutically acceptable carrier. In some embodiments, the bioactive agent delivery particles and bioactive agent are non-immunogens when administered to an individual. Immunogenicity can be measured by methods known in this art. For example, immunogenicity can be assessed by the ELISA method, for example, by detecting the binding of antibodies to the equivalent biologically active agent-delivering particles that have been bound to the immunosorbent plate on the serum obtained from the individual to which the biologically active agent delivery particles have been administered .

<b><i>Instructions</i></b>

The present invention provides methods for administering biologically active agents to individuals. The method of the present invention includes administering a delivery particle as described above, which comprises a lipid-binding polypeptide, a lipid bilayer and a bioactive agent, wherein the inside of the particle contains the hydrophobic surface of the lipid bilayer. Depending on the situation, a therapeutically effective amount of particles is administered, optionally in a pharmaceutically acceptable carrier. Generally speaking, these particles are dish-shaped and have a diameter of about 7 to about 29 nanometers when measured by natural pore-restricted gradient liquid gel electrophoresis. Typically, the bioactive agent contains at least one hydrophobic region, which can be integrated into the hydrophobic region of the lipid bilayer.

The route of administration may vary according to the nature of the bioactive agent to be administered, the individual or the symptoms to be treated. In the case where the individual is a mammal, the administration is generally parenteral. The route of administration includes, but is not limited to, intravenous, intramuscular, subcutaneous, transmucosal, nasal, intrathecal, topical, and transdermal. In a specific embodiment, the particles are administered as an aerosol. The delivery particles can be formulated in a pharmaceutically acceptable form for administration to an individual, as appropriate, in a pharmaceutically acceptable carrier or excipient. The present invention provides a pharmaceutical composition in the form of delivering particles in a solution for parenteral administration. To prepare such a composition, methods known in this art can be used, and any pharmaceutically acceptable carriers, diluents, excipients or other additives normally used in this art can be used.

The delivery particles of the present invention can be made into a pharmaceutical composition by using a suitable pharmaceutical carrier or diluent in combination. For example, the delivery particles can be accelerated to dissolve in solvents commonly used in the preparation of injectable solutions, such as physiological saline, water, or aqueous dextrose. Other suitable pharmaceutical carriers and their formulations are described above<i>Remington's Medical Science</i>middle. This formula can be made up in a sterile vial, which contains delivery particles and optional excipients as appropriate, in the form of dry powder or lyophilized powder. Before use, a physiologically acceptable diluent is added, and the solution is drawn through a syringe for administration to the individual.

The transport particles can also be formulated for controlled release. As used herein, "controlled release" refers to the release of the bioactive agent from the formulation at a rate such that the blood concentration of the agent in the individual is maintained within the therapeutic range over a long period of time, covering a period of several hours, A spectrum of days, weeks or more. The transport particles can be formulated in a controlled bioerodible or non-erodible substrate, many of which are known in the art. The controlled release matrix may include synthetic polymers or copolymers, for example in the form of a hydrogel. Examples of such polymers include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphonates), polyamides, polyurethanes, poly(iminocarbonates) and poly (Phosphazene) and polylactide-co-glycolide (PLGA), a copolymer of poly(lactic acid) and poly(glycolic acid). Substances containing collagen, albumin and fibrinogen can also be used.

The delivery particles can be administered according to the methods described herein to treat a variety of symptoms, including but not limited to bacterial infections, fungal infections, disease states, metabolic disorders, or as preventive drugs, such as preventing bacterial or fungal infections (for example, before or after surgery) Way). Delivery particles can be used, for example, to deliver anti-tumor agents (e.g. chemotherapeutic agents, radionuclides) to tumors. In a specific embodiment, the lipid-binding polypeptide contains a partial group that allows the particle to target a specific tumor. Transport particles can also be used to administer nutrients, which means food or dietary supplements that provide health benefits. In some embodiments, the delivery particles are co-administered with other conventional therapeutic agents, for example as part of a multi-drug "medicine solution", or combined with one or more oral agents, for example for the treatment of fungal infections. The transport particles can also be administered as insecticides or herbicides.

In one aspect, the present invention provides a method of treating fungal infections in an individual. This method includes administering to the individual a therapeutically effective amount of an antifungal agent, in a pharmaceutically acceptable carrier, wherein the antifungal agent is incorporated into a particle comprising a lipid-binding polypeptide and a lipid bilayer, wherein the lipid The inside of the double layer is hydrophobic. In a specific embodiment, the antifungal agent is AmB, which is incorporated into the hydrophobic interior of the lipid bilayer. In some embodiments, the lipid-binding polypeptide is a chimeric protein containing a target moiety and/or a biologically active moiety. In a specific embodiment, the lipid-binding polypeptide comprises a target moiety group yeast α-pairing factor peptide. In another embodiment, the lipid-binding polypeptide comprises the antimicrobial peptide histatin 5.

In another aspect, the present invention provides a method of treating tumors in an individual. This method includes administering a therapeutically effective amount of a chemotherapeutic agent, in a bioactive agent delivery particle as described above, in a pharmaceutically acceptable carrier. In a specific embodiment, the chemotherapeutic agent is camptothecin. The lipid-binding polypeptide component of the delivery particle may contain a targeting moiety so that the particle can target tumor cells. In a specific embodiment, the vascular intestinal peptide (VIP) is linked to the lipid-binding polypeptide. Since breast cancer cells often overexpress this VIP receptor, in a specific embodiment, a bioactive agent delivery particle comprising a camptothecin and a lipid-binding polypeptide-VIP chimera is used in a method of treating breast cancer.

<b><i>Aim</i></b>

The delivery particles of the present invention may include targeting functional groups, for example, to target the particles to a specific cell or tissue type, or to the infectious agent itself. In some embodiments, the particles include a targeting moiety group that is linked to a lipid-binding polypeptide or lipid component. In some embodiments, the bioactive agent incorporated into the particle has the ability to target.

In some embodiments, by designing receptor recognition properties in lipid-binding polypeptides such as apolipoprotein molecules, particles can be targeted to specific cell surface receptors. For example, bioactive agent delivery particles can be targeted to specific cell types that are known to conceal specific types of infectious agents, for example, by modifying the lipid-binding polypeptide component of the particles so that they can interact with receptors on the surface of the targeted cell type effect.

On the one hand, the targeting strategy mediated by the recipient can be used to deliver anti-Leishmania agents to macrophages, which are the main sites for protozoan parasite infections from the genus Leishmania. Examples of such species include Leishmania major, Leishmania dunovanni, and Leishmania brasiliensis. The bioactive agent delivery particles containing anti-leishmania drugs can be targeted at macrophages by changing the lipid-binding peptide composition of the particles to give receptors to the intracellular inclusions of the macrophages. (SR-A) Identification. For example, apolipoproteins that have been chemically or genetically modified to interact with SR-A can be incorporated into delivery particles that contain one or more biological activities that are effective against Leishmania species Agents such as AmB, a pentavalent antimony, and/or hexadecylphosphatidylcholine. Targets containing delivery particles of anti-Leishmania agents specific to macrophages can be used as a way to inhibit the growth and proliferation of Leishmania.

In a specific embodiment, SR-A containing AmB-containing bioactive agent delivery particles are administered to individuals who need to treat Leishmania infections. In another embodiment, another anti-leishmania agent, such as hexadecylphosphatidylcholine, is administered before, at the same time or after the treatment with particles containing amphotericin B.

In some embodiments, the targeting is achieved by modifying the lipid-binding polypeptide (such as apolipoprotein) to be incorporated into the bioactive agent delivery particle, and then imparting SR-A binding ability to the particle. In some embodiments, the target is achieved by changing the charge density of the lipid-binding polypeptide by chemically modifying one or more lysine residues, such as using malondialdehyde, Maleic anhydride or acetic anhydride (see, for example, Goldstein et al. (1979)<i>Proc. Nati. Acad. Sci.</i> 98:241-260). In a specific embodiment, ApoB-100 or its truncated form, such as N-terminal 17% of ApoB-100 (residues 1-782 of apoB-17) is modified by reaction with malondialdehyde. In other embodiments, the apolipoprotein molecule, such as any of the apolipoproteins described herein, can also be modified chemically, such as by acetylation or maleation, and be incorporated. Into the bioactive agent delivery particles containing the anti-Leishmania medicament.

In other specific embodiments, the delivery particles are endowed with SR-A binding ability by causing mutations by position induction, and replacing one or more positively charged amino acids with neutral or negatively charged amino acids. Modification of lipid-binding polypeptides.

In other specific embodiments, SR-A recognition is conferred by preparing a chimeric lipid-binding polypeptide, which contains an N- or C-terminal extension, has a ligand recognized by SR-A, or has a high concentration The amino acid sequence of negatively charged residues. The negatively charged polypeptide extension will not be attracted to the lipid surface of the biologically active agent delivery particle, thus making it easier to access the ligand binding site of the receptor.

<b><i>Method for preparing bioactive agent delivery particles</i></b>

The present invention provides a method of formulating bioactive agent delivery particles. In a specific embodiment, a method is provided, which includes adding a lipid-binding polypeptide molecule to a mixture comprising a lipid that can form a bilayer and a bioactive agent molecule.

In some embodiments, the lipid-bioactive agent mixture also includes a detergent, such as sodium cholate, cholic acid, or octyl glucoside, and the method further includes removing the detergent after the lipid-binding polypeptide has been added. Typically, the cleaning agent is removed by dialysis or gel filtration. In a specific embodiment, the method includes combining a bilayer-forming lipid and a bioactive agent molecule in a solvent to form a bioactive agent mixture, and drying the mixture to remove the solvent (for example, in N₂Under air current and/or by freeze-drying), the dried mixture is brought into contact with the solution containing the detergent to form a lipid-bioactive agent-detergent mixture, the lipid-binding polypeptide molecules are added to the mixture, and then the cleaning agent is removed Agent.

In some embodiments, the particles are made using a microfluidized bed processor. This procedure uses high pressure to force the components together in the reaction chamber.

In some embodiments, the particles are made by sonicating a suspension of lipid vesicles containing a biologically active agent in the presence of a lipid-binding polypeptide such as apolipoprotein.

In other embodiments, the delivery particles are made from a pre-formed vesicle dispersion. Lipids, such as phospholipids, are hydrated with a buffer and dispersed by stirring or vibration. In the dispersion of the lipid bilayer vesicles, the solubilized bioactive agent in a suitable solvent is added to form a lipid-bioactive agent complex. In some embodiments, the solvent is volatile or dialysis to facilitate removal after adding the bioactive agent to the lipid bilayer vesicle dispersion. After further stirring, the lipid-binding polypeptide is added, and the sample is incubated and/or sonicated. Typically, vesicles and apolipoproteins are cultured at or near the specific bilayer-forming lipids or bilayer-forming lipid mixtures used to gel to the liquid crystal phase transition temperature. This phase transition temperature can be measured by the calorimeter method.

It is preferable to use an appropriate lipid composition that can form a bilayer, so that when dispersed in an aqueous medium, the lipid vesicles provide an appropriate environment to transfer the biologically active agent from the carrier solvent to the aqueous environment without causing biological activity Agent precipitation or phase separation. The pre-formed lipid bilayer vesicles are also preferably capable of undergoing the transformation caused by the lipid-binding polypeptide to form the delivery particles of the present invention. Furthermore, the lipid-bioactive agent complex preferably retains the properties of a lipid vesicle, which allows it to be transformed by adding a lipid-binding polypeptide to form bioactive agent delivery particles. The lipid substrate-bioactive agent complex body forms a unique combination of the properties of the lipid-binding polypeptide, which are combined to produce a system, and where appropriate conditions are pH, ionic strength, temperature and lipid-bioactive agent-lipid-binding polypeptide concentration , Is the ternary structural reorganization of these substances, in which the stabilized lipid-binding polypeptide circumscribed by the lipid bilayer is produced as a biologically active agent incorporated into the lipid environment of the bilayer.

The particles produced by any of the above methods can be further purified, such as by dialysis, density gradient centrifugation and/or gel permeation chromatography.

In the preparation method for the formation of bioactive agent delivery particles, it is preferred that at least about 70, more preferably at least about 80, still more preferably at least about 90, and still more preferably at least about 95 percent are used in this process. The bioactive agent is incorporated into the particles.

The present invention provides bioactive agent delivery particles made by any of the above methods. In a specific embodiment, the present invention provides a pharmaceutical composition comprising delivery particles made by any of the above methods and a pharmaceutically acceptable carrier.

<b><i>Storage and stability</i></b>

The particles of the present invention are stable for a long period of time under a variety of conditions (see, for example,<b>Figure 5</b>). The particles or the composition containing the particles of the present invention can be stored at room temperature, frozen (e.g., about 4°C), or frozen (e.g., about -20°C to about -80°C). It can be stored in a solution or dried (e.g., lyophilized). The lyophilized particles can be stored in an inert atmosphere, frozen or in solution at 4°C. The particles can be stored in a liquid medium, such as a buffer (such as a phosphate or other suitable buffer), or in a carrier, such as a pharmaceutically acceptable carrier, for use in the administration method of the biologically active agent to an individual middle. Alternatively, the particles can be stored in a dry, lyophilized form and then reconstituted in a liquid medium before use.

<b><i>Kit</i></b>

The reagents and particles described herein can be packaged in kit form. In one aspect, the present invention provides a kit containing delivery particles and/or reagents that can be used to prepare delivery particles, in suitable packaging. The kits of the present invention individually or collectively contain any of the following substances: lipid-binding polypeptides (such as apolipoproteins), phospholipids, bioactive agents, carriers, reagents, enzymes, host cells and/or growth media for vegetative propagation and / Or express recombinant lipid-binding polypeptide (for example, recombinant apolipoprotein) and/or lipid-binding polypeptide chimera (for example, apolipoprotein chimera), and reagents and/or pharmaceutically acceptable carriers for formulating and delivering particles For voting to individuals.

Each reagent or formulation is supplied in a solid form, a liquid buffer, or a pharmaceutically acceptable carrier, which is suitable for stock or as appropriate for exchange or addition to reactants, cultures or injectable media. Provide proper packaging. As used herein, "package" refers to a system that is used to contain one or more reagents or components (such as delivering particles) in a fixed range for delivery of biologically active agents or one or more for preparation or The solid matrix or material in the method of formulating reagents for delivering particles (such as apolipoprotein molecules, phospholipids, and bioactive agents). Such materials include but are not limited to glass and plastic (such as polyethylene, polypropylene and polycarbonate) bottles, small glass bottles, paper, plastic and plastic foil laminated envelopes, etc.

The kit can optionally provide other components that can be used in the method and the preparation procedure of the present invention, such as buffers, reaction surfaces, or settings for purifying and transporting particles.

In addition, the kit includes markings and/or instructions or explanatory materials as appropriate to provide instructions (meaning mimics) for implementing the method of the present invention, such as the preparation, blending, and/or use of the delivered particles. Although the manual materials typically include written or printed materials, they are not limited to these formats. Any medium capable of storing such instructions and transmitting them to users is intended to be covered by the present invention. Such media include, but are not limited to, electronic storage media (such as magnetic disks, video tapes, cassettes, chips), optical media (such as CDROM), and the like. Such media may include searching for the URL of the Internet location that provides such manual information.

The following examples are intended to illustrate rather than limit the invention.

<b>Instance</b>

<b>Example 1. Preparation and Characterization of ApoA-I-Phospholipid-Amphotericin B Particles</b>

<b><i>Preparation of Recombinant ApoA-I</i></b>

Reorganized Apo-AI line according to Ryan et al. (2003)<i>Protein expression and purification</i>It was prepared as described in 27:98-103 and was used to prepare ApoA-I-phospholipid-AmB particles as described below.

<b><i>Preparation of ApoA-I-phospholipid-AmB particles</i></b>

ApoA-I-phospholipid-AmB particles are made as follows:

The 7:3 molar ratio of two myristyl phospholipid choline (DMPC) and dimyristyl phospholipid glycerol (DMPG) were dissolved in chloroform:methanol (3:1, v/v). To 10 mg of DMPC/DMPG mixture, 0.25 ml of AmB (2 mg/ml; dissolved in acidified chloroform-methanol (3:1, v/v)) was added. Make the mixture in N₂Dry under air flow to produce a film on the container wall. Then, the dried sample was subjected to freeze-drying for 16 hours to remove traces of solvent.

The dried lipid mixture was resuspended in 0.5 ml of Tris-saline buffer (10 mM Tris base, 150 mM NaCl, pH 8), and the mixture was vortexed for 30 seconds.

In this resuspended lipid mixture, 0.5 ml of 22 mM sodium cholate was added to the mixture and vortexed for 3 minutes. The mixture was incubated at 37°C and vortexed every 10 minutes for 1.25 hours, or until the mixture became transparent. To the clear solution, 2 ml of isolated recombinant ApoA-I prepared as described in Example 1 was added at a concentration of 1.5 mg/ml, and the mixture was incubated at 37° C. for another 1 hour. In order to remove the sodium cholate, the sample was exposed to 4 liters of Tris-saline and subjected to dialysis at 4°C for 72 hours, in which the dialysis buffer was changed every 24 hours.

The sample was further purified by density gradient ultracentrifugation. Adjust this solution to a density of 1.30 g/ml by adding solid KBr in 1.5 ml. The sample was transferred to a 3 ml centrifuge tube, covered with saline, and centrifuged in a Beckman L7-55 centrifuge at 275,000xg for 3 hours.

<b><i>Particle stability</i></b>

The particles made according to this procedure are stabilized in freeze-dried form for more than 3 months.

<b><i>Characterization of particles</i></b>

The ApoA-I-phospholipid particles prepared as described above but without AmB added were subjected to UV/visible light scanning and compared with the scanning of particles containing AmB.<b>figure 1</b>Shows the scan of particles that do not contain AmB. The only absorption peak found was the protein absorption peak at about 280 nm.<b>figure 2</b>Display the scan of AmB particles prepared as described above. In addition to the absorption peak at about 280 nm, many other absorption peaks were found in the 300-400 nm spectral region, confirming the presence of AmB. The free state AmB is insoluble in aqueous media, and compared to<b>figure 2</b>Those found in have different spectral properties. Madden et al. (1990)<i>fat</i><i>Quality Chemistry and Physics</i>52:189-98。

Characterization studies have found that ApoA-I, phospholipids and AmB will migrate as individual groups of discrete particles when subjected to density gradient ultracentrifugation.<b>(image 3)</b>. The complex floats to a characteristic density in the gradient fluid, which is determined by the protein/lipid ratio in the particle.

Furthermore, the gradient liquid gel electrophoresis under non-denaturing conditions revealed that the main complexes produced had a uniform size, indicating that the Stokes diameter was 8.5 nanometers<b>(Figure 4)</b>. The analysis of single particles revealed that there was no significant deviation from the initial molar ratios of AmB, phospholipids and apolipoproteins.

<b>Example 2. Bioactive agent delivery particles containing AmB resist the antifungal activity of Saccharomyces cerevisiae</b>

ApoA-I-DMPC/DMPG-AmB particles were prepared as described in Example 1 and used to determine the antifungal activity of the complex. The culture of Saccharomyces cerevisiae is grown in YPD medium in the presence of different amounts of ApoA-I-DMPC/DMPG-AmB particles (0-25 μg AmB/ml). The culture was grown at 30°C for 16 hours, and the degree of growth of the culture was monitored by spectrophotometry. like<b>Figure 8</b>As shown in the article, AmB-containing particles are extremely effective in inhibiting the growth of fungi, in a dose-dependent manner.

<b>Example 3. Long-term stability of bioactive agent delivery particles</b>

Recombinant ApoEN-terminal functional site (ApoE3NT) is based on Fisher et al. (1997) <i>Biochem Cell Biol</i> Made as described in 75:45-53. The particles containing ApoE3NT-AmB were prepared by the cholate dialysis method described in Example 1, and used to evaluate long-term stability.

<b>Figure 5</b>The natural PAGE 4-20% gradient solution slab gel showing the particles is stored in phosphate buffer at 4°C (channel 1), is stored in phosphate buffer at -20°C (channel 2) or is Frozen in phosphate buffer at -80°C, lyophilized and re-dissolved in H before analysis₂O in. The size and mobility of AmB-containing particles are not affected by freezing and thawing, or freeze-drying and re-dissolution, which means that the particles retain their integrity under these conditions. This is a very important parameter for the scaling and long-term storage of AmB particles.

<b>Example 4. Preparation of AmB-containing bioactive agent delivery particles with POPC</b>

The ApoA-I-POPC particles were prepared using the cholate dialysis method described in Example 1. The natural PAGE gradient liquid gel analysis of ApoA-I-POPC particles is shown in<b>Figure 4</b>middle. The particles without AmB are shown in channel 1, and the particles with AmB are shown in channel 2. This gel shows that AmB incorporation into the particles does not change their size. However, this gel shows that the particles containing POPC are of a different size compared to the DMPC/DMPG particles shown in channel 3.

<b>Example 5. Preparation of AmB-containing particles with a microfluidized bed processor</b>

Combine ApoA-I, AmB, egg PC (phospholipid choline), DPPG and cholesterol in a microfluidized bed sample holder, and pass through the reaction chamber of the microfluidized bed processor at 18,000 psi. The resulting solution is collected and characterized by the viewpoints of particle formation, hydrophobic substance incorporation, size, and stability. Obtained AmB-containing particles with a diameter of about 16 nanometers, which are stable to freeze-drying and reconstitution.

<b>Example 6. Using sound vibration to prepare particles containing AmB</b>

The suspension of AmB-containing phospholipid vesicles is prepared in the following manner. A 20-40 mg/ml solution of AmB in DMSO (DMSO) is equivalent to 2.5 mg of AmB and added to the pre-formed It contains 7:3 molar ratio of DMPC:DMPG in an aqueous dispersion of phospholipids. The vesicles were cultured at the phospholipid gel to the liquid phase transition temperature (about 24°C). The addition of 4 mg of apolipoprotein resulted in a decrease in the time dependence of sample turbidity, which was consistent with the formation of AmB-containing bioactive agent delivery particles. Complete sample transparency is achieved at 21-25°C with gentle bath sound vibration for 1-20 minutes, or 4-16 hours without sound vibration at 24°C. The formed particles show &gt;90% AmB incorporation efficiency, which means the percentage of AmB starting material recovered in the conveyed particles, and no material is lost during filtration, centrifugation or dialysis. Other trials have found that similar results can be achieved using AmB concentrations adjusted up to 5 mg/10 mg phospholipids. This procedure was performed equally well with any of the four apolipoproteins tested (ApoA-I, ApoE3NT, Bombyx mori ApoIII, and variant forms of human apolipoprotein AI, which contain C-terminal extensions, including antifungal peptide West Histatin 5).

The density gradient liquid ultracentrifugation containing ApoA-I or ApoE3NT particles found that a single population of particles floated to a characteristic density of 1.21 g/ml, which was consistent with the formation of lipid-protein complexes. The characterization of the fractions obtained after the ultracentrifugation of the density gradient solution revealed that the phospholipids, AmB and apolipoproteins migrated to the same position in the gradient solution, which was consistent with the formation of AmB-containing particles.

A comparison of the relative migration of ApoA-I-AmB-containing particles with known standards on natural PAGE shows that more than 90% of the particles have a Stokes diameter of approximately 8.5 nanometers. This value is similar to the particles produced in the absence of AmB, which means that the addition of this bioactive agent will not significantly change the size distribution of the particles.

As a measure of the overall stability of the particles delivered by the bioactive agent containing ApoA-I-AmB, the particles are frozen or lyophilized at -20°C. Freezing/thawing has no effect on the particle size distribution. Similarly, let the particles undergo lyophilization and redissolve in H₂In O, it does not affect the size distribution or the appearance of the sample.

These data strongly indicate that AmB, phospholipids, and apolipoproteins combine to form a homogeneous population of bioactive agent delivery particles, where AmB is completely integrated into the bilayer part of the particles. Spectrophotometric analysis of AmB-containing particles found that the characteristic combination of absorption peaks in the visible range is consistent with the dissolution-promoting effect of AmB in the bioactive agent delivery particles.

<b>Example 7. Comparison of AmB-containing particles made according to Example 6 and particles made by alternating vibration procedure</b>

Use the method described in Example 6 to incorporate the bioactive agent into the bioactive agent delivery particles and compare it to the incorporation of "new-HDL" particles, which is based on Shouten et al. (1993)<i>Molecular Pharmacology</i> 44:486-492 is prepared as follows: three milligrams of egg yolk phospholipid choline, 0.9 milligrams of cholesterol, and 1.5 milligrams of AmB dissolved in chloroform are mixed in a 20 ml vial, and the solvent is evaporated under a stream of nitrogen. Add 10 ml of sound buffer (10 mM Tris HCl, pH 8.0, 100 mMKCl, 1 mM MEDTA and 0.025% NaN) degassed and saturated with nitrogen₃), and vibrate the contents of the vial with Macrotip (14 micron average yield) under nitrogen flow. The temperature is maintained above 41°C and below 50°C. Stop the sound vibration after 60 minutes and adjust the temperature to 42°C. Continue to vibrate and add 20 mg of apolipoprotein AI dissolved in 2 ml of 4 M urea in ten equal portions over a period of 10 minutes. After all the protein was added, the sound vibration was kept at 42°C for 30 minutes.

Then, the sonic vibrating mixture was centrifuged for 3 minutes to remove large particles and insoluble materials, and the supernatant was analyzed by UV/visible spectrophotometry to evaluate the amount of amphotericin B dissolved in the product particles. The solution was found to be slightly opaque. The sample is scanned from 250 nanometers to 500 nanometers. For comparison, examine the AmB-containing particles produced by the procedure described in Example 6. The result is shown in<b>Picture 12</b>middle. The spectral region (300-500 nm) derived from AmB is very different between the two samples. Although the AmB-containing bioactive agent delivery particles produced using the proposed scheme in Example 6 have the strong characteristic absorbance maximum that shows the dissolution of AmB and the incorporation of the particles into the particles (Madden et al., as cited above) (<b>Figure 12B</b>), but the sample made by Schouten et al. did not lead to the maximum value of these characteristic spectra (<b>Figure 12A</b>). In fact, the obtained spectra are very similar to those reported by Madden et al. as previously reported on the aqueous dispersion of AmB in the absence of lipids. Therefore, using this procedure, AmB was not incorporated into the lipid particles, but the procedure described in Example 6 resulted in significant AmB incorporation.

<b>Example 8. Comparison of antifungal activity between AmB bioactive agent delivery particles and liposome AmB formulations</b>

Formula of ApoA-I-AmB particles and AmB commercially available liposomes made according to Example 6<img file="TW201106988A_D0002.tif" />Its antifungal activity is related to its inhibition of yeast<i>Saccharomyces cerevisiae</i>The ability to grow is compared.<b>Picture 10</b>The data in the data show that the ApoA-I-AmB bioactive agent delivery particle system is more than formulated into<img file="TW201106988A_D0003.tif" />The same amount of AmB more effectively inhibits<i>Saccharomyces cerevisiae</i>Grow. The ApoA-I-AmB bioactive agent delivery particle achieves 90% growth inhibition at 1 microgram/ml, but the degree of inhibition requires 25 micrograms/ml<img file="TW201106988A_D0004.tif" />。

ApoA-I-AmB particles and<img file="TW201106988A_D0005.tif" />Its antifungal activity is also used in the whole cell test of microtiter medium, targeting two species of pathogenic fungi<i>Candida albicans and Aspergillus fumigatus</i>Compare. The particles without AmB were also tested as a control group. The result is shown in<b>Table 1</b>middle.

<tables><img file="TW201106988A_D0006.tif" /></tables>

The results obtained found that the AmB-containing bioactive agent delivery particles are in comparison with the<img file="TW201106988A_D0007.tif" />At lower concentrations, it can effectively resist pathogenic fungal species. Control particles lacking AmB are not effective. Containing AmB bioactive agent delivery particles show the right<i>Candida albicans</i>ED₉₀(The concentration found under 90% growth inhibition) is at a concentration of 0.1 μg/ml, but 0.8 μg/ml is required<img file="TW201106988A_D0008.tif" />To achieve the same degree of growth inhibition. Correct<i>Aspergillus fumigatus</i>, Bioactive agent delivery particles containing AmB inhibit 90% of fungal growth at a concentration of 0.2μg/ml, but 1.6μg/ml is required<img file="TW201106988A_D0009.tif" />To achieve the same effect.

In another experiment, delivery particles containing AmB bioactive agent containing apolipoporphyrin III as a lipid-binding polypeptide, and<img file="TW201106988A_D0010.tif" />Compare its inhibition of three pathogenic fungal species<i>Candida albicans</i>、<i>Aspergillus fumigatus and new cryptococcal yeast</i><i>mother</i>The ability to grow. The data is shown in<b>Table 2</b>middle.

<tables><img file="TW201106988A_D0011.tif" /></tables>

Bioactive agent delivery particles containing AmB inhibit 90% at 0.03μg/ml<i>Candida albicans</i>Grow. by<img file="TW201106988A_D0012.tif" />Get the corresponding ED₉₀It is 0.4 μg/ml. exist<i>Aspergillus fumigatus</i>In this case, the bioactive agent delivery particles containing AmB inhibit 90% of fungal growth at 0.1 μg/ml, but a concentration of 2.5 μg/ml is required.<img file="TW201106988A_D0013.tif" />To achieve the same effect. In a similar way, the particles containing AmB are<img file="TW201106988A_D0014.tif" />Effective inhibition at five times lower AmB concentration<i>Cryptococcus neoformans</i>Grow.

All samples tested can be dissolved in the RPMI medium used in the experiment, and no precipitation or interference was found in any sample tested against any fungal species. These data indicate that the formulation of AmB in the bioactive agent delivery particles of the present invention has more effective antifungal activity than the formulation of liposomes.

<b>Example 9. Incorporation of camptothecin into bioactive agent delivery particles</b>

The bioactive agent delivery particles containing camptothecin were prepared as follows: DMPC:DMPG (total 5 mg) of 7:3 molar ratio was dispersed in a buffer (20 mM sodium phosphate, pH 7.0), and vortexed 1 minute to produce a dispersion of phospholipid bilayer vesicles. Add ten microliters of a 10 mg/ml solution of camptothecin in DMSO to the phospholipid bilayer dispersion. Then two mg of recombinant human apolipoprotein AI (0.5 ml of 4 mg/ml solution in 20 mM sodium phosphate, pH 7.0) was added, and then the sample was subjected to acoustic vibration. Then, the clarified sample was centrifuged at 13,000×g for 3 minutes, and the supernatant was recovered and stored at 4°C.

The fluorescence spectrum of camptothecin particles is shown in<b>Picture 11</b>middle.

The luminescence value is obtained on the PerkinElmerLS50B luminescence spectrometer at an excitation wavelength of 360 nanometers, using the emission monitored from 400 to 600 nanometers. The blue shift (<b>Figure 11A</b>), and camptothecin (<b>Figure 11B</b>) Comparison indicates that the drug is more hydrophobic in the environment where the delivery particles are located in the micelles.

<b>Example 10. Freeze Fracture Electron Microscopy of Delivery Particles Containing AmB Bioactive Agent</b>

The AmB bioactive agent delivery particle preparation for freeze-fracture electron microscopy was prepared as follows: DMPC:DMPG (7:3 mol ratio) AmB bioactive agent delivery particle (3 mg/ (Ml protein) sample, using sandwich technology and liquid nitrogen cooled propane for quenching. Before processing, the cryo-fixed samples were stored in liquid nitrogen for less than 2 hours. This fracture procedure was carried out in JOELJED-900 freezing etching equipment, and the exposed fracture plane was covered with Pt at an angle of 25-35 degrees for 30 seconds, and carbon was used for 35 seconds (2 kV/60-80 mA, 1 ×10^-5Trust). Replicas made in this way are made with dense fuming HNO₃Clean up for 24 hours, then repeat stirring at least 5 times with new chloroform/methanol (1:1 volume ratio). The duplicates cleaned in this way are examined on the JOEL100CX or PhilipsCM10 electron microscope.

The electron micrograph obtained from the freeze-fracture of the AmB-containing particles as described above is shown in<b>Picture 9</b>middle. Electron micrographs taken from several freeze-fractured preparations show that small protein-lipid complexes are present in high concentrations. The apparent diameter ranges from about 20 to 60 nanometers, of which the high frequency is about 40 nanometers. The apparent diameter of the particles, as observed by freeze-fracture electron microscopy, is greater than the value obtained by natural pore-limited gradient liquid gel electrophoresis. This difference may be due to the shadowing of the sample processing, or the dyeing process used to visualize the particles by electron microscopy.

The substantially spherical composite does not show concave or convex fracture surfaces (individual shadows on the front and back of the structure), which is characteristic of liposomes. Furthermore, no evidence on the structure of micelles was found.

<b>Example 11. In vivo evaluation of antifungal activity of delivery particles containing AmB bioactive agent in immunocompetent mice</b>

The in vivo antifungal activity of delivery particles containing AmB bioactive agent was evaluated as follows:

<b><i>animal</i></b>

House female BALB/c mice (20-25 grams) between six and eight weeks old and keep them under standard laboratory conditions.

<b><i>Toxicity Research</i></b>

Let the mice in groups of three receive a dose (for example, 1, 2, 5, 10 or 15 mg/kg AmB), in the bioactive agent delivery particles containing AmB, or the control particles without AmB, 10 mM sodium phosphate is buffered in saline at pH 7.4. A single dose is administered intraperitoneally in a volume of 0.1 ml. Preliminary studies have shown that the bioactive agent delivery particles are completely soluble under these conditions.

After the injection, observe any general reactions of the mouse, such as abnormal movement or posture, difficulty breathing, erect hair or inability to obtain food or drink. Observation of abnormalities or mortality starts immediately after administration, and lasts for seven days twice a day. The weight is recorded every day for the same period.

<b><i>The efficacy of delivery particles containing AmB bioactive agent in the treatment of systemic Cryptococcus</i></b>

Measure the therapeutic range of AmB particles and compare it with<img file="TW201106988A_D0015.tif" />For comparison, as follows:

The clinical isolates of Cryptococcus neoformans, which are susceptible to AmB, were cultured and made into inoculums to be⁶Conidia/ml concentration for infection. Each mouse is tied under general anesthesia and receives 1×10 in 0.05 ml of regular saline solution intracranially⁵Conidia. The antifungal agent is administered intraperitoneally in a volume of 0.1 ml daily for 5 days, starting 2 hours after infection. The dosage of AmB used is determined based on the aforementioned toxicity studies. One treatment group of mice received<img file="TW201106988A_D0016.tif" />, A treatment group received AmB-containing bioactive agent delivery particles, and a control group received no treatment.

Infected mice are monitored twice a day, and any signs of disease or death are recorded for up to 28 days. The weight is recorded every day for the same period. Dying animals that were unable to move normally or take food or drink were euthanized. Based on the results of these studies, a second set of studies was conducted to confirm and reproduce these findings. The dose of AmB used and the number of rats in the control and treatment groups can be adjusted to reflect the knowledge gained from the aforementioned experiments.

<b><i>Determination of tissue fungal load</i></b>

One day after the last day of treatment, the mice were sacrificed. Remove the kidneys and brain aseptically and weigh them. Homogenize the tissue and continuously dilute it in a regular salt solution. The homogenate was cultured on a PDA (potato dextrose agar) plate for 48 hours to determine the colony forming unit (CFU). Determine the fungal load CFU/gram of tissue.

<b><i>Statistical Analysis</i></b>

The difference between survival and average CFU in the kidney or brain is compared in an appropriate manner using statistical tests.

<b><i>Pharmacokinetic studies</i></b>

Blood, liver, kidney, lung and cerebrospinal fluid samples are intravenously injected with AmB bioactive agent delivery particles at doses of 0.8 and 2.0 mg/kg.<img file="TW201106988A_D0017.tif" />Later, at 10 minutes, 2, 8 and 24 hours, collected from infected mice. While the mouse is under general anesthesia, collect whole blood from the axillary vessels. Perform thoracotomy, and infuse the tissue sample with regular saline solution, and then surgically remove it. The tissue was homogenized with methanol containing 1-amino-4-nitronaphthalene. The supernatant of the serum and tissue homogenate is stored until analysis. The concentration of AmB in each sample was determined by high performance liquid chromatography (HPLC), as described in Granich et al. (1986)<i>Antimicrob. Agent Chemother</i> 29:584-88. In short, the serum sample (0.1 ml) was combined with 1.0 ml methanol containing 1.0 mg of internal standard 1-amino-4-nitronaphthalene per ml, and mixed by vortexing. After centrifugation, the supernatant was dried under reduced pressure, and then redissolved with 0.2 ml of methanol for injection on the HPLC column (C₁₈Reverse phase) on. The weighed wet tissue sample is homogenized with a glass homogenizer and centrifuged in 10 volumes of methanol containing 5.0 milligrams per milliliter. The mobile phase is a mixture of acetonitrile and 10 mM sodium acetate buffer (pH 4.0; 11:17 (volume/volume)) at a flow rate of 1.0 ml/min. The concentration of AmB is measured by the ratio of the peak height of AmB to the internal standard.

<b>Example 12. Targeting of bioactive agent delivery particles containing camptothecin to tumor cells</b>

Preparation of biologically active agent delivery agent particles, which have the target portion of the vascular intestinal peptide (VIP) group, which is connected to the lipid-binding polypeptide component.

The lipid-binding polypeptide component containing camptothecin particles can be produced in a recombinant form in Escherichia coli, which has been transformed by a plasmid vector that hides the coding sequence of the lipid-binding polypeptide. For example, recombinant human apolipoprotein AI (apoA-I) can be used. The E. coli cells hiding the apoA-I expression plasmid were cultured in the culture medium at 37°C. When the optical density of the culture reached 0.6 at 600 nm, apoA-I synthesis was induced by the addition of isopropylthiogalactoside (0.5 mM final concentration). After further culturing for 3 hours, the bacteria were pelletized by centrifugation and collapsed by sound vibration. The cell lysate was centrifuged at 20,000xg for 30 minutes at 4°C, and apoA-I was isolated from the supernatant.

The recombinant lipid-binding polypeptide chimeric system is made by designing apoA-I to include N-terminal and/or C-terminal peptide extensions, which correspond to 28 amino acid neuropeptides, which affect the vasogut peptide ( VIP). ApoA-I-VIP chimera can be used to produce bioactive agent delivery particles composed of phospholipid, camptothecin and apoA-I-VIP chimera.

For example, apoA-I-VIP chimera can be constructed by synthesizing complementary oligonucleotide primers corresponding to the coding sequence of the VIP sequence with terminal HindIII and XbaI positions. This oligonucleotide (~100 base pairs) is tempered to produce double-stranded DNA with the desired "sticky ends", and the next generation asexually propagates in a plasma vector containing the ApoA-I coding sequence, which has Properly placed HindIII and XbaI restriction enzyme positions. After ligation, transformation, and selection of the ortho chimera constructs, the plasmid DNA is isolated and subjected to automated dideoxy chain termination sequence analysis. After confirming that the sequence corresponds to the predicted one of the desired chimera, the production of the recombinant apoA-I-VIP chimera was performed in E. coli, as described above for the wild-type apoA-I. Then, for the purified recombinant chimera, gel electrophoresis, mass spectroscopy and its ability to produce the bioactive agent delivery particles of the present invention were evaluated in a manner similar to wild-type apoA-I as described in Example 8. .

The ApoA-I-VIP chimera-camptothecin bioactive agent particles can be used in breast cancer cell growth inhibition studies to measure the extent to which lipid particles are targeted. For example, the human breast cancer cell line MCF-7 was obtained from the U.S. culture type collection and kept at 37°C in a humidified 5% CO₂In an incubator, make a monolayer culture in denatured Eagle's medium supplemented with 10% fetal bovine serum and antibiotics penicillin and streptomycin. The isolated wild-type apoA-I or apoA-I-VIP chimera is radioiodinated, and incorporated into the camptothecin-containing bioactive agent delivery particle of the present invention, and cultured together with the cell. After the labeled camptothecin lipid particles and the cultured MCF-7 cells were cultured at 4°C, the radioactivity of the cell binding was measured. The ability of VIP competitive apoA-I-VIP chimera or bioactive agent delivery particle-associated apoA-I-VIP chimera to bind to MCF cells is determined in the competitive binding assay. The cell binding data was evaluated by Scatchard analysis. The degree of internalization of MCF-7 cells of apoA-I-VIP chimera bioactive agent delivery particles is when it is incubated with radioiodinated apoA-I-VIP chimera-containing bioactive agent delivery particles at 37°C to evaluate. After incubation and washing, the soluble radioactivity of trichloroacetic acid is measured to provide a measure of lipid-binding polypeptide degradation.

The growth inhibition and cytotoxicity studies of particles delivered with different biologically active agents are evaluated by the detection of asexual reproduction sources. The exponentially growing cells were resuspended in the culture medium, and the number of cells was measured using an electronic counter. Alternatively, MCF-7 asexual reproduction and growth of camptothecin-apoA-I-VIP chimera bioactive agent delivery particle inhibition can reduce the³⁵S-methionine absorption is based on the evaluation. The cell liquid was inoculated in three replicates in a petri dish. After incubation, the specific lipid particles were added from the stock solution to the petri dish to achieve a final concentration of 0, 0.1, 1, 5, 10, 50, 100 and 250 nM camptothecin. After a specific time interval ranging from 0 to 72 hours, the medium is removed by aspiration and new medium is added. At each drug concentration, the percentage of survival with different exposure times was measured from the ratio of the number of cone blues that excluded cells and compared with the results obtained from control particles lacking camptothecin.

<b>figure 1</b>Depict the UV/visible absorbance spectrum of ApoA-I-phospholipid particles made according to Example 1 without biologically active agent at 250-450 nm;

<b>figure 2</b>Depict the UV/visible absorbance spectrum of ApoA-I-phospholipid-AmB particles made according to Example 1 at 250-450 nm;

<b>image 3</b>Draw the graph of the fraction of ApoA-I-phospholipid-AmB particles after ultracentrifugation in density gradient solution versus protein concentration. The particles were made as described in Example 2 and adjusted to a density of 1.3 g/ml by adding KBr. The solution is in a discontinuous gradient solution and centrifuged at 275,000xg for 5 hours at 10°C. After centrifugal separation, the contents of the pipe fittings are fractionated from the top, and the protein content in each fraction is determined;

<b>Figure 4</b>It depicts the natural polyacrylamide gel electrophoresis (PAGE) analysis of apolipoprotein AI (ApoA-I)-phospholipid particles on a 4-20% acrylamide gradient slab gel. The particles are made of ApoA-I and two different lipid preparations DMPC/DMPG or palmitoyl oleyl phospholipid choline (POPC). The gel was stained with Coomassie blue. Channel 1: ApoA-IPOPC particles; Channel 2: ApoA-I-POPC-AmB particles; Channel 3: ApoA-I-DMPC/DMPG-AmB particles. The relative creep of the size standard is shown on the left;.

<b>Figure 5</b>It depicts the comparison of the effect of different storage conditions on the stability and structural integrity of ApoEN-terminal functional site (ApoE3NT)-DMPC/DMPG-AmB particles. The particles are separated by density ultracentrifugation, and then subjected to electrophoresis on a natural PAGE 4-20% gradient slab gel. The gel is stained with amide black. Channel 1: The particles are stored in a phosphate buffer at 4°C for 24 hours; Channel 2: The particles are stored in a phosphate buffer at -20°C for 24 hours; Channel 3: The particles are lyophilized and stored at Freeze at -80°C for 24 hours, then dissolve in H₂O in. The relative creep of the size standard is shown on the left;

<b>Image 6</b>It outlines the shape of the biologically active agent delivery particles (<b>Figure 6B</b>) And molecular organisms (<b>Figure 6A</b>)；

<b>Figure 7</b>This is an overview of the chimeric lipid-binding polypeptide and its incorporation into bioactive agent delivery particles. The chimeric protein may contain a targeting moiety (<b>Figure 7A</b>) Or part of the group with the desired biological activity (<b>Figure 7B</b>)。<b>Figure 7C</b>A brief description of the incorporation of the chimeric polypeptide shown in Figures 7A and 7B into the biologically active agent delivery particles;

<b>Figure 8</b>A graphical depiction of the bioactive agent containing AmB in the transport particle resistance culture<i>Saccharomyces cerevisiae</i>The antifungal activity is as described in Example 2.

<b>Picture 9</b>Is a freeze-fracture electron micrograph of the delivery particles containing AmB bioactive agent prepared as described in Example 10;

<b>Picture 10</b>Show ApoA-I-DMPC/DMPG-AmB particles and<img file="TW201106988A_D0018.tif" />inhibition<i>Saccharomyces cerevisiae</i>The comparison between the ability of growth, as described in Example 8;

<b>Picture 11</b>Shows that camptothecin (<b>Curve A</b>) And the bioactive agent containing camptothecin to deliver particles (<b>Curve B</b>) Comparison of fluorescence spectra between, as described in Example 9;

<b>Picture 12</b>Depicting AmB incorporated into lipid particles made as described in Example 7 (<b>Curve A</b>) And the bioactive agent delivery particles prepared as described in Example 6 (<b>Curve B</b>) UV/Visible spectrum comparison; and

<b>Figure 13</b>A diagram of a specific example of a process for preparing particles for the delivery of biologically active agents.

Although the foregoing invention has been described in general detail by way of illustration and examples for the purpose of providing a clear understanding, it will be obvious to those familiar with the art that certain changes and modifications can be implemented without departing from the spirit and scope of the present invention. . Therefore, this description should not be construed as limiting the scope of the present invention, which is described by the appended patent scope.

All publications, patents, and patent applications cited in this article are hereby incorporated in their full text by reference for all purposes, and are as if each individual publication, patent or patent application is shown in detail and individually. So and to the same extent as this article for reference.