WO2015127897A1 - 一种制备中空微米粒子的方法 - Google Patents

一种制备中空微米粒子的方法 Download PDF

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WO2015127897A1
WO2015127897A1 PCT/CN2015/073367 CN2015073367W WO2015127897A1 WO 2015127897 A1 WO2015127897 A1 WO 2015127897A1 CN 2015073367 W CN2015073367 W CN 2015073367W WO 2015127897 A1 WO2015127897 A1 WO 2015127897A1
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aqueous solution
hollow microspheres
preparing hollow
microspheres
hollow
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PCT/CN2015/073367
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English (en)
French (fr)
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赖瑞阳
张高愿
洪子涵
林珀丞
庄明熙
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国玺干细胞应用技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/36—After-treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/22—Lipids, fatty acids, e.g. prostaglandins, oils, fats, waxes
    • A61L2300/222—Steroids, e.g. corticosteroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/25—Peptides having up to 20 amino acids in a defined sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/28—Materials or treatment for tissue regeneration for liver reconstruction
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2207/00—Foams characterised by their intended use
    • C08J2207/10—Medical applications, e.g. biocompatible scaffolds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
    • C08J2367/04—Polyesters derived from hydroxy carboxylic acids, e.g. lactones
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the present invention relates to a hollow microsphere, and in particular to a method of preparing hollow microspheres.
  • the tissue-regenerated scaffold material is a platform that adsorbs cells to form tissue.
  • the scaffold material acts as a temporary barrier between the transplanted cells and the host cells, which is non-toxic and biocompatible.
  • the scaffold material can degrade within the individual when the cells to be transplanted have grown sufficiently at a particular location.
  • the scaffold can be made from synthetic, natural polymers or composite materials thereof.
  • the stent can be fabricated in a variety of configurations and functions.
  • the most commonly used biodegradable polymers include glycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and derivatives and polymers thereof.
  • Natural biodegradable materials include collagen, alginate, hyaluronic acid, gelatin, chitosan, and cellulose. In the manufacture of the stent, various forms of materials such as sponges, gels, cellulose or microbeads may be added.
  • ADSCs were cultured with PLGA scaffolds and supplemented with media that induced differentiation to promote stem cell differentiation.
  • ADSC is cultured on a PLGA scaffold, and the general medium cannot be promoted. Make a lot of hyperplasia. It is shown that the PLGA carrier is disadvantageous for the ADSC attachment growth.
  • ADSCs were cultured with PLGA porous microspheres, supplemented with modified heparin-dopamine (Hep) and lactoferrin (LF) to promote cell growth and differentiation. Quantitative data showed that although the microspheres were modified and modified, the amount of proliferation of the cells on the microspheres was not significant.
  • Hep heparin-dopamine
  • LF lactoferrin
  • the present invention provides a method of preparing hollow microspheres.
  • the hollow microspheres of the present invention are hollow porous, and can culture cells in a large amount and pass through the polypeptide and the target molecule to enhance the function of the carrier.
  • the present invention provides a method of producing hollow microparticles, comprising: (a) providing hollow microspheres; (b) immersing the hollow microspheres in an amine solution to produce an amino group on the surface of the hollow microspheres; (c) adding a polypeptide, an amino group grafted to the surface of the hollow microsphere; and (d) a target molecule added to link the target molecule to the amino group on the surface of the hollow microsphere.
  • the hollow microsphere material comprises polylactic acid, poly(butylene succinate), poly(butylene succinate-co-butylene adipate), poly(self) Butylene dicarboxylate-co-terephthalate), polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvinyl alcohol or mixtures thereof.
  • the step a) comprises: i) providing a biodegradable material emulsion; ii) providing a 0.5% (V/V) or more PVA aqueous solution; iii) cooling the PVA aqueous solution Homogenization; iv) slowly dropping the biodegradable material emulsion into the PVA aqueous solution for emulsification; and v) lyophilizing.
  • the concentration of the PVA aqueous solution in the step ii) is 1% (V/V).
  • the aqueous PVA solution in step iii) is cooled to 10-15 °C.
  • the PVA aqueous solution in the step iii) is homogenized at a number of revolutions of 1000 rpm or less.
  • the amine solution in the step a) comprises an aqueous solution containing hexamethylenediamine, glycine (Glycine), adipic dihydrazide (ADH), polyethylene glycol (Polyethylene glycol). , PEG) aminated aqueous solution, bisamino polyethylene glycol (NH2-PEG-NHS) or bisamino polyethylene glycol (NH2-PEG-NH2) in DMSO.
  • the polypeptide is reacted with a buffer to activate the carboxyl group prior to step (c).
  • the buffer comprises 2-(N-morpholino)ethanesulfonate (MES) buffer, 1-ethyl-(3- An aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or an aqueous solution of N-hydroxysuccinimide (NHS).
  • MES 2-(N-morpholino)ethanesulfonate
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • NHS N-hydroxysuccinimide
  • the polypeptide comprises IKVAV, RGD, YIGSR, REDV, DGEA, VGVAPG, GRGDS, LDV, RGDV, PDSGR, RYWLPR, LGTIPG, LAG, RGDS, RGDF, HHLGGALQAGDV, VTCG, SDGD, GREDVY, GRGDY, GRGDSP, VAPG, GGGGRGDSP, GGGGRGDY, FTLCFD, Poly-Lysine or MAX-1.
  • the target molecule comprises hyaluronic acid, oxidized hyaluronic acid, collagen (Colleagen), glucocorticoid, galectin or osteopontin.
  • the target molecule is 100 to 150 mg.
  • the weight ratio of the target molecule to the hollow microsphere is 1:1.5 to 1:1.
  • FIG. 1 is a flow chart of a method for preparing hollow microparticles of the present invention.
  • Figure 2 is a schematic representation of the preparation of hollow microparticles of the invention.
  • 3a-3d show the external and internal morphology of PLGA microspheres, PLGA-NH2 microspheres, PLGA-NH2-IKVAV, PLGA-NH2-IKVAV-oHA, solid PLGA-IKVAV microspheres of the present invention.
  • FIG. 1 and 2 show a method of producing hollow microparticles of the present invention. It should be noted that, in order to clearly describe the features of the present invention, FIG. 1 and FIG. 2 are only simple illustrations of embodiments of the present invention. In practical applications, the person skilled in the art may add or modify the FIGS. step.
  • the present invention provides a method for producing hollow microspheres, comprising:
  • step S101 hollow microspheres are provided.
  • the hollow microsphere is a kind of fine particles, and the material, shape and size of the particles are not particularly limited.
  • the material of the hollow microspheres should be biodegradable, and the hollow microspheres can be used to culture cells as well as carriers for cells.
  • the materials of the hollow hollow microspheres of the present invention include, but are not limited to, biostable polymers, biodegradable polymers, fullerenes, lipids, or combinations thereof.
  • Biostable refers to a polymer that does not degrade in a living organism.
  • Biodegradable means that it can be used in a part of the human body, such as by exposure to body fluids (such as blood), which can be gradually absorbed and/or eliminated by the body.
  • the hollow microspheres are made of a biodegradable polymer.
  • the biodegradable polymer includes aliphatic polyesters, aliphatic copolymerized lipids, or aliphatic and aromatic copolymerized lipids.
  • the biodegradable polymer is preferably polylactic acid (PLA), poly(butylene succinate) (PBS), poly(butylene succinate-co-butylene adipate) (PBSA) ), poly(butylene adipate-co-terephthalate) (PBAT), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone ( PCL), and polyvinyl alcohol (PVOH) or a combination thereof.
  • PLA polylactic acid
  • PBS poly(butylene succinate)
  • PBSA poly(butylene succinate-co-butylene adipate)
  • PBAT poly(butylene adipate-co-terephthalate)
  • PLGA hollow microspheres are prepared by a secondary emulsification process.
  • the PLGA was dissolved in dichloromethane to be emulsified (first emulsification) to obtain a PLGA emulsion.
  • An aqueous solution of PVA of 0.5% by volume or more is prepared, preferably 0.5 to 1% (V/V).
  • the PVA aqueous solution is cooled to 10-15 ° C, preferably 10 ° C, and the PVA aqueous solution is stirred at a rotation speed of 1000 rpm or less. The speed is preferably from 500 rpm to 1000 rpm.
  • the PVA aqueous solution is preferably from 0.5 to 1% (V/V) to reduce the pore size on the surface of the hollow microspheres.
  • controlling the PVA aqueous solution at a slightly low temperature (about 10-20 ° C) can also reduce the pore size on the surface of the hollow microspheres.
  • the stirring speed is preferably about 500 rpm. Hollow microspheres having a diameter of about 120 ⁇ m can be obtained at this rotation speed. If the rotation speed is too high, the particle size of the hollow microspheres will be too small.
  • the PLGA emulsion was slowly dropped into the stirred 1% (V/V) PVA aqueous solution as a second emulsification, and the second emulsification in the form of water droplets was easier to form a sphere, and the mixture was continuously stirred at 500 rpm.
  • the dichloromethane was volatilized.
  • the PLGA microspheres were obtained by freeze-drying for 3 days, and the PLGA microspheres were sieved to sieve PLGA microspheres having a diameter of about 100-200 ⁇ m.
  • the hollow microspheres are immersed in an amine solution to produce an amino group on the surface of the hollow microspheres (Fig. 2b).
  • the amine solution used in the present invention includes a solution of hexamethylenediamine, Glycine aqueous solution, adipic dihydrazide (ADH) aqueous solution, PEG aminated aqueous solution, NH2-PEG-NHS or NH2-PEG-NH2 in DMSO.
  • ADH adipic dihydrazide
  • PEG aminated aqueous solution NH2-PEG-NHS or NH2-PEG-NH2 in DMSO.
  • the PLGA microspheres of step S101 are immersed in 10% hexamethylenediamine in isopropanol solution, and after reacting for 3 hours, they are centrifuged to obtain microspheres. The microspheres were washed three times with secondary water for vacuum drying. An amino group (NH2) is formed on the surface of the PLGA microsphere, which is a PLGA-NH2 microsphere.
  • the polypeptide is added to graft the polypeptide to the amino group on the surface of the hollow microsphere (eg Figure 2c).
  • the polypeptide Prior to the addition of the polypeptide, the polypeptide can be placed in a buffer to activate the carboxyl group on the polypeptide.
  • Buffers described in the present invention include, but are not limited to, MES, EDC aqueous solution or NHS aqueous solution buffer.
  • the hollow microspheres are then added and the activated carboxyl groups on the polypeptide react with the amino groups to bond.
  • the pH of the buffer may be between 2 and 10, preferably between 3 and 9, more preferably between 4 and 8.
  • the concentration of the polypeptide in the reaction solution is from about 0.01% by weight to 50% by weight, preferably from 1% by weight to 40% by weight, more preferably from 10% by weight to 30% by weight.
  • polypeptide refers to a polypeptide or protein in which two or more amino acids are bonded together by peptide bonds.
  • the polypeptide can be a short chain peptide, oligopeptide or oligomer.
  • the polypeptides of the invention typically have a length of from 2 to 20 amino acids, preferably from 2 to 10 amino acids.
  • Polypeptides of the invention may include, but are not limited to, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.
  • the polypeptide comprises IKVAV, RGD, YIGSR, REDV, DGEA, VGVAPG, GRGDS, LDV, RGDV, PDSGR, RYWLPR, LGTIPG, LAG, RGDS, RGDF, HHLGGALQAGDV, VTCG, SDGD, GREDVY, GRGDY, GRGDSP, VAPG , GGGGRGDSP, GGGGRGDY, FTLCFD, Poly-Lysine or MAX-1, preferably IKVAV.
  • a MES buffer pH 5.5
  • EDC N,N-dimethylaminopropyl carbodiimide
  • NHS hydroxysuccinimide
  • IKVAV is added so that the ratio of IKVAV:EDC:NHS can be about 5:5:1, and after the reaction, PLGA-NH2-IKVAV microspheres are formed.
  • the target molecule is added to link the target molecule to the amino group on the surface of the hollow microsphere that is not bound to the polypeptide (Fig. 2d).
  • a target molecule refers to any substance (eg, a peptide, protein, nucleic acid polymer, aptamer, or small molecule compound) that specifically binds to a particular target.
  • the target can be a tissue, a cell, a cellular structure (eg, a cell), a protein, a peptide, a polysaccharide, or a nucleic acid polymer.
  • the aptamer of the present invention refers to a DNA or RNA molecule randomly obtained from affinity with a target molecule (refer to Cox and Ellington, Bioorg. Med. Chem. 9:2525-2531 (2001); Lee et. Al, Nuc. Acids Res. 32: D95-D100 (2004)).
  • the aptamer can be a nucleic acid, a protein, a small molecule organic compound, a vitamin, an inorganic compound, a cell, and all organisms.
  • the target molecule can be hyaluronic acid, allowing the hollow microspheres to specifically bind to hepatocytes.
  • the target molecule can react with the amino group by itself.
  • oxidized hyaluronic acid (oHA) is used as a target molecule, and an aldehyde group which oxidizes hyaluronic acid reacts with an amino group on a hollow microsphere, and is grafted by formation of an imine bond.
  • the oxidized hyaluronic acid (oHA) is dissolved in water and the alcohol is added slowly.
  • the PLGA-NH2-IKVAV microspheres of the step S105 are added, shaking is performed to form the hollow microspheres of the present invention.
  • the microspheres were washed twice with secondary water, and then soaked in alcohol for 10 minutes, and then vacuum dried.
  • the aldehyde group of the oxidized hyaluronic acid reacts with the amino group on the PLGA-NH2-IKVAV microsphere of the step S105, and is grafted to form the hollow microsphere of the present invention (PLGA-NH2- by the formation of an imine bond). IKVAV-oHA).
  • the hollow microsphere of the invention has a 3D (three-dimensional) hollow structure and can be used for culture and/or delivery cell. Since the 3D cell culture mode can simulate the 3D structural microenvironment in the body and provide more culture space, the culture cost can be reduced and the cell yield can be increased.
  • the polypeptides of the hollow microspheres can be recognized by specific cells to enhance the adhesion of the cells, thereby increasing the amount of cells that can be carried per unit of hollow microspheres. Therefore, the hollow microspheres of the present invention can carry more cells than conventional or commercially available cell carriers.
  • target molecules on the hollow microspheres that can be specifically bound to the desired target, for example, a specific tissue, organ, site or cell.
  • the target molecule is hyaluronic acid such that the hollow microspheres can specifically bind to hepatocytes.
  • the hollow microspheres of the present invention increase the cell loading by using a hollow structure and a polypeptide, and utilize the target molecules to specifically bind the hollow microspheres to a target position, and transplant the cells to a target position.
  • the efficiency of cell transplantation can be significantly enhanced by hollow structures, polypeptides and target molecules.
  • PLGA microspheres were prepared by secondary emulsification. 0.9 g of PLGA was dissolved in 40 ml of dichloromethane (2.25%), and 20 ml of secondary water was added thereto, followed by shaking uniformly for the first emulsification. A 1% PVA aqueous solution was prepared by taking 2.5 g of PVA and 250 ml of secondary water, and after cooling to 10 ° C, a 1% PVA aqueous solution was stirred at 500 rpm. The first emulsion product was slowly dropped into a stirred 1% PVA aqueous solution as a dropper, and stirred at 500 rpm for 2 hours to evaporate the dichloromethane and then stopped.
  • the aqueous solution was removed and washed three times with secondary water.
  • the PLGA microspheres were obtained by freeze drying for 3 days in a freeze dryer.
  • the PLGA microspheres were sieved by a sieve to sieve PLGA microspheres having a diameter of about 100 to 200 ⁇ m. Referring to Fig. 3a, the outer and inner forms of the PLGA microspheres were observed by a scanning electron microscope (SEM), and the inside was hollow and porous.
  • the PLGA microspheres were immersed in a 10% solution of 1,6-hexanediamine in isopropanol, and after reacting for 3 hours, they were centrifuged to obtain microspheres. The microspheres were washed three times with secondary water for vacuum drying. An amino group (NH2) is formed on the surface of the PLGA microspheres as a PLGA-NH2 microsphere. Referring to Figure 3b, the surface of the PLGA-NH2 microspheres is rough.
  • oxidized hyaluronic acid oHA
  • oHA oxidized hyaluronic acid
  • the concentration of the PVA aqueous solution was 0.5%, the rotational speed of the homogenizer was increased to 1000 rpm, and the secondary emulsification was not carried out in the form of water droplets.
  • the results are shown in Table 1.
  • the amount of oxidized hyaluronic acid used was increased from 150 mg to 300 mg, the amount of PLGA-NH2-IKVAV microspheres was increased from 150 mg to 300 mg, and the rotational speed at the time of the reaction was adjusted from 150 rpm to 180 rpm.
  • the results are shown in Table 2.
  • Example 1 150mg hyaluronic acid Difficult to occur microsphere cross-linking Comparative example 1 300mg hyaluronic acid Prone to microsphere cross-linking
  • Example 1 150mg microspheres Difficult to occur microsphere cross-linking
  • Comparative example 1 300mg microspheres Prone to microsphere cross-linking Example 1 150rpm oscillation speed Difficult to occur microsphere cross-linking Comparative example 1 180rpm oscillating speed Prone to microsphere cross-linking

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Abstract

一种制备中空微米粒子的方法，包括：(a)提供中空微球；(b)将所述中空微球浸泡在胺类溶液中，使中空微球的表面产生氨基；(c)加入多肽，且多肽接枝至所述中空微球表面的氨基；以及(d)加入标靶分子，且标靶分子连接至所述中空微球表面未键结的氨基。

Description

一种制备中空微米粒子的方法 技术领域
本发明涉及一种中空微球，且特别有关于中空微球的制备方法。
背景技术
目前，已有针对人工组织移植及再生医学进行各种基础研究和技术开发，例如，干细胞增生与分化的研究、细胞可接受且生物可降解支架的开发、以及各种组织工程工具的建立。以上皆为现今再生医学重要的课题，其中以开发可传送干细胞或组织细胞的支架最为重要。
组织再生的支架材料为一平台，其可吸附细胞以形成组织。支架材料可作为移植细胞与宿主细胞间的暂时性屏障，其无毒且具生物兼容性。此外，当所欲移植的细胞已在特定位置上充足地生长时，支架材料可在个体内降解。
一般来说，支架可由合成、天然聚合物或其复合材料制备。支架可制造成有各种形态和功能的结构。最常使用的生物可降解聚合物包括，乙醇酸(PGA)、聚乳酸(PLA)、聚(乳酸-共-乙醇酸)(PLGA)、聚己内酯(PCL)及其衍生物与聚合物。天然的生物可降解材料包括胶原蛋白、藻酸盐、透明质酸、明胶、脱乙酰壳多醣与纤维素等。在支架的制造中，可添加各种不同形式的材料，如海绵、凝胶、纤维素或微珠等。
Wang,M.,et al揭露以PLGA支架培养ADSC，并辅以诱导分化的培养基促使干细胞分化。ADSC于PLGA支架上培养，一般的培养基无法促 使大量增生。显示PLGA载体不利于ADSC贴附生长的缺点。
Kim SE,et al揭露以PLGA多孔性微球培养ADSC，并辅以改质肝素-多巴胺(Hep)及乳铁蛋白(LF)促使细胞生长及分化。定量数据显示，虽微球经修饰改质，但细胞于微球上之增生数量并不明显。
由上述可知，目前在组织工程技术中仍有许多困难需要克服。例如，细胞培养的空间过小、产量过低、所能携带的细胞量过少、移植成功的机会过低等。因此，业界急需一种新颖的生医材料载体及其制法。
发明内容
有鉴于上述先前技术所存在的问题，本发明提供一种制备中空微球的方法。本发明的中空微球为中空多孔状，可大量培养细胞，并透过多肽与标靶分子，以提升载体功能。
本发明提供一种制造中空微米粒子的方法，包括(a)提供中空微球；(b)将所述中空微球浸泡在胺类溶液中，使中空微球的表面产生氨基；(c)加入多肽，使多肽接枝至所述中空微球表面的氨基；以及(d)加入标靶分子，使标靶分子连接至中空微球表面的氨基。
在本发明实施例中，中空微球材质包括聚乳酸、聚(丁二酸丁二醇酯)、聚(丁二酸丁二醇酯-共-己二酸丁二醇酯)、聚(己二酸丁二醇酯-共-对苯二甲酸酯)、聚乙醇酸、聚(乳酸-共-乙醇酸)、聚己内酯、聚乙烯醇或其混合物。
在本发明实施例中，其中a)步骤包括：i)提供生物分解材质乳化液；ii)提供0.5％(V/V)以上的PVA水溶液；iii)将所述PVA水溶液冷却后 进行均质；iv)将所述生物分解材质乳化液缓慢地滴入所述PVA水溶液，进行乳化；以及v)冷冻干燥。
在本发明实施例中，所述ii)步骤中的PVA水溶液浓度为1％(V/V)。
在本发明实施例中，所述ⅲ)步骤中的PVA水溶液冷却至10-15°C。
在本发明实施例中，所述ⅲ)步骤中的PVA水溶液以1000rpm以下的转速进行均质化。
在本发明实施例中，a)步骤中所述胺类溶液包括含有己二胺、甘氨酸(Glycine)水溶液、己二酸二酰肼(adipic dihydrazide(ADH))水溶液、聚乙二醇(Polyethylene glycol，PEG)胺化水溶液、双氨基聚乙二醇(NH2-PEG-NHS)或双氨基聚乙二醇(NH2-PEG-NH2)的DMSO溶液。
在本发明实施例中，在(c)步骤前，将多肽与缓冲液反应以活化羧基。
在本发明实施例中，所述缓冲液包括2-(N-吗啉代)乙磺酸缓冲液(2-(N-morpholino)ethanesulphonic acid,MES)缓冲液、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,EDC)水溶液或N-羟基丁二酰亚胺(N-hydroxysuccinimide，NHS)水溶液。
在本发明实施例中，所述多肽包括IKVAV、RGD、YIGSR、REDV、DGEA、VGVAPG、GRGDS、LDV、RGDV、PDSGR、RYWLPR、LGTIPG、LAG、RGDS、RGDF、HHLGGALQAGDV、VTCG、SDGD、GREDVY、 GRGDY、GRGDSP、VAPG、GGGGRGDSP、GGGGRGDY、FTLCFD、Poly-Lysine或MAX-1。
在本发明实施例中，所述标靶分子包括透明质酸、氧化透明质酸、胶原蛋白(Colleagen)、糖皮质激素(Glucocorticoid)、半乳糖凝集素(Galectin)或骨桥蛋白(osteopontin)。
在本发明实施例中，所述d)步骤中，标靶分子为100至150mg。
在本发明实施例中，d)步骤中，所述标靶分子与中空微球重量比为1:1.5至1:1。
附图说明
图1为本发明中空微米粒子的制备方法流程图。
图2为制备本发明中空微米粒子的示意图。
图3a-3d为本发明PLGA微球、PLGA-NH2微球、PLGA-NH2-IKVAV、PLGA-NH2-IKVAV-oHA、实心PLGA-IKVAV微球的外部及内部形态。
具体实施方式
图1、2显示本发明中空微米粒子的制造方法。应注意的是，为了清楚描述本发明的特征，图1与图2仅为本发明实施例的简单图示，在实际应用时，此技艺人士可依不同的需求增加或修改图1、2的步骤。
在本发明第范畴中，本发明提供一种中空微球的制造方法，包括：
a)提供中空微球；
b)将所述中空微球浸泡在胺类溶液中，使所述中空微球的表面产生氨基；
c)加入多肽，使所述多肽接枝至所述中空微球表面的氨基，以及
d)加入靶标分子，使所述靶标分子连接至所述中空微球表面未键合的氨基。
参照图1，步骤S101，提供中空微球。此中空微球为一种微粒，颗粒的材料、形状和尺寸并无特别限制。中空微球(如图a所示)的材料应具有生物可分解性，且中空微球可用于培养细胞以及作为细胞的载体。
本发明中空中空微球的材质包括，但不限于，生物稳定性聚合物、生物可降解聚合物、富勒烯、脂质或它们的组合。生物稳定系指在生物体内不会降解的聚合物。生物可分解系指可使用于人体的某部位中，如暴露于体液(如血液)，可逐渐地被人体所吸收及/或消除。
中空微球的材质为生物可分解聚合物。生物可分解聚合物包括脂肪族聚脂类、脂肪族共聚脂类、或脂肪族及芳香族共聚脂类的物质。生物可分解聚合物较佳为聚乳酸(PLA)、聚(丁二酸丁二醇酯)(PBS)、聚(丁二酸丁二醇酯-共-己二酸丁二醇酯)(PBSA)、聚(己二酸丁二醇酯-共-对苯二甲酸酯)(PBAT)、聚乙醇酸(PGA)、聚(乳酸-共-乙醇酸)(PLGA)、聚己内酯(PCL)、及聚乙烯醇(PVOH)或上述组合。
在特定实施例中，以二次乳化法制备PLGA中空微球。将PLGA溶于二氯甲烷中进行乳化(第一次乳化)，获得PLGA乳化物。配制0.5体积％以上的PVA水溶液，较佳为0.5至1％(V/V)。将PVA水溶液冷却至10-15℃，较佳为10℃，并以1000rpm以下的转速搅拌PVA水溶液，转 速较佳为500rpm至1000rpm。在本发明中，PVA水溶液较佳为0.5至1％(V/V)，以降低中空微球表面的孔洞尺寸。另外，将PVA水溶液控制在略低温状态(约10-20℃)同样可降低中空微球表面的孔洞尺寸。搅拌转速较佳为约500rpm。在此转速下可获得直径为约120μm的中空微球。若转速过高会使中空微球的粒径太小。
将PLGA乳化物以滴管缓慢滴入搅拌中的1％(V/V)PVA水溶液，进行第二次乳化，以水滴形式进行第二次乳化较容易形成球状物，并持续以500rpm转速搅拌，使二氯甲烷挥发。
接着，去除水溶液，以二次水清洗3次。以冷冻干燥机，冻干3天获得PLGA微球，以筛网筛PLGA微球，筛出直径约100-200μm之PLGA微球。
参照步骤S103，将中空微球浸泡在胺类溶液中，使中空微球的表面产生氨基(如图2b)。本发明中所使用的胺类溶液包括己二胺、Glycine水溶液、adipic dihydrazide(ADH)水溶液、PEG胺化水溶液、NH2-PEG-NHS或NH2-PEG-NH2的DMSO溶液溶液。本技术领域人士自可依据所使用的胺类溶液选择适当的浸泡的时间及条件。
在特定实施例中，将步骤S101的PLGA微球浸泡于10％之1,6-己二胺的异丙醇溶液中，反应3小时后，离心以获得微球。以二次水清洗微球3次进行真空干燥。PLGA微球表面上形成氨基(NH2)，为PLGA-NH2微球。
参照步骤S105，加入多肽，使多肽接枝至中空微球表面的氨基(如 图2c)。在添加多肽前，可将多肽置于缓冲液中，以活化多肽上的羧基。本发明中所述的缓冲液包括，但不限于MES、EDC水溶液或NHS水溶液缓冲液。接着加入中空微球，多肽上活化的羧基会与氨基反应而键结。缓冲液的pH值可介于2-10之间，较佳为3-9之间，更佳为4-8之间。本技术领域人士自可依据所使用的缓冲液来调整pH值，以活化氨基。多肽在反应溶液中的浓度为约0.01wt％至50wt％，较佳为1wt％至40wt％，更为10wt％至30wt％。
本发明的“多肽”系指2或以上氨基酸以肽键结合在一起的多肽或蛋白质。多肽可为一种短链肽、寡胜肽或寡聚物。本发明多肽的长度一般介于2至20个氨基酸，较佳2至10个氨基酸。本发明多肽可包括，但不限于2、3、4、5、6、7、8或9个氨基酸。在实施例中，多肽包括IKVAV、RGD、YIGSR、REDV、DGEA、VGVAPG、GRGDS、LDV、RGDV、PDSGR、RYWLPR、LGTIPG、LAG、RGDS、RGDF、HHLGGALQAGDV、VTCG、SDGD、GREDVY、GRGDY、GRGDSP、VAPG、GGGGRGDSP、GGGGRGDY、FTLCFD、Poly-Lysine或MAX-1，较佳为IKVAV。
Figure PCTCN2015073367-appb-000001
Figure PCTCN2015073367-appb-000002
在特定实施例中，配制MES缓冲液(pH 5.5)，加入N，N-二甲基氨基丙基碳二亚胺(EDC)与羟基琥珀酰亚胺(NHS)(比例为1：1)，接着加入步骤S103的PLGA-NH2微球，进行反应以活化氨基。
加入IKVAV，使IKVAV：EDC：NHS的比例可为约5：5：1，反应后，形成PLGA-NH2-IKVAV微球。
参照步骤S107，加入标靶分子，使标靶分子连接至中空微球表面未与多肽结合的氨基(如图2d)。
标靶分子是指任何专一性结合至特定目标物的物质(例如，肽、蛋白质、核酸聚合物、适体或小分子化合物)。目标物可为组织、细胞、细胞结构(例如胞器)、蛋白质、胜肽、多醣类、或核酸聚合物。本发明所述的适体是指随机地择自于依据与目标分子亲合力所获得的DNA或RNA分子(参照Cox and Ellington,Bioorg.Med.Chem.9:2525-2531(2001)；Lee et al,Nuc.Acids Res.32:D95-D100(2004))。适体可为核酸、蛋白质、小分子有机化合物、微生素、无机化合物、细胞及所有有机体。
标靶分子可为透明质酸，使中空微球可专一性地与肝细胞结合。标靶分子可自行与氨基反应。例如，以氧化透明质酸(oHA)作为标靶分子，而氧化透明质酸的醛基会与中空微球上的氨基反应，藉由亚胺键的生成而接枝。
在特定实施例中，将氧化透明质酸(oHA)溶于水中，并缓慢地加入酒精。接着，加入步骤S105的PLGA-NH2-IKVAV微球后，进行震荡，以形成本发明中空微球。以二次水清洗微球2次，再分别以酒精浸泡10分钟后进行真空干燥。
在反应中，氧化透明质酸的醛基会与步骤S105的PLGA-NH2-IKVAV微球上的氨基反应，藉由亚胺键的生成而接枝形成本发明的中空微球(PLGA-NH2-IKVAV-oHA)。
本发明的中空微球具有3D(三维)中空结构，可用于培养及/或传送 细胞。由于3D细胞培养模式可仿真体内的3D结构微环境，以及提供更多的培养空间，因此可降低培养成本并增加细胞产量。
中空微球的多肽可被特定细胞所辨识，以提升细胞的贴附力，进而增加每单位中空微球所能携带的细胞量。所以相对于传统或市售的细胞载体，本发明中空微球所能携带的细胞量更多。
另一方面，在中空微球上更具有标靶分子，可专一性地结合至所欲的目标上，例如，特定组织、器官、部位或细胞。在实施例中，标靶分子为透明质酸，使中空微球可专一性地与肝细胞结合。
综上所述，本发明的中空微球利用中空结构与多肽增加细胞负载量，并利用标靶分子使中空微球专一性地结合至目标位置，将细胞移植至目标位置。藉由中空结构、多肽与标靶分子，可显著地提升细胞移植的效力。
实施例
1.中空微球的制备
1-1、PLGA微球的制备
以二次乳化法制备PLGA微球。取0.9g PLGA溶于40ml二氯甲烷(2.25％)中，再加入20ml二次水后均匀震荡进行第一次乳化。取2.5g的PVA与250ml的二次水配制1％PVA水溶液，冷却至10℃后，以500rpm搅拌1％PVA水溶液。将上述第一次乳化产物以滴管缓慢滴入搅拌中的1％PVA水溶液，以500rpm搅拌2小时使二氯甲烷挥发后停止。去除水溶液，以二次水清洗3次。以冷冻干燥机，冻干3天获得PLGA微球， 以筛网筛PLGA微球，筛出直径约100-200μm之PLGA微球。参照第3a图，以扫描式电子显微镜(SEM)观察PLGA微球外部及内部形态，内部呈中空多孔状。
1-2、PLGA微球的氨基改质
将PLGA微球浸泡于10％的1,6-己二胺的异丙醇溶液中，反应3小时后，离心以获得微球。以二次水清洗微球3次进行真空干燥。在PLGA微球表面上形成氨基(NH2)，为PLGA-NH2微球。参照第3b图，PLGA-NH2微球表面呈现粗糙状。
1-3、PLGA-IKVAV接枝
配制0.1M的MES缓冲液(pH 5.5)，加入N，N-二甲基氨基丙基碳二亚胺(EDC)与羟基琥珀酰亚胺(NHS)(比例为1：1)，接着加入IKVAV胜肽，反应2小时活化羧基。加入PLHA-NH2微球，使IKVAV：EDC：NHS的比例为5：5：1，反应溶液中IKVAV重量百分浓度约14％，反应24小时后，形成PLGA-NH2-IKVAV微球。参照第3c图，IKVAV覆盖于PLGA-NH2微球上，表面呈现较为平滑状态。
1-4、PLGA-oHA接枝
最后，将150mg氧化透明质酸(oHA)溶于8ml二次水，并缓慢地加入32ml的99.5％酒精。加入150mg的PLGA-NH2-IKVAV微球后，于pH11下，以150rpm震荡24小时。以二次水清洗微球2次，再分别以30％、95％酒精浸泡10分钟后进行真空干燥。氧化透明质酸的醛基与PLGA-NH2-IKVAV微球上氨基反应，藉由亚胺键的生成而接枝形成本发 明之中空微球(PLGA-NH2-IKVAV-oHA)，如第3d图所示。
2.比较例1
2-1、PLGA微球的制备
PVA水溶液的浓度为0.5％，均质机的转速提升至1000rpm，且非以水滴形式进行二次乳化。结果如表1所示。
表1、实施例1与比较例1的比较
Figure PCTCN2015073367-appb-000003
3.比较例2
3-1、PLGA-oHA接枝
氧化透明质酸的使用量由150mg提升至300mg，PLGA-NH2-IKVAV微球的使用量由150mg提升至300mg，且反应时的转速由150rpm调整至180rpm。结果如表2所示。
表2、实施例1与比较例2的比较
  方法 结果
实施例1 150mg的透明质酸 不易发生微球交联
比较例1 300mg的透明质酸 容易发生微球交联
实施例1 150mg的微球 不易发生微球交联
比较例1 300mg的微球 容易发生微球交联
实施例1 150rpm的震荡转速 不易发生微球交联
比较例1 180rpm的震荡转速 容易发生微球交联
所有说明书中所揭示的发明技术特点可以任意方式组合。说明书中揭示的每一技术特点可以提供相同、等同或相似目的的其他方式替换。因此，除非另有特别说明，文中所有揭示的特点均只是等同或相似特点的一般系列的实例。
由上述可知，熟习此技艺者能轻易地了解本发明的必要特征，在不脱离其精神与范围之下能就本发明做许多改变与调整以应用于不同用途与条件。

Claims (14)

  1. 一种制备中空微米粒子的方法，其特征是，包括
    a)提供中空微球；
    b)将所述中空微球浸泡在胺类溶液中，使所述中空微球的表面产生氨基；
    c)加入多肽，且所述多肽接枝到所述中空微球表明的氨基上；以及
    d)加入靶标分子，且所述靶标分子连接至所述中空微球表明未键合的氨基上。
  2. 如权利要求1所述的方法，其特征是，所述中空微球材质包括聚乳酸、聚(丁二酸丁二醇酯)、聚(丁二酸丁二醇酯-共-己二酸丁二醇酯)、聚(己二酸丁二醇酯-共-对苯二甲酸酯)、聚乙醇酸、聚(乳酸-共-乙醇酸)、聚己内酯、聚乙烯醇或其混合物。
  3. 如权利要求1所述的方法，其特征是，a)步骤包括：
    ⅰ)提供生物分解材质乳化液；
    ⅱ)提供0.5％(V/V)以上的PVA水溶液；
    ⅲ)将所述PVA水溶液冷却后进行均质；
    ⅳ)将所述生物分解材质乳化液缓慢地滴入所述PVA水溶液，以进行乳化；以及
    ⅴ)冷冻干燥。
  4. 如权利要求3所述的制备中空微球粒子方法，其特征是，ii)步骤中的 PVA水溶液浓度为1％(V/V)。
  5. 如权利要求3所述的制备中空微球粒子方法，其特征是，ⅲ)步骤中的PVA水溶液冷却至10-15℃。
  6. 如权利要求3所述的制备中空微球粒子方法，其特征是，ⅲ)步骤中的PVA水溶液以1000rpm以下的转速进行均质化。
  7. 如权利要求1所述的制备中空微球粒子方法，其特征是，b)步骤中所述胺类溶液包括含有己二胺、Glycine水溶液、adipic dihydrazide(ADH)水溶液、PEG胺化水溶液、NH2-PEG-NHS或NH2-PEG-NH2的DMSO溶液。
  8. 如权利要求1所述的制备中空微球粒子方法，其特征是，还包括在c)步骤前，将多肽与缓冲液反应以活化羧基。
  9. 如权利要求8所述的制备中空微球粒子方法，其特征是，所述缓冲液包括MES缓冲液、EDC水溶液或NHS水溶液。
  10. 如权利要求8所述的制备中空微球粒子方法，其特征是，所述缓冲液的pH值介于约5-6。
  11. 如权利要求1所述的制备中空微球粒子方法，其特征是，c)步骤中所述多肽包括IKVAV、RGD、YIGSR、REDV、DGEA、VGVAPG、GRGDS、LDV、RGDV、PDSGR、RYWLPR、LGTIPG、LAG、RGDS、RGDF、HHLGGALQAGDV、VTCG、SDGD、GREDVY、GRGDY、GRGDSP、VAPG、GGGGRGDSP、GGGGRGDY、FTLCFD、Poly-Lysine或MAX-1。
  12. 如权利要求1所述的制备中空微球粒子方法，其特征是，d)步骤 中所述标靶分子包括透明质酸、氧化透明质酸、Colleagen、Glucocorticoid、Galectin或osteopontin。
  13. 如权利要求1所述的制备中空微球粒子方法，其特征是，d)步骤中所述标靶分子的浓度为0.38％至0.25％。
  14. 如权利要求1所述的制备中空微球粒子方法，其特征是，d)步骤中所述标靶分子与中空微球重量比为1:1.5至1:1。
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