WO2012072012A1 - 海藻酸盐-壳聚糖酰基衍生物微胶囊制剂及其制备和应用 - Google Patents

海藻酸盐-壳聚糖酰基衍生物微胶囊制剂及其制备和应用 Download PDF

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WO2012072012A1
WO2012072012A1 PCT/CN2011/083023 CN2011083023W WO2012072012A1 WO 2012072012 A1 WO2012072012 A1 WO 2012072012A1 CN 2011083023 W CN2011083023 W CN 2011083023W WO 2012072012 A1 WO2012072012 A1 WO 2012072012A1
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chitosan
alginate
solution
microspheres
microcapsules
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PCT/CN2011/083023
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English (en)
French (fr)
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马小军
于炜婷
谢红国
刘袖洞
谢威杨
郑国爽
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中国科学院大连化学物理研究所
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Priority to EP11844778.8A priority Critical patent/EP2659883A4/en
Priority to AU2011335563A priority patent/AU2011335563B2/en
Priority to RU2013129219/15A priority patent/RU2542509C2/ru
Priority to US13/988,918 priority patent/US20130316007A1/en
Priority to NZ611435A priority patent/NZ611435A/en
Publication of WO2012072012A1 publication Critical patent/WO2012072012A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K9/5073—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 having two or more different coatings optionally including drug-containing subcoatings
    • A61K9/5078—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 having two or more different coatings optionally including drug-containing subcoatings with drug-free core
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K9/5089—Processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Definitions

  • the present invention relates to a microcapsule product, in particular to an alginate-shell polyacyl derivative microcapsule product for living cell entrapment. Background technique
  • the present invention provides an alginate-chitosan acyl derivative microcapsule, and its preparation and use.
  • the invention uses an acylated modified chitosan for the preparation of biological microcapsules, and invents a novel alginate-chitosan acyl derivative polyelectrolyte complex microcapsule product for bioactive substance embedding,
  • the problem of surface roughness and surface charge is solved under the premise of ensuring the strength and immunoisolation performance of the microcapsules.
  • the alginate-chitosan acyl derivative microcapsule preparation of the present invention is prepared by mixing alginate-chitosan acyl derivative microcapsules and an aqueous solution, wherein:
  • the biological microcapsule structure is divided into two parts: a microcapsule membrane and a core: a microcapsule membrane is a polyelectrolyte composite hydrogel membrane formed of chitosan, alginate, chitosan acyl derivative, and the core is a cell alginate. Liquid or hydrogel environment.
  • the microcapsules in the biological microcapsule preparation product of the present invention are spherical microcapsules having a particle diameter of 10 to 2000 ⁇ m; the film thickness is 0.1-100 ⁇ m, and the molecular weight of the alginate constituting the membrane is 10 kDa -2000 kDaC, for example: 50 kDa -200 kDa; 200 kDa -500 kDa; 600 kDa -1000 kDa; 1000 kDa -2000 kDa), chitosan material with a degree of deacetylation of 70-98%, molecular weight of 1 KDa ⁇ 500 KDa (eg: 1 kDa -50 kDa; 10 kDa -100 kDa; 120 kDa -300 kDa; 350kDa-500kDa), chitosan acyl derivative molecular weight lKDa ⁇ 800Kda (eg 1 kD
  • the chitosan acyl derivative of the microcapsule in the biological microcapsule preparation product is N-acylated chitosan, and its monomer structural formula is:
  • -R represents formyl, acetyl, propionyl, butyryl, valeryl or hexanoyl
  • the degree of substitution of the acyl derivative is 10-60%
  • the molecular weight of the chitosan skeleton material is 1-400 KDa
  • the degree of deacetylation is 90-98%.
  • the alginate in the microcapsule membrane component of the biological microcapsule preparation product is a potassium salt or a sodium salt of alginic acid.
  • the alginate gel in the microcapsule core of the biological microcapsule preparation product is one or more alginate hydrogels of divalent metal calcium, barium or zinc, and the alginate liquid is the potassium salt of alginic acid. Or sodium salt solution.
  • the volume ratio of the biological microcapsules to the aqueous solution in the biological microcapsule preparation product is 10: 1-1:100, wherein the aqueous solution is physiological saline, HEPES solution, and the apparent viscosity is 5-2000 cp (25 ° C, which means the apparent measurement) Hyaluronic acid solution of viscosity), sodium alginate solution with an apparent viscosity of 5-2000 cp (25 ° C), dextran solution with an apparent viscosity of 5-2000 cp (25 ° C), apparent viscosity 5-2000 cp (25 °C) glycerol solution, polyethylene glycol solution with an apparent viscosity of 5-2000 cp (25 ° C), polyvinylpyrrolidone solution with an apparent viscosity of 5-2000 cp (25 ° C), apparent viscosity 5- 2000 cp (25 °C) cellulose derivative solution, cyclodextrin solution with an apparent viscosity of 5-2000 c
  • the microcapsule membrane is formed by chitosan, alginate, chitosan acyl derivative by polyelectrolyte complexation reaction to form a hydrogel film, and the preparation steps of the product are as follows: under aseptic conditions,
  • a microspheres 1) preparing alginate gel microspheres embedded with living cells, referred to as A microspheres;
  • step 2) The A microsphere in step 1) is immersed in the chitosan solution, and the volume ratio of the A microsphere to the chitosan solution is 1:1-1:40, and the reaction is 1-60 minutes, at which time sodium alginate is obtained.
  • Chitosan microcapsules, called B microspheres, are taken out and washed with physiological saline;
  • the chitosan solution is prepared by dissolving chitosan in an acetic acid-sodium acetate buffer having a pH of 5.5-7.0, and the chitosan concentration is 0.1-15 g/L ;
  • step 2) The B microspheres in step 2) are immersed in an alkali metal alginate solution (alginate concentration is 0.1-5 g/L), and the volume ratio of B microspheres to alkali metal alginate solution is 1:1. -1: 40, reaction 1-60 minutes, the microcapsules obtained at this time are C microspheres, and are taken out and washed with physiological saline; 4) alternately repeating the steps of step 2) and step 3) 1-5 times, the microcapsules obtained at this time are D microspheres, and are taken out and washed with physiological saline;
  • the preparation method of chitosan acyl derivatives is: 1-20 g/L;
  • the concentration of the chitosan acyl derivative is 0. 1-20 g / L;
  • the chitosan acyl derivative is 0. 1-20 g / L;
  • step 6) immersing the E microspheres in the step 5) into the alkali metal alginate solution, repeating step 3), obtaining the microcapsules of the inner gel core neutralized on the surface as F microspheres;
  • step 6) Immerse the F microspheres in step 6) into the organometallic chelating agent solution to liquefy the alginate gel inside the microcapsules.
  • the volume ratio of the F microspheres to the organometallic chelating agent solution is 1: 1-1: 40 , the reaction is 1-60 minutes, and is taken out and washed with physiological saline. At this time, the microcapsules of the internal liquid core are obtained as G microspheres;
  • the alginate-chitosan acyl derivative microcapsule preparation is prepared.
  • the alginate gel microspheres are one or more of alginate hydrogels of divalent metal calcium, barium or zinc;
  • the alkali metal alginate used to neutralize the surface charge is a potassium salt or a sodium salt having a molecular weight distribution of 10 KDa to 2000 KDa and an alginate concentration of 0.1 to 5 g/L.
  • the organometallic chelating agent solution involved in the liquefaction reaction is 40-70 mm O l / L of sodium citrate or
  • microcapsules in the biomicrocapsule preparation product of the present invention are used for cell embedding.
  • the cells are human or mammalian isolated islet cells, hepatocytes, thyroid cells, parathyroid cells, adrenal medulla cells, cells secreting biologically active substances, cell line cells, genetically engineered cells, stem cells or stem cells. Various cells.
  • novel alginate-shell polymerization of the present invention compared to conventional sodium alginate-polylysine microcapsules (APA microcapsules) and sodium alginate-chitosan microcapsules (ACA microcapsules)
  • APA microcapsules sodium alginate-polylysine microcapsules
  • ACA microcapsules sodium alginate-chitosan microcapsules
  • the microcapsule product of the sugar-chitosan acyl derivative has a surface roughness of the microcapsule film which is significantly lower than that of the APA microcapsule and the ACA microcapsule, and exhibits better biocompatibility.
  • microcapsule film of the product of the invention maintains excellent biocompatibility while taking into consideration superior membrane strength, and can ensure the integrity of the membrane during tissue cell transplantation and cell culture application.
  • the microcapsule membrane of the product of the invention has superior immuno-isolation performance, and can maintain immuno-isolation performance when used for transplantation of xenogenic tissue cells, that is, cells embedded in the microcapsule cannot produce microcapsules, antibody molecules outside the microcapsule, Complement molecules and immune cells cannot enter the microcapsules to kill the cells, and the active components secreted by the cells can freely enter and exit the microcapsules.
  • DRAWINGS Fig. 1 shows the results of comparison of the surface roughness of the alginate-chitosan acyl derivative (AC film, AC film, and AP film) in Example 1 and Comparative Example 1 and Comparative Example 2.
  • Fig. 2 is a photomicrograph of the microcapsules recovered after 1 month of abdominal transplantation of the novel alginate-chitosan-chitosan acyl derivative microcapsule preparation product of Example 1 (the scale is 100 ⁇ in the figure).
  • Fig. 3 is an optical photograph of a microcapsule recovered after 1 month of abdominal transplantation of a conventional ACA microcapsule mouse in Comparative Example 1 (the scale is 100 ⁇ in the figure). detailed description
  • the method of forming alginate gel microspheres is the electrostatic droplet method (Reference: In Vivo Culture of Encapsulated Endo statin- Secreting Chinese Hamster Ovary Cells for Systemic Tumor Inhibition. Human Gene Therapy. 2007, 18: 474-481) Hole extrusion method (Reference: Preparation method of high economic fish microsphere opening bait, Chinese invention patent, 200510136769.7), emulsification-external gelation method (Reference: Preparation of lactic acid bacteria-enclosing alginate beads in Emulsion system: effect of preparation parameters on bead characteristics , Polym.
  • mice After mixing AC « A microcapsules with normal saline in a volume ratio of 1: 2, the mice were implanted into the abdominal cavity of the mouse with a syringe, and recovered one month later. It was found that the saline was washed out in the abdominal cavity of the mice, and the microcapsules were good. Strength, no damage, smooth surface of microcapsules, no fiber wrap on the surface (see Figure 2).
  • Example 1 The calcium alginate gel microsphere prepared in Example 1 was immersed in a chitosan solution (chitosan molecular weight 50 kDa, deacetylation degree 95%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.5) , chitosan concentration is 5g / L), the volume ratio of microspheres to chitosan solution is 1:10, reaction for 20 minutes, washed with physiological saline and then reacted with 0.2% sodium alginate solution for 10 minutes, washed with physiological saline, prepared Into ACA microcapsules.
  • a chitosan solution chitosan molecular weight 50 kDa, deacetylation degree 95%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.5
  • chitosan concentration is 5g / L
  • the volume ratio of microspheres to chitosan solution is 1:10
  • reaction for 20 minutes
  • Example 1 The calcium alginate gel microsphere prepared in Example 1 was immersed in a polylysine solution (polylysine molecular weight 20 kDa, concentration 0.5 g/L), and the volume ratio of the microsphere to the polylysine solution was 1 :10, the reaction was carried out for 20 minutes, washed with physiological saline, and then reacted with a 2 g/L sodium alginate solution for 10 minutes, and washed with physiological saline to prepare APA microcapsules.
  • polylysine solution polylysine molecular weight 20 kDa, concentration 0.5 g/L
  • microspheres are immersed in chitosan (chitosan molecular weight 20kDa, deacetylation degree 90%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.8, chitosan concentration 4g / L) and Acyl-modified chitosan (chitosan backbone molecular weight 60kDa, formyl substitution degree 30%, soluble in acetic acid-sodium acetate buffer pH 6.8, concentration 4g / L) solution, microsphere to solution volume ratio 1 : 10, the reaction was carried out for 20 minutes, washed with physiological saline and then reacted with 0.2% sodium alginate solution for 10 minutes, washed with physiological saline, and prepared into ACC «A microcapsules.
  • chitosan chitosan molecular weight 20kDa, deacetylation degree 90%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.8, chitos
  • ACC «microcapsules embedded with pig liver cells were prepared into an in vitro artificial liver system for animal models of liver failure dogs.
  • the results showed that alanine aminotransferase and aspartate aminotransferase in liver-deficient mice were transplanted 4 days later.
  • the water level returned to normal, the blood ammonia index returned to normal, and the dog's liver failure symptoms were corrected.
  • microspheres are immersed in chitosan (chitosan molecular weight 40kDa, deacetylation degree 98%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.5, concentration 5g / L), 0.2% alginic acid Sodium solution and acetyl modified chitosan (chitosan skeleton molecular weight 60kDa, acetyl substitution degree 50%, dissolved in physiological saline, concentration 5g / L) solution, microsphere to solution volume ratio of 1:10, The reaction was carried out for 20 minutes, washed with physiological saline, and then reacted with a 2 g/L sodium alginate solution for 10 minutes, washed with physiological saline, liquefied with 55 mM sodium citrate, and washed with physiological saline to prepare ACC microcapsules.
  • chitosan chitosan molecular weight 40kDa, deacetylation degree 98%
  • microspheres are successively immersed in chitosan (chitosan molecular weight lOOkDa, deacetylation degree 95%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.0, concentration 5g/L) and propionyl modification Chitosan (molecular weight of chitosan skeleton 20kDa, degree of propionyl substitution 40%, dissolved in PBS buffer, concentration 5g / L), the ratio of microsphere to solution volume is 1:10, reaction for 20 minutes, normal saline After washing, it was reacted with 2 g/L sodium alginate solution for 10 minutes, washed with physiological saline, liquefied with 55 mM sodium citrate, and washed with physiological saline to prepare ACC A micro.
  • chitosan chitosan molecular weight lOOkDa, deacetylation degree 95%, chitosan dissolved in acetic acid-sodium
  • High-alkali electrostatic method was used to prepare calcium alginate gel microspheres embedded with bovine adrenal medulla cells.
  • the microspheres contained 2 ⁇ 10 7 /ml microspheres.
  • microspheres are immersed in chitosan (chitosan molecular weight 10kDa, deacetylation degree 90%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.8, concentration 5g / L) and butyryl modification Chitosan (chitosan skeleton molecular weight 10kDa, butyryl substitution degree 30%, soluble in chitosan dissolved in acetic acid-sodium acetate buffer pH 6.8, concentration 5g / L) solution, microsphere and solution volume The ratio was 1:10, and the reaction was carried out for 20 minutes.
  • chitosan chitosan molecular weight 10kDa, deacetylation degree 90%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.8, concentration 5g / L
  • butyryl modification Chitosan chitosan skeleton molecular weight 10kDa, butyryl substitution degree 30%, soluble in chitosan
  • Sodium alginate solution (20g/L) is mixed in a volume ratio of 1:1, used for intracranial fixed-point transplantation of monkeys in Parkinson's disease model, Parkinson's symptoms such as hemiplegia of diseased monkeys are corrected, ACC T A microcapsules After six months of transplantation, the ACC T A microcapsules with intact morphology were recovered and there was no fibrosis on the surface.
  • microspheres are immersed in chitosan (chitosan molecular weight 70kDa, deacetylation degree 98%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.3, concentration 5g / L), 0.2% alginic acid Sodium solution and valeryl modified chitosan (chitosan skeleton molecular weight 20kDa, valeryl substitution degree 40%, soluble in HEPES buffer, concentration 5g / L) solution, microsphere to solution volume ratio of 1:10
  • the reaction was carried out for 20 minutes, washed with physiological saline, and then reacted with a 2 g/L sodium alginate solution for 10 minutes, and washed with physiological saline to prepare ACC A microcapsules.
  • the microspheres have a cell content of 5 X 10 7 /ml microspheres.
  • microspheres are immersed in chitosan (chitosan molecular weight 20kDa, deacetylation degree 92%, chitosan dissolved in pH 6.5 acetic acid-sodium acetate buffer, concentration 5g / L) and hexanoyl modification Chitosan (chitosan skeleton molecular weight 20kDa, hexanoyl substitution degree 50%, dissolved in physiological saline, concentration 5g / L) solution, microsphere to solution volume ratio of 1:10, reaction 20 minutes, physiological saline washing After that, it was reacted with 2 g/L sodium alginate solution for 10 minutes, washed with physiological saline, and prepared into ACC A microcapsules.
  • chitosan chitosan molecular weight 20kDa, deacetylation degree 92%, chitosan dissolved in pH 6.5 acetic acid-sodium acetate buffer, concentration 5g / L
  • ACC aBt A microcapsules prepared by recombinant endostatin-encapsulated CHO cells and apparent viscosity 600cp (25 °C) Glycerol solution 50% (V/V) After mixing in a volume ratio of 1:5, it is used for abdominal transplantation of melanoma model mice, and the tumor of the diseased rats is significantly reduced.
  • ACC A microcapsule transplantation After two months of recovery, the ACC aR A microcapsules with intact morphology were recovered and there was no fibrosis on the surface.
  • microspheres are immersed in chitosan (chitosan molecular weight 40kDa, deacetylation degree 98%, chitosan dissolved in acetic acid-sodium acetate buffer pH 6.5, concentration 5g / L), 0.2% alginic acid Sodium solution and acetyl modified chitosan (chitosan skeleton molecular weight 60kDa, acetyl substitution degree 50%, dissolved in physiological saline, concentration 5g / L) solution, microsphere to solution volume ratio of 1:10, The reaction was carried out for 20 minutes, washed with physiological saline, and then reacted with a 2 g/L sodium alginate solution for 10 minutes, washed with physiological saline, liquefied with 55 mM sodium citrate, and washed with physiological saline to prepare ACC microcapsules.
  • chitosan chitosan molecular weight 40kDa, deacetylation degree 98%
  • the prepared ACC ⁇ A microcapsules are mixed with an apparent viscosity of 400 cp (25 ° C ) polyethylene glycol (100 g / L) solution in a volume ratio of 1: 2, and used for cell therapy in a diabetic rat model.
  • an apparent viscosity of 400 cp (25 ° C ) polyethylene glycol (100 g / L) solution in a volume ratio of 1: 2, and used for cell therapy in a diabetic rat model.
  • the blood glucose level of the rats returned to normal level 1 day after transplantation, and the symptoms of diabetes were significantly improved.
  • the microcapsules were found to be intact, the surface of the microcapsules was smooth, and the surface was free of fibrosis. And the islet cells in the microcapsules remained positive for insulin dithizone staining.

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Abstract

本发明涉及一种海藻酸盐-壳聚糖酰基衍生物微胶囊制剂产品，由海藻酸盐-壳聚糖酰基衍生物微胶囊和水溶液混合而成，其中：生物微胶囊结构分为微胶囊膜与内核两部分：微胶囊膜由壳聚糖、海藻酸盐、壳聚糖酰基衍生物形成的聚电解质复合水凝胶膜，内核为含有细胞的海藻酸盐液体或水凝胶环境。

Description

海藻酸盐 -壳聚糖酰基衍生物微胶囊制剂及其制备和应用 技术领域
本发明涉及一种微胶囊产品， 具体地说是一种用于活细胞包埋的海藻酸盐 -壳聚 糖酰基衍生物微胶囊产品。 背景技术
20世纪 60年代， Chang报道了半透膜微胶囊，指出用其包埋蛋白质、酶等生物活性 物质和细胞，可保持生物物质活性 [Chang TMS. Semipermeable microcapsules, Science, 1964, 146： 524-525] 。20世纪 80年代初， Lim和 Sun针对组织 /细胞功能缺损性疾病 (；如 糖尿病)，成功制备了海藻酸钠 /α-聚赖氨酸 lginate/ct-polylysine)半透膜微胶囊 （简称 α-ΑΡΑ微胶囊） ，包封 Wistar大鼠胰岛细胞并移植入糖尿病 WistarLewis大鼠体内，分 泌释放胰岛素以调控血糖量 [ Lim F , Sun A M. Microencapsulated islets bioartificial endocrine pancreas, Science, 1980， 210： 908-910]。 由此推动了微囊化技术相关材料和 制备方法研究的快速发展，在细胞移植、 药物释放和基因治疗等生物医学领域的临床 前研究中得到广泛应用 [Wang W， Liu XD , Ma XJ , et al. Microencapsulation using natural polysaccharides for drug delivery and cell implantation, J . Mater. Chem., 2006 , 16 :3252-3267] 在众多的生物微胶囊中， 海藻酸钠-聚赖氨酸微胶囊使用较多， 但由 于聚赖氨酸价格昂贵 (300-400 US $ /g),加之材料固有的生物相容性差，具有一定毒性， 这极大地限制了海藻酸钠 -聚赖氨酸微胶囊在临床上的应用 [Strand B L， Ryan TL， Veld P I , et al . Cell Transplant., 2001, 10:263-275]。 壳聚糖是取自天然的多糖材料， 因 其成本低， 成膜性能好， 膜机械强度高， 而被研究者看好用于聚赖氨酸替代品， 用于 细胞包埋用微胶囊的制备 [L. Baruch, M. Machluf, Alginate-chitosan complex coacervation for cell encapsulation: Effect on mechanical properties and on long-term viability, Biopolymers 82(2006): 570-579]。但现有用于细胞包埋的壳聚糖微胶囊因其具 有很大的表面粗糙度和表面电荷， 导致移植体内后易引起蛋白吸附，进一步激发机体 纤维化反应。 发明内容
针对上述问题， 本发明提出一种海藻酸盐-壳聚糖酰基衍生物微胶囊及其制备和 应用。
技术方案：
本发明将酰基化修饰的壳聚糖用于生物微胶囊的制备，发明了一种新型的用于生 物活性物质包埋的海藻酸盐 -壳聚糖酰基衍生物聚电解质络合微胶囊产品， 在保证微 胶囊强度和免疫隔离性能的前提下，解决表面粗糙度和表面电荷的问题。本发明的海 藻酸盐 -壳聚糖酰基衍生物微胶囊制剂， 由海藻酸盐 -壳聚糖酰基衍生物微胶囊和水溶 液混合而成， 其中：
生物微胶囊结构分为微胶囊膜与内核两部分： 微胶囊膜由壳聚糖、海藻酸盐、 壳 聚糖酰基衍生物形成的聚电解质复合水凝胶膜，内核为含有细胞的海藻酸盐液体或水 凝胶环境。 本发明的生物微胶囊制剂产品中的微胶囊为粒径 10-2000 微米的球形微胶囊;膜 厚度在 0.1-100微米， 组成膜的海藻酸盐分子量 10 kDa -2000 kDaC例如： 50 kDa -200 kDa; 200 kDa -500 kDa ;600 kDa -1000 kDa; 1000 kDa -2000 kDa), 壳聚糖材料的脱乙 酰度 70-98%， 分子量为 1 KDa〜500 KDa (例如： 1 kDa -50 kDa; 10 kDa -100 kDa ;120 kDa -300 kDa; 350kDa-500kDa), 壳聚糖酰基衍生物分子量 lKDa〜800Kda (例如： 1 kDa -50 kDa; 10 kDa -100 kDa ;120 kDa -300 kDa; 350kDa-500kDa), 壳聚糖、 海藻酸 盐、 壳聚糖酰基衍生物三者质量比为 0:1 :0.1-10:1 :10， 内核中海藻酸盐浓度在 0.1-50g/L ， 内核中细胞占据内核的体积百分比为 10-98%。
生物微胶囊制剂产品中微胶囊的壳聚糖酰基衍生物为 N-酰基化壳聚糖， 其单体 结构式为:
Figure imgf000003_0001
其中， -R代表甲酰、乙酰、丙酰、丁酰、戊酰或己酰，酰基衍生物的取代度 10-60%， 壳聚糖骨架材料的分子量为 1-400 KDa, 脱乙酰度为 90-98%。
生物微胶囊制剂产品中微胶囊膜成分中的海藻酸盐为海藻酸的钾盐或钠盐。 生物微胶囊制剂产品中微胶囊内核中海藻酸盐凝胶为二价金属钙、钡或锌中的一 种或二种以上的海藻酸盐水凝胶， 海藻酸盐液体为海藻酸的钾盐或钠盐溶液。
生物微胶囊制剂产品中生物微胶囊与水溶液的体积比为 10: 1-1 :100, 其中水溶 液为生理盐水、 HEPES溶液、 表观粘度 5-2000cp (25°C，是指时测定的表观粘度） 的 透明质酸溶液、表观粘度 5-2000cp (25°C )的海藻酸钠溶液、表观粘度 5-2000cp (25°C ) 的葡聚糖溶液、表观粘度 5-2000cp (25°C )的丙三醇溶液、表观粘度 5-2000cp (25°C ) 的聚乙烯乙二醇溶液、 表观粘度 5-2000cp (25 °C ) 的聚乙烯吡咯烷酮溶液、 表观粘度 5-2000cp (25 °C ) 的纤维素衍生物溶液、 表观粘度 5-2000cp (25 °C ) 的环糊精溶液、 表观粘度 5-2000cp (25 °C )的淀粉溶液或表观粘度 5-2000cp (25 °C )的淀粉衍生物溶 液中的一种或二种以上的混合物。
生物微胶囊制剂产品中，微胶囊膜由壳聚糖、 海藻酸盐、 壳聚糖酰基衍生物通过 聚电解质络合反应形成水凝胶膜， 产品的制备步骤为： 在无菌条件下，
1 ) 制备包埋有活细胞的海藻酸盐凝胶微球， 称之为 A微球；
2) 将步骤 1 ) 中的 A微球浸入壳聚糖溶液中， A微球与壳聚糖溶液体积比为 1 :1-1 :40, 反应 1-60分钟， 此时得到海藻酸钠-壳聚糖微胶囊， 称之为 B微球， 取出 用生理盐水洗涤；
壳聚糖溶液的配制方法是： 壳聚糖溶于 pH为 5.5-7.0的醋酸-醋酸钠缓冲液， 壳 聚糖浓度为 0.1-15 g/L;
3 )将步骤 2)中的 B微球浸入碱金属海藻酸盐溶液中 (海藻酸盐浓度为 0.1-5 g/L), B微球与碱金属海藻酸盐溶液体积比范围为 1 : 1-1： 40， 反应 1-60分钟， 此时得到 的微胶囊为 C微球， 取出用生理盐水洗涤； 4)交替重复步骤 2)和步骤 3 ) 的过程 1-5次， 此时得到的微胶囊为 D微球， 取 出用生理盐水洗涤；
5 ) 将步骤 1 ) 或 2) 或 3 ) 或 4) 中的 A或 B或 C或 D微球浸入到壳聚糖酰基 衍生物溶液中， 微球与壳聚糖酰基衍生物溶液体积比范围为 1 : 1-1： 40， 反应 1-60 分钟， 此时得到内部凝胶核心的微胶囊， 称之为 E微球， 取出用生理盐水洗涤； 壳聚糖酰基衍生物的配制方法是： 壳聚糖酰基衍生物溶于生理盐水、 HEPES 缓 冲液、 PBS缓冲液， 或 pH为 5.5-7.0的醋酸-醋酸钠缓冲液， 壳聚糖酰基衍生物浓度 为 0. 1-20 g/L;
6)将步骤 5 )中的 E微球浸入到碱金属海藻酸盐溶液中， 重复步骤 3 )， 得到表 面中和的内部凝胶核心的微胶囊为 F微球；
7) 将步骤 6) 中的 F微球浸入有机金属螯合剂溶液中， 液化微胶囊内部的海藻 酸盐凝胶， F微球与有机金属螯合剂溶液体积比范围为 1 : 1-1： 40， 反应 1-60分钟， 取出用生理盐水洗涤， 此时得到内部液态核心的微胶囊为 G微球；
8) 将步骤 5 ) 或 6) 或 7) 得到的 E或 F或 G微球与上述水溶液混合后， 即制 备成海藻酸盐 -壳聚糖酰基衍生物微胶囊制剂。
海藻酸盐凝胶微球为二价金属钙、 钡或锌中的一种或二种以上的海藻酸盐水凝 胶；
用于中和表面电荷的碱金属海藻酸盐为钾盐或钠盐， 分子量分布为 10KDa〜 2000KDa, 海藻酸盐浓度为 0.1-5g/L。
参与液化反应的有机金属螯合剂溶液为 40-70mmOl/L 的柠檬酸钠或
50-200mmol/L的 EDTA。
本发明的生物微胶囊制剂产品中微胶囊用于细胞的包埋。
其中，细胞为人或哺乳动物来源的离体的胰岛细胞、 肝细胞、 甲状腺细胞、 甲状 旁腺细胞、 肾上腺髓质细胞具有分泌生物活性物质功能的细胞， 细胞系细胞， 基因工 程细胞， 干细胞或干细胞分化的各种细胞。
本发明的有益效果：
1. 与传统的海藻酸钠-聚赖氨酸微胶囊 （APA微胶囊） 和海藻酸钠 -壳聚糖微胶 囊 (ACA微胶囊)相比， 本发明的这种新型海藻酸盐-壳聚糖 -壳聚糖酰基衍生物微胶囊 产品，其微胶囊膜的表面粗糙度显著低于 APA微胶囊和 ACA微胶囊， 显示出更佳的 生物相容性。
2.本发明产品的微胶囊膜在保持优良生物相容性的同时， 兼顾了优越的膜强度， 能保证作为组织细胞移植、 细胞培养应用过程中膜的完整性。
3.本发明产品的微胶囊膜具有优越的免疫隔离性能，用于异种组织细胞移植时， 能保持免疫隔离性能， 即微囊内包埋的细胞不能出微胶囊， 微囊外的抗体分子、补体 分子、免疫细胞不能进入微胶囊内杀死细胞， 同时细胞代谢分泌的活性成分能自由进 出微胶囊。
4. 本发明产品的制备过程条件温和， 壳聚糖酰基衍生物可溶于生理盐水中， 有 利于细胞的活性保持。 附图说明 图 1为实施例 1和比较例 1及比较例 2中海藻酸盐 -壳聚糖酰基衍生物 （AC 膜及 AC膜、 AP膜的表面粗糙度比较结果。
图 2为实施例 1中新型海藻酸盐-壳聚糖 -壳聚糖酰基衍生物微胶囊制剂产品小鼠 腹腔移植 1个月后回收的微胶囊光学照片 （图中标尺为 100μηι)。
图 3为比较例 1中传统的 ACA微胶囊小鼠腹腔移植 1个月后回收的微胶囊光学 照片 （图中标尺为 100μηι)。 具体实施方式
形成海藻酸盐凝胶微球的方式为静电液滴法 （参考文献： In Vivo Culture of Encapsulated Endo statin- Secreting Chinese Hamster Ovary Cells for Systemic Tumor Inhibition. Human Gene Therapy. 2007, 18: 474-481 ) 锐孔挤出法 （参考文献： 一种高 经济鱼类微球开口饵料的制备方法， 中国发明专利， 200510136769.7)、 乳化-外部凝 胶 化 法 ( 参 考 文 献 ： Preparation of lactic acid bacteria-enclosing alginate beads in emulsion system: effect of preparation parameters on bead characteristics ， Polym. Bull, 2009, 63： 599-607)、 乳化-内部凝胶化法 （参考文献： 乳化-内部凝胶 化工艺制备固定化酵母微胶囊， 化工学报， 2009， 60(3)： 710-717) 或膜乳化法 （参 考文献： Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal gelation。 Journal of Applied Polymer Science,2003 , 87 ( 5 )： 848-852)。
实施例 1
1 ) 无菌条件下通过高压静电法制备海藻酸钙凝胶微球。
2) 将微球浸入乙酰基改性壳聚糖溶液中 （壳聚糖骨架分子量 50kDa， 乙酰基取 代度 40%， 溶液由生理盐水配制， 浓度 5g/L)， 微球与壳聚糖溶液体积比为 1 :10， 反 应 20分钟， 生理盐水洗涤后再与 2g/L海藻酸钠溶液反应 10分钟， 生理盐水洗涤， 制备成 AC Bt A微胶囊。
3 ) 制备成的 AC 聚电解质络合膜用表面轮廓仪测定膜的表面粗糙度， 结果 显示膜表面粗糙度最小 42 ± 9nm， 明显低于同样方法制备的比较例中的 APA膜和 ACA膜 （见图 1 )
4)将 AC « A微胶囊与生理盐水按照 1 : 2的体积比混合后， 用注射器植入小鼠 腹腔， 一个月后回收， 发现生理盐水冲洗小鼠腹腔即可冲刷下来， 微胶囊具有良好强 度， 无破损， 微胶囊表面光滑， 表面无纤维化包裹现象 （见图 2)。
比较例 1
1 ) 将实施例 1 中制备的海藻酸钙凝胶微球浸入壳聚糖溶液中 （壳聚糖分子量 50kDa， 脱乙酰度 95%， 壳聚糖溶于 pH为 6.5的醋酸-醋酸钠缓冲液， 壳聚糖浓度为 5g/L)， 微球与壳聚糖溶液体积比为 1 :10， 反应 20分钟， 生理盐水洗涤后再与 0.2% 海藻酸钠溶液反应 10分钟， 生理盐水洗涤， 制备成 ACA微胶囊。
2)制备成的 ACA聚电解质络合膜用表面轮廓仪测定膜的表面粗糙度，结果显示 膜表面粗糙度为 157±20明显高于实施例 1结果 AC S A聚电解质络合膜（见图 1 )。
3 ) 将 ACA微胶囊与生理盐水按照 1 : 2的体积比混合后， 用注射器植入小鼠腹 腔， 一个月后回收， 发现生理盐水冲洗小鼠腹腔难以将 ACA微胶囊冲刷下来， 微胶 囊表面呈现明显的纤维化包裹现象 （见图 3 )。
比较例 2
1 )将实施例 1中制备的海藻酸钙凝胶微球浸入聚赖氨酸溶液中 (聚赖氨酸分子量 20kDa, 浓度 0.5g/L)， 微球与聚赖氨酸溶液体积比为 1 :10， 反应 20分钟， 生理盐水 洗涤后再与 2g/L海藻酸钠溶液反应 10分钟， 生理盐水洗涤， 制备成 APA微胶囊。
2)制备成的 APA聚电解质络合膜用表面轮廓仪测定膜的表面粗糙度， 结果显示 膜表面粗糙度为 161 ±26，明显高于实施例 1结果 AC S A聚电解质络合膜（见图 1 )。
实施例 2
1 ) 锐孔挤出法制备包埋有猪肝细胞的海藻酸钙凝胶微球， 微球中细胞含量 5 X 107/ml微球。
2) 将微球先后浸入壳聚糖 （壳聚糖分子量 20kDa， 脱乙酰度 90%， 壳聚糖溶于 pH为 6.8的醋酸-醋酸钠缓冲液， 壳聚糖浓度为 4g/L) 和甲酰基改性壳聚糖 （壳聚糖 骨架分子量 60kDa， 甲酰基取代度 30%， 溶于 pH为 6.8的醋酸-醋酸钠缓冲液， 浓度 为 4g/L) 溶液中， 微球与溶液体积比为 1 :10， 反应 20分钟， 生理盐水洗涤后再与 0.2%海藻酸钠溶液反应 10分钟， 生理盐水洗涤， 制备成 ACC «A微胶囊。
3 )制备成的包埋有猪肝细胞的 ACC «微胶囊制备成体外人工肝系统，用于肝衰 狗的动物模型， 结果显示， 移植 4天后， 肝衰小鼠的谷丙转氨酶、 谷草转氨酶水平均 恢复到正常水平， 血氨指标恢复正常， 狗的肝衰症状得以纠正， ACC «A微胶囊在 人工肝系统保持形态完整， 血液灌流后未发现蛋白吸附现象。
实施例 3
1 )高压静电法制备包埋有猪胰岛细胞的海藻酸钙凝胶微球，每个微球中含有 1-2 个胰岛。
2) 将微球先后浸入壳聚糖 （壳聚糖分子量 40kDa， 脱乙酰度 98%， 壳聚糖溶于 pH为 6.5的醋酸-醋酸钠缓冲液， 浓度为 5g/L)， 0.2%海藻酸钠溶液和乙酰基改性壳 聚糖 （壳聚糖骨架分子量 60kDa， 乙酰基取代度 50%， 溶于生理盐水中， 浓度 5g/L) 溶液中， 微球与溶液体积比为 1 :10， 反应 20分钟， 生理盐水洗涤后再与 2g/L海藻酸 钠溶液反应 10分钟， 生理盐水洗涤， 55mM柠檬酸钠液化后， 生理盐水洗涤， 制备 成 ACC 微胶囊。
3 ) 制备成的 ACC 微胶囊与 HEPES溶液按照 1 : 5的体积比混合后， 用于 糖尿病大鼠模型的细胞治疗，通过腹腔移植， 大鼠血糖水平在移植后 1天即恢复正常 水平， 糖尿病症状得以显著改善， 体内移植 6个月后回收， 发现微胶囊完整， 微胶囊 表面光滑， 表面无纤维化包裹现象， 且微囊内胰岛细胞保持胰岛素双硫腙染色阳性。
实施例 4
1 ) 高压静电法制备包埋有大鼠甲状腺细胞的海藻酸钙凝胶微球， 微球中细胞含 量 3 X 107/ml微球。
2)将微球先后浸入壳聚糖（壳聚糖分子量 lOOkDa, 脱乙酰度 95%， 壳聚糖溶于 pH为 6.0的醋酸-醋酸钠缓冲液， 浓度为 5g/L) 和丙酰基改性壳聚糖 （壳聚糖骨架分 子量 20kDa， 丙酰基取代度 40%， 溶于 PBS缓冲液中， 浓度 5g/L) 溶液中， 微球与 溶液体积比为 1 :10， 反应 20分钟， 生理盐水洗涤后再与 2g/L海藻酸钠溶液反应 10 分钟， 生理盐水洗涤， 55mM柠檬酸钠液化后， 生理盐水洗涤， 制备成 ACC A微 胶囊。
3)制备成的包埋有大鼠甲状腺细胞的 ACC ¾Α微胶囊与表观粘度 500cp(25°C) 透明质酸溶液（20g/L)按照 1: 1的体积比混合后， 用于异种大鼠甲低模型三角肌移 植， 疾病大鼠的甲低症状得以纠正， T3 Τ4水平恢复正常， ACC Α微胶囊移植三 个月后回收， 回收到形态保持完整的 ACC A微胶囊， 表面没有纤维化现象。
实施例 5
1) 高压静电法制备包埋有牛肾上腺髓质细胞的海藻酸钙凝胶微球， 微球中细胞 含量 2X107/ml微球。
2) 将微球先后浸入壳聚糖 （壳聚糖分子量 10kDa， 脱乙酰度 90%， 壳聚糖溶于 pH为 6.8的醋酸-醋酸钠缓冲液， 浓度为 5g/L) 和丁酰基改性壳聚糖 （壳聚糖骨架分 子量 10kDa， 丁酰基取代度 30%， 溶于壳聚糖溶于 pH为 6.8的醋酸-醋酸钠缓冲液， 浓度为 5g/L) 溶液中， 微球与溶液体积比为 1:10， 反应 20分钟， 生理盐水洗涤后再 与 2g/L海藻酸钠溶液反应 10分钟， 生理盐水洗涤， 55mM柠檬酸钠液化后， 生理盐 水洗涤， 制备成 ACC A微胶囊。
3)制备成的包埋有牛肾上腺髓质细胞的 ACCT A微胶囊与表观粘度 1000cp(25
V)海藻酸钠溶液（20g/L)按照 1: 1的体积比混合后， 用于帕金森疾病模型猴的颅 内定点移植， 疾病猴的偏瘫等帕金森症状得以纠正， ACC T A微胶囊移植六个月后 回收， 回收到形态保持完整的 ACCT A微胶囊， 表面没有纤维化现象。
实施例 6
1) 高压静电法制备包埋有牛肾上腺髓质细胞的海藻酸钙凝胶微球， 微球中细胞 含量 lX107/ml微球。
2) 将微球先后浸入壳聚糖 （壳聚糖分子量 70kDa， 脱乙酰度 98%， 壳聚糖溶于 pH为 6.3的醋酸-醋酸钠缓冲液， 浓度为 5g/L)， 0.2%海藻酸钠溶液和戊酰基改性壳 聚糖 （壳聚糖骨架分子量 20kDa， 戊酰基取代度 40%， 溶于 HEPES缓冲液中， 浓度 5g/L) 溶液中， 微球与溶液体积比为 1:10， 反应 20分钟， 生理盐水洗涤后再与 2g/L 海藻酸钠溶液反应 10分钟， 生理盐水洗涤， 制备成 ACC A微胶囊。
3) 制备成的包埋有牛肾上腺髓质细胞的 ACC «A微胶囊与与表观粘度 800 cp (25 °C)葡聚糖溶液（30g/L)按照 1: 1的体积比混合后， 用于顽固性疼痛模型大鼠 的脊髓蛛网膜下腔移植， 疾病大鼠的疼痛症状得以纠正， 肢体抽动次数显著降低， ACCAstA微胶囊移植六个月后回收， 回收到形态保持完整的 ACC« A微胶囊，表面 没有纤维化现象。
实施例 7
1)高压静电法制备包埋有重组血管内皮细胞生长抑制因子 (endostatin)的 CHO细 胞的海藻酸钙凝胶微球， 微球中细胞含量 5 X 107/ml微球。
2) 将微球先后浸入含有壳聚糖 （壳聚糖分子量 20kDa， 脱乙酰度 92%， 壳聚糖 溶于 pH为 6.5的醋酸-醋酸钠缓冲液， 浓度为 5g/L)和己酰基改性壳聚糖（壳聚糖骨 架分子量 20kDa， 己酰基取代度 50%， 溶于生理盐水中， 浓度 5g/L) 溶液中， 微球 与溶液体积比为 1:10， 反应 20分钟， 生理盐水洗涤后再与 2g/L藻酸钠溶液反应 10 分钟， 生理盐水洗涤， 制备成 ACC A微胶囊。
3) 制备成的包埋有重组 endostatin的 CHO细胞的 ACC aBtA微胶囊与表观粘度 600cp (25 °C ) 丙三醇溶液 50% (V/V) 按照 1 : 5的体积比混合后， 用于黑色素瘤模 型鼠的腹腔移植， 疾病大鼠的肿瘤明显缩小， ACC A微胶囊移植两个月后回收， 回收到形态保持完整的 ACC aRA微胶囊， 表面没有纤维化现象。
实施例 8
1 )高压静电法制备包埋有猪胰岛细胞的海藻酸钙凝胶微球，每个微球中含有 1-2 个胰岛。
2) 将微球先后浸入壳聚糖 （壳聚糖分子量 40kDa， 脱乙酰度 98%， 壳聚糖溶于 pH为 6.5的醋酸-醋酸钠缓冲液， 浓度为 5g/L)， 0.2%海藻酸钠溶液和乙酰基改性壳 聚糖 （壳聚糖骨架分子量 60kDa， 乙酰基取代度 50%， 溶于生理盐水中， 浓度 5g/L) 溶液中， 微球与溶液体积比为 1 :10， 反应 20分钟， 生理盐水洗涤后再与 2g/L海藻 酸钠溶液反应 10分钟， 生理盐水洗涤， 55mM柠檬酸钠液化后， 生理盐水洗涤， 制 备成 ACC 微胶囊。
3 ) 制备成的 ACC ^A微胶囊与与表观粘度 400cp (25 °C ) 聚乙二醇 （100g/L) 溶液按照 1 : 2的体积比混合后， 用于糖尿病大鼠模型的细胞治疗， 通过腹腔移植， 大鼠血糖水平在移植后 1天即恢复正常水平， 糖尿病症状得以显著改善， 体内移植 6 个月后回收， 发现微胶囊完整， 微胶囊表面光滑， 表面无纤维化包裹现象， 且微囊内 胰岛细胞保持胰岛素双硫腙染色阳性。

Claims

权 利 要 求 书
1、 海藻酸盐 -壳聚糖酰基衍生物微胶囊制剂， 包括海藻酸盐 -壳聚糖酰基衍生物 微胶囊、 或由海藻酸盐 -壳聚糖酰基衍生物微胶囊和水溶液混合而成， 其中：
生物微胶囊结构分为微胶囊膜与内核两部分： 微胶囊膜由壳聚糖、海藻酸盐、 壳 聚糖酰基衍生物形成的聚电解质复合水凝胶膜，内核为含有细胞的海藻酸盐液体或水 凝胶环境。
2、 按照权利要求 1所述的生物微胶囊制剂， 其特征在于： 产品中的微胶囊为粒 径 10-2000微米的球形微胶囊； 膜厚度在 0.1-100微米， 组成膜的海藻酸盐分子量 10 kDa〜2000 kDa， 壳聚糖材料的脱乙酰度 70-98%， 分子量为 1 kDa〜500 kDa， 壳聚糖 酰基衍生物分子量 lkDa〜800kDa， 壳聚糖、 海藻酸盐、 壳聚糖酰基衍生物三者质量 比为 0:1 :0.1-10:1 :10; 内核中海藻酸盐浓度在 0.1-50g/L。
3、 按照权利要求 1或 2所述的生物微胶囊制剂， 其特征在于： 产品中微胶囊的 壳聚糖酰基衍生物为 N-酰基化壳聚糖， 其单体结构式为：
Figure imgf000009_0001
其中， -R代表甲酰、乙酰、丙酰、丁酰、戊酰或己酰，酰基衍生物的取代度 10-60%， 壳聚糖骨架材料的分子量为 1-400 KDa， 脱乙酰度为 90-98%。
4、 按照权利要求 1或 2所述的生物微胶囊制剂， 其特征在于： 产品中微胶囊膜 成分中的海藻酸盐为海藻酸的钾盐或钠盐。
5、 按照权利要求 1或 2所述的生物微胶囊制剂， 其特征在于： 产品中微胶囊内 核中海藻酸盐凝胶为二价金属钙、钡或锌中的一种或二种以上的海藻酸盐水凝胶，海 藻酸盐液体为海藻酸的钾盐或钠盐溶液。
6、 按照权利要求 1所述的生物微胶囊制剂， 其特征在于： 产品中生物微胶囊与 水溶液的体积比为 10: 1-1 :100, 其中水溶液为生理盐水、 HEPES 溶液、 表观粘度 5-2000cp (25 °C ) 的透明质酸溶液、 表观粘度 5-2000cp (25 °C ) 的海藻酸钠溶液、 表 观粘度 5-2000cp (25 °C ) 的葡聚糖溶液、 表观粘度 5-2000cp (25 °C ) 的丙三醇溶液、 表观粘度 5-2000cp (25 °C ) 的聚乙烯乙二醇溶液、 表观粘度 5-2000cp (25 °C ) 的聚乙 烯吡咯烷酮溶液、表观粘度 5-2000cp (25°C )的纤维素衍生物溶液、表观粘度 5-2000cp
(25 °C )的环糊精溶液、表观粘度 5-2000cp (25°C )的淀粉溶液或表观粘度 5-2000cp (25°C ) 的淀粉衍生物溶液中的一种或二种以上的混合物。
7、 一种权利要求 1所述生物微胶囊制剂的制备方法， 其特征在于： 微胶囊膜由 壳聚糖、海藻酸盐、 壳聚糖酰基衍生物通过聚电解质络合反应形成水凝胶膜， 产品的 制备步骤为： 在无菌条件下，
1 ) 制备包埋有活细胞的海藻酸盐凝胶微球， 称之为 A微球；
2) 将步骤 1 ) 中的 A微球浸入壳聚糖溶液中， A微球与壳聚糖溶液体积比为 1 :1-1 :40, 反应 1-60分钟， 此时得到海藻酸钠-壳聚糖微胶囊， 称之为 B微球， 取出 用生理盐水洗涤；
壳聚糖溶液的配制方法是： 壳聚糖溶于 pH为 5.5-7.0的醋酸-醋酸钠缓冲液， 壳 聚糖浓度为 0.1-15 g/L;
3 )将步骤 2)中的 B微球浸入碱金属海藻酸盐溶液中 (海藻酸盐浓度为 0.1-5 g/L),
B微球与碱金属海藻酸盐溶液体积比范围为 1 : 1-1： 40， 反应 1-60分钟， 此时得到 的微胶囊为 C微球， 取出用生理盐水洗涤；
4)交替重复步骤 2)和步骤 3 ) 的过程 1-5次， 此时得到的微胶囊为 D微球， 取 出用生理盐水洗涤；
5 ) 将步骤 1 ) 或 2) 或 3 ) 或 4) 中的 A或 B或 C或 D微球浸入到壳聚糖酰基 衍生物溶液中， 微球与壳聚糖酰基衍生物溶液体积比范围为 1 : 1-1： 40， 反应 1-60 分钟， 此时得到内部凝胶核心的微胶囊， 称之为 E微球， 取出用生理盐水洗涤； 壳聚糖酰基衍生物的配制方法是： 壳聚糖酰基衍生物溶于生理盐水、 HEPES 缓 冲液、 PBS缓冲液， 或 pH为 5.5-7.0的醋酸-醋酸钠缓冲液， 壳聚糖酰基衍生物浓度 为 0. 1-20 g/L;
6)将步骤 5 )中的 E微球浸入到碱金属海藻酸盐溶液中， 重复步骤 3 )， 得到表 面中和的内部凝胶核心的微胶囊为 F微球；
7) 将步骤 6) 中的 F微球浸入有机金属螯合剂溶液中， 液化微胶囊内部的海藻 酸盐凝胶， F微球与有机金属螯合剂溶液体积比范围为 1 : 1-1： 40， 反应 1-60分钟， 取出用生理盐水洗涤， 此时得到内部液态核心的微胶囊为 G微球；
8) 将步骤 5 ) 或 6) 或 7) 得到的 E或 F或 G微球与权利要求 6中的水溶液混 合后， 即制备成海藻酸盐-壳聚糖酰基衍生物微胶囊制剂。
8. 按照权利要求 7所述微胶囊的制备方法， 其特征在于： 海藻酸盐凝胶微球为 二价金属钙、 钡或锌中的一种或二种以上的海藻酸盐水凝胶；
步骤 3 ) 和 6) 用于中和表面电荷的碱金属海藻酸盐为钾盐或钠盐， 分子量分布 为 10KDa〜2000KDa， 海藻酸盐浓度为 0.1-5g/L。
9. 按照权利要求 7所述微胶囊的制备方法， 其特征在于： 参与液化反应的有机 金属螯合剂溶液为 40-70mmol/L的柠檬酸钠或 50-200mmol/L的 EDTA。
10. 一种权利要求 1所述的应用， 其特征在于： 产品中微胶囊用于细胞的包埋。
11. 按照权利要求 10所述的应用， 其特征在于： 所述细胞为人或哺乳动物来源 的离体的胰岛细胞、 肝细胞、 甲状腺细胞、 甲状旁腺细胞、 肾上腺髓质细胞具有分泌 生物活性物质功能的细胞， 细胞系细胞， 基因工程细胞， 干细胞或干细胞分化的各种 细胞。
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EP2659883A4 (en) 2014-09-03
AU2011335563A1 (en) 2013-06-27
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