CN102101036A - Alginate-epsilon-polylysine microcapsules and preparation and application thereof - Google Patents
Alginate-epsilon-polylysine microcapsules and preparation and application thereof Download PDFInfo
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- CN102101036A CN102101036A CN2010105401202A CN201010540120A CN102101036A CN 102101036 A CN102101036 A CN 102101036A CN 2010105401202 A CN2010105401202 A CN 2010105401202A CN 201010540120 A CN201010540120 A CN 201010540120A CN 102101036 A CN102101036 A CN 102101036A
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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
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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
- A61K9/5052—Proteins, e.g. albumin
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/10—Complex coacervation, i.e. interaction of oppositely charged particles
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- 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
- A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
- A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
Abstract
The invention relates to a bioactive substance included alginate microcapsule product. The invention is characterized in that: the microcapsule product is a spherical microcapsule with diameter of 100 to 1,000 microns, and the structure of the microcapsule product comprises a microcapsule film and a core, wherein the microcapsule film is a polyelectrolyte composite hydrogel film formed by electrostatic complexing reaction of two macromolecular materials, namely alginate and epsilon-polylysine; and the core is a liquid or hydrogel environment containing a bioactive substance. The microcapsule product is mainly applied to cell transplantation, cell culture and included carriers of bioactive substances such as protein, polypeptide, enzyme, nucleic acid and the like.
Description
Technical field
The present invention relates to a kind of microcapsule product, specifically a kind of alginate-epsilon-polylysine microcapsule product that is used for the bioactivator embedding.
Background technology
The sixties in 20th century, Chang has reported the pellicle microcapsules, points out with bioactivator and cells such as its embedding protein, enzymes, can keep biological substance activity [Chang TMS.Semipermeable microcapsules, Science, 1964,146:524-525].Early 1980s, Lim and Sun are at tissue/cell functional impairment disease (as diabetes), sodium alginate/α-polylysine (pellicle microcapsules of alginate/ α-polylysine) (being called for short α-APA microcapsules) have successfully been prepared, seal the Wistar rat Islet cells and be implanted in the diabetes WistarLewis rat body, the secretion uelralante is with regulating and controlling blood sugar amount [Lim F, Sun A M.Microencapsulated islets bioartificial endocrine pancreas, Science, 1980,210:908-910].Promoted the fast development of microencapsulation technology associated materials and preparation method's research thus, [Wang W is used widely in the preclinical study of biomedical sectors such as cell transplantation, medicine release and gene therapy, Liu XD, Ma XJ, et al.Microencapsulation using natural polysaccharides for drug delivery and cell implantation, J.Mater.Chem., 2006,16:3252-3267].In numerous bio-microcapsules, sodium alginate/polylysine microcapsules use more, its microcapsule membrane material is the synthetic α-polylysine (amido link between the lysine residue is to be formed by alpha-amido and α-carboxyl condensation) of chemical method, but because α-polylysine costs an arm and a leg (300-400US$/g), the intrinsic biocompatibility of material is poor in addition, has certain toxicity, this has greatly limited sodium alginate/α-polylysine microcapsules application [Strand B L clinically, Ryan TL, Veld P I, et al.Cell Transplant., 2001,10:263-275].Japanese scholar Shima in 1977 and Sakai prepare a kind of polymer of being made up of a plurality of lysine residue homotype monomers through the fermentation streptomyces albus, amido link is to be formed by connecting by α-carboxyl and epsilon-amino, so be called epsilon-polylysine ([the Shoji shima. of ε-PLL), Polylysine produced by Streptomyces.Agric Biol Chem., 1977,41:1807-1809].As a kind of natural microbial metabolism product, its cost is low, environmental friendliness, can be decomposed into lysine in vivo and digested and assimilated fully, without any toxic and side effect [Hiraki J, Ichikawa T, Ninomiya S, et al.Use of ADME studies to confirm the safety of epsilon-polylysine as a preservative in food.Regulatory Toxicology and Pharmacology, 2003,37:328-340], authenticated (the CAS registration number is 28211-04-3) by U.S. FDA in 2004.Because epsilon-polylysine has high molecular characteristic, there are a large amount of primary amino radicals in the strand, amino protonated in solution, having a large amount of positive charges becomes cationic polymer, can form alginate/epsilon-polylysine compound under electrostatic interaction with the polyanion alginate.Substitute α-polylysine preparation with epsilon-polylysine and be used for the microcapsule product of bioactivator embeddings such as cell, protein, enzyme or nucleic acid, the application of carrying out biomedical sector aspects such as cell transplantation, cell cultivation, medicine release still belongs to blank.
Summary of the invention
At the problems referred to above, the present invention proposes alginate-epsilon-polylysine microcapsules and preparation and application.
Technical scheme:
The present invention adopts epsilon-polylysine to substitute the preparation that α-polylysine is used for bio-microcapsule, reach the equal performance of α-polylysine bio-microcapsule, a kind of novel alginate that is used for the bioactivator embedding-epsilon-polylysine microcapsule product has been proposed, microcapsule structure is divided into microcapsule membrane and kernel two parts: wherein, and the polyelectrolyte compound water congealing glued membrane that microcapsule membrane is formed by alginate, epsilon-polylysine; Kernel is alginate liquid or the alginate hydrogel environment that contains bioactivator.
Microcapsule product among the present invention is the spherical microcapsule of particle diameter 100-1000 micron; Film thickness is at the 1-100 micron, and the alginate molecular weight 10kDa～2000kDa of component film (for example: 50kDa-200kDa; 200kDa-500kDa; 600kDa-1000kDa; 1000kDa-2000kDa), the molecular weight of epsilon-polylysine is at 1kDa～500kDa, (for example: 1kDa-50kDa; 10kDa-100kDa; 120kDa-300kDa; 35kDa-500kDa), epsilon-polylysine and sodium alginate mass ratio are 0.1-10; Alginate concentration is at 1-50g/L in the kernel, and to occupy the percent by volume of kernel be 10-98% to bioactivator in the kernel, and actively keeps more than 90%.
Epsilon-polylysine in the microcapsule product is high polymerization polyvalent cation homopolymers, and molecular weight is 1KDa～500KDa, and amido link is to be formed by connecting by α-carboxyl and epsilon-amino, and its structural formula is:
Wherein n is 5-3500.
Alginate in the film component of microcapsule product is the sylvite or the sodium salt of alginic acid.
The alginate gel is the alginate hydrogel of divalent metal calcium, barium or zinc in the kernel of microcapsule product, and alginate solution is the sylvite or the sodium salt solution of alginic acid.
Microcapsule membrane forms aquagel membrane by alginate, two kinds of macromolecular materials of epsilon-polylysine by the polyelectrolyte complex reaction in the above-mentioned microcapsule product, and the preparation method of microcapsule product comprises one-step method or two-step method;
Wherein, the preparation process of one-step method is: the alginate solution that will be mixed with bioactivator joins in the mixed liquor of bivalent cation and epsilon-polylysine in the mode of drop, is prepared into the ε-AP microcapsules of gel state;
The preparation process of two-step method is:
1) preparation is embedded with the alginate gel micro-ball of bioactivator, is referred to as the A microballoon;
2) the A microballoon in the step 1) is added in the epsilon-polylysine solution, the coated film that epsilon-polylysine is used for the alginate capsule slowly-releasing, the A microballoon is 1: 1～1: 40 with the epsilon-polylysine liquor capacity than scope, reacted 1-60 minute, reaction temperature obtains ε-AP microcapsules taking-up this moment and washs with physiological saline at 0-37 ℃.
With step 2) or ε-AP microcapsules of one-step method preparation immerse in the alkali metal alginate solution, alkali metal alginate anion and ε-remaining unreacted amino cationoid reaction in AP microcapsule membrane surface, ε-AP microcapsules are 1: 1～1: 40 with the alkali metal alginate liquor capacity than scope, reacted 1-60 minute, reaction temperature is at 0-37 ℃, obtain ε-APA microcapsules this moment, take out and wash with physiological saline.
ε-AP the microcapsules or the ε-APA microcapsules of two-step method or one-step method preparation are immersed in the organic metal chelating agent solution, the alginate gel of liquefaction microcapsules inside, ε-APA microcapsules and organic metal chelating agent solution volume ratio scope are 1: 1～1: 40, reacted 1-60 minute, reaction temperature is at 0-37 ℃, taking-up is washed with physiological saline, obtains the ε-AP or the ε-APA microcapsules of interior liquid core this moment.
Wherein, the epsilon-polylysine molecular weight that participates in film formation reaction is 1KDa～500KDa, the compound method of solution is: epsilon-polylysine is dissolved in physiological saline, HEPES buffer solution or the PBS buffer solution physiological buffer system that pH is 5.5-7.4, and epsilon-polylysine concentration is 0.1-50g/L.
The alginate gel micro-ball is one or two or more kinds alginate hydrogel in divalent metal calcium, barium or the zinc;
Be used for and the alkali metal alginate of surface charge is sylvite or sodium salt, molecular weight distribution is 10KDa～2000KDa, and alginate concentration is 0.1-5g/L.
The organic metal chelating agent solution that participates in liquefaction reaction is the natrium citricum of 40-70mmol/L or the EDTA of 50-200mmol/L.
Microcapsule product of the present invention is used for the embedding of bioactivators such as cell, medicine, enzyme or nucleic acid.
Wherein, cell is behaved or the islet cells, liver cell, thyroid cell, parathyroid gland cell, the adrenal medullary cell that exsomatize in mammal (mouse, ox, pig, dog etc.) source have the cell of secreting the bioactivator function, cell line cell, genetically engineered cell, the various cells of stem cell or stem cell differentiation.
Medicine is elemene, vitamin E, evening primrose wet goods oil-soluble small-molecule drug, or protein and peptide drugs such as interferon, endostatin, EPO, insulin, growth factor.
Enzyme is industrial enzymes products such as lipase, amylase, dehydrogenase.
Nucleic acid is the nucleic acid drug that DNA, siRNA etc. have the gene therapy effect.
Beneficial effect of the present invention:
1, compares with traditional sodium alginate/α-polylysine/sodium alginate micro gel capsule (α-APA microcapsules), this novel alga hydrochlorate of the present invention-epsilon-polylysine microcapsule product, the surface roughness of its microcapsule membrane significantly is lower than α-APA microcapsules, demonstrates better biocompatibility.
2, compare with traditional sodium alginate/α-polylysine/sodium alginate micro gel capsule (α-APA microcapsules), epsilon-polylysine of the present invention (ε-PLL) be mostly to obtain by microbial fermentation, cost is low, environmental friendliness, the degradation in vivo product is a lysine and being digested and assimilated fully, without any toxic and side effect, safe in utilization.By contrast, α-polylysine that traditional microcapsules prepare usefulness is that chemical synthesis obtains, and cost exceeds 600 times than epsilon-polylysine.
3, the microcapsule membrane of product of the present invention has superior film-strength, can guarantee the integrality as film in histocyte transplanting, cell cultivation, the pharmaceutical carrier application process.
4, the microcapsule membrane of product of the present invention has superior function of immune isolation, when being used for heteroplasm's cell transplantation, can keep function of immune isolation, be that the cell of embedding can not go out microcapsules in the micro-capsule, outer antibody molecule, complement molecule, the immunocyte of micro-capsule can not enter cell killing in the microcapsules, and the active component of the secretion of cellular metabolism simultaneously can free in and out microcapsules.
5, used alginate microcapsules of the present invention and epsilon-polylysine solution and alginate solution carry out self assembly by electrostatic interaction, the reaction condition gentleness, and the activity that helps cell/protein/enzyme/life active compounds such as nucleic acid keeps.
Description of drawings
Fig. 1 is the cellular morphology optical photograph when cultivating Chinese hamster ovary celI in ε among the embodiment 1-APA microcapsules; (scale is 100 μ m among the figure);
Fig. 2 is the cellular morphology optical photograph when cultivating Chinese hamster ovary celI in α in the comparative example 1-APA microcapsules; (scale is 100 μ m among the figure).
The specific embodiment
The mode that forms the alginate drop is a static sessile drop method (list of references: In Vivo Culture of Encapsulated Endostatin-Secreting Chinese Hamster Ovary Cells for Systemic Tumor Inhibition.Human Gene Therapy.2007,18:474-481), orifice extrusion molding (list of references: a kind of preparation method of microsphere open-top bait for high-economic fish, Chinese invention patent, 200510136769.7), emulsification-outside gelling technique (list of references: Preparation of lactic acid bacteria-enclosing alginate beads in emulsion system:effect of preparation parameters on bead characteristics, Polym.Bull., 2009,63:599-607), (list of references: emulsification-internal gelation technology prepares the fixed yeast microcapsules in emulsification-internal gelation method, the chemical industry journal, 2009,60 (3): 710-717) or film emulsion process (list of references: Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with interna
Embodiment 1
1) utilizes high-pressure electrostatic method [Qu Beibei, Yu Weiting, Liu hole etc. of tucking inside the sleeve, the high-pressure electrostatic legal system is equipped with the calcium alginate micro gel bead of monodispersity, chemical industry journal, 2005,56 (8): 1547] preparation is embedded with the calcium alginate gel bead of Chinese hamster ovary celI, and cell initial inoculation density is 1 * 10
6/ mL sodium alginate soln.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.01% (w/v), pH5.5 is in epsilon-polylysine/normal saline solution, the CHO/ calcium alginate gel bead is 1: 40 with ε-PLL liquor capacity ratio, reacted 60 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules being immersed concentration is in sodium alginate/0.9% (w/v) the NaCl solution of 0.05% (w/v), CHO/ ε-AP microcapsules and sodium alginate soln volume ratio are 1: 40, reacted 10 minutes, and obtained ε-APA microcapsules, physiological saline washing 3 times.
4) the above-mentioned ε that makes-APA microcapsules are immersed in the sodium citrate solution of 55mmol/L, CHO/ ε-APA microcapsules and sodium citrate solution volume ratio are 1: 10, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside, be prepared into the ε-APA microencapsulation Chinese hamster ovary celI (Fig. 1) of interior liquid core, the microcapsules good sphericity, smooth surface.
5) microencapsulated cell is cultivated in the CO2 incubator, condition of culture list of references [Optimization of the Seeding Density in Microencapsulated Recombinant CHO Cell Culture.Ying Zhang, Jing Zhou, Xulang Zhang, et.al., Chemical and Biochemical Engineering Quarterly, 2008,22 (1): 105-111].
6) detect Chinese hamster ovary celI growing state in ε-APA microcapsules with mtt assay, find the cell well-grown, suitable with comparative example 1 effect.
Embodiment 2
1) preparation is embedded with hepatocellular calcium alginate gel bead, and cell initial inoculation density is 1 * 10
6/ mL sodium alginate soln.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.1% (w/v), pH6.5 is in epsilon-polylysine/normal saline solution, liver cell/calcium alginate gel bead is 1: 20 with ε-PLL liquor capacity ratio, reacted 15 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules being immersed concentration is in sodium alginate/0.9% (w/v) the NaCl solution of 0.1% (w/v), liver cell/ε-AP microcapsules and sodium alginate soln volume ratio are 1: 20, reacted 15 minutes, and obtained ε-APA microcapsules, physiological saline washing 3 times.
4) the above-mentioned ε that makes-APA microcapsules are immersed in the sodium citrate solution of 55mmol/L, HepG2/ ε-APA microcapsules and sodium citrate solution volume ratio are 1: 10, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the ε-APA microencapsulated hepatocyte cell of interior liquid core.
5) [the microencapsulation human liver cell is transplanted the therapeutic action to the chmice acute hepatic failure, Wang Qiuyan, Li Shuanyue to the preparation list of references of hepatic failure model mouse, Liu Jing, Yang Jingxian, the king is Ma Xiaojun, Yuan Quan, China's clinical rehabilitation, 2006,10:54-57], the glutamic-oxalacetic transaminease 590nkat/L of modeling mouse, glutamic-pyruvic transaminase 890nkat/L.
6) above-mentioned preparation is embedded with hepatocellular sodium alginate-epsilon-polylysine microcapsule product is used for hepatic failure model by intraperitoneal transplantation treatment, the result shows, transplant after 4 days, decline glutamic-pyruvic transaminase, the glutamic-oxalacetic transaminease level of mouse of liver all returns to normal level, and the Mouse Liver symptom that declines is significantly improved.
Embodiment 3
1) preparation is embedded with the calcium alginate gel bead of embryonic stem cell (ES), and cell initial inoculation density is 1 * 10
7/ mL sodium alginate soln.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.2% (w/v), pH7.4 is in epsilon-polylysine/normal saline solution, the ES/ calcium alginate gel bead is 1: 5 with ε-PLL liquor capacity ratio, reacted 15 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules being immersed concentration is in sodium alginate/0.9% (w/v) the NaCl solution of 0.2% (w/v), ES/ ε-AP microcapsules and sodium alginate soln volume ratio are 1: 5, reacted 10 minutes, and obtained ε-APA microcapsules, physiological saline washing 3 times.
4) the above-mentioned ε that makes-APA microcapsules are immersed in the sodium citrate solution of 55mmol/L, ES/ ε-APA microcapsules and sodium citrate solution volume ratio are 1: 10, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the ε-APA microencapsulation ES cell of interior liquid core.
5) condition of culture reference literature [Proliferation and Differentiation of Mouse Embryonic Stem Cells in APA Microcapsule:a model for studying the interaction between Stem cells and their niche, Xiuli Wang, Wei Wang, Juan Ma, et al., Biotechnology Progress, 2006,22 (3): 791-800], embryonic stem cell is had significant proliferation in micro-capsule, reaches 1 * 10
8/ mL microcapsules.
Embodiment 4
1) preparation is embedded with the calcium alginate gel bead of islet cells.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.2% (w/v), pH6.5 is in epsilon-polylysine/normal saline solution, pancreas islet/calcium alginate gel bead is 1: 10 with ε-PLL liquor capacity ratio, reacted 15 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules being immersed concentration is that pancreas islet/ε-AP microcapsules and sodium alginate soln volume ratio are 1: 10 in sodium alginate/HEPES solution of 0.1% (w/v), reacts 15 minutes, obtains ε-APA microcapsules, physiological saline washing 3 times.
4) the above-mentioned ε that makes-APA microcapsules are immersed in the sodium citrate solution of 55mmol/L, pancreas islet/ε-APA microcapsules and sodium citrate solution volume ratio are 1: 10, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the ε-APA microencapsulation islet cells of interior liquid core.
5) sodium alginate that is embedded with islet cells of above-mentioned preparation-epsilon-polylysine microcapsule product immerses in the FITC-IgG solution, and laser confocal scanning microscope is observed in the micro-capsule does not have fluorescently-labeled antibody (FITC-IgG) molecular diffusion people microcapsules.
6) sodium alginate that the is embedded with islet cells-epsilon-polylysine microcapsule product of above-mentioned preparation is used for the cell therapy of diabetes rat model, pass through intraperitoneal transplantation, the rat blood sugar level was promptly recovered normal level in back 1 day in transplanting, diabetic symptom is able to remarkable improvement, transplant after 6 months in the body and reclaim, the discovery microcapsules are complete, the no obvious cell infiltration in surface, and islet cells still keeps good excreting insulin function in the micro-capsule.
Embodiment 5
1) preparation is embedded with the calcium alginate gel bead of bacillus coli DH 5 alpha, and cell initial inoculation density is 5 * 10
6/ mL sodium alginate soln.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.5% (w/v), pH7.0 is in epsilon-polylysine/HEPES solution, Escherichia coli/calcium alginate gel bead is 1: 3 with ε-PLL liquor capacity ratio, reacted 15 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules being immersed concentration is in sodium alginate/0.9% (w/v) the NaCl solution of 0.2% (w/v), Escherichia coli/ε-AP microcapsules and sodium alginate soln volume ratio are 1: 10, reacted 15 minutes, and obtained ε-APA microcapsules, physiological saline washing 3 times.
4) the above-mentioned ε that makes-APA microcapsules are immersed in the EDTA solution of 100mmol/L, Escherichia coli/ε-APA microcapsules are 1: 10 with EDTA liquor capacity ratio, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the ε-APA microencapsulation Escherichia coli of interior liquid core.
5) with microencapsulation Escherichia coli condition of culture list of references [the colibacillary research of alginate microcapsules immobilization, Fu Yingli, hawk, Liu hole of tucking inside the sleeve, Yu Weiting, Wang Yili, Yu Xingju, Ma Xiaojun, bioengineering journal, 2002,18:239-241]
6) above-mentioned preparation be embedded with the colibacillary High Density Cultivation that colibacillary sodium alginate-epsilon-polylysine microcapsule product is used for recombinant epo, be used for the production of high value biochemical drug EPO.
Embodiment 6
1) preparation is embedded with the calcium alginate gel bead of plant cell-yew cell, and cell initial inoculation density is 1 * 10
6/ mL sodium alginate soln.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.5% (w/v), pH7.4 is in epsilon-polylysine/PBS solution, yew cell/calcium alginate gel bead is 1: 5 with ε-PLL liquor capacity ratio, reacted 1 minute, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules being immersed concentration is in sodium alginate (Mw500KDa)/0.9% (w/v) the NaCl solution of 0.5% (w/v), yew cell/ε-AP microcapsules and sodium alginate soln volume ratio are 1: 5, reacted 1 minute, obtain ε-APA microcapsules, physiological saline washing 3 times.
4) under 37 ℃ of temperature conditions, the above-mentioned ε that makes-APA microcapsules are immersed in the EDTA solution of 55mmol/L, yew cell/ε-APA microcapsules and sodium citrate solution volume ratio are 1: 10, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the ε-APA microencapsulation yew cell of interior liquid core.
5) sodium alginate that the is embedded with yew cell-epsilon-polylysine microcapsule product of above-mentioned preparation is used for the production of culture plant cell and the purple triol of high value biochemical drug.
Embodiment 7
1) utilizes emulsion process [X.D.Liu, D.C.Bao, W.M.Xue, et al., Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal g
2) under 25 ℃ of temperature conditions, the above-mentioned calcium alginate gel bead that makes is immersed 1% (w/v), pH6.5, in epsilon-polylysine/HEPES solution, the BSA/ calcium alginate gel bead is 1: 3 with ε-PLL liquor capacity ratio, reacted 10 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) under 25 ℃ of temperature conditions, the above-mentioned ε that makes-AP microcapsules are immersed in potassium alginate/0.45% (w/v) the NaCl solution of concentration 0.1% (w/v), BSA/ ε-AP microcapsules are 1: 10 with potassium alginate liquor capacity ratio, reacted 10 minutes, obtain ε-APA microcapsules, physiological saline washing 3 times.
4) envelop rate of BSA/ ε-APA microcapsules is near 90%, and in-vitro release rate and medicine carrying amount are proportionate, and is negative correlation with the crosslinking curing degree of ε-PLL, and is suitable with comparative example 2 effects.
Embodiment 8
1) utilizes film emulsion process [X.D.Liu, D.C.Bao, W.M.Xue, et al., Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal gelation, Journal of Applied Polymer Science, 2003,87 (5): 848-852] preparation is embedded with the alginic acid gel of zinc microballoon of dna vaccination, dna vaccination/PBS solution and sodium alginate soln (1.5%w/v) volume ratio are 1: 5, mix as water; Corn oil is oil phase, 10%ZnAc
2Be glue crosslinking agent, carried out gelation reaction 10 minutes.
2) the above-mentioned alginic acid gel of zinc microballoon that makes is immersed 5% (w/v), pH7.4 is in epsilon-polylysine/PBS solution, DNA/ alginic acid gel of zinc microballoon is 1: 1 with ε-PLL liquor capacity ratio, reacted 5 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules are immersed in sodium alginate/0.25% (w/v) the NaCl solution of concentration 0.25% (w/v), DNA/ ε-AP microcapsules and sodium alginate soln volume ratio are 1: 15, reacted 5 minutes, and obtained ε-APA microcapsules, physiological saline washing 3 times.
4) the microencapsulated rate of DNA/ ε-APA can reach 70%; In-vitro release rate and medicine carrying amount are proportionate, and are negative correlation with the crosslinking curing degree of ε-PLL, and be suitable with comparative example 3 effects.
Embodiment 9
1) at sessile drop method [matsumoto S., Kobayashi H., Takahima Y., Prodution of monodispersed capsules.J.Microencapsulation, 1986,3,25-31] preparation is embedded with the alginic acid barium gel micro-ball of beta-glucosidase, and carried out gelation reaction 30 minutes.(glucuroide and alginate mass ratio 1: 6)
2) the above-mentioned alginic acid barium gel micro-ball that makes is immersed 3% (w/v), pH5.5 is in epsilon-polylysine/HEPES solution, beta-glucosidase/alginic acid barium gel micro-ball is 1: 2 with ε-PLL liquor capacity ratio, reacted 1 minute, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules are immersed in potassium alginate/0.1% (w/v) the NaCl solution of concentration 0.5% (w/v), beta-glucosidase/ε-AP microcapsules are 1: 5 with potassium alginate liquor capacity ratio, reacted 1 minute, and obtained ε-APA microcapsules, physiological saline washing 3 times.
4) in the synthesizing alkyl polyglycoside catalyzed reaction of ε-APA microencapsulation beta-glucosidase in catalytic capability and the comparative example 4 effect suitable.
Embodiment 10
1) utilize emulsification-internal gelation preparation to be embedded with saccharomycetic calcium alginate gel bead, cell initial inoculation density is 5 * 10
6/ mL sodium alginate soln.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.25% (w/v), pH7.0 is in epsilon-polylysine/HEPES solution, saccharomycete/calcium alginate gel bead is 1: 15 with ε-PLL liquor capacity ratio, reacted 15 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned ε that makes-AP microcapsules are immersed in the sodium citrate solution of 55mmol/L, ε-AP microcapsules and sodium citrate solution volume ratio are 1: 8, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the ε-AP microencapsulation saccharomycete of interior liquid core.
4) the saccharomycetic sodium alginate of being embedded with of above-mentioned preparation-epsilon-polylysine microcapsule product is used for the production of high value biochemical drug.
Embodiment 11
1) preparation is embedded with the calcium alginate gel bead of elemene.
2) under 25 ℃ of temperature conditions, the above-mentioned calcium alginate gel bead that makes is immersed 1% (w/v), pH6.5, in epsilon-polylysine/HEPES solution, elemene/calcium alginate gel bead is 1: 3 with ε-PLL liquor capacity ratio, reacted 10 minutes, and made ε-AP microcapsules, then with physiological saline washing 3 times.
3) envelop rate of elemene/ε-AP microcapsules is near 90%, and in-vitro release rate and medicine carrying amount are proportionate.
4) sodium alginate that the is embedded with elemene-epsilon-polylysine microcapsule product of above-mentioned preparation is used for the tumor post-operation localized pulverization and prevents tumor recurrence.
Embodiment 12
1) the small-molecule drug vitamin E is mixed according to volume ratio with sodium alginate soln at 1: 5.
2) above-mentioned mixed liquor is ejected into CaCl by the orifice extrusion molding
2In the mixed liquor of epsilon-polylysine, one-step method is prepared into the sodium alginate-epsilon-polylysine microcapsules that are embedded with vitamin E, wherein, and CaCl
2With CaCl in the mixed liquor of epsilon-polylysine
2Concentration is 3%, and epsilon-polylysine concentration is 5%.
3) the microencapsulated rate of the sodium alginate-epsilon-polylysine that is embedded with vitamin E that is prepared into can realize the slowly-releasing of vitamin E greater than 90%, is released to zero level and discharges.
Embodiment 13
The calcium alginate flat sheet membrane for preparing is immersed in respectively in α-polylysine and the epsilon-polylysine solution, and taking-up is immersed in the sodium alginate soln after cleaning again, prepares α-APA film and ε-APA film respectively.And measuring the surface roughness of two kinds of films with surperficial white light instrument, the result shows: α-PLL film=161.59 ± 57.11; δ-PLL film=43.244 ± 18.71.The roughness of epsilon-polylysine film is starkly lower than α-polylysine film (method list of references: Langmiur, 2010,26 (8): 5587-5594).
Comparative example 1
1) utilizes high-pressure electrostatic method [Qu Beibei, Yu Weiting, Liu hole etc. of tucking inside the sleeve, the high-pressure electrostatic legal system is equipped with the calcium alginate micro gel bead of monodispersity, chemical industry journal, 2005,56 (8): 1547] preparation is embedded with the calcium alginate gel bead of Chinese hamster ovary celI, and carries out gelation reaction 30 minutes.(concentration of cell is 1 * 10
6/ mL)
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.01% (w/v), pH7.0 is in α-polylysine/normal saline solution, the CHO/ calcium alginate gel bead is 1: 20 with α-PLL liquor capacity ratio, reacted 20 minutes, and made α-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned α that makes-AP microcapsules being immersed concentration is in sodium alginate/0.9% (w/v) the NaCl solution of 0.01% (w/v), CHO/ α-AP microcapsules and sodium alginate soln volume ratio are 1: 20, reacted 20 minutes, and obtained α-APA microcapsules, 3 times (Fig. 1) of physiological saline washing.
4) the above-mentioned α that makes-APA microcapsules are immersed in the sodium citrate solution of 55mmol/L, CHO/ α-APA microcapsules and sodium citrate solution volume ratio are 1: 5, reacted 5 minutes, the calcium alginate gel of liquefaction microcapsules inside is prepared into the α-APA microcapsules (Fig. 2) of interior liquid core.
5) microencapsulated cell is cultivated according to embodiment 1 condition, detected Chinese hamster ovary celI growing state in α-APA microcapsules, find the cell well-grown with mtt assay.
Comparative example 2
1) utilizes emulsion process [X.D.Liu, D.C.Bao, W.M.Xue, et al., Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal gelation, Journal of Applied Polymer Science, 2003,87 (5): 848-852] preparation is embedded with the calcium alginate gel bead of bovine serum albumin(BSA) (BSA), and (bovine serum albumin(BSA) and alginate solution mix (mass ratio 1: 6) as water; Corn oil is oil phase, 15%CaAc
2Be glue crosslinking agent, carried out gelation reaction 10 minutes.
2) the above-mentioned calcium alginate gel bead that makes is immersed 0.1% (w/v), pH6.5 is in α-polylysine/HEPES solution, the BSA/ calcium alginate gel bead is 1: 10 with α-PLL liquor capacity ratio, reacted 10 minutes, and made α-AP microcapsules, then with physiological saline washing 3 times.
3) under 25 ℃ of temperature conditions, the above-mentioned α that makes-AP microcapsules are immersed in potassium alginate/0.45% (w/v) the NaCl solution of concentration 0.1% (w/v), BSA/ α-AP microcapsules are 1: 10 with potassium alginate liquor capacity ratio, reacted 10 minutes, obtain α-APA microcapsules, physiological saline washing 3 times.
4) in-vitro release rate of BSA/ α-APA microcapsules and medicine carrying amount are proportionate, and are negative correlation with the crosslinking curing degree of α-PLL, meet one-level release dynamics equation.
Comparative example 3
1) utilizes emulsion process [X.D.Liu, D.C.Bao, W.M.Xue, et al., Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal gelation, Journal of Applied Polymer Science, 2003,87 (5): 848-852] preparation is embedded with the alginic acid zinc microcapsules of dna vaccination, dna vaccination PBS solution and sodium alginate soln (1.5%w/v) volume ratio is 1: 5, mix, as water; Corn oil is oil phase, 10%ZnAc
2Be glue crosslinking agent, carried out gelation reaction 10 minutes.
2) the above-mentioned alginic acid zinc microcapsules that make are immersed 0.25% (w/v), pH7.4 is in α-polylysine/PBS solution, DNA/ alginic acid gel of zinc little with α-PLL liquor capacity ratio be 1: 15, reacted 5 minutes, and made α-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned α that makes-AP microcapsules are immersed in sodium alginate/0.25% (w/v) the NaCl solution of concentration 0.25% (w/v), DNA/ α-AP microcapsules and sodium alginate soln volume ratio are 1: 15, reacted 5 minutes, and obtained α-APA microcapsules, physiological saline washing 3 times.
4) DNA/ α-APA microcapsules in-vitro release rate and medicine carrying amount are proportionate, and are negative correlation with the crosslinking curing degree of α-PLL, meet one-level release dynamics equation.
Comparative example 4
1) at sessile drop method [matsumoto S., Kobayashi H., Takahima Y., Prodution of monodispersed capsules.J.Microencapsulation, 1986,3,25-31] preparation is embedded with the alginic acid barium gel micro-ball of beta-glucosidase, and carried out gelation reaction 30 minutes.(glucuroide and alginate mass ratio 1: 6)
2) the above-mentioned alginic acid barium gel micro-ball that makes is immersed 0.5% (w/v), pH5.5, in α-polylysine/HEPES solution, beta-glucosidase/alginic acid barium gel micro-ball is 1: 5 with α-PLL liquor capacity ratio, reacted 1 minute, make α-AP microcapsules, then with physiological saline washing 3 times.
3) the above-mentioned α that makes-AP microcapsules are immersed in potassium alginate/0.1% (w/v) the NaCl solution of concentration 0.5% (w/v), beta-glucosidase/α-AP microcapsules are 1: 5 with potassium alginate liquor capacity ratio, reacted 1 minute, and obtained α-APA microcapsules, physiological saline washing 3 times.
4) synthesizing alkyl polyglycoside catalyzed reaction: reaction substrate is a 1g glucose, 10ml laruyl alcohol, 2ml acetate buffer solution (pH=5); Catalyst is α-APA microencapsulation beta-glucosidase (being converted into pure enzyme 0.01g), but places the dodecyl glucosides of 50 ℃ of water-bath synthesis of high purity.Improved the utilization rate of rate of catalysis reaction and enzyme, catalyst is recyclable.
Claims (13)
1. alginate-epsilon-polylysine microcapsules, it is characterized in that: microcapsule structure is divided into microcapsule membrane and kernel two parts: wherein, the polyelectrolyte compound water congealing glued membrane that microcapsule membrane is formed by alginate, epsilon-polylysine; Kernel is alginate liquid or the alginate hydrogel environment that contains bioactivator.
2. according to the described microcapsules of claim 1, it is characterized in that: microcapsule product is the spherical microcapsule of particle diameter 10-2000 micron; Film thickness is at the 0.1-100 micron, and the alginate molecular weight 10kDa～2000kDa of component film, the molecular weight of epsilon-polylysine are at 1kDa～500kDa, and epsilon-polylysine and sodium alginate mass ratio are 0.1-10; Alginate concentration is at 1-50g/L in the kernel, and to occupy the percent by volume of kernel be 10-98% to bioactivator in the kernel.
3. according to claim 1 or 2 described microcapsules, it is characterized in that: the epsilon-polylysine in the microcapsule product is high polymerization polyvalent cation homopolymers, molecular weight is 1～500KDa, and amido link is to be formed by connecting by α-carboxyl and epsilon-amino, and its structural formula is:
4. according to claim 1 or 2 described microcapsules, it is characterized in that: the alginate in the film component of microcapsule product is the sylvite or the sodium salt of alginic acid.
5. according to claim 1 or 2 described microcapsules, it is characterized in that: the alginate gel is one or two or more kinds the alginate hydrogel in divalent metal calcium, barium or the zinc in the kernel of microcapsule product, and alginate solution is the sylvite or the sodium salt solution of alginic acid.
6. the preparation method of the described microcapsules of claim 1, it is characterized in that: microcapsule membrane forms aquagel membrane by alginate, two kinds of macromolecular materials of epsilon-polylysine by the polyelectrolyte complex reaction, and the preparation method of product comprises one-step method or two-step method;
Wherein, the preparation process of one-step method is: the alginate solution that will be mixed with bioactivator joins in the mixed liquor of bivalent cation and epsilon-polylysine in the mode of drop, is prepared into the ε-AP microcapsules of gel state;
The preparation process of two-step method is:
1) preparation is embedded with the alginate gel micro-ball of bioactivator, is referred to as the A microballoon;
2) the A microballoon in the step 1) is added in the epsilon-polylysine solution, the coated film that epsilon-polylysine is used for the alginate capsule slowly-releasing, the A microballoon is 1: 1～1: 40 with the epsilon-polylysine liquor capacity than scope, reacted 1-60 minute, reaction temperature obtains ε-AP microcapsules taking-up this moment and washs with physiological saline at 0-37 ℃.
7. according to the preparation method of the described microcapsules of claim 6, it is characterized in that:
Will be by the step 2 in the two-step method) or ε-AP microcapsules of one-step method preparation immerse in the alkali metal alginate solution, alkali metal alginate anion and ε-remaining unreacted amino cationoid reaction in AP microcapsule membrane surface, ε-AP microcapsules are 1: 1～1: 40 with the alkali metal alginate liquor capacity than scope, reacted 1-60 minute, reaction temperature is at 0-37 ℃, obtain ε-APA microcapsules this moment, take out and wash with physiological saline.
8. according to the preparation method of claim 6 or 7 described microcapsules, it is characterized in that:
ε-APA the microcapsules that obtain in the ε-AP microcapsules of two-step method in the claim 6 or one-step method preparation or the claim 7 are immersed in the organic metal chelating agent solution, the alginate gel of liquefaction microcapsules inside, ε-APA microcapsules and organic metal chelating agent solution volume ratio scope are 1: 1～1: 40, reacted 1-60 minute, reaction temperature is at 0-37 ℃, taking-up is washed with physiological saline, obtains the ε-AP or the ε-APA microcapsules of interior liquid core this moment.
9. according to the preparation method of the described microcapsules of claim 6, it is characterized in that: the epsilon-polylysine molecular weight that participates in film formation reaction is 1KDa～500KDa, the compound method of solution is: epsilon-polylysine is dissolved in physiological saline, HEPES buffer solution or the PBS buffer solution physiological buffer system that pH is 5.5-7.4, and epsilon-polylysine concentration is 0.1-50g/L.
10. according to the preparation method of claim 6 or 7 described microcapsules, it is characterized in that: the alginate gel micro-ball is one or two or more kinds alginate hydrogel in divalent metal calcium, barium or the zinc;
Be used for and the alkali metal alginate of surface charge is sylvite or sodium salt, molecular weight distribution is 10KDa～2000KDa, and alginate concentration is 0.1-5g/L.
11. the preparation method according to the described microcapsules of claim 7 is characterized in that: the organic metal chelating agent solution that participates in liquefaction reaction is the natrium citricum of 40-70mmol/L or the EDTA of 50-200mmol/L.
12. the described microcapsules of claim 1 are used for the embedding of cell, medicine, enzyme or biological nucleic acid active material.
13. be used for the embedding of bioactivators such as cell, medicine, enzyme or nucleic acid according to the described microcapsules of claim 12, it is characterized in that: described cell is behaved or the islet cells, liver cell, thyroid cell, parathyroid gland cell, the adrenal medullary cell that exsomatize in mammal source have the cell of secreting the bioactivator function, cell line cell, genetically engineered cell, the various cells of stem cell or stem cell differentiation;
Described medicine is oil-soluble small-molecule drug elemene, vitamin E, evening primrose oil, or protein and peptide drugs interferon, endostatin, EPO, insulin, growth factor;
Described enzyme is industrial enzymes product lipase, amylase or dehydrogenase;
Described nucleic acid is nucleic acid drug DNA or the siRNA with gene therapy effect.
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Application publication date: 20110622 |