CN1201858C - Prepn of biocompatible SA/CS-CaCl2/PMCG microcapsule - Google Patents
Prepn of biocompatible SA/CS-CaCl2/PMCG microcapsule Download PDFInfo
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- CN1201858C CN1201858C CN 03129151 CN03129151A CN1201858C CN 1201858 C CN1201858 C CN 1201858C CN 03129151 CN03129151 CN 03129151 CN 03129151 A CN03129151 A CN 03129151A CN 1201858 C CN1201858 C CN 1201858C
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 39
- 239000001110 calcium chloride Substances 0.000 title abstract description 6
- 229910001628 calcium chloride Inorganic materials 0.000 title abstract description 6
- 239000002775 capsule Substances 0.000 claims abstract description 81
- 239000000243 solution Substances 0.000 claims abstract description 64
- 239000012535 impurity Substances 0.000 claims abstract description 36
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000000661 sodium alginate Substances 0.000 claims abstract description 35
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 35
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- -1 polymethylene Polymers 0.000 claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000007787 solid Substances 0.000 claims abstract description 23
- 239000011259 mixed solution Substances 0.000 claims abstract description 16
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 14
- 239000008367 deionised water Substances 0.000 claims description 33
- 229910021641 deionized water Inorganic materials 0.000 claims description 33
- 239000003960 organic solvent Substances 0.000 claims description 29
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 26
- 229920000447 polyanionic polymer Polymers 0.000 claims description 26
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 22
- 238000002360 preparation method Methods 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 14
- 230000008859 change Effects 0.000 claims description 12
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 239000012467 final product Substances 0.000 claims description 11
- 238000012805 post-processing Methods 0.000 claims description 11
- 238000001556 precipitation Methods 0.000 claims description 11
- 230000035484 reaction time Effects 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000001509 sodium citrate Substances 0.000 claims description 11
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 11
- 230000003068 static effect Effects 0.000 claims description 11
- 238000001291 vacuum drying Methods 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- 229960004756 ethanol Drugs 0.000 claims description 4
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 9
- 244000005700 microbiome Species 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 3
- 230000035699 permeability Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000004113 cell culture Methods 0.000 abstract description 2
- 150000003384 small molecules Chemical class 0.000 abstract description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 abstract 1
- 239000001913 cellulose Substances 0.000 abstract 1
- 229920002678 cellulose Polymers 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 229910052938 sodium sulfate Inorganic materials 0.000 abstract 1
- 235000011152 sodium sulphate Nutrition 0.000 abstract 1
- 230000000813 microbial effect Effects 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 102100026735 Coagulation factor VIII Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 235000001465 calcium Nutrition 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003989 dielectric material Substances 0.000 description 1
- GQOKIYDTHHZSCJ-UHFFFAOYSA-M dimethyl-bis(prop-2-enyl)azanium;chloride Chemical compound [Cl-].C=CC[N+](C)(C)CC=C GQOKIYDTHHZSCJ-UHFFFAOYSA-M 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- SUPCQIBBMFXVTL-UHFFFAOYSA-N ethyl 2-methylprop-2-enoate Chemical compound CCOC(=O)C(C)=C SUPCQIBBMFXVTL-UHFFFAOYSA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000008004 immune attack Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
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- Medicinal Preparation (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Materials For Medical Uses (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Abstract
The present invention discloses a method for preparing an SA/CS-CaCl2/PMCG microcapsule with biocompatibility. The multi-component system is prepared from sodium alginate (SA), cellulose sodium sulfate (CS), calcium chloride and polymethylene dicarbamidine chlorine hydride (PMCG), the SA and CS mixed solution is dropped into calcium chloride solution to form a solid pre-capsule, the solid pre-capsule is transferred into the PMCG to react, and the microcapsule is obtained. The commercial raw material PMCG is high-viscosity water solution containing 35% of PMCG, wherein the high-viscosity water solution contains small-molecule impurities with toxicity to microorganisms. The impurities are precipitated by ethanol, are centrifugated, are dried by vacuum, are freeze-dried, etc. thus, harmful substances contained in the impurities are completely eliminated. The capsule prepared by the present invention can independently adjust the capsule parameters such as capsule size, membrane thickness, mechanical strength and permeability. Aiming at various requirements of practical application, the characteristic of capsules can be adjusted and optimized. The capsule has good biocompatibility, and is a cell culture immobilizing system with the advantages of prospects and good performance.
Description
Technical field
The present invention relates to a kind of four component microcapsule preparation methods with biocompatibility.Take this to carry out the cultivation of microorganism cells.
Background technology
The bio-microcapsule technology is the new and high technology that drives and grow up for organ transplantation.Treat such as various human diseasess such as hormone or potein deficiencys with Transplanted cells also very limited because the very fast meeting of the cell of being transplanted is destroyed by the acceptor immunity system.Overcome this obstacle, secretion hormone or proteinic cell will be wrapped in the semi-permeable membranes avoiding the immune attack of acceptor, but will allow entering and the discharge of required cellular products of small molecules (as oxygen and nitrogen etc.) that cellular function or existence plays an important role simultaneously again.And the semi-permeable membranes of bio-microcapsule just has this immune isolated function.Therefore, the preparation of bio-microcapsule and application thereof have become one of the research focus in fields such as biological medicine engineering, protein chemistry, incretology.Simultaneously, as the important component part of immobilized cell technique, its Application Areas has related to a plurality of fields such as biology, enzyme engineering, biochemical engineering, polymer chemistry, catalytic chemistry, medical diagnosis, environmental purification and Energy production.Therefore, the bio-microcapsule technology is studied, its importance is self-evident.
Up to the present, having developed some can be the immobilized micro-capsule system of biological substance, for example by Lim and Sun in sodium alginate/poly-L-Methionin/sodium alginate capsule (APA capsule) system of invention in 1980, but the APA capsule is a kind of many electrolyte composites of fragility, its physical strength is relatively poor relatively, and the costing an arm and a leg etc. of poly-L-Methionin, other is also just like systems such as methacrylic acid ethyl ester/methyl methacrylate (HEMA/MMA) and alginate calciums, but when it is used, be subjected to various condition effect, brought many inconvenience.Another important microcapsule system is Ushercell (NaCS) and the Poly Dimethyl Diallyl Ammonium Chloride (PDMDAAC) that grows up the eighties in 20th century) microcapsule, this is two kinds of polymer dielectrics, can make microcapsule system by simple preparation method, some successful application examples have been arranged at present by the membrane-enclosed hollow of one deck 20-100mm porous.Above-mentioned microcapsule system is present most representative example.These systems are bicomponent system, all with wherein a kind of compound is relevant for all film parameters, if optimize one of them parameter, will influence other parameter, this character that can not independently adjust capsule parameter (as physical strength or permeability) has seriously limited the practical application of these systems.Up to now, do not occur both having had excellent biological compatibility and enough physical strengths as yet, can overcome above circumscribed ideal system simultaneously again.
A kind of multi-component microcapsule system once appearred the beginning of this century, by sodium alginate (SA)/Ushercell (CS)-calcium chloride (CaCl
2The capsule that)/polymethylene two guanidine hydrogenchloride (PMCG) four components constitute, but document is not reported the preparation process that it is concrete, simultaneously because the impure or component of component collocation improper, make resulting microcapsule have toxicity, therefore can't be used for cell cultures as bio-microcapsule to microorganism etc.
Summary of the invention
The purpose of this invention is to provide a kind of four component microcapsule preparation methods with biocompatibility.
Its step is as follows:
1) with dehydrated alcohol, Virahol and acetone organic solvent are respectively 0.5: 1～3: 1 proportioning thorough mixing with volume ratio with polymethylene two guanidine hydrochloride aqueous solutions, static 25～80 minutes, obtain three kinds of mixed solutions, with three kinds of mixed solutions centrifugal 5～20min under 4000～8000rpm respectively, collecting precipitation, and with corresponding organic solvent washing throw out 2～4 times, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last throw out was descended the polymethylene two guanidine hydrogenchloride of the small molecular weight impurity that is removed dry 24～72 hours in 37～70 ℃ in vacuum drying oven;
2) be that 0.5%～4% sodium alginate and weight percent are 0.5%～5% Ushercell thorough mixing with weight percent, both volume ratios are 0.2～5, this mixture is the polyanion solution of body series, it is to be cured in 0.5%～5% calcium chloride solution that polyanion solution is splashed into weight percent by syringe, be 5～80 minutes set time, obtain solid pre-capsule, with the solid pre-capsule of deionized water wash, solid pre-capsule being changed over to weight percent again and be 1%～5% removes in the polymethylene two guanidine hydrogen chloride solutions of small molecular weight impurity and reacts, reaction times is 3～35 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind the deionized water wash capsule, change in the sodium citrate solution of 0.5M and placed 5～30 minutes, take out capsule, after using the deionized water wash capsule once more, be to preserve in 0.9% sodium chloride solution at weight percent.
Advantage of the present invention:
1) can independently adjust the film parameter;
At the different requirements of practical application, capsular characteristic parameter, for example; Capsule size, thickness, physical strength and permeability can be adjusted and optimize;
2) biocompatibility with the polycomponent microcapsule of the preparation of the PMCG after purifying obviously improves, and has excellent biological compatibility, is suitable for microbial immobilization and cultivates.
3) the microcapsule rete for preparing under condition of the present invention is very thin, and physical strength is excellent.
The capsule thickness is about 20～150mm, generally can bear 3～7 newton's positive pressure, have in addition can bear positive pressure up to 10 newton.Such physical strength can make capsule be unlikely to damaged with the shearing force that produces owing to stirring in the opposing bio-reactor completely.
4) capsular chemical property is stable
Capsule of the present invention is not had the substance dissolves of sequestering action by citric acid, phosphoric acid etc. to calcium ion, is insoluble to most organic solvent (as ethanol, acetone etc.) yet.Change also not obvious to potential of hydrogen.
5) preparation condition gentleness, cost of material is cheap, is convenient to use and popularization.
Description of drawings
Fig. 1 is the comparison synoptic diagram with the cultivation of microcapsule immobilization intestinal bacteria with the free cultivation of the PMCG preparation of PMCG that purifies and not purification, and among the figure: zero free cultivation ■ immobilization is cultivated (PMCG after purifying) ▲ immobilization and cultivated (commercially available PMCG)
Fig. 2 is the comparison synoptic diagram with the cultivation of microcapsule immobilized brewing yeast with the free cultivation of the PMCG preparation of PMCG that purifies and not purification, and among the figure: zero free cultivation ■ immobilization is cultivated (PMCG after purifying) ▲ immobilization and cultivated (commercially available PMCG)
Embodiment
In order to overcome above-mentioned limitation, the present invention is at sodium alginate (SA)/Ushercell (CS)-calcium chloride (CaCl
2On the basis of)/polymethylene two guanidine hydrogenchloride (PMCG) microcapsule, analyzed the reason that wherein may bring toxic substance into, developed the effective purifying technique of a cover, encystation raw material with serious toxicity has been carried out purifying, and carried out reasonably combined between component, and not only made microcapsule with better biocompatibility, be used for the cultivation of microorganism cells, and the capsule that makes thus can independently adjust its parameter, for example; Capsule size, thickness, physical strength and transmission characteristic.At the different requirements of practical application, capsular characteristic can be adjusted and optimize.For achieving the above object, the present invention takes the following step:
1) with dehydrated alcohol, Virahol and acetone and other organic solvent respectively with the proportioning thorough mixing of the PMCG aqueous solution with 0.5: 1～3: 1, static 25～80 minutes, with mixed solution centrifugal 5～20min under 4000～8000rpm, collecting precipitation, and with corresponding organic solvent washing 2～4 times, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last that throw out is following dry 24～72 hours in 37～70 ℃ in vacuum drying oven;
2) with 0.5%～4% sodium alginate (SA) and 0.5%～5% Ushercell (CS) thorough mixing, both ratios are 0.2～5, this mixture is the polyanion solution of body series, polyanion solution splashed in 0.5%～5% calcium chloride solution by syringe be cured, be 5～80 minutes set time, obtain solid pre-capsule, with the pre-capsule of deionized water wash, again it is changed in polymethylene two guanidine hydrogenchloride (PMCG) solution after 1%～5% the purification and react, reaction times is 3～35 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 5～30 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
Below in conjunction with example the present invention is elaborated:
1) with dehydrated alcohol, Virahol and acetone and the PMCG aqueous solution proportioning thorough mixing with 1: 1～2.5: 1, static 30～60 minutes, with mixed solution centrifugal 10～15min under 5000～7000rpm, collecting precipitation, and with corresponding organic solvent washing 2～3 times.Again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last with throw out in vacuum drying oven in 40～60 ℃ dry 30～48 hours down, standby.
2) with 1.5%～3% sodium alginate (SA) and 1.5%～4% Ushercell (CS) thorough mixing, both ratios are 0.4～3, this mixture is the polyanion solution of body series, polyanion solution splashed in 2.5%～4% calcium chloride solution by syringe be cured, be 10～40 minutes set time, obtain solid pre-capsule, with the pre-capsule of deionized water wash, again it is changed in 1.5%～3% the polymethylene of removing small molecular weight impurity two guanidine hydrogenchloride (PMCG) solution and react, reaction times is 5～30 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 8～15 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
The preparation material therefor of novel biology microcapsule of the present invention is as follows:
1) Ushercell (CS) is prepared by Zhejiang University's bio-engineering research.
2) sodium alginate (SA) is available from the biochemical institute in Shanghai.
3) polymethylene two guanidine hydrogenchloride (PMCG), available from U.S. Scientific Polymer Products company, other reagent such as calcium chloride etc. are commercially available analytical reagent.
Provide different examples below the present invention be described:
1) at normal temperatures, with dehydrated alcohol and the PMCG aqueous solution proportioning thorough mixing with 2: 1, static 30 minutes, with mixed solution centrifugal 15min under 6000rpm, collecting precipitation, and, again throw out is carried out lyophilize, to remove residual ethanol and other small molecular weight impurity with absolute ethanol washing 3 times.At last with throw out in vacuum drying oven in 50 ℃ dry 48 hours down, standby;
2) with 2% sodium alginate (SA) and 3.5% Ushercell (CS) thorough mixing, both ratios are 1.5, this mixture is the polyanion solution of body series, polyanion solution splashed in 3% calcium chloride solution by syringe be cured, be 15 minutes set time, obtain solid pre-capsule, with the pre-capsule of deionized water wash, again it is changed in 2% the polymethylene of removing small molecular weight impurity two guanidine hydrogenchloride (PMCG) solution and react, reaction times is 15 minutes, the gained capsule gets final product after aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 10 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
The capsule median size that makes thus is 2.5mm, and physical strength reaches 7 newton approximately, and thickness is about 100mm.
Embodiment 2
1) with dehydrated alcohol and the PMCG aqueous solution proportioning thorough mixing with 0.5: 1, static 25 minutes, with mixed solution centrifugal 5min under 4000rpm, collecting precipitation, and, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity with corresponding organic solvent washing 2 times, at last throw out was descended the polymethylene two guanidine hydrogenchloride (PMCG) of the small molecular weight impurity that is removed dry 24 hours in 37 ℃ in vacuum drying oven;
2) with 0.5% sodium alginate (SA) and 0.5% Ushercell (CS) thorough mixing, both ratios are 0.2, this mixture is the polyanion solution of body series, polyanion solution splashed in 0.5% calcium chloride solution by syringe be cured, be 5 minutes set time, obtain solid pre-capsule, with the solid pre-capsule of deionized water wash, it being changed over to 1% removes in polymethylene two guanidine hydrogenchloride (PMCG) solution of small molecular weight impurity and reacts again, reaction times is 3 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 5 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
Embodiment 3
1) with dehydrated alcohol, Virahol and acetone and other organic solvent respectively with the proportioning thorough mixing of the PMCG aqueous solution with 3: 1, static 80 minutes, with mixed solution centrifugal 20min under 8000rpm, collecting precipitation, and with corresponding organic solvent washing 4 times, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last throw out was descended the polymethylene two guanidine hydrogenchloride (PMCG) of the small molecular weight impurity that is removed dry 72 hours in 70 ℃ in vacuum drying oven;
2) with 4% sodium alginate (SA) and 5% Ushercell (CS) thorough mixing, both ratios are 5, this mixture is the polyanion solution of body series, polyanion solution splashed in 5% calcium chloride solution by syringe be cured, be 80 minutes set time, obtain solid pre-capsule, with the solid pre-capsule of deionized water wash, it being changed over to 5% removes in polymethylene two guanidine hydrogenchloride (PMCG) solution of small molecular weight impurity and reacts again, reaction times is 35 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 30 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
Embodiment 4
1) with dehydrated alcohol, Virahol and acetone and other organic solvent respectively with the proportioning thorough mixing of the PMCG aqueous solution with 0.5: 1, static 80 minutes, with mixed solution centrifugal 5min under 8000rpm, collecting precipitation, and with corresponding organic solvent washing 3 times, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last throw out was descended the polymethylene two guanidine hydrogenchloride (PMCG) of the small molecular weight impurity that is removed dry 24 hours in 70 ℃ in vacuum drying oven;
2) with 0.5% sodium alginate (SA) and 5% Ushercell (CS) thorough mixing, both ratios are 5, this mixture is the polyanion solution of body series, polyanion solution splashed in 0.5% calcium chloride solution by syringe be cured, be 80 minutes set time, obtain solid pre-capsule, with the solid pre-capsule of deionized water wash, it being changed over to 1% removes in polymethylene two guanidine hydrogenchloride (PMCG) solution of small molecular weight impurity and reacts again, reaction times is 35 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 30 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
Embodiment 5
1) with dehydrated alcohol, Virahol and acetone and other organic solvent respectively with the proportioning thorough mixing of the PMCG aqueous solution with 3: 1, static 25 minutes, with mixed solution centrifugal 5min under 4000rpm, collecting precipitation, and, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity with corresponding organic solvent washing 4 times, at last throw out was descended the polymethylene two guanidine hydrogenchloride (PMCG) of the small molecular weight impurity that is removed dry 72 hours in 37 ℃ in vacuum drying oven;
2) with 4% sodium alginate (SA) and 0.5% Ushercell (CS) thorough mixing, both ratios are 0.2, this mixture is the polyanion solution of body series, polyanion solution splashed in 5% calcium chloride solution by syringe be cured, be 5 minutes set time, obtain solid pre-capsule, with the solid pre-capsule of deionized water wash, it being changed over to 5% removes in polymethylene two guanidine hydrogenchloride (PMCG) solution of small molecular weight impurity and reacts again, reaction times is 3 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind deionized water wash, change in the sodium citrate solution of 0.5M and placed 5 minutes, take out capsule, with behind the deionized water wash, in 0.9% sodium chloride solution, preserve once more.
Implementation condition is as follows to the influence of experimental result in the implementation process:
1) in the purifying technique different organic solvents to the influence of PMCG refining effect
Adopted multiple organic solvents such as dehydrated alcohol, Virahol and acetone in the experiment, wherein, the alcoholic acid refining effect is the most desirable.If adopt other organic solvents such as Virahol or acetone, finally can not remove the impurity that contains among the PMCG fully.Be in particular in: the capsule as if the PMCG preparation of removing small molecular weight impurity with Virahol or acetone and other organic solvent carries out microorganism culturing such as immobilized brewing yeast, and microorganism growth is had the obvious suppression effect.And when carrying out microbial immobilized cultivations with the bio-microcapsule that ethanol is removed the PMCG preparation of small molecular weight impurity, microbial growth is similar with the culturing process of dissociating accordingly.
2) purifying technique of raw material PMCG is to the influence of microcapsule biocompatibility
The purifying technique of raw material PMCG is the vital factor of decision microcapsule biocompatibility.If directly do not use and prepare this microcapsule through the PMCG that purifies, then the biocompatibility of gained microcapsule is very poor, be in particular in: if use by not carrying out the immobilization cultivation such as microorganisms such as yeast saccharomyces cerevisiae, intestinal bacteria through the microcapsule embedded of PMCG preparation of purifying, these microbial growths are subjected to severe inhibition, almost can't grow; And when carrying out microbial immobilized cultivations with the bio-microcapsule of the PMCG preparation of removing small molecular weight impurity, microbial growth is similar with the culturing process of dissociating accordingly, illustrates that these microcapsule have good biocompatibility.Corresponding experiment result is seen Fig. 1 and Fig. 2 respectively.Must complete this purification of use route, the finally good microcapsule of obtained performance.If only with organic deposition or Freeze Drying Technique, can't make final microcapsule have excellent biological compatibility.
3) character of Ushercell (CS) is to the influence of capsule outward appearance and preparation process thereof
By discovering to different degree of substitution (DS) Ushercell (CS) (model is respectively A3, A4, A5, A6), under same concentrations, substitution value is big more, its soltion viscosity is just more little, in addition, the size of CS strength of solution directly influences the size of its soltion viscosity, and can its soltion viscosity size be form outward appearance one of the deciding factor of microcapsule preferably.If viscosity is too high, then sphericity is bad; Otherwise then the capsule film does not reach required physical strength.Xuan Ding model is the raw material of A6 Ushercell as body series at last, and under preferable condition, the concentration of CS solution is 3.5%.
4) concentration of polyanion solution and composition thereof
Polyanion solution is that in comparatively ideal composition, both ratios are approximately 1.5 by sodium alginate (SA) and Ushercell (CS) solution composition, and the total concn of polyanion solution is 3%.Increase the content of SA, cause the capsule physical strength to reduce, but capsular transparency and sphericity can increase; On the contrary,, capsular transparency, sphericity and slipperiness are had negative impact, but the capsule thickness can increase, cause the also corresponding increase of capsule physical strength if increase the content of CS.But during the too high levels of CS, then can cause the reduction of physical strength again.Experimental result shows that sodium alginate in the polyanion solution (SA) is 0.4～3 with the preferred proportion scope of Ushercell (CS).
Claims (3)
1. four component microcapsule preparation methods with biocompatibility is characterized in that its step is as follows:
1) with dehydrated alcohol, Virahol and acetone organic solvent are respectively 0.5: 1～3: 1 proportioning thorough mixing with volume ratio with polymethylene two guanidine hydrochloride aqueous solutions, static 25～80 minutes, obtain three kinds of mixed solutions, with three kinds of mixed solutions centrifugal 5～20min under 4000～8000rpm respectively, collecting precipitation, and with corresponding organic solvent washing throw out 2～4 times, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last throw out was descended the polymethylene two guanidine hydrogenchloride of the small molecular weight impurity that is removed dry 24～72 hours in 37～70 ℃ in vacuum drying oven;
2) be that 0.5%～4% sodium alginate and weight percent are 0.5%～5% Ushercell thorough mixing with weight percent, both volume ratios are 0.2～5, this mixture is the polyanion solution of body series, it is to be cured in 0.5%～5% calcium chloride solution that polyanion solution is splashed into weight percent by syringe, be 5～80 minutes set time, obtain solid pre-capsule, with the solid pre-capsule of deionized water wash, solid pre-capsule being changed over to weight percent again and be 1%～5% removes in the polymethylene two guanidine hydrogen chloride solutions of small molecular weight impurity and reacts, reaction times is 3～35 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind the deionized water wash capsule, change in the sodium citrate solution of 0.5M and placed 5～30 minutes, take out capsule, after using the deionized water wash capsule once more, be to preserve in 0.9% sodium chloride solution at weight percent.
2. four component microcapsule preparation methods with biocompatibility according to claim 1 is characterized in that its step is as follows:
1) with dehydrated alcohol, Virahol and acetone and polymethylene two guanidine hydrochloride aqueous solutions are 1: 1～2.5: 1 proportioning thorough mixing with volume ratio, static 30～60 minutes, obtain three kinds of mixed solutions, with three kinds of mixed solutions centrifugal 10～15min under 5000～7000rpm respectively, collecting precipitation, and with corresponding organic solvent washing throw out 2～3 times, again throw out is carried out lyophilize, to remove residual organic solvent and other small molecular weight impurity, at last throw out was descended the polymethylene two guanidine hydrogenchloride of the small molecular weight impurity that is removed dry 30～48 hours in 40～60 ℃ in vacuum drying oven;
2) be that 1.5%～3% sodium alginate and weight percent are 1.5%～4% Ushercell thorough mixing with weight percent, both volume ratios are 0.4～3, this mixture is the polyanion solution of body series, it is to be cured in 2.5%～4% calcium chloride solution that polyanion solution is splashed into weight percent by syringe, be 10～40 minutes set time, obtain solid pre-capsule, with the pre-capsule of deionized water wash, solid pre-capsule being changed over to weight percent again and be 1.5%～3% removes in the polymethylene two guanidine hydrogen chloride solutions of small molecular weight impurity and reacts, reaction times is 5～30 minutes, the gained capsule gets final product through aftertreatment, post-processing step is as follows: behind the deionized water wash capsule, change in the sodium citrate solution of 0.5M and placed 8～15 minutes, take out capsule, after using the deionized water wash capsule once more, be to preserve in 0.9% sodium chloride solution at weight percent.
3. four component microcapsule preparation methods with biocompatibility according to claim 1 and 2 is characterized in that its step is as follows:
1) with dehydrated alcohol and polymethylene two guanidine hydrochloride aqueous solutions is 2: 1 proportioning thorough mixing with volume ratio, static 30 minutes, obtain three kinds of mixed solutions, with three kinds of mixed solutions centrifugal 15min under 6000rpm respectively, collecting precipitation, and with absolute ethanol washing throw out 3 times, again throw out is carried out lyophilize, to remove residual ethanol and other small molecular weight impurity, at last throw out was descended the polymethylene two guanidine hydrogenchloride of the small molecular weight impurity that is removed dry 48 hours in 50 ℃ in vacuum drying oven;
2) be that 2% sodium alginate and weight percent are 3.5% Ushercell thorough mixing with weight percent, both volume ratios are 1.5, this mixture is the polyanion solution of body series, it is to be cured in 3% calcium chloride solution that polyanion solution is splashed into weight percent by syringe, be 15 minutes set time, obtain solid pre-capsule, with the pre-capsule of deionized water wash, solid pre-capsule being changed over to weight percent again and be 2% removes in the polymethylene two guanidine hydrogen chloride solutions of small molecular weight impurity and reacts, reaction times is 15 minutes, the gained capsule gets final product after aftertreatment, post-processing step is as follows: behind the deionized water wash capsule, change in the sodium citrate solution of 0.5M and placed 10 minutes, take out capsule, after using the deionized water wash capsule once more, be to preserve in 0.9% sodium chloride solution at weight percent.
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