WO2014206043A1 - Application method of cyclodextrin multi-aldehyde cross-linking agent - Google Patents
Application method of cyclodextrin multi-aldehyde cross-linking agent Download PDFInfo
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- WO2014206043A1 WO2014206043A1 PCT/CN2013/090019 CN2013090019W WO2014206043A1 WO 2014206043 A1 WO2014206043 A1 WO 2014206043A1 CN 2013090019 W CN2013090019 W CN 2013090019W WO 2014206043 A1 WO2014206043 A1 WO 2014206043A1
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- WO
- WIPO (PCT)
- Prior art keywords
- cyclodextrin
- collagen
- polyaldehyde
- cross
- modified collagen
- Prior art date
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- 229920000858 Cyclodextrin Polymers 0.000 title claims abstract description 135
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 239000003431 cross linking reagent Substances 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 17
- 108010035532 Collagen Proteins 0.000 claims abstract description 136
- 102000008186 Collagen Human genes 0.000 claims abstract description 136
- 229920001436 collagen Polymers 0.000 claims abstract description 136
- 239000007864 aqueous solution Substances 0.000 claims abstract description 34
- 239000000512 collagen gel Substances 0.000 claims abstract description 26
- 238000001035 drying Methods 0.000 claims abstract description 8
- 238000007710 freezing Methods 0.000 claims abstract description 3
- 230000008014 freezing Effects 0.000 claims abstract description 3
- 229920001744 Polyaldehyde Polymers 0.000 claims description 96
- 239000000243 solution Substances 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- -1 cyclodextrin poly-aldehyde Chemical class 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 abstract description 8
- 239000002253 acid Substances 0.000 abstract 1
- 239000011259 mixed solution Substances 0.000 abstract 1
- 238000002715 modification method Methods 0.000 abstract 1
- 239000001116 FEMA 4028 Substances 0.000 description 47
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 47
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 47
- 229960004853 betadex Drugs 0.000 description 47
- 238000004132 cross linking Methods 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 26
- 239000000463 material Substances 0.000 description 19
- 239000000706 filtrate Substances 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 14
- 125000003172 aldehyde group Chemical group 0.000 description 11
- CJNZAXGUTKBIHP-UHFFFAOYSA-N 2-iodobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1I CJNZAXGUTKBIHP-UHFFFAOYSA-N 0.000 description 9
- 239000004971 Cross linker Substances 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 8
- 150000001408 amides Chemical class 0.000 description 7
- 230000015556 catabolic process Effects 0.000 description 7
- 238000006731 degradation reaction Methods 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 230000007071 enzymatic hydrolysis Effects 0.000 description 7
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 7
- 238000001914 filtration Methods 0.000 description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical class O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 238000003760 magnetic stirring Methods 0.000 description 6
- 239000007800 oxidant agent Substances 0.000 description 6
- 230000001590 oxidative effect Effects 0.000 description 6
- 239000003880 polar aprotic solvent Substances 0.000 description 6
- 238000001291 vacuum drying Methods 0.000 description 6
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 5
- 238000010382 chemical cross-linking Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002329 infrared spectrum Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 150000001299 aldehydes Chemical class 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical group OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000002262 Schiff base Substances 0.000 description 2
- 150000004753 Schiff bases Chemical class 0.000 description 2
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 2
- 229940043377 alpha-cyclodextrin Drugs 0.000 description 2
- 238000005452 bending Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229940097362 cyclodextrins Drugs 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 231100000956 nontoxicity Toxicity 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 229920002085 Dialdehyde starch Polymers 0.000 description 1
- 239000004593 Epoxy Chemical class 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000005057 Hexamethylene diisocyanate Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000012669 compression test Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000000113 differential scanning calorimetry Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 1
- 108010020199 glutaraldehyde-cross-linked collagen Proteins 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003464 sulfur compounds Chemical class 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/738—Cyclodextrins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
- C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K5/00—Use of organic ingredients
- C08K5/04—Oxygen-containing compounds
- C08K5/07—Aldehydes; Ketones
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/16—Cyclodextrin; Derivatives thereof
Definitions
- the present invention relates to a cyclodextrin and a collagen crosslinker, and in particular to a method for applying a cyclodextrin polyaldehyde crosslinker.
- Collagen is a biological natural high molecular substance, English name collages collagen exists in the book
- Collagen In all cell animals, it is the most abundant type of protein in the body, present in almost all tissues, and is an extracellular matrix component.
- Collagen has a special three-stranded helical peptide chain structure that imparts many good biological and chemical properties to collagen.
- Collagen has good biocompatibility, biodegradability, low antigenicity; has cell adaptation, cell proliferation and promotes platelet aggregation; has good solubility, non-toxicity, unique fiber formation characteristics ; and easy to modify and a wide range of sources. Therefore, as a very important biological material, collagen is widely used in wound dressings, artificial skin, beauty, drug release, tissue engineering scaffolds and cell culture. The value of collagen is receiving more and more attention.
- the main crosslinking agents in chemical crosslinking are: glutaraldehyde, carbodiimide (EDC), epoxy compounds, genipin, glyoxylic acid, sulfur compounds, hexamethylene diisocyanate, acyl azide and diphenyl. Phosphate, dialdehyde starch, etc.
- the above chemical cross-linking agents can effectively improve the mechanical strength and stability of collagen, etc., but also all have some disadvantages through cross-linking, for example, some may cause cytotoxicity, some may cause color of collagen materials, Some have calcification potential, Some do not significantly improve some of the disadvantages of collagen materials.
- Cyclodextrin is a cyclic oligosaccharide composed of 6 ⁇ 8 D-(+) glucopyranose units through ⁇ -1, 4-glycosidic linkages, corresponding to ⁇ , ⁇ , ⁇ cyclodextrin, cyclodextrin
- the model of the molecule is like a cone-shaped cylinder with an inner hollow to the top.
- the polar hydroxyl group is located at the edge of the cyclodextrin unit, and the secondary and primary polar hydroxyl groups protrude from the edge, so that hydrophilic groups are distributed both above and below, and are hydrophilic. .
- a sugar-oxygen bridge atom is arranged inside the cyclodextrin cavity. Since the non-bonded electrons of the oxygen atom are directed to the center, the electron density in the cavity is high; the hydrogen atoms on the glucopyranose ring C-3, C-5 are located in the cavity and cover the oxygen atom of the glycoside. The interior of the cavity becomes a hydrophobic space.
- the hydrophobic cavity can form a clathrate by hydrophobic interaction, hydrogen bonding, van der Waals force, etc., and thus exhibits special adsorption properties. Cyclodextrins form stable inclusion complexes with many organic compounds, halogens and certain inorganic compounds.
- Cyclodextrins can act as sustained release agents for the substances to be included, and they have no significant toxic side effects to humans. The advantages of cyclodextrin make it widely available in all areas of biomedical science. Summary of the invention
- an object of the present invention is to provide a method for applying a cyclodextrin polyaldehyde cross-linking agent, wherein a cyclodextrin polyaldehyde cross-linking agent is used for modifying collagen.
- the cyclodextrin polyaldehyde cross-linking agent does not produce cytotoxicity while improving the mechanical strength and stability of collagen.
- a method for applying a cyclodextrin polyaldehyde crosslinking agent wherein the cyclodextrin polyaldehyde crosslinking agent is used for modifying a collagen; wherein the chemical structure of the cyclodextrin polyaldehyde crosslinking agent is:
- the cyclodextrin polyaldehyde crosslinker is used for the preparation of porous sponge-like modified collagen.
- the preparation method of the porous sponge-like modified collagen comprises the following steps:
- the modified collagen solution obtained in the step (1) is kept at 35 to 38 ° C for 15 to 20 minutes, then the modified collagen solution is transferred to a mold, and the mold is frozen at -15 to 25 ° C. ⁇ 50h, preparing a modified collagen gel;
- the mass concentration of the aqueous solution of the cyclodextrin polyaldehyde crosslinking agent in the step (1) is 0.075% to 0.6%, and the mass concentration of the aqueous collagen solution is 0.6%.
- the reaction temperature in the step (1) is 4 °C.
- the modified collagen solution in step (2) is incubated in a 37 ° C water bath at a freezing temperature of -20 ° C.
- the drying method described in the step (3) is freeze drying.
- the present invention has the following advantages and beneficial effects:
- the present invention uses a cyclodextrin polyaldehyde having an aldehyde group of 2 or 3 as a crosslinking agent; since the cyclodextrin polyaldehyde crosslinking agent has the advantages of non-toxicity and good crosslinking effect, it is particularly suitable for Modification of collagen.
- the cyclodextrin polyaldehyde having 2 or 3 aldehyde groups is used for the modification of collagen, and the modified collagen has high cross-linking efficiency, and the modified collagen material has greatly improved thermal stability and markedly enhanced degradation resistance.
- the present invention cross-links cyclodextrin polyaldehyde with collagen, which can not only make up for some shortcomings of collagen, improve the performance of collagen materials, but also avoid the introduction of cytotoxic chemical cross-linking agents such as glutaraldehyde. It not only maintains some of the original properties of collagen, but also introduces some excellent properties of cyclodextrin, such as special rigid structure, inclusion coordination, molecular recognition and assembly, which greatly expands the application range of collagen.
- Figure 1 is a schematic diagram of a synthetic route of a cyclodextrin polyaldehyde
- Figure 2 is a schematic diagram of cyclodextrin polyaldehyde cross-linked collagen
- Figure 3 is an infrared spectrum (FTIR) of ⁇ -cyclodextrin and ⁇ -cyclodextrin polyaldehyde;
- Figure 4 is a nuclear magnetic resonance spectrum (1H-NMR) of ⁇ -cyclodextrin polyaldehyde
- Figure 5 is an infrared spectrum (FTIR) of ⁇ -cyclodextrin polyaldehyde and collagen before and after cross-linking;
- FTIR infrared spectrum
- Figure 6 is a graph showing the degree of crosslinking of ⁇ -cyclodextrin polyaldehyde crosslinked collagen
- Figure 7 is a DSC chart before and after cross-linking of ⁇ -cyclodextrin polyaldehyde with collagen
- Figure 8 is a graph showing the mechanical properties of ⁇ -cyclodextrin polyaldehyde before and after cross-linking with collagen
- Figure 9 is a graph showing the degree of enzymatic hydrolysis before and after cross-linking of ⁇ -cyclodextrin polyaldehyde with collagen. detailed description
- cyclodextrin is oxidized to a cyclodextrin polyaldehyde derivative, i.e., a polyfunctional compound, as a crosslinking agent by using 2-iodobenzoic acid (hydrazine).
- a cyclodextrin polyaldehyde derivative i.e., a polyfunctional compound
- 2-iodobenzoic acid hydrazine
- the aldehyde group of the cyclodextrin polyaldehyde is reacted with the active amino group on the collagen to form a Schiff base to crosslink the modified collagen, so that the mechanical properties, thermal stability and degradation resistance of the collagen are greatly improved.
- the present invention is suitable for ⁇ -cyclodextrin, ⁇ -cyclodextrin and cyclodextrin.
- the method for synthesizing the cyclodextrin polyaldehyde crosslinking agent of the present invention comprises the following steps:
- the yield of ⁇ -cyclodextrin polyaldehyde was 90%.
- the cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used.
- Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
- the modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 18 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
- FT-IR analysis was performed on a NICOLET 760-type infrared spectrophotometer, and the sample preparation was performed by KBr tableting.
- the NMR analysis was performed using a Bruker 400 MHz nuclear magnetic resonance apparatus, and the solvent was deuterated DMSO.
- the cross-linking degree of ⁇ -cyclodextrin polyaldehyde-modified collagen was determined by the ninhydrin method using a glycine solution as a standard curve, and the UV absorbance value was measured at 570 nm using a LAMBDA950 ultraviolet spectrophotometer.
- the content of aldehyde group in the synthesized ⁇ -cyclodextrin polyaldehyde derivative is determined by the "alkali consumption method", and the specific steps are as follows: Accurately weigh a certain amount of ⁇ -cyclodextrin polyaldehyde derivative in a 250 ml Erlenmeyer flask Accurately add O.Olmol/L sodium hydroxide solution 30, and then place the conical flask in a 70 ° C water bath for 4 min, then quickly rinse it with tap water to cool it, then add 0.01 mol/L H 2 S0 4 solution. 20 ml and 30 ml of distilled water, add phenolphthalein indicator, shake well to make it evenly mixed, and titrate to the end point. Calculated as follows:
- G sample quality, g ;
- 3376 cnr 1 is the stretching vibration peak of -OH
- 2926 cnr 1 is the -CH 2 stretching vibration peak
- 1158 cnr 1 is the COC stretching vibration peak
- 1081 cnr 1 is the C-0-H bending vibration peak
- 1030 ( ⁇ 1_ 1 is ( -0 and 0 -0 coupled vibration peak
- 707 cnr 1 is the ring vibration peak.
- Figure 4 is a NMR spectrum of the ⁇ -cyclodextrin polyaldehyde of the present example, and it can be seen from the spectrum that ⁇ 9.70 (s, 1H), 9.53 (s, 1H), 5.68 (s, 14H), 4.94 ( s, 2H), 4.84 (s, 5H), 4.43 (s, 4H), 3.64 (s, 24H), 3.56 (s, 14H where ⁇ 9.70 (s, IH), 9.53 (s, IH) is aldehyde
- the characteristic peak of hydrogen that is, the product formed by the present invention does have the formation of an aldehyde group, and the ratio of the area of ⁇ 4.94 to the peak area of ⁇ 4.84 is 2: 5 to 3:
- the ⁇ -cyclodextrin polyaldehyde synthesized in the present invention has between 2 and 3 aldehyde groups per mole of aldehyde groups, and has 2 to 3 reactive groups, which is suitable for use as a crosslinking agent.
- FIG. 5 is an FTIR chart of the ⁇ -cyclodextrin polyaldehyde aqueous solution having a mass concentration of 0.45% before and after crosslinking with collagen in the present embodiment
- 33 1 8 CHT 1 is an amide oxime band peak ( ⁇ - ⁇ stretching vibration:)
- 3071 cnr 1 is the amide B band peak (N-H stretching vibration:)
- 1549 cnr 1 is the amide II band peak (N-H bending vibration), from the figure It can be seen that the position and intensity of the amide I band associated with the collagen triple helix conformation are substantially unchanged, and there is still a high absorption peak, indicating that the triple helix structure of the collagen after cross-linking is not destroyed.
- the absorption peak intensity of the amide B band and the amide I band decreased after cross-linking, that is, the amino group content decreased, indicating that ⁇ -cyclodextrin dialdehyde was cross-linked with collagen.
- Figure 6 is a graph showing the degree of crosslinking of the ⁇ -cyclodextrin polyaldehyde crosslinked collagen of the present embodiment, wherein Col-C-1 to Col-C-5 are different concentrations of ⁇ -cyclodextrin polyaldehyde crosslinked collagen
- the degree of cross-linking the concentration of ⁇ -cyclodextrin polyaldehyde is 0.075%, 0.15%, 0.30%, 0.45% and 0.60%, respectively.
- concentration of ⁇ -cyclodextrin polyaldehyde increases, the degree of crosslinking increases. , the highest reached about 91%.
- Col-G-6 is the cross-linking degree of 0.25% glutaraldehyde cross-linked collagen, the degree of cross-linking is 81.26%, and the aldehyde content of 0.25% glutaraldehyde is about 0.3%.
- the aldehyde content in ⁇ -cyclodextrin polyaldehyde About 4 times, but the degree of cross-linking is not as high as 0.3% ⁇ -cyclodextrin polyaldehyde cross-linked collagen, so ⁇ -cyclodextrin polyaldehyde is a cross-linking efficiency. Agent.
- Figure 7 is a graph showing the mass concentration of 0.45% ⁇ -cyclodextrin polyaldehyde aqueous solution in the present embodiment before and after cross-linking with collagen
- the heat denaturation temperature is the temperature at which the triple helix structure unique to collagen is completely destroyed.
- the thermal denaturation temperature of pure collagen is 71.4 °C.
- the heat-denatured temperature of the collagen modified by ⁇ -cyclodextrin polyaldehyde crosslinking was 88.3 ° C, which was 16.9 ° C higher than that of pure collagen.
- the heat-denatured temperature of the collagen modified by crosslinking with glutaraldehyde was 82.3 ° C, which was 10.9 ° C higher than that of pure collagen. It can be seen that ⁇ -cyclodextrin polyaldehyde cross-linked collagen can significantly improve the thermal stability of collagen, even more effective than glutaraldehyde-modified collagen, which again proves that collagen and ⁇ -cyclodextrin dialdehyde occur.
- Cross-linking The increase of the interaction between collagen molecules makes the activity of the peptide chain constrained, and the difficulty of phase change increases. The heat energy and temperature required for denaturation increase, and the thermal stability of the material increases, which can expand the application range of the material.
- Fig. 8 is a graph showing the mechanical properties of the ⁇ -cyclodextrin polyaldehyde and collagen before and after cross-linking of the present embodiment.
- the collagen hydrogel is mainly subjected to compressive stress in many fields of application, and thus the collagen hydrogel was subjected to a compression test.
- a cylindrical collagen gel (about 6 mm in diameter and 15 mm in height) was compression-tested at 1 mm/min using a biomechanical testing machine, and linear fitting was performed using the initial straight line portion of the obtained stress-strain curve. For compression modulus. It can be seen from the figure that the compressive modulus of pure collagen is only about 0.6 KPa.
- FIG. 9 is a graph showing the enzymatic stability of the ⁇ -cyclodextrin polyaldehyde and collagen before and after cross-linking of the present embodiment, and the relative enzymatic hydrolysis degree of the pure collagen is determined to be 100%, as shown in the figure, along with the cross-linking agent
- concentration increases, the degree of enzymatic hydrolysis of the modified collagen material decreases in turn.
- concentration of the cross-linking agent the smaller the degree of enzymatic hydrolysis, indicating that the higher the cross-linking agent concentration, the better the cross-linking effect, and the more resistant the collagen material has. Strong.
- the concentration of the new cross-linking agent is 0.6%
- the relative enzymatic hydrolysis degree is 28.68%
- the degree of enzymatic hydrolysis is significantly lower than that of pure collagen, which indicates that the novel cross-linking is very effective in improving the bio-stability of collagen and its degradation rate. Can be greatly reduced.
- the new cross-linking agent cross-links with collagen, forming a series of chemical bonds in the molecule and between molecules, and even forming a network cross-linking between the molecules, thereby making it more stable.
- the relative enzymatic hydrolysis degree of the collagen material cross-linked with glutaraldehyde is about 30.05%, and it is known that the anti-enzymatic ability of the collagen material modified by the novel cross-linking agent is slightly better than that of glutaraldehyde.
- a brief description of the new cross-linking agent can effectively improve the anti-enzymatic ability of collagen, It slows down the degradation time of collagen, which is mainly due to the inability of the enzyme to fully contact the collagen molecules due to cross-linking. The degradation of the enzyme can only be carried out on the surface of the collagen material, thus slowing down the degradation of collagen.
- 0.5 mmol of the purified Y-cyclodextrin was weighed into a 50 ml flask, and 5 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.2 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, the mixture was reacted under magnetic stirring at 25 ° C for 30 hours.
- DMSO polar aprotic solvent dimethyl sulfoxide
- the mixture was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.
- the cyclodextrin polyaldehyde yield was determined to be 87%.
- the cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used.
- Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
- the modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 20 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
- Example 3 0.5 mmol of the purified ⁇ -cyclodextrin was weighed into a 50 ml flask, and 8 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.8 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, the mixture was reacted under magnetic stirring at 25 ° C for 30 hours.
- DMSO polar aprotic solvent dimethyl sulfoxide
- the mixture was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.
- the cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used.
- Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
- the modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 18 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
- the cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used.
- Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
- the modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
- the cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. different density
- the cyclodextrin polyaldehyde aqueous solution was modified with collagen as follows:
- the modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
- the cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used.
- Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
- the modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, Preparing a modified collagen gel;
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Abstract
Provided is an application method of a cyclodextrin multi-aldehyde cross-linking agent. In the cyclodextrin multi-aldehyde cross-linking agent, there are 2-3 acid groups per mole on average, which are used to modify collagen, the modification method comprising: mixing the aqueous solution of the cyclodextrin multi-aldehyde cross-linking agent and the collagen aqueous solution, freezing the obtained mixed solution in a mold to prepare modified collagen gel, taking out of the modified collagen gel after the mold is unfrozen at room temperature, and drying the modified collagen gel to obtain porous spongy modified collagen.
Description
一种环糊精多醛交联剂的应用方法 Application method of cyclodextrin polyaldehyde crosslinking agent
技术领域 Technical field
本发明涉及环糊精和胶原蛋白交联剂, 具体涉及一种环糊精多醛交联剂的 应用方法。 说 背景技术 The present invention relates to a cyclodextrin and a collagen crosslinker, and in particular to a method for applying a cyclodextrin polyaldehyde crosslinker. Background technology
胶原蛋白是一种生物天然高分子物质, 英文学名 collages胶原蛋白存在于 书 Collagen is a biological natural high molecular substance, English name collages collagen exists in the book
所有细胞动物体内, 是体内含量最多的一类蛋白质, 存在于几乎所有组织中, 是细胞外基质成分。 胶原蛋白拥有特殊的 3股螺旋肽链结构, 赋予胶原蛋白许 多良好的生物和化学特性。 胶原蛋白具有良好的生物相容性, 生物可降解性, 低抗原性; 具有细胞适应性、 细胞增殖左右和促进血小板凝聚的作用; 具有较 好的可溶解性, 无毒性, 独特的纤维形成特性; 以及易于改性和来源广泛等优 点。 因此, 胶原蛋白作为一种非常重要的生物材料, 被广泛应用与伤口敷料、 人工皮肤、 美容、 药物缓释、 组织工程支架和细胞培养等方面, 胶原蛋白的价 值越来越受到人们的重视。 In all cell animals, it is the most abundant type of protein in the body, present in almost all tissues, and is an extracellular matrix component. Collagen has a special three-stranded helical peptide chain structure that imparts many good biological and chemical properties to collagen. Collagen has good biocompatibility, biodegradability, low antigenicity; has cell adaptation, cell proliferation and promotes platelet aggregation; has good solubility, non-toxicity, unique fiber formation characteristics ; and easy to modify and a wide range of sources. Therefore, as a very important biological material, collagen is widely used in wound dressings, artificial skin, beauty, drug release, tissue engineering scaffolds and cell culture. The value of collagen is receiving more and more attention.
但是, 从动物组织中提取的纯胶原蛋白存在着力学强度不高, 作为组织修 复材料不能承受体内的力学环境, 在体内降解速度过快和热稳定性不够等问题。 因此人们通过各种方法对胶原蛋白进行改性, 包括物理交联、 化学交联等方法。 其中用的比较多的是化学交联。 在化学交联中主要的交联剂有: 戊二醛、 碳化 二亚胺 (EDC)、 环氧化合物、 京尼平、 乙醛酸、 含硫化合物、 己二异氰酸酯、 酰基叠氮及二苯基磷酸盐、 双醛淀粉等。 上述化学交联剂都可以比较有效的提 高胶原蛋白的力学强度和稳定性等, 但也都通过交联或多或少产生一些缺点, 比如有些会产生细胞毒性、 有些会使胶原材料有颜色、 有些则有钙化潜力、 有
些不能明显改善胶原材料的某些缺点等。 However, the pure collagen extracted from animal tissues has low mechanical strength, and the tissue repairing material cannot withstand the mechanical environment in the body, and the degradation rate in the body is too fast and the thermal stability is insufficient. Therefore, people have modified collagen by various methods, including physical crosslinking, chemical crosslinking and the like. Among them, more is chemical cross-linking. The main crosslinking agents in chemical crosslinking are: glutaraldehyde, carbodiimide (EDC), epoxy compounds, genipin, glyoxylic acid, sulfur compounds, hexamethylene diisocyanate, acyl azide and diphenyl. Phosphate, dialdehyde starch, etc. The above chemical cross-linking agents can effectively improve the mechanical strength and stability of collagen, etc., but also all have some disadvantages through cross-linking, for example, some may cause cytotoxicity, some may cause color of collagen materials, Some have calcification potential, Some do not significantly improve some of the disadvantages of collagen materials.
环糊精是由 6〜8个 D-(+)吡喃葡萄糖单元通过 α-1, 4-糖苷键相结合的环状低 聚糖, 分别对应 α、 β、 Υ环糊精, 环糊精分子的模型像一个内空去顶的锥型圆 筒。 在环糊精分子的环形结构中, 极性羟基位于环糊精单位的边缘, 仲位和伯 位极性羟基从边缘伸出, 所以上下以及外部都分布有亲水基团, 具有亲水性。 环糊精空腔内部排列着配糖氧桥原子。 由于氧原子的非键合电子指向中心, 使 空腔内具有很高的电子密度; 吡喃葡萄糖环 C-3, C-5上的氢原子位于空腔内并 覆盖了配糖氧原子, 使空腔内部成为疏水性空间。 该疏水空腔可通过疏水作用 力、 氢键、 范德华力等作用形成包合物, 因而表现出特殊的吸附性能。 环糊精 能与许多有机化合物、 卤素及某些无机化合物形成稳定的包合复合物。 它像分 子袋一样可以使许多化合物或化合物的某些基团包合起来, 改善被包合物质的 水溶性、 稳定性以及其他理化性质。 环糊精对于被包含的物质可以起到缓释剂 的作用, 且其对人没有明显的毒副作用。 环糊精的优点使其能广泛应用与生物 医学的各个领域。 发明内容 Cyclodextrin is a cyclic oligosaccharide composed of 6~8 D-(+) glucopyranose units through α-1, 4-glycosidic linkages, corresponding to α, β, Υcyclodextrin, cyclodextrin The model of the molecule is like a cone-shaped cylinder with an inner hollow to the top. In the ring structure of the cyclodextrin molecule, the polar hydroxyl group is located at the edge of the cyclodextrin unit, and the secondary and primary polar hydroxyl groups protrude from the edge, so that hydrophilic groups are distributed both above and below, and are hydrophilic. . Inside the cyclodextrin cavity, a sugar-oxygen bridge atom is arranged. Since the non-bonded electrons of the oxygen atom are directed to the center, the electron density in the cavity is high; the hydrogen atoms on the glucopyranose ring C-3, C-5 are located in the cavity and cover the oxygen atom of the glycoside. The interior of the cavity becomes a hydrophobic space. The hydrophobic cavity can form a clathrate by hydrophobic interaction, hydrogen bonding, van der Waals force, etc., and thus exhibits special adsorption properties. Cyclodextrins form stable inclusion complexes with many organic compounds, halogens and certain inorganic compounds. It can enclose certain compounds or certain groups of compounds like a molecular bag to improve the water solubility, stability and other physical and chemical properties of the encapsulated material. Cyclodextrins can act as sustained release agents for the substances to be included, and they have no significant toxic side effects to humans. The advantages of cyclodextrin make it widely available in all areas of biomedical science. Summary of the invention
为解决上述现有技术的不足之处, 本发明的目的在于提供一种环糊精多醛 交联剂的应用方法, 该方法中将环糊精多醛交联剂用于对胶原的改性, 环糊精 多醛交联剂在提高胶原的力学强度和稳定性的同时, 不会产生细胞毒性。 In order to solve the above deficiencies of the prior art, an object of the present invention is to provide a method for applying a cyclodextrin polyaldehyde cross-linking agent, wherein a cyclodextrin polyaldehyde cross-linking agent is used for modifying collagen. The cyclodextrin polyaldehyde cross-linking agent does not produce cytotoxicity while improving the mechanical strength and stability of collagen.
为实现上述发明目的, 本发明采用如下技术方案: In order to achieve the above object, the present invention adopts the following technical solutions:
一种环糊精多醛交联剂的应用方法, 所述环糊精多醛交联剂用于对胶原的 改性; 其中所述环糊精多醛交联剂的化学结构为:
A method for applying a cyclodextrin polyaldehyde crosslinking agent, wherein the cyclodextrin polyaldehyde crosslinking agent is used for modifying a collagen; wherein the chemical structure of the cyclodextrin polyaldehyde crosslinking agent is:
优选的, 所述环糊精多醛交联剂用于多孔海绵状改性胶原的制备。
优选的, 所述多孔海绵状改性胶原的制备方法包括如下歩骤:Preferably, the cyclodextrin polyaldehyde crosslinker is used for the preparation of porous sponge-like modified collagen. Preferably, the preparation method of the porous sponge-like modified collagen comprises the following steps:
( 1 ) 将环糊精多醛交联剂水溶液与胶原水溶液混合, 并在 2〜6 °C反应 20〜30h, 得到改性胶原溶液; (1) mixing a cyclodextrin polyaldehyde crosslinking agent aqueous solution with an aqueous collagen solution, and reacting at 2 to 6 ° C for 20 to 30 hours to obtain a modified collagen solution;
(2)将歩骤(1 )得到的改性胶原溶液在 35〜38°C保温 15〜20min, 然后将 改性胶原溶液转移至模具中, 再将模具在 -15〜- 25°C冷冻 40〜50h, 制备改性胶 原凝胶; (2) The modified collagen solution obtained in the step (1) is kept at 35 to 38 ° C for 15 to 20 minutes, then the modified collagen solution is transferred to a mold, and the mold is frozen at -15 to 25 ° C. ~50h, preparing a modified collagen gel;
(3 ) 将模具在室温下放置 4〜6h后将改性胶原凝胶取出, 并将其干燥得到 所述多孔海绵状改性胶原。 (3) The mold was taken out at room temperature for 4 to 6 hours, and the modified collagen gel was taken out and dried to obtain the porous sponge-like modified collagen.
优选的,歩骤(1 )中环糊精多醛交联剂水溶液的质量浓度为 0.075%〜0.6%, 胶原水溶液的质量浓度为 0.6%。 Preferably, the mass concentration of the aqueous solution of the cyclodextrin polyaldehyde crosslinking agent in the step (1) is 0.075% to 0.6%, and the mass concentration of the aqueous collagen solution is 0.6%.
优选的, 歩骤 (1 ) 中反应温度为 4°C。 Preferably, the reaction temperature in the step (1) is 4 °C.
优选的, 歩骤(2) 中改性胶原溶液在 37°C水浴中保温, 冷冻温度为 -20°C。 优选的, 歩骤 (3 ) 中所述的干燥方法为冷冻干燥。 Preferably, the modified collagen solution in step (2) is incubated in a 37 ° C water bath at a freezing temperature of -20 ° C. Preferably, the drying method described in the step (3) is freeze drying.
相对于现有技术, 本发明具有如下优点及有益效果: Compared with the prior art, the present invention has the following advantages and beneficial effects:
( 1 )本发明使用醛基个数为 2或 3的环糊精多醛做交联剂; 由于该环糊精 多醛交联剂具有无毒性、 交联效果好的优点, 特别适用于对胶原的改性。 将醛 基个数为 2或 3的环糊精多醛用于胶原的改性, 改性胶原交联效率高, 并且改 性后的胶原材料热稳定性大大提高、 耐降解性能显著增强。 (1) The present invention uses a cyclodextrin polyaldehyde having an aldehyde group of 2 or 3 as a crosslinking agent; since the cyclodextrin polyaldehyde crosslinking agent has the advantages of non-toxicity and good crosslinking effect, it is particularly suitable for Modification of collagen. The cyclodextrin polyaldehyde having 2 or 3 aldehyde groups is used for the modification of collagen, and the modified collagen has high cross-linking efficiency, and the modified collagen material has greatly improved thermal stability and markedly enhanced degradation resistance.
(2)本发明将环糊精多醛与胶原蛋白交联, 既可以弥补胶原蛋白的一些缺 点, 改善胶原材料的性能, 同时也避免了引入戊二醛等有细胞毒性的化学交联 剂, 不仅保持了胶原原有的一些特性, 还引入了一些环糊精的优良性能, 如特 殊刚性结构, 包合配位特性, 分子识别和组装等性能, 大大扩展了胶原的应用 范围。 附图说明 (2) The present invention cross-links cyclodextrin polyaldehyde with collagen, which can not only make up for some shortcomings of collagen, improve the performance of collagen materials, but also avoid the introduction of cytotoxic chemical cross-linking agents such as glutaraldehyde. It not only maintains some of the original properties of collagen, but also introduces some excellent properties of cyclodextrin, such as special rigid structure, inclusion coordination, molecular recognition and assembly, which greatly expands the application range of collagen. DRAWINGS
图 1是环糊精多醛的合成路线示意图;
图 2是环糊精多醛交联胶原原理图; Figure 1 is a schematic diagram of a synthetic route of a cyclodextrin polyaldehyde; Figure 2 is a schematic diagram of cyclodextrin polyaldehyde cross-linked collagen;
图 3是 β-环糊精和 β-环糊精多醛的红外光谱图 (FTIR); Figure 3 is an infrared spectrum (FTIR) of β-cyclodextrin and β-cyclodextrin polyaldehyde;
图 4是 β-环糊精多醛的核磁共振图 (1H-NMR); Figure 4 is a nuclear magnetic resonance spectrum (1H-NMR) of β-cyclodextrin polyaldehyde;
图 5是 β-环糊精多醛与胶原交联前后的红外光谱图 (FTIR); Figure 5 is an infrared spectrum (FTIR) of β-cyclodextrin polyaldehyde and collagen before and after cross-linking;
图 6是 β-环糊精多醛交联胶原的交联度测试图; Figure 6 is a graph showing the degree of crosslinking of β-cyclodextrin polyaldehyde crosslinked collagen;
图 7是 β-环糊精多醛与胶原交联前后的 DSC图; Figure 7 is a DSC chart before and after cross-linking of β-cyclodextrin polyaldehyde with collagen;
图 8是 β-环糊精多醛与胶原交联前后的力学性能图; Figure 8 is a graph showing the mechanical properties of β-cyclodextrin polyaldehyde before and after cross-linking with collagen;
图 9是 β-环糊精多醛与胶原交联前后的酶解度图。 具体实施方式 Figure 9 is a graph showing the degree of enzymatic hydrolysis before and after cross-linking of β-cyclodextrin polyaldehyde with collagen. detailed description
下面结合实施例与附图对本发明作进一歩详细的描述, 但本发明的实施方 式不限于此。 The present invention will be described in detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.
本发明的原理如图 1和 2所示, 利用 2-碘酰基苯甲酸(ΙΒΧ)将环糊精氧化 成环糊精多醛衍生物, 即一种多官能团化合物, 作为交联剂。 然后用环糊精多 醛的醛基与胶原上的活性氨基反应生成希夫碱来交联改性胶原, 使胶原的力学 性能、 热稳定性能和耐降解性能大大的提高。 本发明适合 α-环糊精、 β-环糊精和 环糊精。 The principle of the present invention is shown in Figures 1 and 2, wherein cyclodextrin is oxidized to a cyclodextrin polyaldehyde derivative, i.e., a polyfunctional compound, as a crosslinking agent by using 2-iodobenzoic acid (hydrazine). Then, the aldehyde group of the cyclodextrin polyaldehyde is reacted with the active amino group on the collagen to form a Schiff base to crosslink the modified collagen, so that the mechanical properties, thermal stability and degradation resistance of the collagen are greatly improved. The present invention is suitable for α-cyclodextrin, β-cyclodextrin and cyclodextrin.
本发明环糊精多醛交联剂的合成方法包括如下歩骤: The method for synthesizing the cyclodextrin polyaldehyde crosslinking agent of the present invention comprises the following steps:
( 1 )将 0.5mmol纯化后的环糊精加入 5〜8ml二甲基亚砜中、 并将环糊精完 全溶解, 得到混合物; (1) 0.5 mmol of the purified cyclodextrin is added to 5 to 8 ml of dimethyl sulfoxide, and the cyclodextrin is completely dissolved to obtain a mixture;
(2) 向歩骤 (1 ) 所得的混合物中加入 2.2〜2.8当量的 2-碘酰基苯甲酸,并 在保持搅拌、 25〜30°C的条件下反应 30〜48小时,然后对反应液过滤、保留滤液; (2) adding 2.2 to 2.8 equivalents of 2-iodobenzoic acid to the mixture obtained in the step (1), and reacting for 30 to 48 hours while maintaining stirring at 25 to 30 ° C, and then filtering the reaction solution. Keep the filtrate;
(3 ) 将歩骤 (2) 中得到的滤液滴入到丙酮中, 并过滤、 保留沉淀;(3) Drop the filtrate obtained in the step (2) into acetone, and filter and retain the precipitate;
(4)将歩骤(3 ) 中得到的沉淀在 30〜40°C下真空干燥 24h, 然后再用去离 子水溶解; (4) The precipitate obtained in the step (3) is dried under vacuum at 30 to 40 ° C for 24 hours, and then dissolved in deionized water;
(5 )将歩骤(4)所得的溶液过滤、 保留滤液, 将滤液冷冻干燥 48h, 得到
所述环糊精多醛交联剂。 (5) The solution obtained in the step (4) is filtered, the filtrate is retained, and the filtrate is freeze-dried for 48 hours to obtain The cyclodextrin polyaldehyde crosslinker.
实施例 1 Example 1
(一) 制备环糊精多醛交联剂: (1) Preparation of cyclodextrin polyaldehyde crosslinker:
称取 0.5mmol纯化后的 β-环糊精于 50ml烧瓶中, 向烧瓶中加入 7ml极性非 质子溶剂二甲基亚砜(DMSO), 磁力搅拌环糊精至溶解完全后, 向其中加入 2.4 当量的温和氧化剂 2-碘酰基苯甲酸(IBX)。然后在磁力搅拌下在 25°C下反应 48 小时, 反应结束后过滤, 将滤液滴入到 150ml的丙酮中, 将析出来的物质过滤 得沉淀, 将沉淀用真空干燥箱在 35°C下真空干燥 24h, 最后将烘干后的样品用 50ml去离子水溶解, 过滤除掉杂质, 滤液经冷冻干燥得浅黄色样品。 0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.4 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 25 ° C for 48 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 h, the dried sample was finally dissolved in 50 ml of deionized water, and the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a pale yellow sample.
经检测, β-环糊精多醛产率为 90%。 The yield of β-cyclodextrin polyaldehyde was 90%.
(二) 改性胶原: (2) Modified collagen:
将歩骤 (一) 制备的环糊精多醛交联剂配制成 5种质量浓度为 0.075%、 0.15%、 0.30%、 0.45%、 0.60%的环糊精多醛水溶液, 然后使用这 5种不同浓度 的环糊精多醛水溶液分别按如下歩骤对胶原进行改性: The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
( 1 )将环糊精多醛水溶液与质量浓度为 0.6%的胶原水溶液均匀混合,并在 4°C冰箱里反应 24h, 得到改性胶原溶液; (1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;
(2) 将歩骤 (1 ) 得到的改性胶原溶液置于 37°C水浴中 18min, 并用吸管 将改性胶原溶液滴入一定形状的模具中, 随后将模具放在 -20°C冰箱中冷冻 48h, 制备改性胶原凝胶; (2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 18 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
(3 )将模具在室温下放置 6h后将改性胶原凝胶取出, 然后通过冷冻干燥机 对改性胶原凝胶进行干燥, 得到多孔海绵状改性胶原。 (3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen.
(三)实施例 1的 β-环糊精多醛及多孔海绵状改性胶原产物经过冷冻干燥后, 进行表征分析。 (III) The β-cyclodextrin polyaldehyde and the porous sponge-like modified collagen product of Example 1 were subjected to characterization by freeze-drying.
FT-IR分析在 NICOLET760形红外分光光度计上进行, 样品制备采用 KBr 压片法。 -NMR分析采用 Bruker400MHz核磁共振仪, 溶剂为氘代 DMSO。 β- 环糊精多醛改性胶原的 DSC分析在美国 ΡΕ公司生产的 DIAMOND DSC型差示
扫描量热仪上进行,准确称取 5〜10mg样品于 DSC坩埚中,以空坩埚作为参比, 从 35 °C加热至 210°C, 升温速率为 5 °C/min, 样品室的氮气流量为 20mL/min。 β-环糊精多醛改性胶原的交联度测定采用茚三酮法, 以甘氨酸溶液作标准曲线, 用 LAMBDA950型紫外分光光度计在 570nm处测定其紫外吸光度值。 合成的 β- 环糊精多醛衍生物中醛基含量的测定利用 "碱消耗法", 具体歩骤是: 准确称取 一定质量的 β-环糊精多醛衍生物于 250ml锥形瓶中, 精确加入 O.Olmol/L的氢氧 化钠溶液 30, 将锥形瓶置于 70°C水浴中加热 4min后, 迅速用自来水冲洗使之 冷却, 然后加入 0.01mol/L的 H2S04溶液 20ml和蒸水 30ml, 加入酚酞指示剂, 充分振荡使其混合均匀, 滴定至终点。 计算公式如下: FT-IR analysis was performed on a NICOLET 760-type infrared spectrophotometer, and the sample preparation was performed by KBr tableting. The NMR analysis was performed using a Bruker 400 MHz nuclear magnetic resonance apparatus, and the solvent was deuterated DMSO. DSC analysis of β-cyclodextrin polyaldehyde modified collagen DIAMOND DSC type differential produced by American company Scanning calorimeter, accurately weigh 5~10mg sample in DSC坩埚, use air as reference, heat from 35 °C to 210 °C, heating rate is 5 °C/min, nitrogen flow in sample chamber It is 20 mL/min. The cross-linking degree of β-cyclodextrin polyaldehyde-modified collagen was determined by the ninhydrin method using a glycine solution as a standard curve, and the UV absorbance value was measured at 570 nm using a LAMBDA950 ultraviolet spectrophotometer. The content of aldehyde group in the synthesized β-cyclodextrin polyaldehyde derivative is determined by the "alkali consumption method", and the specific steps are as follows: Accurately weigh a certain amount of β-cyclodextrin polyaldehyde derivative in a 250 ml Erlenmeyer flask Accurately add O.Olmol/L sodium hydroxide solution 30, and then place the conical flask in a 70 ° C water bath for 4 min, then quickly rinse it with tap water to cool it, then add 0.01 mol/L H 2 S0 4 solution. 20 ml and 30 ml of distilled water, add phenolphthalein indicator, shake well to make it evenly mixed, and titrate to the end point. Calculated as follows:
每摩尔 β—环糊精多醛中醛基个数= ( CiVi-2C2V2 ) I ( G/1131 ) Number of aldehyde groups per mole of β-cyclodextrin polyaldehyde = ( CiVi-2C 2 V2 ) I ( G/1131 )
式中: CI—— NaOH溶液的浓度, mol/L; Where: CI - concentration of NaOH solution, mol / L;
VI——消耗 NaOH溶液的体积, L; VI - the volume of the NaOH solution consumed, L;
C2—— H2S04溶液的浓度, mol/L; C2——concentration of H2S04 solution, mol/L;
V2——消耗 H2S04溶液的体积, L; V2 - the volume of the H2S04 solution consumed, L;
G——样品质量, g; G——sample quality, g ;
1131一一环糊精多醛的分子质量, g/mol。 Molecular mass of 1131-cyclodextrin polyaldehyde, g/mol.
经过 3次重复实验后得到结论: 平均每摩尔 β-环糊精多醛中醛基的个数约为 2.5 个。 图 3为本实施例的 β-环糊精多醛和 β-环糊精的 FTIR图, 对比两图谱可以很 明显的看出 β-环糊精多醛在 1733.93 cnr1处有一新生成的峰, 此峰为醛基的特征 峰, 说明合成的产物中有醛基生成。另外, 图中 3376 cnr1为 -OH的伸縮振动峰, 2926 cnr1为 -CH2伸縮振动峰, 1158 cnr1为 C-O-C伸縮振动峰, 1081 cnr1为 C-0-H 弯曲振动峰, 1030 (^1_1为( -0和0 -0耦合振动峰, 707 cnr1为环振动峰。 可见 这些 β-环糊精多醛的吸收峰都与 β-环糊精的红外光谱图无明显区别,这表明利用 2-碘酰基苯甲酸 (ΙΒΧ) 氧化 β-环糊精合成多醛类衍生物并没有破坏 β-环糊精的 特殊结构, 这使得合成的 β-环糊精多醛衍生物保持了 β-环糊精的许多优良性能。
图 4是本实施例的 β-环糊精多醛的 -NMR谱图, 从图谱中可以看出 δ 9.70 (s, 1H), 9.53 (s, 1H), 5.68 (s, 14H), 4.94 (s, 2H), 4.84 (s, 5H), 4.43 (s, 4H), 3.64 (s, 24H), 3.56 (s, 14H 这其中 δ 9.70 (s, IH), 9.53 (s, IH)为醛基氢的特征峰, 即本发 明生成的产物中确实有醛基的生成, 而且 δ 4.94与 δ 4.84峰面积之比为 2: 5〜3:After three repeated experiments, it was concluded that the average number of aldehyde groups per mole of β-cyclodextrin polyaldehyde was about 2.5. 3 is a FTIR diagram of β-cyclodextrin polyaldehyde and β-cyclodextrin of the present embodiment, and it can be clearly seen from the two spectra that the β-cyclodextrin polyaldehyde has a newly formed peak at 1733.93 cnr 1 This peak is a characteristic peak of the aldehyde group, indicating that an aldehyde group is formed in the synthesized product. In addition, in the figure, 3376 cnr 1 is the stretching vibration peak of -OH, 2926 cnr 1 is the -CH 2 stretching vibration peak, 1158 cnr 1 is the COC stretching vibration peak, and 1081 cnr 1 is the C-0-H bending vibration peak, 1030 ( ^1_ 1 is ( -0 and 0 -0 coupled vibration peak, 707 cnr 1 is the ring vibration peak. It can be seen that the absorption peaks of these β-cyclodextrin polyaldehydes are not significantly different from the infrared spectrum of β-cyclodextrin. This indicates that the oxidation of β-cyclodextrin with 2-iodobenzoic acid (ΙΒΧ) to synthesize polyaldehyde derivatives does not destroy the specific structure of β-cyclodextrin, which allows the synthesis of β-cyclodextrin polyaldehyde derivatives to remain. Many excellent properties of β-cyclodextrin. Figure 4 is a NMR spectrum of the β-cyclodextrin polyaldehyde of the present example, and it can be seen from the spectrum that δ 9.70 (s, 1H), 9.53 (s, 1H), 5.68 (s, 14H), 4.94 ( s, 2H), 4.84 (s, 5H), 4.43 (s, 4H), 3.64 (s, 24H), 3.56 (s, 14H where δ 9.70 (s, IH), 9.53 (s, IH) is aldehyde The characteristic peak of hydrogen, that is, the product formed by the present invention does have the formation of an aldehyde group, and the ratio of the area of δ 4.94 to the peak area of δ 4.84 is 2: 5 to 3:
4之间, 这表明本发明合成的 β-环糊精多醛中每摩尔的醛基数在 2〜3之间, 有 2〜3个活性基团, 适合做交联剂使用。 Between 4, this indicates that the β-cyclodextrin polyaldehyde synthesized in the present invention has between 2 and 3 aldehyde groups per mole of aldehyde groups, and has 2 to 3 reactive groups, which is suitable for use as a crosslinking agent.
图 5是本实施例的质量浓度为 0.45%的 β-环糊精多醛水溶液与胶原交联前后 的 FTIR图, 33 1 8CHT1为酰胺 Α带峰 (Ν— Η伸縮振动:), 3071 cnr1为酰胺 B带 峰 (N— H伸縮振动:), 1655 cnr1为酰胺 I带峰 (C= 0伸縮振动:), 1549 cnr1为酰 胺 II带峰 (N— H弯曲振动),从图中可以看出与胶原三螺旋构象相关的酰胺 I带 的位置和强度基本没有变化, 而且仍有较高的吸收峰, 表明交联后胶原的三股 螺旋结构没有被破坏。而酰胺 B带和酰胺 I带的吸收峰强度在交联后有所减少, 即氨基含量减少, 这表明 β-环糊精二醛与胶原发生了交联作用。另外, 由于希夫 碱的 C=N伸縮振动峰约 1660 cm"1被胶原的酰胺 I带的峰遮蔽,所以在红外图谱 中并没有发现新的吸收峰。 5 is an FTIR chart of the β-cyclodextrin polyaldehyde aqueous solution having a mass concentration of 0.45% before and after crosslinking with collagen in the present embodiment, and 33 1 8 CHT 1 is an amide oxime band peak (Ν-Η stretching vibration:), 3071 cnr 1 is the amide B band peak (N-H stretching vibration:), 1655 cnr 1 is the amide I band peak (C= 0 stretching vibration:), 1549 cnr 1 is the amide II band peak (N-H bending vibration), from the figure It can be seen that the position and intensity of the amide I band associated with the collagen triple helix conformation are substantially unchanged, and there is still a high absorption peak, indicating that the triple helix structure of the collagen after cross-linking is not destroyed. The absorption peak intensity of the amide B band and the amide I band decreased after cross-linking, that is, the amino group content decreased, indicating that β-cyclodextrin dialdehyde was cross-linked with collagen. In addition, since the C=N stretching vibration peak of the Schiff base is about 1660 cm" 1, it is blocked by the peak of the amide I band of the collagen, so no new absorption peak is found in the infrared spectrum.
图 6是本实施例的 β-环糊精多醛交联胶原的交联度测试图, 其中 Col-C-1到 Col-C-5是不同浓度的 β-环糊精多醛交联胶原的交联度, β-环糊精多醛浓度依次 为 0.075%、 0.15%、 0.30%、 0.45%和 0.60%, 随着 β-环糊精多醛浓度的增加,交 联度也随之增加, 最高达到了 91%左右。 当交联剂浓度较低的时, 随着交联剂 浓度的提高, 交联度增加的比较快, 而当交联剂浓度达到 0.45%时, 再增加交联 剂的浓度, 交联度则变化不大, 这主要是当交联剂浓度超过 0.45%时, 胶原中的 自由氨基基本反应完全。 Col-G-6是 0.25%戊二醛交联胶原的交联度, 交联度为 81.26%, 而 0.25%戊二醛的醛含量约为 0.3%β-环糊精多醛中醛基含量的 4倍左 右, 但其交联度却还没有 0.3%β-环糊精多醛交联胶原的交联度高, 所以 β-环糊 精多醛是一种交联效率较好的交联剂。 Figure 6 is a graph showing the degree of crosslinking of the β-cyclodextrin polyaldehyde crosslinked collagen of the present embodiment, wherein Col-C-1 to Col-C-5 are different concentrations of β-cyclodextrin polyaldehyde crosslinked collagen The degree of cross-linking, the concentration of β-cyclodextrin polyaldehyde is 0.075%, 0.15%, 0.30%, 0.45% and 0.60%, respectively. As the concentration of β-cyclodextrin polyaldehyde increases, the degree of crosslinking increases. , the highest reached about 91%. When the concentration of the cross-linking agent is low, the degree of cross-linking increases rapidly as the concentration of the cross-linking agent increases, and when the cross-linking agent concentration reaches 0.45%, the concentration of the cross-linking agent is increased, and the degree of cross-linking is The change is not large, mainly because when the concentration of the cross-linking agent exceeds 0.45%, the free amino group in the collagen is substantially completely reacted. Col-G-6 is the cross-linking degree of 0.25% glutaraldehyde cross-linked collagen, the degree of cross-linking is 81.26%, and the aldehyde content of 0.25% glutaraldehyde is about 0.3%. The aldehyde content in β-cyclodextrin polyaldehyde About 4 times, but the degree of cross-linking is not as high as 0.3% β-cyclodextrin polyaldehyde cross-linked collagen, so β-cyclodextrin polyaldehyde is a cross-linking efficiency. Agent.
图 7是本实施例的质量浓度为 0.45%的 β-环糊精多醛水溶液与胶原交联前后
的 DSC图,热变性温度为胶原特有的三螺旋结构完全破坏的温度。从图中可知, 纯胶原的热变性温度为 71.4°C。用 β-环糊精多醛交联改性后的胶原的热变性温度 为 88.3 °C, 比纯胶原的热变性温度提高了 16.9°C。 用戊二醛交联改性后的胶原 的热变性温度为 82.3 °C, 比纯胶原的热变性温度提高了 10.9°C。 由此可知 β-环糊 精多醛交联胶原可以显著的提高胶原的热稳定性, 甚至比戊二醛改性胶原更加 有效,这也再次证明胶原与 β-环糊精二醛之间发生了交联。胶原分子间作用力的 增加使得肽链的活动受到约束, 发生相变化的难度增大, 表现为变性所需的热 能和温度增加, 材料的热稳定性增加, 从而可以扩大材料的应用范围。 Figure 7 is a graph showing the mass concentration of 0.45% β-cyclodextrin polyaldehyde aqueous solution in the present embodiment before and after cross-linking with collagen In the DSC chart, the heat denaturation temperature is the temperature at which the triple helix structure unique to collagen is completely destroyed. As can be seen from the figure, the thermal denaturation temperature of pure collagen is 71.4 °C. The heat-denatured temperature of the collagen modified by β-cyclodextrin polyaldehyde crosslinking was 88.3 ° C, which was 16.9 ° C higher than that of pure collagen. The heat-denatured temperature of the collagen modified by crosslinking with glutaraldehyde was 82.3 ° C, which was 10.9 ° C higher than that of pure collagen. It can be seen that β-cyclodextrin polyaldehyde cross-linked collagen can significantly improve the thermal stability of collagen, even more effective than glutaraldehyde-modified collagen, which again proves that collagen and β-cyclodextrin dialdehyde occur. Cross-linking. The increase of the interaction between collagen molecules makes the activity of the peptide chain constrained, and the difficulty of phase change increases. The heat energy and temperature required for denaturation increase, and the thermal stability of the material increases, which can expand the application range of the material.
图 8是本实施例的 β-环糊精多醛与胶原交联前后的力学性能图,胶原水凝胶 在很多应用领域中主要受到压应力, 因此对胶原水凝胶进行了压縮测试。 将圆 柱体胶原凝胶(直径约为 6mm,高 15mm)用生物力学试验机以 lmm/min对其进 行压縮测试, 用得到的应力-应变曲线初始的直线部分进行线性拟合, 直线斜率 定为压縮模量。 从图中可以看出纯胶原的压縮模量只有约 0.6KPa,随着环糊精多 醛交联剂的浓度的增加, 其压縮强度也不断增加, 最高达到了 3.522KPa, 比纯 胶原提高了近 6倍。 可见用环糊精多醛交联后其压縮强度明显提高, 力学性能 十丄幽日 J fJinU。 Fig. 8 is a graph showing the mechanical properties of the β-cyclodextrin polyaldehyde and collagen before and after cross-linking of the present embodiment. The collagen hydrogel is mainly subjected to compressive stress in many fields of application, and thus the collagen hydrogel was subjected to a compression test. A cylindrical collagen gel (about 6 mm in diameter and 15 mm in height) was compression-tested at 1 mm/min using a biomechanical testing machine, and linear fitting was performed using the initial straight line portion of the obtained stress-strain curve. For compression modulus. It can be seen from the figure that the compressive modulus of pure collagen is only about 0.6 KPa. As the concentration of the cyclodextrin polyaldehyde crosslinking agent increases, the compressive strength increases continuously, reaching a maximum of 3.522 KPa, which is better than pure collagen. Increased nearly 6 times. It can be seen that the compressive strength of the cyclodextrin polyaldehyde is significantly improved after cross-linking, and the mechanical properties are ten-day J FJinU.
图 9是本实施例的 β-环糊精多醛与胶原交联前后的酶解稳定性图,将纯胶原 的相对酶解度定为 100%, 从图中可以看出随着交联剂浓度的增加, 改性胶原材 料的酶解度依次减少, 交联剂浓度越高, 酶解度越小, 表明交联剂浓度越高, 交联效果越好,胶原材料的耐酶解能力越强。其中当新型交联剂浓度为 0.6%时, 相对酶解度为 28.68%, 其酶解度明显低于纯胶原, 这表明新型交联非常有效的 提高了胶原的生物稳定性, 使其降解速率得以大大降低。 同时说明新交联剂与 胶原发生交联反应, 在分子内及分子间形成了一系列的化学键, 甚至在分子间 形成了网状交联, 从而使其更稳定。 从上图还可知用戊二醛交联的胶原材料的 相对酶解度约为 30.05%, 可知新型交联剂改性的胶原材料的抗酶解能力比戊二 醛的还略好, 这进一歩说明了新型交联剂能有效的提高胶原的抗酶解能力, 延
缓了胶原的降解时间, 这主要是由于交联作用使酶无法和胶原分子充分接触, 酶的降解作用只能在胶原材料的表面进行, 因此减缓了胶原的降解。 实施例 2 9 is a graph showing the enzymatic stability of the β-cyclodextrin polyaldehyde and collagen before and after cross-linking of the present embodiment, and the relative enzymatic hydrolysis degree of the pure collagen is determined to be 100%, as shown in the figure, along with the cross-linking agent When the concentration increases, the degree of enzymatic hydrolysis of the modified collagen material decreases in turn. The higher the concentration of the cross-linking agent, the smaller the degree of enzymatic hydrolysis, indicating that the higher the cross-linking agent concentration, the better the cross-linking effect, and the more resistant the collagen material has. Strong. When the concentration of the new cross-linking agent is 0.6%, the relative enzymatic hydrolysis degree is 28.68%, and the degree of enzymatic hydrolysis is significantly lower than that of pure collagen, which indicates that the novel cross-linking is very effective in improving the bio-stability of collagen and its degradation rate. Can be greatly reduced. At the same time, it indicates that the new cross-linking agent cross-links with collagen, forming a series of chemical bonds in the molecule and between molecules, and even forming a network cross-linking between the molecules, thereby making it more stable. It can be seen from the above figure that the relative enzymatic hydrolysis degree of the collagen material cross-linked with glutaraldehyde is about 30.05%, and it is known that the anti-enzymatic ability of the collagen material modified by the novel cross-linking agent is slightly better than that of glutaraldehyde. A brief description of the new cross-linking agent can effectively improve the anti-enzymatic ability of collagen, It slows down the degradation time of collagen, which is mainly due to the inability of the enzyme to fully contact the collagen molecules due to cross-linking. The degradation of the enzyme can only be carried out on the surface of the collagen material, thus slowing down the degradation of collagen. Example 2
(一) 制备环糊精多醛交联剂: (1) Preparation of cyclodextrin polyaldehyde crosslinker:
称取 0.5mmol纯化后的 Y -环糊精于 50ml烧瓶中, 向烧瓶中加入 5ml极性 非质子溶剂二甲基亚砜 (DMSO), 磁力搅拌环糊精至溶解完全后, 向其中加入 2.2当量的温和氧化剂 2-碘酰基苯甲酸 (IBX)。 然后在磁力搅拌下在 25°C下反 应 30小时, 反应结束后过滤, 将滤液滴入到 150ml的丙酮中, 将析出来的物质 过滤得沉淀, 将沉淀用真空干燥箱在 35°C下真空干燥 24h, 最后将烘干后的样 品用 50ml去离子水溶解, 过滤除掉杂质, 滤液经冷冻干燥得最终样品。 0.5 mmol of the purified Y-cyclodextrin was weighed into a 50 ml flask, and 5 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.2 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, the mixture was reacted under magnetic stirring at 25 ° C for 30 hours. After the reaction was completed, the mixture was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.
经检测, 环糊精多醛产率为 87%。 The cyclodextrin polyaldehyde yield was determined to be 87%.
(二) 改性胶原: (2) Modified collagen:
将歩骤 (一) 制备的环糊精多醛交联剂配制成 5种质量浓度为 0.075%、 0.15%、 0.30%、 0.45%、 0.60%的环糊精多醛水溶液, 然后使用这 5种不同浓度 的环糊精多醛水溶液分别按如下歩骤对胶原进行改性: The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
( 1 )将环糊精多醛水溶液与质量浓度为 0.6%的胶原水溶液均匀混合,并在 4°C冰箱里反应 24h, 得到改性胶原溶液; (1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;
(2) 将歩骤 (1 ) 得到的改性胶原溶液置于 37°C水浴中 20min, 并用吸管 将改性胶原溶液滴入一定形状的模具中, 随后将模具放在 -20°C冰箱中冷冻 48h, 制备改性胶原凝胶; (2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 20 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
(3 )将模具在室温下放置 6h后将改性胶原凝胶取出, 然后通过冷冻干燥机 对改性胶原凝胶进行干燥, 得到多孔海绵状改性胶原。 实施例 3
称取 0.5mmol纯化后的 β-环糊精于 50ml烧瓶中, 向烧瓶中加入 8ml极性非 质子溶剂二甲基亚砜(DMSO), 磁力搅拌环糊精至溶解完全后, 向其中加入 2.8 当量的温和氧化剂 2-碘酰基苯甲酸(IBX)。然后在磁力搅拌下在 25°C下反应 30 小时, 反应结束后过滤, 将滤液滴入到 150ml的丙酮中, 将析出来的物质过滤 得沉淀, 将沉淀用真空干燥箱在 35°C下真空干燥 24h, 最后将烘干后的样品用 50ml去离子水溶解, 过滤除掉杂质, 滤液经冷冻干燥得最终样品。 (3) After the mold was left at room temperature for 6 hours, the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 3 0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 8 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.8 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, the mixture was reacted under magnetic stirring at 25 ° C for 30 hours. After the reaction was completed, the mixture was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.
经检测, 环糊精多醛最终产率达到了 91%。 The final yield of cyclodextrin polyaldehyde was 91%.
(二) 改性胶原: (2) Modified collagen:
将歩骤 (一) 制备的环糊精多醛交联剂配制成 5种质量浓度为 0.075%、 0.15%、 0.30%、 0.45%、 0.60%的环糊精多醛水溶液, 然后使用这 5种不同浓度 的环糊精多醛水溶液分别按如下歩骤对胶原进行改性: The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
( 1 )将环糊精多醛水溶液与质量浓度为 0.6%的胶原水溶液均匀混合,并在 4°C冰箱里反应 24h, 得到改性胶原溶液; (1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;
(2) 将歩骤 (1 ) 得到的改性胶原溶液置于 37°C水浴中 18min, 并用吸管 将改性胶原溶液滴入一定形状的模具中, 随后将模具放在 -20°C冰箱中冷冻 48h, 制备改性胶原凝胶; (2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 18 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
(3 )将模具在室温下放置 6h后将改性胶原凝胶取出, 然后通过冷冻干燥机 对改性胶原凝胶进行干燥, 得到多孔海绵状改性胶原。 实施例 4 (3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 4
(一) 制备环糊精多醛交联剂: (1) Preparation of cyclodextrin polyaldehyde crosslinker:
称取 0.5mmol纯化后的 α-环糊精于 50ml烧瓶中, 向烧瓶中加入 7ml极性非 质子溶剂二甲基亚砜(DMSO), 磁力搅拌环糊精至溶解完全后, 向其中加入 2.6 当量的温和氧化剂 2-碘酰基苯甲酸(IBX)。然后在磁力搅拌下在 28°C下反应 30 小时, 反应结束后过滤, 将滤液滴入到 150ml的丙酮中, 将析出来的物质过滤 得沉淀, 将沉淀用真空干燥箱在 35°C下真空干燥 24h, 最后将烘干后的样品用
50ml去离子水溶解, 过滤除掉杂质, 滤液经冷冻干燥得最终样品。 0.5 mmol of the purified α-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.6 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 28 ° C for 30 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. Dry for 24h, and finally use the dried sample Dissolve in 50 ml of deionized water, remove impurities by filtration, and freeze the filtrate to obtain the final sample.
经检测, 环糊精多醛最终产率达到了 90%。 After testing, the final yield of cyclodextrin polyaldehyde reached 90%.
(二) 改性胶原: (2) Modified collagen:
将歩骤 (一) 制备的环糊精多醛交联剂配制成 5种质量浓度为 0.075%、 0.15%、 0.30%、 0.45%、 0.60%的环糊精多醛水溶液, 然后使用这 5种不同浓度 的环糊精多醛水溶液分别按如下歩骤对胶原进行改性: The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
( 1 )将环糊精多醛水溶液与质量浓度为 0.6%的胶原水溶液均匀混合,并在 4°C冰箱里反应 24h, 得到改性胶原溶液; (1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;
(2) 将歩骤 (1 ) 得到的改性胶原溶液置于 37°C水浴中 15min, 并用吸管 将改性胶原溶液滴入一定形状的模具中, 随后将模具放在 -20°C冰箱中冷冻 48h, 制备改性胶原凝胶; (2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
(3 )将模具在室温下放置 6h后将改性胶原凝胶取出, 然后通过冷冻干燥机 对改性胶原凝胶进行干燥, 得到多孔海绵状改性胶原。 实施例 5 (3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 5
(一) 制备环糊精多醛交联剂: (1) Preparation of cyclodextrin polyaldehyde crosslinker:
称取 0.5mmol纯化后的 β-环糊精于 50ml烧瓶中, 向烧瓶中加入 7ml极性非 质子溶剂二甲基亚砜(DMSO), 磁力搅拌环糊精至溶解完全后, 向其中加入 2.4 当量的温和氧化剂 2-碘酰基苯甲酸(IBX)。然后在磁力搅拌下在 30°C下反应 48 小时, 反应结束后过滤, 将滤液滴入到 150ml的丙酮中, 将析出来的物质过滤 得沉淀, 将沉淀用真空干燥箱在 35°C下真空干燥 24h, 最后将烘干后的样品用 50ml去离子水溶解, 过滤除掉杂质, 滤液经冷冻干燥得最终样品。 0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.4 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 30 ° C for 48 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.
经检测, 环糊精多醛最终产率达到了 92%。 The final yield of cyclodextrin polyaldehyde was 92%.
(二) 改性胶原: (2) Modified collagen:
将歩骤 (一) 制备的环糊精多醛交联剂配制成 5种质量浓度为 0.075%、 0.15%、 0.30%、 0.45%、 0.60%的环糊精多醛水溶液, 然后使用这 5种不同浓度
的环糊精多醛水溶液分别按如下歩骤对胶原进行改性: The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. different density The cyclodextrin polyaldehyde aqueous solution was modified with collagen as follows:
( 1 )将环糊精多醛水溶液与质量浓度为 0.6%的胶原水溶液均匀混合,并在 4°C冰箱里反应 24h, 得到改性胶原溶液; (1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;
(2) 将歩骤 (1 ) 得到的改性胶原溶液置于 37°C水浴中 15min, 并用吸管 将改性胶原溶液滴入一定形状的模具中, 随后将模具放在 -20°C冰箱中冷冻 48h, 制备改性胶原凝胶; (2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;
(3 )将模具在室温下放置 6h后将改性胶原凝胶取出, 然后通过冷冻干燥机 对改性胶原凝胶进行干燥, 得到多孔海绵状改性胶原。 实施例 6 (3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 6
(一) 制备环糊精多醛交联剂: (1) Preparation of cyclodextrin polyaldehyde crosslinker:
称取 0.5mmol纯化后的 β-环糊精于 50ml烧瓶中, 向烧瓶中加入 7ml极性非 质子溶剂二甲基亚砜(DMSO), 磁力搅拌环糊精至溶解完全后, 向其中加入 2.6 当量的温和氧化剂 2-碘酰基苯甲酸(IBX)。然后在磁力搅拌下在 30°C下反应 48 小时, 反应结束后过滤, 将滤液滴入到 150ml的丙酮中, 将析出来的物质过滤 得沉淀, 将沉淀用真空干燥箱在 35°C下真空干燥 24h, 最后将烘干后的样品用 50ml去离子水溶解, 过滤除掉杂质, 滤液经冷冻干燥得最终样品。 0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.6 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 30 ° C for 48 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.
经检测, 环糊精多醛最终产率达到了 93%。 The final yield of cyclodextrin polyaldehyde was 93%.
(二) 改性胶原: (2) Modified collagen:
将歩骤 (一) 制备的环糊精多醛交联剂配制成 5种质量浓度为 0.075%、 0.15%、 0.30%、 0.45%、 0.60%的环糊精多醛水溶液, 然后使用这 5种不同浓度 的环糊精多醛水溶液分别按如下歩骤对胶原进行改性: The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:
( 1 )将环糊精多醛水溶液与质量浓度为 0.6%的胶原水溶液均匀混合,并在 4°C冰箱里反应 24h, 得到改性胶原溶液; (1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;
(2) 将歩骤 (1 ) 得到的改性胶原溶液置于 37°C水浴中 15min, 并用吸管 将改性胶原溶液滴入一定形状的模具中, 随后将模具放在 -20°C冰箱中冷冻 48h,
制备改性胶原凝胶; (2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, Preparing a modified collagen gel;
(3 )将模具在室温下放置 6h后将改性胶原凝胶取出, 然后通过冷冻干燥机 对改性胶原凝胶进行干燥, 得到多孔海绵状改性胶原。 (3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen.
上述实施例为本发明较佳的实施方式, 但本发明的实施方式并不受上述实 施例的限制, 其他的任何未背离本发明的精神实质与原理下所作的改变、 修饰、 替代、 组合、 简化, 均应为等效的置换方式, 都包含在本发明的保护范围之内。
The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and modifications may be made without departing from the spirit and scope of the invention. Simplifications, which are equivalent replacement means, are included in the scope of the present invention.
Claims
1、 一种环糊精多醛交联剂的应用方法, 其特征在于, 所述环糊精多醛交联 剂用于对胶原的改性; 其中所述环糊精多醛交联剂的化学结构为:
1. An application method of cyclodextrin polyaldehyde cross-linking agent, characterized in that the cyclodextrin poly-aldehyde cross-linking agent is used to modify collagen; wherein the cyclodextrin poly-aldehyde cross-linking agent is The chemical structure is:
2、 根据权利要求 1所述的应用方法, 其特征在于, 所述环糊精多醛交联剂 用于多孔海绵状改性胶原的制备。 2. The application method according to claim 1, characterized in that the cyclodextrin polyaldehyde cross-linking agent is used for the preparation of porous sponge-like modified collagen.
3、 根据权利要求 2所述的应用方法, 其特征在于, 所述多孔海绵状改性胶 原的制备方法包括如下歩骤: 3. The application method according to claim 2, characterized in that the preparation method of the porous sponge-like modified collagen includes the following steps:
( 1 ) 将环糊精多醛交联剂水溶液与胶原水溶液混合, 并在 2〜6 °C反应 20〜30h, 得到所述改性胶原溶液; (1) Mix the cyclodextrin polyaldehyde cross-linking agent aqueous solution and the collagen aqueous solution, and react at 2 to 6°C for 20 to 30 hours to obtain the modified collagen solution;
(2 )将歩骤(1 )得到的改性胶原溶液在 35〜38°C保温 15〜20min, 然后将 改性胶原溶液转移至模具中, 再将模具在 -15〜- 25 °C冷冻 40〜50h, 制备改性胶 原凝胶; (2) Insulate the modified collagen solution obtained in step (1) at 35~38°C for 15~20 minutes, then transfer the modified collagen solution to the mold, and then freeze the mold at -15~-25°C for 40 ~50h, prepare modified collagen gel;
( 3 ) 将模具在室温下放置 4〜6h后将改性胶原凝胶取出, 并将其干燥得到 多孔海绵状改性胶原。 (3) Place the mold at room temperature for 4 to 6 hours, take out the modified collagen gel, and dry it to obtain porous sponge-like modified collagen.
4、 根据权利要求 3所述的应用方法, 其特征在于, 歩骤 (1 ) 中环糊精多 醛交联剂水溶液的质量浓度为 0.075%〜0.6%, 胶原水溶液的质量浓度为 0.6%。 4. The application method according to claim 3, characterized in that, in step (1), the mass concentration of the cyclodextrin polyaldehyde cross-linking agent aqueous solution is 0.075%~0.6%, and the mass concentration of the collagen aqueous solution is 0.6%.
5、 根据权利要求 3所述的应用方法, 其特征在于, 歩骤 (1 ) 中反应温度 为 4°C。 5. The application method according to claim 3, characterized in that the reaction temperature in step (1) is 4°C.
6、 根据权利要求 3所述的应用方法, 其特征在于, 歩骤 (2 ) 中改性胶原 溶液在 37°C水浴中保温, 冷冻温度为 -20°C。 6. The application method according to claim 3, characterized in that in step (2), the modified collagen solution is insulated in a 37°C water bath, and the freezing temperature is -20°C.
7、 根据权利要求 3所述的应用方法, 其特征在于, 歩骤 (3 ) 中所述的干 燥方法为冷冻干燥。
7. The application method according to claim 3, characterized in that the drying method described in step (3) is freeze drying.
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