CN116535540B - 季铵化改性甲壳素及其海绵、多孔微球的制备方法、用途 - Google Patents
季铵化改性甲壳素及其海绵、多孔微球的制备方法、用途 Download PDFInfo
- Publication number
- CN116535540B CN116535540B CN202210093853.9A CN202210093853A CN116535540B CN 116535540 B CN116535540 B CN 116535540B CN 202210093853 A CN202210093853 A CN 202210093853A CN 116535540 B CN116535540 B CN 116535540B
- Authority
- CN
- China
- Prior art keywords
- chitin
- quaternized
- modified chitin
- quaternized modified
- aqueous solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920002101 Chitin Polymers 0.000 title claims abstract description 187
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 239000004005 microsphere Substances 0.000 title abstract description 19
- 239000000463 material Substances 0.000 claims abstract description 37
- 230000035945 sensitivity Effects 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000006467 substitution reaction Methods 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 230000002439 hemostatic effect Effects 0.000 claims abstract description 17
- 208000005189 Embolism Diseases 0.000 claims abstract description 13
- 230000023597 hemostasis Effects 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 54
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
- 239000007864 aqueous solution Substances 0.000 claims description 37
- 239000002245 particle Substances 0.000 claims description 19
- -1 2, 3-epoxypropyl triethyl ammonium bromide Chemical compound 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 17
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 15
- 239000004202 carbamide Substances 0.000 claims description 15
- 239000008367 deionised water Substances 0.000 claims description 15
- 229910021641 deionized water Inorganic materials 0.000 claims description 15
- 230000021736 acetylation Effects 0.000 claims description 12
- 238000006640 acetylation reaction Methods 0.000 claims description 12
- 230000000844 anti-bacterial effect Effects 0.000 claims description 12
- 239000002994 raw material Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 9
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
- 230000008014 freezing Effects 0.000 claims description 9
- 230000004048 modification Effects 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 8
- 238000004132 cross linking Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- PUVAFTRIIUSGLK-UHFFFAOYSA-M trimethyl(oxiran-2-ylmethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC1CO1 PUVAFTRIIUSGLK-UHFFFAOYSA-M 0.000 claims description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 230000001112 coagulating effect Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000005956 quaternization reaction Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 239000004094 surface-active agent Substances 0.000 claims description 6
- 230000006196 deacetylation Effects 0.000 claims description 5
- 238000003381 deacetylation reaction Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
- 206010038727 Respiratory tract haemorrhage Diseases 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 238000006386 neutralization reaction Methods 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- HPLFXTUAVXJDPJ-UHFFFAOYSA-M (3-chloro-2-hydroxypropyl)-triethylazanium;chloride Chemical compound [Cl-].CC[N+](CC)(CC)CC(O)CCl HPLFXTUAVXJDPJ-UHFFFAOYSA-M 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 239000004593 Epoxy Substances 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- YQFOHQBSENCNTR-UHFFFAOYSA-M oxiran-2-ylmethyl(tripropyl)azanium;chloride Chemical compound [Cl-].CCC[N+](CCC)(CCC)CC1CO1 YQFOHQBSENCNTR-UHFFFAOYSA-M 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 238000001694 spray drying Methods 0.000 claims description 3
- 238000002560 therapeutic procedure Methods 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 abstract description 3
- 238000010382 chemical cross-linking Methods 0.000 abstract description 3
- 239000003431 cross linking reagent Substances 0.000 abstract description 3
- 239000003937 drug carrier Substances 0.000 abstract description 3
- 239000012567 medical material Substances 0.000 abstract description 2
- 229920001661 Chitosan Polymers 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 206010018910 Haemolysis Diseases 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000008186 active pharmaceutical agent Substances 0.000 description 6
- 230000008588 hemolysis Effects 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 230000007704 transition Effects 0.000 description 6
- 238000002834 transmittance Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 241000191967 Staphylococcus aureus Species 0.000 description 5
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000010257 thawing Methods 0.000 description 4
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000015271 coagulation Effects 0.000 description 3
- 238000005345 coagulation Methods 0.000 description 3
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 3
- 239000000017 hydrogel Substances 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 230000005311 nuclear magnetism Effects 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 2
- 208000009344 Penetrating Wounds Diseases 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- PRXRUNOAOLTIEF-ADSICKODSA-N Sorbitan trioleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCC\C=C/CCCCCCCC)[C@H]1OC[C@H](O)[C@H]1OC(=O)CCCCCCC\C=C/CCCCCCCC PRXRUNOAOLTIEF-ADSICKODSA-N 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229920005615 natural polymer Polymers 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 206010010214 Compression fracture Diseases 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004176 ammonification Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000000607 artificial tear Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000011236 particulate material Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000035440 response to pH Effects 0.000 description 1
- 230000006903 response to temperature Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 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 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
- LTVDFSLWFKLJDQ-UHFFFAOYSA-N α-tocopherolquinone Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)(O)CCC1=C(C)C(=O)C(C)=C(C)C1=O LTVDFSLWFKLJDQ-UHFFFAOYSA-N 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
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0036—Porous materials, e.g. foams or sponges
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/08—Polysaccharides
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/204—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with nitrogen-containing functional groups, e.g. aminoxides, nitriles, guanidines
- A61L2300/208—Quaternary ammonium compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/23—Carbohydrates
- A61L2300/232—Monosaccharides, disaccharides, polysaccharides, lipopolysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Materials Engineering (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Surgery (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明公开了一种季铵化改性甲壳素及其海绵、多孔微球的制备方法、用途,先将甲壳素加入到碱性尿素的冷冻低温混合水溶液中制得均相的甲壳素溶液,加入季铵化试剂搅拌反应,经后处理和干燥得到具有温度或pH敏感性的季铵化改性甲壳素,基于该改性甲壳素进一步可以制成海绵和多孔微球。本发明方法制备工艺简单,产品取代度均匀易控,产率高,且反应条件温和,为环境友好的绿色工艺;甲壳素基本上未降解,具有较高的乙酰度,制得的季铵化甲壳素具有pH敏感性或温度敏感性,利用本发明改性甲壳素制备多孔微球或者多孔止血海绵,不使用化学交联剂,材料体内可吸收,具有良好的生物相容性和生物降解性,可作为药物载体、止血和栓塞等植入医用材料使用。
Description
技术领域
本发明属于属于天然高分子改性和材料科学领域,涉及一种甲壳素改性方法,具体涉及一种季铵化改性甲壳素、甲壳素海绵、多孔微球的制备及用途。
背景技术
甲壳素是β(1-4)-N-乙酰-D-葡萄糖胺残基的多聚糖,它是从藻类、真菌类的细胞壁及虾、蟹、昆虫的壳中提取的天然多糖,是仅次于纤维素的第二大可再生天然高分子,每年自然界生物合成量多达100亿吨。由于甲壳素的单体组成与细胞外基质的成分相似,为N-乙酰氨基葡萄糖,所以其具有非常好的生物相容性、可降解性和低毒性。以其为代表的甲壳素基材料已经被广泛用于制备支架,海绵,膜及水凝胶等,然而,由于甲壳素分子内和分子间存在大量氢键,较高的结晶度使其不溶于酸碱和其他有机溶剂,从而限制了甲壳素的应用。通过改性获得的甲壳素衍生物能赋予其新的性能并提高其附加值。
吴奕光等将β-甲壳素分散在异丙醇中,在浓NaOH溶液条件下将3-氯-2-羟丙基三甲基氯化铵与甲壳素合成了季铵化甲壳素,由于反应是在非均相条件下进行的,所以存在取代位点不均一的问题[Chen Q, Wu Y, Pu Y, et al. Synthesis andcharacterization of quaternized β-chitin[J]. Carbohydrate research, 2010, 345(11): 1609-1612.]。杜予民等将甲壳素与季铵盐试剂(主要为2,3-环氧丙基三甲基氯化铵)在NaOH/尿素体系下经过均相反应后,能形成含有季铵基的水溶性阳离子聚合物其取代均一,极大拓展了甲壳素衍生物的应用领域 [Ding F ,Shi X ,Li X , et al.Homogeneous synthesis and characterization of quaternized chitin in NaOH/ureaaqueous solution[J]. Carbohydrate Polymers, 2012, 87(1):422-426;中国发明专利CN102276757 B]。在此基础上,蔡杰等在KOH/尿素体系在温和的反应条件下使β-甲壳素衍生物的乙酰氨基能最大程度保留,反应中的脱乙酰程度很低,在羟基上引入季铵基团,使其在水中溶解性较好,并对其抗菌性能进行了系统研究,结果表明局部的正电荷能明显抑制细菌生长,具有较好的抗菌性[Xu H ,Fang Z ,Tian W , et al. Green Fabrication ofAmphiphilic Quaternized β‐Chitin Derivatives with Excellent Biocompatibilityand Antibacterial Activities for Wound Healing[J]. Advanced Materials, 2018,30(29):e1801100;中国发明专利CN105622780A]。刘海清等将水溶性季铵化甲壳素和甲壳素纳米晶须混合制备的止血海绵也表现出优异的止血性能,该材料的吸水倍率可以达到130倍,可用于不可压缩的贯穿伤模型[Fang Y, Xu Y, Wang Z, et al. 3D porouschitin sponge with high absorbency, rapid shape recovery, and excellentantibacterial activities for noncompressible wound[J]. Chemical EngineeringJournal, 2020, 388: 124169.]。甲壳素与羧基化试剂在氢氧化钠-尿素均相水体系中可以制得具有pH敏感性和温度敏感性的羧基甲壳素[中国发明专利申请公开说明书CN201310641249.6]。甲壳素和羟丙基化试剂同样在低浓度碱液中可以制备出具有温度敏感性的羟丙基甲壳素,该方法制备得到的甲壳素衍生物可以作为人工泪液、可注射水凝胶和药物载体材料的应用[中国专利CN103951764A]。温敏性羟丁基壳聚糖和温敏性羟戊基壳聚糖等温敏材料的制备和应用也是作为温敏材料的典型材料,这种材料经过设计后可以在特定的温度和pH下发生相的转变,并且不会引入其他有机溶剂,极大拓展了其应用领域[中国发明专利CN200810033699.6;中国发明专利CN201210220246.0]。但是对于季铵化改性甲壳素的研究和应用主要在水溶性的季铵化衍生物(一般指室温下可以溶于水),而对于具有温度或pH的响应的季铵化改性甲壳素的制备和研究却未有报道。
发明内容
本发明针对现有季铵化改性甲壳素应用的局限性,提供了一种工艺简单的均相制备具有pH敏感性或温度敏感性季铵化甲壳素及其制备方法和应用:反应条件温和,有利于大规模工业化生产;产品取代度分布均一易控,产率高,甲壳素基本不降解。
本发明提供如下技术方案:
一种具有温度或pH敏感性的季铵化改性甲壳素,该季铵化改性甲壳素的单元结构式示意图为:
R1为-H或者-COCH3;
R2为H、-CH2C(OH)CH2NCl(CH3)3、-CH2C(OH)CH2NCl(CH2CH3)3及-CH2C(OH)CH2N+(CH3)2(CH2)mCH3中的任意一种;
R3为H、-CH2C(OH)CH2NCl(CH3)3、-CH2C(OH)CH2NCl(CH2CH3)3及-CH2C(OH)CH2N+(CH3)2(CH2)mCH3中的任意一种;
m是1-12的正整数;
所述季铵化改性甲壳素的乙酰度为0.79~0.94,季铵化取代度为0.06~0.23。
优选的,所述季铵化改性甲壳素的乙酰度为0.79~0.94,季铵化取代度为0.06~0.20。
更优的,所述季铵化改性甲壳素的乙酰度为0.79~0.94,季铵化取代度为0.06~0.18。
更优的,所述季铵化改性甲壳素的乙酰度为0.79~0.94,季铵化取代度为0.06~0.15。
本发明还提供一种季铵化改性甲壳素的制备方法,包括如下步骤:
步骤1、将原料甲壳素在低温下溶解形成甲壳素水溶液,均相甲壳素水溶液的浓度为质量份0.2~9%;
步骤2、往甲壳素水溶液中加入季铵化试剂在0~40oC下搅拌均相反应,进行季铵化改性;
步骤3、将反应溶液干燥后得到季铵化改性甲壳素。
优选的,步骤1中,具体方式为:将原料甲壳素加入到碱性尿素的冷冻低温混合水溶液中混合均匀,并于-30~-12oC下冷冻8~96小时,再于1~25oC下搅拌制得均相甲壳素水溶液。
优选的,步骤2中,均相反应时间为6~71小时,所述季铵化试剂的加入量为甲壳素结构单元摩尔数的0.5~4倍。
优选的,步骤1中,所述的原料甲壳素为脱乙酰度低于20%的粉状甲壳素。
更优的,步骤1中,原料甲壳素脱乙酰度低于10%。
优选的,步骤1中,原料甲壳素的重均分子量为5×104~5×106。
优选的,步骤2中,所述的季铵化试剂为2,3-环氧丙基三甲基氯化铵、3-氯-2-羟丙基三甲基氯化铵、3-氯-2-羟丙基三乙基氯化铵、2,3-环氧丙基三乙基氯化铵、2,3-环氧丙基三乙基溴化铵、2,3-环氧丙基三丙基氯化铵、短烷基碳原子数为m的环氧基短烷基链季铵盐以及氯羟丙基短烷基季铵盐的任意一种或者几种的混合物,m为1-12的正整数。
优选的,步骤2中,所述季铵化试剂的加入量为甲壳素结构单元摩尔数的1~2倍。
优选的,所述碱性尿素的冷冻低温混合水溶液为可溶性碱:尿素:纯水=5~20:3~12:68~91的混合液。
优选的,所述的可溶性碱为氢氧化钠、氢氧化钾和氢氧化锂中的任意一种或几种。
优选的,步骤3中,干燥前,对季铵化改性甲壳素溶液用稀酸调节pH值到中性,用纯水透析或者沉淀过滤洗涤除去尿素和盐等小分子物质,之后通过冷冻干燥或者加热干燥得到季铵化改性甲壳素;
优选的,所述的稀酸为盐酸、硫酸、硝酸、甲酸、乙酸、柠檬酸中的一种或几种。
本发明还提供一种季铵化改性甲壳素多孔材料的制备方法,包括如下步骤:
S1、在0-20oC低温下,将上述制备方法得到的季铵化改性甲壳素配置成碱性的水溶液;
S2、将步骤S1配置的季铵化改性甲壳素溶液加入分散相媒介中,通过中和升温、物理交联法或者喷雾干燥法制得的温敏季铵化改性甲壳素颗粒;
S3、洗涤纯化温敏季铵化改性甲壳素颗粒,然后用去离子水浸泡使其充分吸水溶胀,之后冷冻干燥,制得季铵化改性甲壳素多孔颗粒材料。
优选的,所述分散相媒介为含有表面活性剂的有机分散相或含有聚乙二醇的水分散相,在升温物理交联过程中通过酸性溶液将反应体系中和;所制得的季铵化改性甲壳素多孔颗粒材料的形状为球形或椭球型。
本发明还提供一种上述所制备的季铵化改性甲壳素多孔材料的用途,其特征在于:制成药物载体材料,用于止血、术前栓塞、消化道和呼吸道出血及恶性肿瘤的介入治疗的非永久性栓塞、栓塞减肥。
本发明还提供一种季铵化改性甲壳素止血海绵的制备方法,包括如下步骤:
M1、在0-20oC低温下,将上述制备方法得到的季铵化改性甲壳素配置成碱性的水溶液;
M 2、将步骤M1配置的季铵化改性甲壳素溶液脱泡后倒入模具中冷冻成冰;
M 3、将季铵化改性甲壳素冰块浸泡在凝固浴中,制得再生海绵;
M 4、用去离子水洗涤纯化再生海绵,脱水干燥,制得季铵化改性甲壳素止血海绵。
优选的,步骤M 2中,所述冷冻成冰的温度为-18°C~ -30°C。
优选的,步骤M 3中,所述凝固浴为50% ~ 100%甲醇/水溶液、50% ~ 100%乙醇/水溶液或50% ~ 100%丙酮/水溶液中的任意一种.
优选的,步骤M 3中,所述凝固浴温度为-30°C ~ 4°C;所述在凝固浴中浸泡的时长为1 ~ 7天。
本发明还提供一种上述所制备的季铵化改性甲壳素止血海绵的用途,所述季铵化甲壳素止血海绵材料用于伤口止血抗菌。
本发明方法可制得具有pH敏感性或温度敏感性季铵化改性甲壳素,由于其价廉可生物降解,可广泛用于其他医药和食品加工等领域,具有较高的应用开发价值。本发明的反应介质是使用较低浓度的可溶性碱和尿素的混合水溶液,整个过程一直到后处理未使用有机溶剂,反应条件温和,产品取代度分布均一,产率高,有利于大规模生产。
与现有技术相比,本发明方法具有显著的技术进步和特点:
第一,本发明方法是在甲壳素的均相水溶液中制备低脱乙酰度具有pH敏感性或温度敏感性季铵化改性甲壳素,制备工艺简单,反应条件温和,反应过程易控,产品取代度均匀,产率高,且环境友好,利于大规模工业化生产。
第二,本方法制备的具有pH敏感性或温度敏感性季铵化改性甲壳素,可以低温0-20oC水溶液里溶解,中和升温形成凝胶,具有较好的抗菌性;可以通过物理交联制备多孔颗粒微球材料和多孔海绵,制备过程不需要使用化学交联剂,环境友好。
第三,制备的上述材料具有较好的抗菌性,可应用于药物载体材料,止血、术前栓塞、消化道和呼吸道出血及恶性肿瘤的介入治疗的非永久性栓塞、栓塞减肥等植入医用材料及止血海绵材料用于深且窄的伤口、不可压迫性伤口及贯穿性伤口的止血。
附图说明
图1为本发明实施例1中合成的季铵化甲壳素T2在25℃下在20 wt% DCl/D2O的核磁氢谱图。
图2为实施例6中动态光散射测试季铵化甲壳素T2在不同温度时(溶液浓度 10mg/mL,pH =0,7或14)光散射强度的变化。
图3为实施例6中紫外可见分光光度计在600纳米波长测得季铵化甲壳素T2和T5(10 mg/mL)在不同pH(T5右图和T2左图的pH图例标识一样)时不同温度时的透过率的变化图。
图4为实施例7中紫外可见分光光度计在600纳米波长测得季铵化甲壳素T2和T5(10 mg/mL,37℃)在不同pH时的透过率的变化。
图5为实施例8中pH敏感性和温度敏感性季铵化甲壳素T2和水溶性季铵化甲壳素T5对大肠杆菌和金黄色葡萄球菌的抗菌性能,且以中性条件下壳聚糖为阴性对照组。图中A1~A5分别对应空白组,T2,壳聚糖,T5和链霉素五种样品对大肠杆菌的抗菌效果;B1~B5分别对应空白组,T2,壳聚糖,T5和青霉素五种样品对金黄色葡萄球菌的抗菌效果。
图6为实施例9中季铵化甲壳素T2在不同浓度时的溶血率。
图7为实例10季铵化甲壳素T1,T2和T5利用双水相法制备的微球显微照片图。
具体实施方式
下面将结合实例进一步说明本发明,但它们不是对权利要求的限制。
实施例1均相季铵化改性甲壳素的制备
将5克甲壳素加入到预先冷冻的含有11 wt%氢氧化钠和4 wt%尿素的245克水溶液中,在-24℃下冷冻6 h后,取出在室温下机械搅拌解冻,再重复冷冻解冻即可得到溶解的2 wt% 甲壳素水溶液。再根据表1加入不同比例的2,3-环氧丙基三甲基氯化铵,分别在2~5oC下搅拌充分,使其溶解,然后根据表1在5~15oC下搅拌反应24~ 48小时,控制反应液为均相透明体系。之后,将体系冷却至2℃,并用3M的盐酸调节pH值到中性,使用纯水透析洗涤,除去其中尿素和盐类小分子,冷冻干燥得到海绵蓬松状季铵化改性甲壳素,通过核磁氢谱1HNMR,在3.15ppm 处出现N+(CH3)3的吸收峰,说明该产品已被季铵化,且反应产物取代度分布均一。通过核磁氢谱计算得到其乙酰度DA和季铵化取代度DS。由表1可知,这些季铵化改性甲壳素乙酰度都高于0.81,收率在86%以上。
表1 不同投料比的均相季铵化改性甲壳素
由核磁氢谱计算出产物的季铵基团取代度DS和乙酰度DA; *5℃下的反应时间,#15℃下的反应时间
其中T1-T8均可以溶解在低温0-20oC较强酸性(1.0 M HCl溶液)和较强碱性(1.0M NaOH溶液)水溶液中,产物T5和T8在去离子水中可以溶解为完全透明的季铵化甲壳素水溶液(10 mg/ml)。
本实施例中采用增加氢氧化钠质量比到20%和增加尿素质量比达12%制备的混合水溶液用于溶解甲壳素,采用氢氧化钾和/或氢氧化锂代替或者部分代替氢氧化钠,对后面的反应和处理没有明显的影响;后处理调节pH值的酸可用硫酸、 硝酸、甲酸、乙酸或柠檬酸代替盐酸使用,其浓度大小没有影响;采用沉淀过滤洗涤代替纯水透析,加热干燥代替冷冻干燥,对产品也没有明显的影响。例如采用加入1:1的2,3-环氧丙基三甲基氯化铵,分别得到季铵化甲壳素冷冻干燥样品,分别记为S1,S2和S3,进行核磁制样,测得其取代度DS和乙酰度DA,分别为DS:0.06、0.07和0.08,DA:0.79、0.87和0.86。
实施例2 均相季铵化改性甲壳素的制备
配制91g含11% NaOH,4% 尿素的水溶液,在-12~-30℃下冷冻72小时,解冻后于5℃下加入9 g甲壳素,配制9%甲壳素溶液,按照物质的量比1:2加入2,3-环氧丙基三甲基氯化铵,在0℃下机械搅拌反应71h,调节溶液pH值为7左右,用去离子水透析后冷冻干燥得海绵状季铵化甲壳素,编号为T9样品。
实施例3均相季铵化改性甲壳素的制备
配制99.8g含11% NaOH,4% 尿素的水溶液,在-12~-20℃下冷冻72小时,解冻后于1~25℃下加入0.2g甲壳素,配制0.2 wt%甲壳素溶液,按照物质的量比0.5:1加入2,3-环氧丙基三甲基氯化铵,在40℃下机械搅拌6 h,调节溶液pH值为7左右,用去离子水透析后冷冻干燥得海绵状季铵化甲壳素,编号为T10样品。
实施例4 高温不均匀反应(对比例)
将2克甲壳素加入到98克的氢氧化钠,尿素和纯水(质量比为11:4:85)的混合水溶液混匀,在-12~-20℃下冷冻72小时,解冻后于5~12℃下充分搅拌制得2wt%的甲壳素均相溶液。加入1.42g (接近1:1) 2,3-环氧丙基三甲基氯化铵,并升温到40℃反应7 h,整个体系开始为乳白色浊液不是很均匀,说明该反应为非均相体系。同样的反应液升温到80℃下反应3 h,整个体系在反应过程中为乳白色浊液,不是很均匀。
实施例5 高浓度甲壳素不溶解(对比例)
将实施例1中相同的原料甲壳素15克加入到85克的纯水,氢氧化钠和纯水(质量比为11:4:85)的混合水溶液混匀,在-12~-20℃下冷冻72小时,解冻后于5~12℃下充分搅拌以期制备15wt%甲壳素溶液,发现甲壳素无法完全溶解,减少甲壳素的浓度到10 wt%甲壳素溶液也没法得到均匀的流动的溶液。
通过实施例4和实施例5可知,并不是所有均相甲壳素水溶液浓度都可以制得,季铵化改性反应温度范围也不是所有温度都可以保持季铵化改性反应均相进行的。只有质量浓度为0.2~9%时才能得到甲壳素均相水溶液的,只有在0~40oC下才能进行甲壳素水溶液的季铵化改性均相反应,优化反应温度在1~25oC可进行均相甲壳素水溶液季铵化改性。
实施例6温敏性
使用实例1中制得季铵化改性甲壳素T2,分别称取20 mg,在低温4℃下加入1.0 MNaOH 和1.0 M HCl 2.0 mL使其完全溶解以配制pH=0和14的溶液,同时称取20 mg的T2溶于1 mL的1.0 M NaOH溶液并用2.0 M HCl溶液和0.1 M HCl溶液调节pH至7,季铵化改性甲壳素浓度为10 mg/mL并利用动态光散射仪升温时相对光散射强度的变化,升温范围为5-65℃,平衡时间10s,升温间隔为5℃,升温速率为15 min/5℃,测试结果如图2所示。由该图可知,这种季铵化改性甲壳素具有温度敏感性,在pH=7时的转变温度约为25℃。同样将实施例1中制得的季铵化改性甲壳素T1,T3和T4按照上述方法进行测试,结果表明其均具有温度敏感性,且其对应的在pH=7时的转变温度分别为5℃,40℃和50℃,当然这里的升温速度也会影响其转变温度值的。同样通过测试季铵化改性甲壳素水溶液在600 nm的透过率随着升温而发生改变(图3),说明样品T1-T4及T6和T7都具有温敏性,实施例1中S1、S2和S3三个样品也具有温度敏感性,实施例中2的T9和实施例3中T10样品也具有温度敏感性,当然这里的升温速度、样品浓度及pH都会影响其转变温度值的,而T5和T8的水溶液一直透明,透过率不随温度升高而变化,没有温敏性。这些表明调节季铵化甲壳素的乙酰度和取代度在合适的乙酰度和取代度范围,乙酰度为0.79-0.94和取代度为0.06-0.23,其具有温度敏感性,如果用于人体医药,那么转变温度一般不超过40,所以最佳季铵基团取代度DS不超过0.18。
专利CN105622780A公开的技术中季铵化取代度摘要中记载为0.10-1.95,实施例中最低为0.16,最高为0.43,季铵化甲壳素具有水溶性,但是根据相同作者公开发表的论文[Xu H ,Fang Z ,Tian W , et al. Green Fabrication of Amphiphilic Quaternizedβ‐Chitin Derivatives with Excellent Biocompatibility and AntibacterialActivities for Wound Healing[J]. Advanced Materials, 2018, 30(29):e1801100],只有在季铵化取代度DS在 0.20-0.43的改性甲壳素才是全部水里溶解的。根据本发明结果可知,取代度达到0.23时,还具有温敏性,也就是低于50℃还是溶于水,但高于50℃就不溶于水,这不符合CN105622780A中高溶解性的要求,因此,专利CN105622780A记载的取代度范围0.10-1.95超过其要解决技术问题的范围,另外取代度测试方法的不同也会影响其取代度的具体数值,所以本发明与专利CN105622780A实际上是甲壳素改性完全相反的应用方向。本发明得到季铵化改性甲壳素的乙酰度为0.79~0.94,季铵化取代度为0.06~0.23的产品具有温敏性或者pH敏感性,没有温敏性或者pH敏感性,也就是无法进行本发明的上述医药应用。
实施例7 pH敏感性
称取30 mg 实例1中制得的季铵化甲壳素T2和T5,分别在低温4℃下加入1.0 MNaOH或1.0 M HCl 3 mL 完全溶解,然后用1 M HCl溶液缓慢调低聚合物的溶液pH到12、10、8和7,或者用1.0 M NaOH溶液缓慢调节酸性聚合物的pH到2、4和6同时控制溶液温度在4℃左右,并使用紫外可见分光光度计在600纳米波长测试相应pH溶液在37℃时的透过率,结果如图4。由该图可知,T2在中性环境下透过率最低,且在1MNaOH 或1 M HCl能完全溶解,但随着pH慢慢接近中性,则析出越来越明显,说明这种季铵化甲壳素T2具有pH敏感性,而T5溶液则一直比较透明,不具有这种pH敏感性质。类似的,样品T1-T4及T6和T7都具有pH敏感性,实施例中2的T9和实施例3中T10样品也具有pH敏感性,而T5和T8的水溶液一直透明,没有pH敏感性。
实施例1中采用3-氯-2-羟丙基三甲基氯化铵、3-氯-2-羟丙基三乙基氯化铵、2,3-环氧丙基三乙基氯化铵、2,3-环氧丙基三乙基溴化铵、2,3-环氧丙基三丙基氯化铵、其它短烷基碳原子数为m的环氧基短烷基链季铵盐或氯羟丙基短烷基季铵盐的一种或者任何几种的混合物 (这里的m是1-12的正整数)代替2,3-环氧丙基三甲基氯化铵制得的季铵化改性甲壳素也都不影响其温敏和pH敏感特性。
实施例8 抗菌性试验
使用实例1中制备的T2,T5对其进行抗菌性实验[Xu H ,Fang Z ,Tian W , etal. Green Fabrication of Amphiphilic Quaternized β‐Chitin Derivatives withExcellent Biocompatibility and Antibacterial Activities for Wound Healing[J].Advanced Materials, 2018, 30(29):e1801100;国家卫生行业标准WS/T 650—2019]。设置去离子水为空白组,T2,壳聚糖,T5和抗生素链霉素和青霉素(其对应为大肠杆菌,和金黄色葡萄球菌的阳性对照),样品初始浓度为200 μg/mL(均用灭菌后的去离子水配制)。首先将菌悬液稀释至8x104CFU/mL,然后将菌液取100 μl加入到对应的10 mL溶液环境中,按照菌液:样品悬液1:100 ( v/v)比例进行稀释,并在220 rpm,37℃摇床中震荡共培养10 h,然后取100 μL共培养液涂板,将涂板后的样品置于恒温恒湿箱中培养24 h(37℃,98%),观察菌落的生长情况,每组实验重复5次,并按照平板计数法计算样品的杀菌效果,结果显示T2在200μg/mL时对大肠杆菌和金黄色葡萄球菌的杀菌率均可以达到100%。实验结果如图5所示,说明T2和T5的抗菌性优于壳聚糖,季铵化甲壳素均具有较好的广谱抗菌性。对T2的进一步的最小抑菌浓度试验(抑菌率为90%以上的最低浓度MIC)的结果分别为: 对大肠杆菌的MIC: 8 mg/L, 对金黄色葡萄球菌的MIC:32 mg/L。
实施例9溶血测试
将实例1中制备的T2和T5进行溶血实验测试。首先配制3.8 wt %柠檬酸钠溶液,按照9:1(v/v)的比例将大鼠活血与抗凝剂混合均匀,得到抗凝全血;随后制备5%(v/v)红细胞悬液(RBCs):在3000 rpm离心10min,去除上清液血清,并用生理盐水洗涤多次,直至透明,然后用生理盐水配制成5%的红细胞悬液;材料的准备,设置纯的生理盐水为阴性对照,不同浓度T2和T5的溶血实验,设置Triton x-100 0.1%(v/v)为阳性对照组,在5 ml离心管中加入1ml以上样品,随后加入1ml 5% RBCs,在37℃下孵育1h,然后再3000 rpm离心5min,取上清液在540 nm处测量其吸光度。溶血率计算公式为:
HR=(As-An)/(Ap-An)× 100%
其中As、An、Ap分别为样品、生理盐水、阳性实验组在540 nm处的吸光值。每组实验平行重复五次,测试结果为平均值±标准偏差(SD)值。结果如图6所示,可以看出在不同浓度(0-1 mg/mL)的T2作用下其溶血率均低于5%,说明这种材料不会使红细胞破裂,表现出血液相容性,在生物医用材料领域是比较安全的材料。
实施例10 季铵化改性甲壳素的多孔颗粒材料的制备和应用
使用实例1中的T1,T2和T5,将其与PEG-10k分别溶于1M NaOH溶液中,制得2 wt%T1,T2和T5水溶液和30 wt% PEG溶液。分别将T1,T2和T5溶液2 mL与PEG溶液20 mL在冰浴条件下以1:10 (v/v)的投料比混合,搅拌10~30 min。随后用60℃的油浴代替冰水浴,同时用1.0 M HCl将体系中和至中性。中和升温物理交联使颗粒微球固化20~30min,不使用任何化学交联剂。生成的季铵化改性甲壳素微球分散体系用去离子水反复洗涤,得到吸水溶胀的样品通过显微镜观察如图7。由该图可以看出由温敏性的季铵化甲壳素T1和T2样品可以制得微球,而无温敏性的T5无法制备出微球形态。然后将成球的样品通过冷冻干燥得到季铵化甲壳素干燥微球,并对其在生理盐水中的饱和吸水倍率进行测量。结果可知T1微球在生理盐水中的饱和吸水倍率为18倍,而T2制备的微球在生理盐水中吸水倍率为25倍,较高的溶胀倍率是比较理想的止血材料。
同样的,在低温下将具有温度敏感性或者pH敏感性的季铵化改性甲壳素T2配置成碱性的2 wt%水溶液2 mL,将其加入含有表面活性剂1.0 g的司盘85的有机分散相媒介异辛烷20 mL中并置于冰水浴中,用70℃的油浴代替冰水浴,并用1.0 M HCl中和,通过中和升温、物理交联法制得温敏季铵化改性甲壳素颗粒微球,最后使用80%乙醇及去离子水洗涤、冷冻干燥即得。改变有机相溶剂异辛烷为石蜡油、植物油等,改变表面活性剂司盘85为其它表面活性剂如司盘60,改变表面活性剂在有机相里的浓度(0.2~5%),改变季铵化改性甲壳素的乙酰度(0.79~0.94)和取代度(0.06~0.23)及其浓度0.5~5wt%,改变季铵化改性甲壳素溶液与有机相溶液体积比(1:1~1:20)等,均可制备得到季铵化改性甲壳素微球。
同样的,在低温0-20oC下将具有温度敏感性或者pH敏感性的季铵化改性甲壳素T2配置成碱性的2 wt%水溶液,采用喷雾干燥法可以制得多孔颗粒,洗涤纯化该季铵化改性甲壳素颗粒,用去离子水浸泡使其充分吸水溶胀,之后冷冻干燥,制得季铵化改性甲壳素多孔颗粒材料。
这些季铵化改性甲壳素多孔颗粒材料或者微球,可以应用于药物载体材料,止血、术前栓塞、消化道和呼吸道出血及恶性肿瘤的介入治疗的非永久性栓塞、栓塞减肥等植入医用材料。
实施例11温敏季铵化改性甲壳素的止血海绵材料的制备和应用
将实例1中T1,T2和T3样品每组称取200 mg,然后加入一定体积 1M NaOH溶液,在低温0-20oC下使其充分溶解呈澄清透明状,配制5 wt%季铵化甲壳素溶液,低温离心脱泡加入到长度为12 mm,直径8 mm的圆柱形模具中,然后置于-30℃中进行冷冻凝固,将其置于-30℃下的95wt%乙醇水溶液凝固浴中浸泡3-7天,制得再生海绵,然后将样品用去离子水清洗,随后冷冻干燥制备出季铵化甲壳素海绵。将上述样品参照国家行业标准YY/T 1511-2017进行吸水倍率实验,在30 s,15 min,1 h测量其吸水倍率,每组设置5个重复组,结果表明T1,T2和T3的吸水倍率分别可达到15倍,18倍和23倍,且在30s以内就能达到饱和吸水量。同时以T1为实验组测试其吸水平衡后的压缩强度,设置载荷速度50 mm/min,实验重复4次,测得其压缩断裂强度为322 kPa。同时以T1海绵和T2海绵为例测试其全血凝血指数(BCI),设置4个重复组[Song F, Kong Y, Shao C, et al. Chitosan-based multifunctionalflexible hemostatic bio-hydrogel[J]. Acta Biomaterialia, 2021, 136: 170-183.],将 50μL全血滴加到 20 mg 干燥的海绵上,放在37°C水浴下60 rpm的摇床里孵育5min,随后加入10 mL去离子水并吸取上清液测540 nm的吸光度以计算凝血指数。将50 μL全血加入 10 mL 去离子水中作为空白对照组。结果显示T1海绵和T2海绵的BCI 分别为26%和17%,远低于已经批准上市的医用纱布和明胶海绵分别为100%和91%。这表明该类温敏季铵化改性甲壳素海绵材料具备优良的吸水性、止血性和力学强度,可望适用于深且窄的伤口、不可压迫性伤口及贯穿性伤口的止血。但是使用没有温敏性的T5材料不能通过上述物理交联过程制得止血海绵材料。
将季铵化甲壳素溶液浓度调整为1-10%,冷冻成冰的温度改为-18°C~ -30°C,凝固浴温度改为-30°C ~ 4°C,凝固浴改为50% ~ 100%甲醇/水溶液、50% ~ 100%乙醇/水溶液或50% ~ 100%丙酮/水溶液中的任意一种,凝固浴中浸泡的时长为1 ~ 7天,都可以制得性能良好的季铵化甲壳素止血海绵材料。
以上所述的具体实施方式, 对本发明的目的、 技术方案和有益效果进行了进一步的详细说明,所应理解的是, 以上所述仅为本发明的具体实例而已, 并不用于限制本发明,凡在本发明的精神和原则之内, 所做的任何修改、 等同替换、改进等, 均应包含在本发明的保护范围之内。
Claims (10)
1.一种具有温度或pH敏感性的季铵化改性甲壳素,其特征在于:该季铵化改性甲壳素的单元结构式示意图为:
R1为-H或者-COCH3;
R2为H、-CH2C(OH)CH2NCl(CH3)3、-CH2C(OH)CH2NCl(CH2CH3)3及-CH2C(OH)CH2N+ (CH3)2(CH2) mCH3 中的任意一种;
R3为H、-CH2C(OH)CH2NCl(CH3)3、-CH2C(OH)CH2NCl(CH2CH3)3及-CH2C(OH)CH2N+ (CH3)2(CH2) mCH3 中的任意一种;
其中,m=1;所述季铵化改性甲壳素的乙酰度为0.79~0.94,季铵化取代度为0.06~0.23;所述季铵化改性甲壳素采用的原料甲壳素的重均分子量为5×104~5×106。
2.一种权利要求1所述季铵化改性甲壳素的制备方法,其特征在于,包括如下步骤:
步骤1、将原料甲壳素在低温下溶解形成甲壳素水溶液,均相甲壳素水溶液的浓度为质量份0.2~9%;
步骤2、往甲壳素水溶液中加入季铵化试剂在0~40oC下搅拌均相反应,进行季铵化改性;
步骤3、将反应溶液干燥后得到季铵化改性甲壳素。
3.根据权利要求2所述季铵化改性甲壳素的制备方法,其特征在于,步骤1中,具体方式为:将原料甲壳素加入到碱性尿素的冷冻低温混合水溶液中混合均匀,并于-30~-12oC下冷冻8~96小时,再于1~25oC下搅拌制得均相甲壳素水溶液;
步骤2中,均相反应时间为6~71小时,所述季铵化试剂的加入量为甲壳素结构单元摩尔数的0.5~4倍。
4.根据权利要求3所述季铵化改性甲壳素的制备方法,其特征在于,步骤1中,所述的原料甲壳素为脱乙酰度低于20%的粉状甲壳素;
步骤2中,所述的季铵化试剂为2,3-环氧丙基三甲基氯化铵、3-氯-2-羟丙基三甲基氯化铵、3-氯-2-羟丙基三乙基氯化铵、2,3-环氧丙基三乙基氯化铵、2,3-环氧丙基三乙基溴化铵、2,3-环氧丙基三丙基氯化铵、短烷基碳原子数为m的环氧基短烷基链季铵盐以及氯羟丙基短烷基季铵盐的任意一种或者几种的混合物,m为1。
5.根据权利要求3所述季铵化改性甲壳素的制备方法,其特征在于,步骤1中,原料甲壳素脱乙酰度低于10%;
步骤2中,所述季铵化试剂的加入量为甲壳素结构单元摩尔数的1~2倍;
所述碱性尿素的冷冻低温混合水溶液为可溶性碱:尿素:纯水=5~20:3~12:68~91的混合液;
所述的可溶性碱为氢氧化钠、氢氧化钾和氢氧化锂中的任意一种或几种;
步骤3中,干燥前,对季铵化改性甲壳素溶液用稀酸调节pH值到中性,用纯水透析或者沉淀过滤洗涤除去尿素和盐等小分子物质,之后通过冷冻干燥或者加热干燥得到季铵化改性甲壳素;
所述的稀酸为盐酸、硫酸、硝酸、甲酸、乙酸、柠檬酸中的一种或几种。
6.一种季铵化改性甲壳素多孔材料的制备方法,其特征在于,包括如下步骤:
S1、在低温下,将权利要求2-5任意一项所述制备方法得到的季铵化改性甲壳素配置成碱性的水溶液,所述低温为0-20oC;
S2、将步骤S1配置的季铵化改性甲壳素溶液加入分散相媒介中,通过中和升温、物理交联法或者喷雾干燥法制得的温敏季铵化改性甲壳素颗粒;
S3、洗涤纯化温敏季铵化改性甲壳素颗粒,然后用去离子水浸泡使其充分吸水溶胀,之后冷冻干燥,制得季铵化改性甲壳素多孔颗粒材料。
7.根据权利要求6所述季铵化改性甲壳素多孔材料的制备方法,其特征在于:所述分散相媒介为含有表面活性剂的有机分散相或含有聚乙二醇的水分散相,在升温物理交联过程中通过酸性溶液将反应体系中和;所制得的季铵化改性甲壳素多孔颗粒材料的形状为球形或椭球型。
8.一种权利要求6或7所制备的季铵化改性甲壳素多孔材料的用途,其特征在于:用于作为止血、术前栓塞、消化道和呼吸道出血及恶性肿瘤的介入治疗的非永久性栓塞、栓塞减肥药物的载体。
9.一种季铵化改性甲壳素止血海绵的制备方法,其特征在于,包括如下步骤:
M1、在0-20oC低温下,将权利要求2-5任意一项所述制备方法得到的季铵化改性甲壳素配置成碱性的水溶液;
M 2、将步骤M1配置的季铵化改性甲壳素溶液脱泡后倒入模具中冷冻成冰;
M 3、将季铵化改性甲壳素冰块浸泡在凝固浴中,制得再生海绵;
M 4、用去离子水洗涤纯化再生海绵,脱水干燥,制得季铵化改性甲壳素止血海绵。
10.一种权利要求9所制备的季铵化改性甲壳素止血海绵的用途,其特征在于:所述季铵化甲壳素止血海绵材料用于制备伤口止血抗菌材料。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210093853.9A CN116535540B (zh) | 2022-01-26 | 2022-01-26 | 季铵化改性甲壳素及其海绵、多孔微球的制备方法、用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210093853.9A CN116535540B (zh) | 2022-01-26 | 2022-01-26 | 季铵化改性甲壳素及其海绵、多孔微球的制备方法、用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116535540A CN116535540A (zh) | 2023-08-04 |
CN116535540B true CN116535540B (zh) | 2024-04-12 |
Family
ID=87456556
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210093853.9A Active CN116535540B (zh) | 2022-01-26 | 2022-01-26 | 季铵化改性甲壳素及其海绵、多孔微球的制备方法、用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116535540B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0400364A2 (en) * | 1989-05-17 | 1990-12-05 | Farmhispania S.A. | New process for preparing chitosan and its derivatives containing quaternary ammonium groups |
CN102276757A (zh) * | 2011-07-29 | 2011-12-14 | 武汉大学 | 一种甲壳素季铵盐的制备方法 |
CN103834045A (zh) * | 2013-11-07 | 2014-06-04 | 浙江科技学院 | 一种双向可逆温敏凝胶及其制备方法 |
CN109966542A (zh) * | 2019-02-26 | 2019-07-05 | 福建师范大学 | 甲壳素基可注射止血密封胶的制备方法 |
-
2022
- 2022-01-26 CN CN202210093853.9A patent/CN116535540B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0400364A2 (en) * | 1989-05-17 | 1990-12-05 | Farmhispania S.A. | New process for preparing chitosan and its derivatives containing quaternary ammonium groups |
CN102276757A (zh) * | 2011-07-29 | 2011-12-14 | 武汉大学 | 一种甲壳素季铵盐的制备方法 |
CN103834045A (zh) * | 2013-11-07 | 2014-06-04 | 浙江科技学院 | 一种双向可逆温敏凝胶及其制备方法 |
CN109966542A (zh) * | 2019-02-26 | 2019-07-05 | 福建师范大学 | 甲壳素基可注射止血密封胶的制备方法 |
Also Published As
Publication number | Publication date |
---|---|
CN116535540A (zh) | 2023-08-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ravishankar et al. | Advances in chitosan-based hydrogels: Evolution from covalently crosslinked systems to ionotropically crosslinked superabsorbents | |
Amirian et al. | In-situ crosslinked hydrogel based on amidated pectin/oxidized chitosan as potential wound dressing for skin repairing | |
Fang et al. | Synthesis of superabsorbent polymers based on chitosan derivative graft acrylic acid-co-acrylamide and its property testing | |
Kundu et al. | Cellulose hydrogels: Green and sustainable soft biomaterials | |
Ibrahim et al. | Polysaccharide-based polymer gels and their potential applications | |
Tan et al. | Collagen cryogel cross-linked by naturally derived dialdehyde carboxymethyl cellulose | |
Ma et al. | Advances in cellulose-based superabsorbent hydrogels | |
Zhou et al. | An investigation of chitosan and its derivatives on red blood cell agglutination | |
Li et al. | Preparation and characterization of acid resistant double cross-linked hydrogel for potential biomedical applications | |
Peng et al. | Preparation of chitosan/gelatin composite foam with ternary solvents of dioxane/acetic acid/water and its water absorption capacity | |
El Knidri et al. | Chitin and chitosan: chemistry, solubility, fiber formation, and their potential applications | |
JPS6228697B2 (zh) | ||
CN107118361B (zh) | 一种丝素蛋白/羧甲基壳聚糖复合凝胶及其制备方法 | |
Sharma et al. | Borax mediated synthesis of a biocompatible self-healing hydrogel using dialdehyde carboxymethyl cellulose-dextrin and gelatin | |
CN114524950B (zh) | 一种疏水药物载体水凝胶及其制备方法和应用 | |
Song et al. | Effects of degree of deacetylation on hemostatic performance of partially deacetylated chitin sponges | |
Liao et al. | A fungal chitin derived from Hericium erinaceus residue: Dissolution, gelation and characterization | |
Gao et al. | Synergistic effect of hydrogen bonds and chemical bonds to construct a starch-based water-absorbing/retaining hydrogel composite reinforced with cellulose and poly (ethylene glycol) | |
Thakur et al. | Synthetic chemistry of cellulose hydrogels-A review | |
Chen et al. | Developing slow release fertilizer through in-situ radiation-synthesis of urea-embedded starch-based hydrogels | |
Wei et al. | Preparation and characterization of starch-cellulose interpenetrating network hydrogels based on sequential Diels-Alder click reaction and photopolymerization | |
Hu et al. | Highly antibacterial hydrogels prepared from amino cellulose, dialdehyde xylan, and Ag nanoparticles by a green reduction method | |
CN105343886B (zh) | 一种抑菌性壳聚糖药物载体及其制备方法 | |
JP2004059618A (ja) | 多糖類複合体及びその製造方法 | |
WO2001055220A1 (en) | Chitosan condensation products, their preparation and their uses |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |