WO2023143540A1 - 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 - Google Patents
一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 Download PDFInfo
- Publication number
- WO2023143540A1 WO2023143540A1 PCT/CN2023/073618 CN2023073618W WO2023143540A1 WO 2023143540 A1 WO2023143540 A1 WO 2023143540A1 CN 2023073618 W CN2023073618 W CN 2023073618W WO 2023143540 A1 WO2023143540 A1 WO 2023143540A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- film
- membrane
- hydrogel
- dehydration
- unidirectional
- Prior art date
Links
- 239000000017 hydrogel Substances 0.000 title claims abstract description 124
- 230000018044 dehydration Effects 0.000 title claims abstract description 98
- 238000006297 dehydration reaction Methods 0.000 title claims abstract description 98
- 238000000034 method Methods 0.000 title claims abstract description 40
- 229920001002 functional polymer Polymers 0.000 title claims abstract description 13
- 239000012528 membrane Substances 0.000 claims abstract description 205
- 239000000243 solution Substances 0.000 claims abstract description 53
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000007864 aqueous solution Substances 0.000 claims abstract description 42
- 229920000642 polymer Polymers 0.000 claims abstract description 34
- 229920006254 polymer film Polymers 0.000 claims abstract description 19
- 239000000203 mixture Substances 0.000 claims abstract description 14
- 239000000463 material Substances 0.000 claims abstract description 13
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 229920001940 conductive polymer Polymers 0.000 claims abstract description 9
- 238000011065 in-situ storage Methods 0.000 claims abstract description 5
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 4
- 108010022355 Fibroins Proteins 0.000 claims description 130
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 77
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 76
- 239000005018 casein Substances 0.000 claims description 28
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 28
- 235000021240 caseins Nutrition 0.000 claims description 28
- 238000002360 preparation method Methods 0.000 claims description 25
- 238000000502 dialysis Methods 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- 239000011148 porous material Substances 0.000 claims description 15
- 239000003431 cross linking reagent Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 239000000835 fiber Substances 0.000 claims description 7
- 235000018102 proteins Nutrition 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 241000255789 Bombyx mori Species 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 229920005615 natural polymer Polymers 0.000 claims description 5
- 229920002521 macromolecule Polymers 0.000 claims description 4
- 239000004014 plasticizer Substances 0.000 claims description 4
- 235000004443 Ricinus communis Nutrition 0.000 claims description 3
- 229920001872 Spider silk Polymers 0.000 claims description 3
- 238000010382 chemical cross-linking Methods 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 230000003204 osmotic effect Effects 0.000 claims description 3
- 239000000049 pigment Substances 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- 229920000298 Cellophane Polymers 0.000 claims description 2
- 239000004677 Nylon Substances 0.000 claims description 2
- 239000000919 ceramic Substances 0.000 claims description 2
- 108010025899 gelatin film Proteins 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 229920001778 nylon Polymers 0.000 claims description 2
- 239000002313 adhesive film Substances 0.000 claims 1
- 229920002401 polyacrylamide Polymers 0.000 claims 1
- -1 3D scaffolds Substances 0.000 abstract description 9
- 239000012620 biological material Substances 0.000 abstract description 7
- 241000894006 Bacteria Species 0.000 abstract description 4
- 239000000446 fuel Substances 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 239000002861 polymer material Substances 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000002977 biomimetic material Substances 0.000 abstract description 2
- 238000012377 drug delivery Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- 239000002131 composite material Substances 0.000 description 30
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 26
- 230000000694 effects Effects 0.000 description 23
- 230000008961 swelling Effects 0.000 description 18
- 239000000843 powder Substances 0.000 description 16
- 108010046334 Urease Proteins 0.000 description 14
- 241000588724 Escherichia coli Species 0.000 description 13
- 229960004679 doxorubicin Drugs 0.000 description 13
- 238000001704 evaporation Methods 0.000 description 13
- 230000008020 evaporation Effects 0.000 description 13
- 230000012010 growth Effects 0.000 description 13
- 238000010586 diagram Methods 0.000 description 10
- 230000035755 proliferation Effects 0.000 description 10
- 238000002441 X-ray diffraction Methods 0.000 description 9
- 239000013078 crystal Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 229920005597 polymer membrane Polymers 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 238000002329 infrared spectrum Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 229920002472 Starch Polymers 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- 229920000704 biodegradable plastic Polymers 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000008107 starch Substances 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 6
- 239000004743 Polypropylene Substances 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 210000002950 fibroblast Anatomy 0.000 description 5
- 238000002844 melting Methods 0.000 description 5
- 230000008018 melting Effects 0.000 description 5
- 229920000128 polypyrrole Polymers 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108010093096 Immobilized Enzymes Proteins 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 238000005266 casting Methods 0.000 description 4
- 230000003203 everyday effect Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 229920001155 polypropylene Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000008707 rearrangement Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000013268 sustained release Methods 0.000 description 4
- 239000012730 sustained-release form Substances 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- 108091005507 Neutral proteases Proteins 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 108010013296 Sericins Proteins 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000013068 control sample Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000005470 impregnation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000001728 nano-filtration Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 229920003169 water-soluble polymer Polymers 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 101100327917 Caenorhabditis elegans chup-1 gene Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 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 2
- 238000009864 tensile test Methods 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 238000009777 vacuum freeze-drying Methods 0.000 description 2
- AOBIOSPNXBMOAT-UHFFFAOYSA-N 2-[2-(oxiran-2-ylmethoxy)ethoxymethyl]oxirane Chemical compound C1OC1COCCOCC1CO1 AOBIOSPNXBMOAT-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000003170 Bronchiolo-Alveolar Adenocarcinoma Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 239000011837 N,N-methylenebisacrylamide Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000722270 Regulus Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000011173 biocomposite Substances 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000005422 blasting Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- MBDICAOEPMIKPN-UHFFFAOYSA-L calcium ethanol dichloride hydrate Chemical compound O.C(C)O.[Cl-].[Ca+2].[Cl-] MBDICAOEPMIKPN-UHFFFAOYSA-L 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000113 differential scanning calorimetry Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000004070 electrodeposition Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 108091005899 fibrous proteins Proteins 0.000 description 1
- 102000034240 fibrous proteins Human genes 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 239000013081 microcrystal Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- MUSLHCJRTRQOSP-UHFFFAOYSA-N rhodamine 101 Chemical compound [O-]C(=O)C1=CC=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MUSLHCJRTRQOSP-UHFFFAOYSA-N 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000008149 soap solution Substances 0.000 description 1
- 239000001476 sodium potassium tartrate Substances 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- PCTXBFQNMDKOSP-UHFFFAOYSA-M sodium;(2-carboxyphenyl) sulfate Chemical compound [Na+].OS(=O)(=O)OC1=CC=CC=C1C([O-])=O PCTXBFQNMDKOSP-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002076 thermal analysis method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- 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
- C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
- C08J5/18—Manufacture of films or sheets
-
- 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
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
- C08L89/005—Casein
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L93/00—Compositions of natural resins; Compositions of derivatives thereof
-
- 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
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- 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
- C08J2393/00—Characterised by the use of natural resins; Derivatives thereof
-
- 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
- C08J2429/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal, or ketal radical; Hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Derivatives of such polymer
- C08J2429/02—Homopolymers or copolymers of unsaturated alcohols
- C08J2429/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids
Definitions
- the invention relates to the preparation of polymer membranes, polymer hydrogel membranes, polymer composite/crosslinked membranes, polymer membranes or polymer hydrogel membranes of immobilized enzymes/polypeptides/drugs/active factors/microorganisms prepared by water-soluble polymers and a preparation method thereof, in particular to a preparation method of a polymer film/hydrogel film with excellent mechanical strength, good biocompatibility, and flexibility and a multifunctional composite/cross-linked film, belonging to biological materials, biomimetic materials, and Wearable devices, drug sustained release and delivery, microbial immobilization, separation membranes, medical tissue engineering materials and other fields.
- Silk is a protein-based natural polymer fiber produced by the silkworm. It is composed of silk fibroin and sericin that coats its fibers and acts as an adhesive. For decades, the extended application of silk protein, especially silk fibroin, has received great attention, mainly because of its unique molecular structure, physical and chemical properties and excellent biocompatibility.
- the silk fibroin from which the outer sericin has been removed can be dissolved in a high-concentration neutral salt solution such as lithium bromide or calcium chloride, and can be dialyzed with water to obtain an aqueous solution of regenerated silk fibroin.
- This water-soluble natural polymer solution can be widely used in biomaterials in various application scenarios after subsequent processing.
- the silk fibroin molecule is composed of light chain (27.5 kDa) and heavy chain (about 390 kDa) in a 1:1 molar ratio with a single disulfide bond, and its molecular weight is as high as 410 kDa.
- silk fibroin fiber is a kind of fibrous protein with amorphous and double crystal structure, and its molecular chain is composed of random coil, ⁇ -helix, type II ⁇ -turn and antiparallel ⁇ -sheet structure.
- the previously mentioned Silk I and Silk II crystal structures are now more likely to be considered to be the latter two, respectively.
- This regenerated liquid silk fibroin can be easily transformed from its random coils and Silk I to a more stable Silk II structure through physical or chemical treatment, so that it can be processed into various forms of biomaterials with stable performance.
- Regenerated liquid silk fibroin can be prepared by physical methods such as shearing, foaming, ultrasonic wave, etc. to prepare silk fibroin in the form of hydrogel, powder, etc., and processed into silk with different crystal structures by electrodeposition, laser radiation, plasma, electric field, etc. Film, glue, hydrogel and other forms.
- the most conventional preparation method of film-like silk fibroin is to spread the regenerated liquid silk fibroin on a polypropylene plate for evaporation and dehydration.
- the silk fibroin film formed at or below room temperature is often mostly soluble in water, so it is often called casting type or casting type.
- Water Soluble Silk Fibroin Film SFM-E.
- Silk I can be transformed into an antiparallel ⁇ -sheet Silk II silk membrane (SFM-EM).
- the original stretched and transparent state is transformed into a milky white wrinkled silk fibroin film, which is brittle in the dry state and slightly elastic in the wet state, but its overall mechanical properties are poor, and its practical application is greatly limited.
- this film When this film is exposed to low-temperature water vapor treatment under reduced pressure, which is often called “annealing", it can be made into a water-insoluble silk film with good performance and increased Silk I structure content (see literature: Hu et al. Biomacromolecules 2011 , 12: 1686-1696). Both temperature and humidity will affect the structure of silk fibroin film during casting. When it is slowly dried below 9°C, it can become a water-soluble amorphous silk film.
- the single-component silk fibroin film prepared by the prior art has shortcomings such as poor mechanical properties, optical properties, elasticity, toughness, and poor water absorption.
- the above slow drying method takes a long time to prepare the water-insoluble silk fibroin film, the drying speed is difficult to control, and the tensile strength is obviously reduced to 1.4 ⁇ 0.1 MPa, which is 45% and 36% of the silk fibroin film treated by methanol impregnation and water vapor annealing respectively. . Therefore, the stability and operability of the existing method to prepare high-performance silk membranes with more Silk I structures still need to be improved.
- Polyvinyl alcohol is a water-soluble polymer that can be dissolved or swelled in water to form an aqueous solution or dispersion system. Its molecular structure contains a large number of hydrophilic groups - hydroxyl groups. PVA is easy to make hydrogel, chemically inactive, non-toxic, good biocompatibility, biodegradable, good mechanical properties, high water absorption, easy to process and shape, widely used in agriculture, forestry, medicine, Daily chemicals, environmental protection, biological materials, especially medical tissue engineering materials and other fields.
- PVA's linear polymer structure and a large number of hydroxyl groups in the side chains bring the above-mentioned excellent properties, but there are also disadvantages such as easy swelling or even dissolution in water and poor mechanical strength of its hydrogel film, which cannot meet the needs of practical applications. All requirements in . Therefore, it is necessary to modify the PVA hydrogel film to improve its mechanical and water resistance properties, as well as to broaden its functions such as flame retardancy, antibacterial properties, and optical and electrical properties. has been widely applied.
- PVA film modification is usually divided into two categories: physical treatment and chemical crosslinking.
- the former mainly refers to blending with other organic or inorganic substances, nanocomposite, heat treatment, etc. These methods are simple to operate and can easily expand the functionality of PVA hydrogel membranes.
- the latter mainly refers to the use of crosslinking or grafting methods before and after PVA film formation to form chemical bonds between the internal polymer chains to form a crosslinked network, thereby significantly improving the mechanical properties of polyvinyl alcohol hydrogel membranes (PVAHM). , thermal stability and water resistance.
- heat treatment modification is a relatively simple method to adjust the internal crystallinity of PVA film and improve its performance. The mechanical properties, thermal stability, water holding and water resistance properties have been greatly improved.
- freeze-thaw cycle method is the most common and typical method to promote the formation of hydrogen bonds and increase the crystallinity to improve the properties of materials.
- freezing at -20°C or below limits the polymer chains to form crystal nuclei
- thawing at 25°C allows crystals to grow, and then increases the number of freeze-thaw cycles to perfect the crystal structure and increase the degree of crystallization.
- the microcrystals formed during freeze-thaw cycles can also be regarded as physical cross-linking points to cross-link PVA polymer chains.
- the existing PVA hydrogels prepared by physical methods have poor mechanical properties, are difficult to withstand the load imposed on the human body, and have poor biocompatibility, tensile strength and elongation at break. Difficult to meet severe conditions of use.
- the existing technology has made some progress in mechanical properties, the function is relatively single and lacks biological functions.
- the present invention aims at the deficiencies in the existing polymer membranes, polymer hydrogel membranes and multifunctional biological composite materials in terms of green processing, operation controllability, functionality and mechanical properties, etc., and adopts the technology of unidirectional nanopore dehydration,
- the water molecules in the polymer aqueous solution are caused to dehydrate in one direction through the nanopores of the filter membrane, causing the molecules of the polymer to be arranged in an orderly manner, providing a flexible, transparent, and mechanically strong polymer film/polymer hydrogel film, multi- Functional biocomposite material, conductive material and preparation method thereof.
- the technical scheme to realize the object of the present invention is: provide a kind of preparation method based on the functional macromolecule membrane/hydrogel membrane of unidirectional nanopore dehydration, polymer aqueous solution or its mixture solution are from the sample hole of container top Add it into the mold, the mold uses the nanoporous filter membrane as the bottom container, and seals the sample hole with a sealing cover; the water molecules in the solution are dehydrated downward through the nanoporous filter membrane, and on the upper surface of the nanoporous filter membrane A polymer film/hydrogel film is obtained.
- the technical scheme of the invention includes the functional polymer membrane/hydrogel membrane based on unidirectional nanopore dehydration obtained by the above preparation method.
- the technical solution of the present invention provides a method for preparing a conductive polymer film/hydrogel film based on unidirectional nanopore dehydration.
- the polymer aqueous solution or its mixture solution is added to the mold from the sample hole on the top of the container.
- Said mold uses the nanoporous filter membrane as the bottom of the container, and the sample hole is sealed with a sealing cover; the water molecules in the solution are dehydrated downward through the nanoporous filter membrane, and the polymer film/water is obtained on the upper surface of the nanoporous filter membrane.
- Gel film performing in-situ polymerization reaction on the obtained polymer film/hydrogel film and pyrrole in an aqueous solution to obtain a polypyrrole-modified conductive polymer film/hydrogel film.
- the technical solution of the present invention includes a conductive polymer film/hydrogel film based on unidirectional nanopore dehydration obtained by the above-mentioned preparation method.
- the natural macromolecule includes casein, one of domestic silkworm silk, spider silk, tussah silk, castor silk or wild silk regenerated liquid silk fibroin; the artificial synthetic macromolecule includes polyvinyl alcohol.
- the technical scheme of the present invention provides a device for preparing a functional polymer membrane/hydrogel membrane based on unidirectional nanopore dehydration, which uses a nanoporous filter membrane as the bottom of the container, and the top of the container is provided with a sample hole and A sealing cover matched with it; the pore size of the nanoporous filter membrane is a dialysis membrane with a molecular weight cut-off of 0.1 to 1000 kDa, or a polymer synthetic membrane with a filter pore ⁇ 50 nm.
- a device for preparing a functional polymer membrane/hydrogel membrane based on unidirectional nanopore dehydration includes a mold frame for installing the device, below the nanoporous filter membrane at the bottom of the container A superabsorbent material or a one-way dehydration accelerator is provided; the one-way dehydration accelerator includes a flow air generating device, a constant temperature and humidity chamber, a negative pressure chamber, and an osmotic pressure difference chamber.
- a device for preparing a functional polymer membrane/hydrogel membrane based on unidirectional nanopore dehydration includes regenerated fiber dialysis membrane, plane dialysis cellophane, ceramic membrane, polypropylene Amide hydrogel membrane, nylon membrane.
- the beneficial effect of the present invention is: 1.
- the water-insoluble ultra-thick silk fibroin film rich in type 2 ⁇ -turning structure provided by the present invention has a wet tensile strength of 14.6 ⁇ 0.6 MPa, a maximum elongation at break of 640 ⁇ 55%, and a swelling rate of About 55%.
- the wet tensile strength of polyvinyl alcohol hydrogel film prepared by unidirectional nanoporous dehydration is 3.0-10.5 MPa, the elongation at break is 342-891%, and the swelling rate is 300-335%.
- the polymer aqueous solution can be blended or biologically connected with other water-soluble polymers, cross-linking agents, plasticizers, enzymes, drugs, pigments, active factors and even microorganisms to prepare excellent mechanical properties.
- Bioactive or conductive polymer composite membrane/composite hydrogel membrane The PVAHM-immobilized Escherichia coli has undergone 40 times of medium change and repeated culture, the bacteria can divide stably and continuously without falling, and the proliferation efficiency remains above 90%. Mouse fibroblast L929 can grow and proliferate well on these polymer materials.
- the new polymer material based on unidirectional nanopore dehydration provided by the present invention can be widely used in medical biomaterials, 3D scaffolds, bionic materials, enzyme/drug delivery and sustained release, wearable electronic devices, bioreactors, microorganisms Fuel cells, engineering bacteria reactors, membrane separation materials and other fields.
- SFM-U silk fibroin membrane
- FIG. 5 is the 4th growth state diagram of L929 cells on silk fibroin membrane (SFM-U) provided by the present invention
- Figure 6 is a diagram of A549 cells provided by the present invention cultured on a doxorubicin slow-release silk film
- Fig. 7 is a comparison chart of the light transmission performance of the conductive silk fibroin-polypyrrole modified film and the casein-PEGDE cross-linked film provided by the present invention
- Fig. 8 is the stress and strain curve diagram of the silk fibroin bioplastic provided by the present invention
- FIG. 9 is a stress and strain curve diagram of the influence of the unidirectional nanopore dehydration temperature on the mechanical properties of PVAHM provided by the present invention
- Figure 10 is the stress-strain curve (a), DSC (b), infrared spectrum (c) and X-ray diffraction pattern ( d)
- Figure 11 is a cross-sectional scanning electron micrograph of a polyvinyl alcohol hydrogel film and a composite/crosslinked film provided by an embodiment of the present invention
- Figure 12 is a diagram of the process of culturing L929 cells on polyvinyl alcohol hydrogel membranes and composite/crosslinked membranes provided by the present invention
- Figure 13 is a diagram of the growth state of L929 cells cultured on the polyvinyl alcohol hydrogel membrane provided by the present invention on the 4th day
- Fig. 14 is a graph showing the proliferation stability of Escherichia coli immobilized in the polyvinyl alcohol hydrogel film provided by the present invention.
- This example provides the preparation of the sample solutions required for each example according to the technical solution of the present invention.
- Polyvinyl alcohol aqueous solution Polyvinyl alcohol powder (CAS: 9002-89-5; Mowiol®PVA-124, viscosity: 54-66 mPa ⁇ S) was purchased from Shanghai Aladdin Company. Suspend the PVA powder with water, heat it on an electric furnace at 90 °C to suspend and mix it, and then place it under high temperature and high pressure at 120 °C for 2 h, and finally prepare a PVA aqueous solution with a concentration of 10 w% for later use.
- This example provides a preparation method of the polymer membrane/hydrogel membrane described in the technical solution of the present invention.
- the preparation of unidirectional nanoporous dehydration polymer membrane or hydrogel membrane is referring to accompanying drawing 1, is the structural representation of the U mold that the unidirectional nanopore dehydration polymer membrane or hydrogel membrane that this embodiment provides;
- the mold is mainly composed of a film-forming cup 1 with an inner cavity diameter of 30 mm or 80 mm, a nanoporous filter membrane 2, a fixed ring 3, a sample injection hole 4, and a rubber plug 5 for the sample injection hole.
- the circular nanoporous filter membrane 2 moistened in water is rotated and fixed by the inner screw of the fixing ring 3, placed on the horizontal mold shelf, and a little distilled water is added through the sample hole 4 with a pipette to test whether the fixed filter membrane 2 is water leakage. If there is water leakage, repeat the above operation until there is no water leakage.
- the mold nanoporous filter membrane which is a polymer membrane or a polymer hydrogel membrane.
- the water-soluble silk film (SFM-E) is formed by evaporation, which needs to be post-treated by immersing it in 80% methanol aqueous solution for 1-2 minutes.
- SFM-EM water-insoluble b-silk fibroin film
- This example provides testing methods for the mechanics, structure and in vitro performance of materials provided by various examples of the technical solutions of the present invention.
- Swelling rate (%) (M n - M 0 )/M 0 ⁇ 100 % (3) Structural analysis Weigh ⁇ 5.0 mg of the polymer film/hydrogel film powder sample, and perform TG, DTG and DSC analysis with a thermogravimetric/differential thermal instrument (SDT2960, TA Company, USA). Parameter settings: the protective gas is nitrogen 100 mL/min, the temperature range is 25 °C to 800 °C, and the heating rate is 10 °C/min. The structural characteristics of polymer film/hydrogel film powder samples were detected by Fourier transform infrared spectrometer (Nicolet 6700, Thermo Fisher, USA).
- X'Pert-Pro MPD X-ray diffraction spectrometer
- mouse fibroblast L929 was selected as the test object to observe the growth, proliferation and growth of mouse fibroblasts on polymer membranes/hydrogel membranes and other membranous samples, as well as the stability of cells. Morphology, cell engraftment and biocompatibility of these samples were assessed.
- the biological medium in this experiment was DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. All operations in the cell culture process are carried out in ultra-clean workbenches. The environment of the cell incubator was set at a constant temperature of 37°C and a carbon dioxide concentration of 5%.
- the cell culture solution to be tested was transferred to a new 96-well plate, and placed in a microplate reader to measure the absorbance value of the cell culture solution at 450 nm, with 20 parallels in each group.
- This example provides a silk film with excellent mechanical properties prepared by unidirectional nanopore dehydration.
- the thickness of the formed film becomes thicker. That is to say, the time for the unidirectional nanopore dehydration to form silk fibroin film is prolonged, and its tensile strength and elongation at break will be significantly reduced. It can be observed from Table 1 that the silk fibroin film that can be formed within 1 day has the strongest mechanical properties of 11.8 MPa, and the maximum elongation at break is 490%.
- the thickest silk fibroin film formed by unidirectional nanopore dehydration at 4°C was 0.236 mm, and the lowest tensile strength was 7.91 MPa, which was almost half of the thickness at 70°C; while the elongation at break was the largest at 25°C.
- Evaporated silk fibroin film was prepared by blasting 35 mL of silk fibroin solution of the same concentration in a polypropylene circular box with an area of 50 cm 2 , and then soaked in 80% methanol aqueous solution for 1–2 min to become a b-formed silk fibroin membrane (SFM-E).
- Silk fibroin membrane was used as a control sample. According to the method of Example 3, the mechanical properties were tested by stretching with a stretcher, and the measurement was repeated 15 times, and the average value and standard deviation ( ⁇ SD) were calculated.
- the experimental results show that the silk fibroin film formed by unidirectional nanopore dehydration (SFM-U) and the control evaporated silk film (SFM-EM) have great differences in film thickness and tensile properties (Table 6).
- the thickness of SFM-U was almost proportional to the amount of silk fibroin solution added; the thickness of the silk film prepared by 35 mL of 3% silk fibroin solution was 0.19 mm, while that of ordinary SFM-EM was only 0.10 mm, the former was 1.9 times that of the latter. This fully shows that the internal structure of the unidirectional nanoporous dehydrated silk membrane is much looser than that of the evaporation type.
- the tensile force of SFM-U is 1.8 times stronger than that of the evaporated film, reaching 7.26 N; as the thickness of the dehydrated film increases, its tensile force also increases proportionally; since the tensile strength is related to the film thickness, the three unidirectional
- the nanoporous dehydrated silk membrane is also similar to the evaporation membrane, both of which are 13.60 MPa.
- the elongation-at-break properties of the last two films differ even more, with ⁇ 100% elongation for the evaporated film, while the elongation for the three SFM-U films is 4–6 times higher.
- FIG. 2 it is the silk fibroin membrane stress and the strain curve contrast chart provided in this embodiment;
- SFM-EM Evaporation silk fibroin membrane + methanol impregnation treatment
- SFM-U One-way nanoporous dehydration method preparation
- the elongation force is 1.8 times higher than that of the evaporated membrane; the two membranes are both about 13.6 MPa in terms of tensile strength, and the average elongation at break of the three unidirectional nanoporous dehydration membranes is more than 5 times that of the evaporated membrane.
- This example provides the structural characteristics and characterization of a silk fibroin membrane prepared by unidirectional nanopore dehydration.
- Infrared spectrum of silk fibroin film usually has two main absorption peaks, the amide I band (1638 cm -1 ) caused by the typical C–O stretching vibration in the protein molecule and the amide II band (1545 cm -1 ) caused by the N–H vibration.
- FIG. 3a is the infrared spectrum diagram of the silk film provided by the present invention; in the figure, the evaporated silk film (SFM-E) becomes a water-insoluble silk film (SFM-EM) after methanol impregnation; one-way Silk fibroin membrane (SFM-U) prepared by nanoporous dehydration method.
- the SFM-U formed by unidirectional nanopore dehydration is obviously different from the above SFM-E, and a strong peak appears at 1640.2 cm -1 , which fully demonstrates that the silk fibroin membrane structure formed by unidirectional nanopore dehydration follows a type II ⁇ -transition The so-called silk I structure is dominant.
- the experimental sample silk film was prepared by dehydrating 5.0 mL 30 mg/mL regenerated silk fibroin solution through unidirectional nanopores for 24 h ( SFM-U).
- the other two control samples are the water-soluble SFM-E prepared by evaporation on the PP plate under the same conditions for 12 h and the water-insoluble SFM-EM prepared by immersing in 80% methanol aqueous solution for 1-2 min.
- FIG. 3b it is the X-ray diffraction spectrogram of the silk fibroin membrane provided by the embodiment of the present invention; -EM); silk fibroin membrane prepared by unidirectional nanoporous dehydration method (SFM-U). From the X-ray diffraction spectrum in Figure 3b, it can be observed that the silk film prepared by conventional evaporation (SFM-E) only has a very broad peak around 21.84°, indicating that this is the typical amorphous structure of the soluble silk film. picture. However, the diffraction peaks of the silk fibroin film caused by methanol treatment appear at 9.11°, 20.44°, and 24.14°.
- the silk film has a Silk II structure dominated by b-fold.
- the diffraction peaks of the silk film prepared by the unidirectional nanopore dehydration method in the present invention are completely different. They appear at 12.01°, 19.87°, 24.11°, 28.06°, 32.40° and 36,81°, respectively.
- This example provides the enzymatic properties and biocompatibility of a silk fibroin membrane prepared by unidirectional nanopore dehydration.
- FIG. 4a it is the in vitro neutral protease degradation diagram of the silk fibroin membrane provided by the embodiment of the present invention.
- the SFM-U prepared by unidirectional nanopore dehydration can The degradation is about 20%.
- the weight loss rate increases.
- the weight has remained less than 3%.
- the above results show that the water stability of this SFM-U is excellent, but its unique The secondary structure is easily degraded by neutral protease, and the material prepared by the invention is very suitable for biodegradable implants in vivo.
- the mouse fibroblast L929 was cultured on the unidirectional nanoporous dehydrated silk fibroin membrane (SFM-U), and the L-929 on the common culture dish and on the silk membrane was measured by CCK8 quantitative method every day cell viability.
- SFM-U unidirectional nanoporous dehydrated silk fibroin membrane
- Figure 4b is the growth curve of L929 cells on the silk membrane provided by the embodiment of the present invention. From it, it can be seen that the cells were cultured on the silk membrane formed by unidirectional nanopore dehydration on the third day, and L- The 929 cells had already appeared adherent growth, and then the cell attachment gradually increased. After 5 days of culture, the cells still grew normally, covering almost the entire silk membrane surface, and showing a typical fibroblast morphology.
- This example provides the characteristics of a silk fibroin plasticized/composite/crosslinked membrane prepared by unidirectional nanopore dehydration.
- Multifunctional silk fibroin-glycerin plasticized film SFM-G
- silk fibroin-PEGDE crosslinked film SFM-PEGDE
- silk fibroin-rhodamine 101 composite film SFM-R101
- SFM-G plasticized film is softer than SFM-U without plasticizer, the mechanical properties of silk fibroin-PEGDE crosslinked film are stronger than that of SFM-U, and SFM-R101 is light red under sunlight, and the 365 mm ultraviolet It is bright red under light, which is mainly the fluorescent color emitted by R101; under sunlight, SFM-G and SFM-PEGDE are the same as SFM-U, showing translucent milky white, and they are light blue under ultraviolet light, which is silk fibroin The rendered color.
- the stress-strain curve of SFM-G is similar to that of unplasticized SFM-U, but the tensile strength is significantly enhanced (18.36 MPa), and the elongation at break (382%) is slightly lower.
- SFM-PEGDE The mechanical properties of SFM-PEGDE were significantly better than that of uncrosslinked SFM-U.
- the stress-strain curve of SFM-R101 is similar to that of SFM-U, but the tensile strength (13.59 MPa) and elongation at break (380%) are slightly lower than those of SFM-U silk fibroin film.
- This example provides a preparation of immobilized urease silk membrane prepared by unidirectional nanopore dehydration.
- a dialysis membrane with a molecular weight cut-off of ⁇ 10 kDa is selected as the bottom of the mold.
- Immobilized urease silk membrane SFM-Urease
- the method for the determination of urease activity is as follows: take 5ml of urea (0.3M) solution and heat it in a water bath at 37°C for 5 minutes, put a certain amount of urease or an immobilized urease silk film (0.2 g) soaked overnight into the urea solution, and continue to React in a water-bath shaker at 37°C at 110 rpm for 10 min; after the reaction is complete, take an appropriate amount of the reaction solution and dilute it to 200 ⁇ L with double-distilled water, then add 10 ⁇ L of 10% sodium potassium tartrate and 20 ⁇ L of Nessler’s reagent in sequence , mixed thoroughly, and left to react for 15 minutes, then measure the absorbance value at 420 nm; finally, substitute the absorbance value into the ammonium concentration standard curve (0, 0.1, 0.2, 0.3, 0.4, 0.5 mL of 0.25 M ammonium sulfate solution) The ammonium ion concentration was calculated, and the catalyzed
- the active recovery rate of nine kinds of different contents urease in the immobilized urease silk film shown in table 7, take the active determination value of free urease as 100%, then calculate the active recovery of immobilized enzyme by the linear equation of the free urease standard curve of establishment Rate. It can be found from the table that the urease content in the silk film gradually increases from 1.5 U to 25 U, and the activity recovery rate of the immobilized enzyme also changes accordingly. When the silk fibroin/urease ratio reaches 1:4, that is, 0.2 g silk When the plain film contains 0.8 U urease, the recovery rate of enzyme activity can reach 75%.
- This example provides a doxorubicin sustained-release silk film prepared by unidirectional nanopore dehydration.
- a dialysis membrane with a molecular weight cut-off of ⁇ 10 kDa is selected as the bottom of the mold.
- SFM-DOX doxorubicin sustained-release silk fibroin membrane
- A549 cell culture method punch SFM-U and SFM-DOX membranes into a disc structure of a certain size, rinse with PBS for 5 minutes, then soak in 75% ethanol for 10 minutes, repeat the above steps three times, and irradiate with ultraviolet light for 30 minutes. min. Then put the membrane into a 12-well plate, add F12 medium, and put it in an incubator overnight. Rinse the silk membrane with PBS, add the A549 cells to be subcultured and the membrane into a 12-well plate, add F12 medium containing serum, and shake well. Put the 12-well plate into the incubator, and observe the cell status regularly every day.
- FIG. 6A it is respectively the exterior photograph of DOX-immobilized silk film doxorubicin slow-release of 5 kinds of concentrations provided by the embodiment of the present invention
- a, b, c, d and e represent 0.2 mg, 0.4 mg respectively , 0.6 mg, 0.8 mg and 1.0 mg/g DOX.
- Fig. 6B which are respectively the culture photos of A549 cells on SFM-DOX (0.6 mg/g) provided in the examples of the present invention on the 4th day. From Figure 6A, it can be found that the color of silk fibroin membranes of these five membranes increases with the increase of DOX concentration, from light pink to red.
- the transparency of the silk fibroin film was not affected by adding DOX.
- the human alveolar adenocarcinoma basal epithelial cell A549 was used as a model, and the antitumor activity test of the immobilized DOX silk membrane was carried out in vitro, and the results are shown in Figure 6B.
- Figure 6Ba shows the state of the cells on the 4th day of culture.
- A549 cells were observed to die on the second day on SFM-DOX containing DOX, and almost all of them died on the fourth day (Fig. 6Bb). This result shows that the doxorubicin, which has broad-spectrum anti-cancer effect, can still inhibit tumor growth after being made into a slow-release silk film.
- This example provides a conductive silk fibroin-polypyrrole modified membrane prepared by unidirectional nanopore dehydration.
- a dialysis membrane with a molecular weight cut-off of ⁇ 10 kDa is selected as the bottom of the mold.
- 5 mL of 3.0% regenerated liquid silk fibroin aqueous solution was placed in a unidirectional nanopore dehydration mold (30 mm in diameter), placed in an environment of 25 °C and 35% RH, and removed from the mold after unidirectional nanopore dehydration for 10 to 16 h Silk fibroin membrane (SFM-U).
- This example provides a casein cross-linked hydrogel membrane prepared by directional nanopore dehydration.
- a dialysis membrane with a molecular weight cut-off of ⁇ 10 kDa is selected as the bottom of the mold.
- From the injection hole (4) on the top of the mold add 5 mL of 3% Casein aqueous solution containing 0.025 mL PEGDE into the mold (30 mm in diameter), and block the injection hole (4) with a rubber stopper (5) after adding, Place it horizontally on a mold frame at room temperature (25°C) or relative humidity (35%RH).
- a fan under the mold to make the air flow quickly, and then dehydrate the unidirectional nanopores for 10 to 16 hours.
- the control sample—evaporated casein cross-linked film prepared by casting method described in Example 2 (Fig. 7c).
- FIG 7 it is a photo of the appearance of the casein cross-linked hydrogel film (c) provided by the embodiment of the present invention and the casein cross-linked hydrogel film (d) formed by ordinary evaporation.
- the conventionally evaporated casein-PEGDE crosslinked film is uneven, wrinkled, and the thickness is very thin ( ⁇ 0.15 mm) and uneven ( Figure 7d); while the casein-PEGDE crosslinked film prepared by the method of the present invention
- the membrane (left in the figure) is flat, very transparent, elastic, and uniform in thickness, with a thickness of 0.55 mm, more than 2 to 3 times that of the former (Figure 7c).
- This example provides a silk fibroin bioplastic prepared by unidirectional nanopore dehydration.
- This silk fibroin bioplastic has good biocompatibility, and is not easy to be degraded by protease or digested by gastric juice in vivo and in vitro, and is suitable for the scene application of medical tissue engineering materials.
- FIG 8 is the SFB formed by stretching SFM-U 5 times and the control sample SFM-EM provided by the embodiment of the present invention.
- the silk fibroin film formed by ordinary evaporation and treated with methanol (SFM-EM) was the experimental control group, and the tensile strength of the silk film formed by unidirectional nanopore dehydration was similar to that of the evaporated film, which was about 13.60 MPa. But the elongation at break is more than 5 times that of the former.
- this experiment designed and prepared another dehydrated silk fibroin stretched film, that is, when the dehydrated film was mechanically stretched to 500% (not broken), it stopped stretching and kept it for several minutes.
- the nanoporous dehydrated silk fibroin membrane (SFM ⁇ 5) which is stretched 5 times, is milky white. It is not easy to break and break in the dry state, and it is as hard as plastic. The performance before and after stretching in the dry state is completely different. Therefore, it is also called silk fibroin bioplastic (SFB) here.
- the stretched dehydration membrane (SFM ⁇ 5) was also wet and then subjected to a tensile test. As shown in the figure, its tensile strength reached 51.05 MPa, which was 3.3 times that of the original dehydration membrane. The elongation correspondingly decreased significantly by only 50%.
- This example provides a polyvinyl alcohol hydrogel film prepared by directional nanopore dehydration and its mechanical properties.
- Example 2 and Figure 1 add 35.0 mL of 10% polyvinyl alcohol (PVA) aqueous solution into the mold, and add The micro-fan accelerated the dehydration of the unidirectional nanopores, and after 12-16 hours of dehydration, a polyvinyl alcohol hydrogel membrane (PVAHM) with an area of 50.0 cm 2 was obtained.
- PVA polyvinyl alcohol hydrogel membrane
- the PVAHM in Table 8 is cut into a rectangular strip film according to the state when it is just taken off from the mold, and the mechanical properties of the wet film are measured according to the test method of the mechanical properties in Example 3.
- the tensile properties of PVAHM are obviously affected by different thicknesses. The thicker the hydrogel film, the stronger the tensile force, ranging from 1 to 50 N; the greater the elongation at break, ranging from 350% to 800%.
- Different volumes of PVA solutions will form hydrogel films with different thicknesses. As the volume and thickness increase, the maximum load Fmax of PVA hydrogel films will gradually increase.
- the elongation at break BE is positively correlated with the volume of PVA.
- the tensile strength of PVAHM is close to 3.0 MPa, and the elongation at break also reaches 400%; when the temperature rises from 40°C to 55°C, the tensile strength and elongation at break of PVAHM formed by dehydration of oriented nanopores The rate increases by 2 to 3 times at the same time; when the temperature rises to 70°C, these two indicators reach their peak values, which are 10.5 MPa and 890% respectively.
- the mechanical properties of PVAHM provided by the present invention are affected by temperature changes.
- the PVAHM formed by unidirectional nanopore dehydration at 25°C has good mechanical properties, the tensile strength reaches 2.95 MPa, and the elongation at break also reaches 407% at this time; when the unidirectional dehydration is performed, the temperature is increased to At 70°C, the dehydration time is significantly shortened by 1/3, and not only the tensile strength is greatly increased, it is 3.6 times stronger; the elongation at break is even increased by 40%. Therefore, the mechanical properties of this hydrogel mold can be adjusted by the temperature during preparation. Referring to Figure 9b, it is the PVAHM stress and strain curves formed by unidirectional nanopore dehydration at 25°C and 70°C provided by the present invention.
- This example provides a PVA composite/crosslinked hydrogel membrane prepared by directional nanopore dehydration.
- Table 9 shows the effect of casein incorporation on the mechanical properties of PVA-CA composite hydrogel membranes. It can be found that the more casein is incorporated into the elongation, the greater the effect on the tensile force of the composite hydrogel membrane; on the contrary, the elongation at break increases with the increase of casein content. It can be observed from Table 10 that the PVA-DOS composite hydrogel film was prepared by mixing PVA and starch (DOS) in different proportions. The rate has increased slightly. It can be found from Table 11 that the addition of MBA in the PVA crosslinked hydrogel film has an effect on its mechanical properties. The more MBA is added, the stronger the tensile force is, and the elongation at break increases obviously, while the swelling rate decreases slightly. When the PVA crosslinked film containing 0.5% MBA has a tensile force of 14.67 N, the elongation at break reaches about 460%.
- the film is very hard.
- the diameters of PVA film, PVA-10%CA, PVA-10%DOS and PVA-0.2%MBA stretched from 80 mm to 104 mm, 106 mm, respectively. mm, 105 mm and 103 mm. Its swelling rate increased significantly. However, the degree of transparency, stability in water, and mechanical properties of these hydrogel films were not significantly affected.
- FIG. 9b it is the stress and strain curve of four kinds of PVAHM, PVA-10%CA, PVA-10%DOS, PVA-0.2%MBA provided by the present invention.
- Figure 10 it can be seen from Figure 10 that, compared with the PVA hydrogel film alone ( Figure 10a), the PVA-composite hydrogel film formed by incorporating 10% casein or starch in PVA has the same mechanical properties as the PVA hydrogel film alone. Compared with PVA hydrogel, there is no significant change in tensile strength except for a slight increase in elongation at break.
- the thermal performance, infrared spectrum and X-ray diffraction pattern of PVA hydrogel film/composite/cross-linked hydrogel film refer to accompanying drawing 10b, are four kinds of PVAHM, PVA-10%CA, PVA provided by the present invention Thermal analysis DSC spectra of -10%DOS, PVA-0.2%MBA and its commercially available PVA powder. It can be observed from Figure 10b that the DSC spectrum of PVA powder has two melting peaks at 223.9 and 293.7°C, and the latter melting peak is stronger. It may indicate that there are strong hydrogen bonds in the PVA chain or between the chains in the powder.
- FIG. 10c it is the infrared spectrum of four samples of PVAHM, PVA-10%CA, PVA-10%DOS and PVA-0.2%MBA provided by the present invention. From Figure 10c, it can be observed that the infrared spectrum of the PVA-powder sample has a broad absorption peak around 3285.6 cm -1 , which is the O–H stretching vibration peak. The O–H stretching vibration peak of the hydrogel film formed by unidirectional dehydration of nanopores is obviously enhanced, and the absorption peak shifts to a higher wave number at 3273.7 cm -1 , indicating that the binding ability of hydrogen bonds is obviously enhanced.
- Figure 10d is the XRD patterns of four PVA hydrogel films.
- the PVA powder sample has obvious diffraction peaks at 11.28°, 19.52°, 22.53° and 40.53° of 2 ⁇ , especially the strongest diffraction peak at 19.52°.
- the formed polyvinyl alcohol hydrogel film When the PVA aqueous solution is dehydrated in one direction through nanopores, the formed polyvinyl alcohol hydrogel film, whether adding other proteins, polysaccharides or cross-linking agents or a separate component hydrogel film without adding any substances, has caused PVA Intra-chain or inter-chain molecular rearrangement, more or stronger hydrogen bonds appear between intra-chain or inter-chain hydroxyl groups, and two small diffraction peaks at 11.28° and 22.53° of 2 ⁇ almost disappear, while at 19.52° The strong diffraction peaks did not change significantly, but the 2 ⁇ shifted slightly to 19.34°.
- PVAHM Electron microscope observation of PVA hydrogel film/composite/crosslinked hydrogel film is referring to accompanying drawing 11, is PVAHM (a), PVA-10%CA (b), PVA-10%DOS ( c) and PVA-0.2%MBA (d) cross-sectional SEM images of four hydrogel membranes.
- the microstructure of individual PVAHMs can be clearly observed from Fig. 11, showing a typical sponge-like porous structure.
- the network distribution is uniform, and the pore size is between 0.5 and 1.5 ⁇ m. After adding 10% casein (CA), the network pore size becomes 5-10 times larger.
- CA casein
- the polyvinyl alcohol cross-linked membrane with 0.2% MBA cross-linking agent also has a network structure, and its pore size is much smaller than that of the single polyvinyl alcohol hydrogel membrane, and the hydrogel network is denser.
- the PVA composite hydrogel membrane (PVA-CA) added with 10% casein was a bit milky white, and the light transmittance was slightly poor, which affected the optical microscope imaging, but the growth density of L929 cells was similar on the 4th day.
- the light transmittance of PVA-10%DOS is better than that of PVA-10%CA film, and the growth state and density of cells are almost the same as those of the control group.
- the cell growth rate, density and state of PVA-0.2%MBA cross-linked membrane were similar to those of PVA-CA membrane. Referring to Figure 13, it is the growth state of L929 cells cultured on the polyvinyl alcohol hydrogel membrane provided by the present invention on the 4th day.
- This example provides an immobilized Escherichia coli polyvinyl alcohol hydrogel membrane prepared by unidirectional nanopore dehydration and its proliferation stability.
- Example 2 the dialysis membrane with a molecular weight cut-off of ⁇ 10 kDa is used as the bottom of the mold.
- EC Escherichia coli
- PVA-Escherichia coli solution in the mold cup has been dehydrated to form a sheet, which is immersed in sterile water to remove the filter.
- a water-insoluble, soft, elastic, and transparent polyvinyl alcohol-Escherichia coli hydrogel composite film (PVA-EC) was obtained after the membrane.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Manufacturing & Machinery (AREA)
- Materials Engineering (AREA)
- Dispersion Chemistry (AREA)
- Manufacture Of Macromolecular Shaped Articles (AREA)
- Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)
Abstract
一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置。通过模具顶部加样孔将高分子水溶液或它的混合物溶液加入到以纳米孔滤膜为底部的容器，密闭加样孔后，溶液中的水分子经滤膜纳米孔向下单向脱水，在滤膜上得到各向异性的高分子膜/水凝胶膜；其还可与吡咯在水溶液中进行原位聚合反应，得到一种聚吡咯修饰的导电高分子膜/水凝胶膜。本发明采用单向纳米孔脱水的模具，绿色加工得到了分子排列有序的高聚物新材料，具有柔性、透明、力学性能强的特点，可广泛应用于医用生物材料、3D支架、仿生材料、酶／药物投递与缓释、可穿戴电子设备、生物反应器、微生物燃料电池、工程菌反应器、膜分离材料等领域。
Description
本发明涉及水溶性高分子制备高分子膜、高分子水凝胶膜、高分子复合/交联膜、固定化酶/多肽/药物/活性因子/微生物的高分子膜或高分子水凝胶膜及其制备方法，尤其涉及一种机械强度优良、生物相容性好、柔性的高分子膜/水凝胶膜及其多功能复合/交联膜的制备方法，属于生物材料、仿生材料、可穿戴设备、药物缓释与投递、微生物固定化、分离膜、医用组织工程材料等领域。
目前，利用半透过性膜、微孔滤膜、中空纤维膜等脱水过程中，通过压力差或渗透压差使水分子穿过微孔达到水溶液浓缩或二相分离的目的。高分子水溶液的脱水干燥一般利用喷雾干燥技术脱水制成粉末或颗粒，而对于那些敏感性的生物活性物质如微生物、蛋白酶、多肽药物、活性因子等用真空冷冻干燥或低温冷冻喷雾干燥脱水制成粉末或颗粒，是各向同性的，其结构与理化性能大都不会有明显的差异。
蚕丝是家蚕生产的一种以蛋白质为基础的天然高分子纤维，它是由丝素纤维蛋白和被覆在其纤维周围的起胶粘作用的丝胶蛋白组成。数十年来，丝蛋白特别是丝素蛋白的扩展应用受到了人们的极大重视，这主要是其具有独一无二的分子结构、理化性能以及优异的生物相容性。去除外层丝胶的丝素纤维蛋白能溶解在高浓度中性盐溴化锂或氯化钙等溶液中，用水透析后可以获得再生丝素水溶液。这种一种水溶性的天然高分子溶液，经过后续的加工可以广泛地应用于各种应用场景的生物材料。
丝素分子由轻链（27.5 kDa）和重链（约390 kDa）以1:1摩尔比二者之间以单个二硫键结合而成的分子量高达410 kDa。现在一般认为，丝素纤维是一种具有无定形的和双晶型结构的纤维蛋白，分子链由无规卷曲、a-螺旋、II型β-转折和反平行β-折叠结构组成。以前所说的Silk I和Silk II晶体结构现在更倾向于认为分别是后两者。这种再生液体丝素通过物理或化学处理使其分子很容易从其无规卷曲和Silk I转变成更稳定的Silk II结构，从而加工成各种形态的性能稳定的生物材料。再生液体丝素可通过物理方法如剪切、发泡、超声波等来制备水凝胶、粉末等形态的丝素，用电沉积、激光辐射、等离子体、电场等方法加工成为不同晶体结构的丝素膜、胶水、水凝胶等多种形态。
膜状丝素最常规的制备方法是将再生液体丝素平铺在聚丙烯板上蒸发脱水，在室温或室温以下形成的丝素膜往往大部分溶于水，故常称为浇铸型或流延型水溶性丝素膜(SFM-E)。当这种膜经过物理或化学处理如浸入有机溶剂如甲醇中片刻或者加湿机械拉伸就能将Silk I转变成反向平行β-折叠的Silk II构造的丝素膜(SFM-EM)。原来伸展的和透明的状态转变成一种乳白色皱折的丝素膜，干态脆而湿态稍有弹性，但总体力学性能差，实际应用大大受限。当这种膜暴露于减压状态下的低温水蒸汽处理常称为“退火”，可以制成Silk I结构含量增加的水不溶性的性能良好的丝素膜 (参见文献：Hu et al. Biomacromolecules 2011, 12: 1686-1696)。浇铸成膜时温度和湿度都会对丝素膜的结构产生影响，当低于9℃缓慢干燥能成为水溶性的无定形丝素膜，20-26℃能蒸发形成水不溶的Silk I结构的丝素膜，高于40℃蒸发形成水不溶的丝素膜呈Silk II结构(参见文献：Kawahara et al. Macromol Mater Eng 2006, 291: 458-462)。再生液体丝素的冻-融、深冻-浅冻处理或者定向的冷冻都可以直接将液态丝素转变成不溶于水的Silk I构造增多的多孔固态丝素。盛有丝素溶液的盒盖留小孔使其盒内水份缓慢蒸发（多达6天或以上）也可以制成Silk I结构增多的水不溶性的丝素膜(参见文献：Lu et al. Acta Biomaterialia 2010, 6: 1380-1387)。
现有技术制备的单一成份的丝素膜存在着力学性能、光学性能、弹性、韧性、吸水性差等不足。上述缓慢干燥法制备水不溶丝素膜时间沉长，干燥速度难以控制，拉伸强度明显降低到1.4 ±0.1 MPa，分别为甲醇浸渍法和水气退火处理的丝素膜的45%和36%。所以，现有方法制备Silk I构造增多的高性能丝素膜的稳定性、可操作性等方面还有待提高。
聚乙烯醇（PVA）是一种水溶性高分子聚合物，能溶解或溶胀于水中形成水溶液或分散体系，其分子结构中含有大量的亲水基团—羟基。PVA很容易制成水凝胶，在化学性质上不活泼、无毒性、生物相容性好、可生物降解、机械性能良好，吸水量高，易加工成型，广泛应用于农业、林业、医药、日用化工、环保、生物材料尤其医用组织工程材料等领域。PVA线型高分子结构和侧链的大量羟基，带来了上述这些优异的性能，但也存在易在水中溶胀甚至溶解以及其水凝胶膜的机械力学强度差等缺点，无法满足在实际应用中的全部要求。因此，需要对PVA水凝膜进行改性，提高其力学和耐水性能，以及拓宽其阻燃性、抗菌性以及光和电性能等功能，期望在生物材料特别医用生物材料、可穿戴柔性材料等方面得到广泛应用。
PVA膜改性通常分为物理处理和化学交联二大类。前者主要指与其它有机物或无机物共混、纳米复合、热处理等，这些方法操作步骤简单，极易拓展PVA水凝胶膜的功能性。而后者主要指在PVA成膜前后利用交联或接枝方法使其内部的高分子链之间形成化学键而形成交联的网络，从而显著提高聚乙烯醇水凝胶膜(PVAHM)的力学性能、热稳定性和耐水性。这些方法中，热处理改性是一种调整PVA膜内部结晶度进而改善其性能的一种比较简单的方法，其主要原理是促使PVA线型分子链上的许多羟基相互形成氢键，从而使膜的机械性能、热稳定性能、持水和耐水性能有较大提升。其中，冻融循环法是最普遍和最典型的一种促进氢键形成而提高结晶度来提高材料性能的方法。一般在-20℃或以下温度冷冻使高分子链受到限制而形成晶核，在25℃解冻使晶体开始生长，之后通过增加冻融循环的次数来完善晶体结构和提高结晶程度。在冻融循环过程中形成的这种微晶也可看作为物理交联点交联PVA高分子链。一般说来，这种PVAHM的冻融循环次数越多、解冻速度越慢，晶体生长的体积会增大，结晶度会增加，PVA膜的力学性能越好。不过，这也会带来PVAHM拉伸强度和弹性模量的增大而断裂伸长率的降低 (参见文献：Fukumori et al.. J Appl Polym Sci 2014, 131(15), 40578)。
综上所述，现有通过物理方法尤其是冻融循环法制备的PVA水凝胶，力学性能欠佳，难以承受施加于人体的负荷，生物相容性、拉伸强度和断裂伸长率也难以满足严峻的使用条件。另外，现有技术虽然机械性能取得了一定进展，但功能相对单一，缺乏生物功能。这些问题限制了PVA水凝胶在组织工程领域的应用。因此，发展一种在生理条件下粘结效果好、强度高、生物相容性好同时具有活性物质等固定化功能的水凝胶膜，在组织和伤口愈合、创伤敷料、人工肌肉、活性物质甚至微生物固定化、生物骨组织包括软骨的修复和再生等生物医学应用领域具有迫切需求。
本发明针对现有高分子膜、高分子水凝胶膜及多功能生物复合材料在绿色加工、操作可控性、功能性和力学性能等方面存在的不足，采用单向纳米孔脱水的技术，引起高分子水溶液中水分子通过滤膜纳米孔进行单向脱水，引起高聚物的分子有序排列，提供一种柔性、透明、力学性能强的高分子膜/高分子水凝胶膜、多功能生物复合材料、导电材料及其制备方法。
实现本发明目的的技术方案是：提供一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的制备方法，将高分子水溶液或它的混合物溶液从容器顶部的加样孔中加入到模具中，所述模具以纳米孔滤膜为底部的容器，用密封盖将加样孔密闭；溶液中的水分子经纳米孔滤膜向下单向脱水，在纳米孔滤膜上表面得到高分子膜/水凝胶膜。
本发明技术方案包括按上述制备方法得到的基于单向纳米孔脱水的功能性高分子膜/水凝胶膜。
本发明技术方案提供一种基于单向纳米孔脱水的导电高分子膜/水凝胶膜的制备方法，将高分子水溶液或它的混合物溶液从容器顶部的加样孔中加入到模具中，所述模具以纳米孔滤膜为容器的底部，用密封盖将加样孔密闭；溶液中的水分子经纳米孔滤膜向下单向脱水，在纳米孔滤膜上表面得到高分子膜/水凝胶膜；将得到的高分子膜/水凝胶膜与吡咯在水溶液中进行原位聚合反应，得到一种聚吡咯修饰的导电高分子膜/水凝胶膜。
本发明技术方案包括按上述制备方法得到的一种基于单向纳米孔脱水的导电高分子膜/水凝胶膜
本发明所述的高分子水溶液包括天然高分子、人工合成高分子水溶液，或经物理混合或化学交联而成的50 kDa以上的混合高分子水溶液；按质量百分比，高分子的浓度为1～20 %；所述的混合物包括蛋白质、多糖、酶、交联剂、塑化剂、药物、色素、生长因子、微生物；按质量百分比混合物的浓度为0.1～20 % 。
本发明所述的高分子水溶液包括天然高分子、人工合成高分子水溶液，或经物理混合或化学交联而成的50 kDa以上的混合高分子水溶液；按质量百分比，高分子的浓度为1～20 %；所述的混合物包括蛋白质、多糖、酶、交联剂、塑化剂、药物、色素、生长因子、微生物；按质量百分比混合物的浓度为0.1～20 % 。
所述的天然高分子包括酪蛋白，家蚕丝、蜘蛛丝、柞蚕丝、蓖麻蚕丝或野蚕丝的再生液体丝素蛋白中的一种；所述的人工合成高分子包括聚乙烯醇。
本发明技术方案提供一种用于制备基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的装置，它以纳米孔滤膜为容器的底部，容器的顶部设有加样孔和与其配合的密封盖；所述的纳米孔滤膜的孔径为截留0.1 ~ 1000 kDa分子量的透析膜，或滤孔≤50 nm的高分子合成膜。
本发明所述的一种用于制备基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的装置，包括用于安装所述装置的模具架，在容器底部的纳米孔滤膜下方设置高吸水材料或单向脱水加速器；所述的单向脱水加速器包括流动空气产生装置、恒温恒湿箱、负压腔、渗透压差腔。
本发明所述的一种用于制备基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的装置，其纳米孔滤膜包括再生纤维透析膜、平面透析玻璃纸、陶瓷膜、聚丙烯酰胺水凝胶膜、尼龙膜。
与现有技术相比，本发明的有益效果在于：
1.本发明提供的富含2型β-转折结构的水不溶性的超厚丝素膜，其湿态拉伸强度达14.6 ±0.6 MPa，断裂伸长率最高可达640 ±55 %、溶胀率约55 %。单向纳米孔脱水制备的聚乙烯醇水凝胶膜湿态拉伸强度3.0 ~ 10.5 MPa、断裂伸长342 ~ 891%、溶胀率300 ~ 335%。
1.本发明提供的富含2型β-转折结构的水不溶性的超厚丝素膜，其湿态拉伸强度达14.6 ±0.6 MPa，断裂伸长率最高可达640 ±55 %、溶胀率约55 %。单向纳米孔脱水制备的聚乙烯醇水凝胶膜湿态拉伸强度3.0 ~ 10.5 MPa、断裂伸长342 ~ 891%、溶胀率300 ~ 335%。
2．在单向纳米孔脱水之前高分子水溶液可与其他水溶性高分子、交联剂、塑化剂、酶、药物、色素、活性因子甚至微生物等共混或生物连接，制备得到具有优异的力学性能、具有生物活性或导电性的高分子复合膜/复合水凝胶膜。PVAHM固定化的大肠杆菌经过四十次换液重复培养，菌不掉落能稳定地连续分裂，增殖效率仍保持90%以上。小鼠成纤细胞L929在这些高分子材料上都能良好地着生与增殖。
3.本发明提供的基于单向纳米孔脱水得到的高分子新材料可广泛应用于医用生物材料、3D支架、仿生材料、酶／药物投递与缓释、可穿戴电子设备、生物反应器、微生物燃料电池、工程菌反应器、膜分离材料等多种领域。
图1是本发明提供的单向纳米孔脱水模具的结构示意图；
图中，1. 成膜杯；2. 纳米孔滤膜；3. 固定环；4. 加样孔；5. 加样孔密封塞；
图2是本发明实施例提供的丝素膜应力与应变曲线图；
图3是本发明实施例提供的丝素膜的红外光谱(a)和X-射线衍射图谱(b)；
图4是本发明实施例提供的丝素膜(SFM-U)体外中性蛋白酶降解(a)与细胞培养(b)图；
图5是本发明提供的丝素膜(SFM-U)上L929细胞第4d的生长状态图；
图6是本发明提供的A549细胞在阿霉素缓释丝素膜上培养图；
图7是本发明提供的导电丝素-聚吡咯修饰膜和酪蛋白-PEGDE交联膜的透光性能对比图；
图8是本发明提供的丝素生物塑料的应力与应变曲线图；
图9是本发明提供的单向纳米孔脱水温度对PVAHM机械性能影响的应力与应变曲线图；
图10是本发明实施例提供的聚乙烯醇水凝胶膜、复合/交联水凝胶膜的应力应变曲线(a)、DSC(b)、红外光谱(c)与X-射线衍射图谱(d)；
图11是本发明实施例提供的聚乙烯醇水凝胶膜、复合/交联膜的横切面电子扫描电镜图；
图12是本发明提供的聚乙烯醇水凝胶膜、复合/交联膜上L929细胞培养进程图；
图13是本发明提供的聚乙烯醇水凝胶膜上培养第4天的L929细胞生长状态图；
图14是本发明提供的聚乙烯醇水凝胶膜中固定化的大肠杆菌的增殖稳定性曲线图。
图中，1. 成膜杯；2. 纳米孔滤膜；3. 固定环；4. 加样孔；5. 加样孔密封塞；
图2是本发明实施例提供的丝素膜应力与应变曲线图；
图3是本发明实施例提供的丝素膜的红外光谱(a)和X-射线衍射图谱(b)；
图4是本发明实施例提供的丝素膜(SFM-U)体外中性蛋白酶降解(a)与细胞培养(b)图；
图5是本发明提供的丝素膜(SFM-U)上L929细胞第4d的生长状态图；
图6是本发明提供的A549细胞在阿霉素缓释丝素膜上培养图；
图7是本发明提供的导电丝素-聚吡咯修饰膜和酪蛋白-PEGDE交联膜的透光性能对比图；
图8是本发明提供的丝素生物塑料的应力与应变曲线图；
图9是本发明提供的单向纳米孔脱水温度对PVAHM机械性能影响的应力与应变曲线图；
图10是本发明实施例提供的聚乙烯醇水凝胶膜、复合/交联水凝胶膜的应力应变曲线(a)、DSC(b)、红外光谱(c)与X-射线衍射图谱(d)；
图11是本发明实施例提供的聚乙烯醇水凝胶膜、复合/交联膜的横切面电子扫描电镜图；
图12是本发明提供的聚乙烯醇水凝胶膜、复合/交联膜上L929细胞培养进程图；
图13是本发明提供的聚乙烯醇水凝胶膜上培养第4天的L929细胞生长状态图；
图14是本发明提供的聚乙烯醇水凝胶膜中固定化的大肠杆菌的增殖稳定性曲线图。
下面结合附图和实施例对本发明技术方案作进一步的阐述。
本实施例提供按本发明技术方案制备各实施例所需样品溶液的配制。
(1)再生丝素溶液
将蚕茧壳置于70°C热水中浸泡过夜。翌日，以1：40（W/V）浴比，将软化的蚕茧壳继续水煮2 h脱去蚕丝外层大部分丝胶；随后取出脱水后继续以1：40（W/V）浴比转移至0.2 %（w/v）中性皂水溶液中水煮脱胶0.5 h。二次脱胶的丝素纤维用蒸馏水彻底漂洗，于80°C烘箱中烘干4 ~ 5 h，最后置于室内过夜平衡得到丝素纤维。1：25（W/V）浴比用CaCl 2-EtOH-H 2O（1:2:7, mol）氯化钙-乙醇-水三元溶液中65°C摇动溶解3 h，成为丝素钙盐溶液。接着使用截留分子量为8-14 kDa的透析袋在纯水中透析72 h以除去盐离子，最后离心除去杂质和少量蛋白聚集体，获得浓度为3.0 %（W/V）的再生丝素水溶液。
将蚕茧壳置于70°C热水中浸泡过夜。翌日，以1：40（W/V）浴比，将软化的蚕茧壳继续水煮2 h脱去蚕丝外层大部分丝胶；随后取出脱水后继续以1：40（W/V）浴比转移至0.2 %（w/v）中性皂水溶液中水煮脱胶0.5 h。二次脱胶的丝素纤维用蒸馏水彻底漂洗，于80°C烘箱中烘干4 ~ 5 h，最后置于室内过夜平衡得到丝素纤维。1：25（W/V）浴比用CaCl 2-EtOH-H 2O（1:2:7, mol）氯化钙-乙醇-水三元溶液中65°C摇动溶解3 h，成为丝素钙盐溶液。接着使用截留分子量为8-14 kDa的透析袋在纯水中透析72 h以除去盐离子，最后离心除去杂质和少量蛋白聚集体，获得浓度为3.0 %（W/V）的再生丝素水溶液。
(2)聚乙烯醇水溶液
聚乙烯醇粉末（CAS: 9002-89-5; Mowiol®PVA-124,粘度: 54-66 mPa·S）购自上海aladdin公司。将PVA粉末加水悬浮，置于电炉上90℃加热使之悬浮混合，然后置于120℃高温高压中热处理2 h，最终配制成浓度为10 w% PVA水溶液备用。
聚乙烯醇粉末（CAS: 9002-89-5; Mowiol®PVA-124,粘度: 54-66 mPa·S）购自上海aladdin公司。将PVA粉末加水悬浮，置于电炉上90℃加热使之悬浮混合，然后置于120℃高温高压中热处理2 h，最终配制成浓度为10 w% PVA水溶液备用。
本实施例提供本发明技术方案所述的高分子膜/水凝胶膜的制备方法。
(1)单向纳米孔脱水高分子膜或水凝胶膜的制备
参见附图1，为本实施例提供的单向纳米孔脱水高分子膜或水凝胶膜制备U模具的结构示意图；加工模具主要由内腔直径为30 mm或80 mm的成膜杯1，纳米孔滤膜2，固定环3，加样孔4，加样孔橡胶塞5组成。将水中湿润的圆形纳米孔滤膜2通过固定环3内螺牙旋转拧紧固定，放置在水平的模具搁架上，用移液器通过加样孔4加入少许蒸馏水试验固定的滤膜2是否漏水。若漏水重复上述操作直到不漏水为止。
参见附图1，为本实施例提供的单向纳米孔脱水高分子膜或水凝胶膜制备U模具的结构示意图；加工模具主要由内腔直径为30 mm或80 mm的成膜杯1，纳米孔滤膜2，固定环3，加样孔4，加样孔橡胶塞5组成。将水中湿润的圆形纳米孔滤膜2通过固定环3内螺牙旋转拧紧固定，放置在水平的模具搁架上，用移液器通过加样孔4加入少许蒸馏水试验固定的滤膜2是否漏水。若漏水重复上述操作直到不漏水为止。
用移液器将一定体积和浓度的高分子水溶液或混合物水溶液通过加样孔4加入到模具的成膜杯1中，加样完毕用硅胶塞5将加样孔4堵住。然后，将此整个模具搁架放置在一定的温度和湿度的环境条件下，使模具内溶液中水分子通过滤膜的单向纳米孔脱水干燥，数小时或数十小时后形成一块面积为7.0 cm
2或50 cm
2的高分子膜/水凝胶膜及其多功能复合材料。
(2)加速单向纳米孔脱水的方法
按附图1提供的模具和本实施例上述采用的单向纳米孔脱水方法，选用截留0.1~ 10 kDa分子量的透析膜作为模具底部进行安装。从模具顶部加样孔4，加入5 mL或35 mL质量百分浓度为3.0 %的再生液体丝素水溶液，加完用橡皮塞5堵住加样孔4，水平放置在一个模具架上置于室温（25℃）或相对湿度（35%RH）环境下，同时，在模具下方增加风扇使空气快速流动，然后纳米孔单向脱水10 ~ 16 h后，模具杯中的水溶液已经干燥成片状物，从模具纳米孔滤膜上取下形成的膜，即为高分子膜或高分子水凝胶膜。
按附图1提供的模具和本实施例上述采用的单向纳米孔脱水方法，选用截留0.1~ 10 kDa分子量的透析膜作为模具底部进行安装。从模具顶部加样孔4，加入5 mL或35 mL质量百分浓度为3.0 %的再生液体丝素水溶液，加完用橡皮塞5堵住加样孔4，水平放置在一个模具架上置于室温（25℃）或相对湿度（35%RH）环境下，同时，在模具下方增加风扇使空气快速流动，然后纳米孔单向脱水10 ~ 16 h后，模具杯中的水溶液已经干燥成片状物，从模具纳米孔滤膜上取下形成的膜，即为高分子膜或高分子水凝胶膜。
(3) 制备实验对照组样品：高分子蒸发膜/水凝胶蒸发膜
以传统方法将一定体积和浓度的高分子水溶液平铺（浇铸）在水平的聚丙烯平板上，在室温下或恒温恒湿（25℃和50%RH）内通过水分蒸发的形式，蒸发干燥形成的高分子膜（简称蒸发型或延流型高分子膜或水凝胶膜），这些高分子膜或水凝胶膜主要用于实验对照。
以传统方法将一定体积和浓度的高分子水溶液平铺（浇铸）在水平的聚丙烯平板上，在室温下或恒温恒湿（25℃和50%RH）内通过水分蒸发的形式，蒸发干燥形成的高分子膜（简称蒸发型或延流型高分子膜或水凝胶膜），这些高分子膜或水凝胶膜主要用于实验对照。
本实施例对照组样品的高分子膜为丝素蛋白时，蒸发形成的是水溶性的丝素膜（SFM-E），需要后处理即将其浸渍在80%甲醇水溶液中1~2 min，由此形成了不溶于水的b-化丝素膜（SFM-EM），这二种丝素膜均作为实验对照组样品。
本实施例提供按本发明技术方案各实施例提供的材料的力学、结构和体外性能的测试方法。
(1)力学性能
当高分子水溶液经过单向纳米孔脱水形成膜或水凝胶膜后，从直径为30 mm或80 mm模具上刚取下时状态裁切成一定尺寸的长方形条状膜或水凝胶膜。在拉伸性能测定前1天，浸入25°C水中充分湿润（24 h）后，用电子拉力试验机（WH-5000，宁波伟恒检测仪器有限公司）测定。测定时室温维持在25 °C左右，拉伸时有效间距10 mm，当应力-应变曲线开始急剧下降时，停止实验，记录数据。计算平均值和标准差。
当高分子水溶液经过单向纳米孔脱水形成膜或水凝胶膜后，从直径为30 mm或80 mm模具上刚取下时状态裁切成一定尺寸的长方形条状膜或水凝胶膜。在拉伸性能测定前1天，浸入25°C水中充分湿润（24 h）后，用电子拉力试验机（WH-5000，宁波伟恒检测仪器有限公司）测定。测定时室温维持在25 °C左右，拉伸时有效间距10 mm，当应力-应变曲线开始急剧下降时，停止实验，记录数据。计算平均值和标准差。
(2)溶胀率
将高分子膜/水凝胶膜样品切割成3.0 ´ 20 mm长方形条，放入干燥箱，37 °C烘干过夜，称重记为M 0；接着，把烘干后的样品膜浸入含有过量PBS的离心管内，置于37 °C生化培养箱中保温，使其吸水发生溶胀；隔1 h取出，拿滤纸拭去样品表面水分，称重，记为M n；根据如下公式得出样品溶胀率，重复5个样品，计算平均值和标准差（±SD）。
将高分子膜/水凝胶膜样品切割成3.0 ´ 20 mm长方形条，放入干燥箱，37 °C烘干过夜，称重记为M 0；接着，把烘干后的样品膜浸入含有过量PBS的离心管内，置于37 °C生化培养箱中保温，使其吸水发生溶胀；隔1 h取出，拿滤纸拭去样品表面水分，称重，记为M n；根据如下公式得出样品溶胀率，重复5个样品，计算平均值和标准差（±SD）。
溶胀率（%）= (M
n- M
0)/M
0×100 %
(3)结构分析
称取高分子膜/水凝胶膜粉末样品~5.0 mg，用热重/差热联用仪（SDT2960，美国TA公司）进行TG、DTG和DSC分析。参数设定：保护气体为氮气100 mL/min，温度范围25 °C ~ 800 °C，升温速度10 °C/min。使用傅里叶变换红外光谱仪（Nicolet 6700，美国Thermo Fisher）检测高分子膜/水凝胶膜粉末样品的结构特征。取约100 mg溴化钾晶体与1 mg待测样品粉末混合，研磨。取少量混合样品粉末压成薄片后测试。扫描次数16次，分辨率4 cm -1，光谱范围在4000 ~ 1000 cm -1。膜状样品直接用ATR附件测定傅里叶变换红外全反射光谱。粉末状样品用荷兰帕纳科公司X-射线衍射光谱仪（X’Pert-Pro MPD）测试，Cu靶，管压40 kV，管流50 mA，λ= 1.5406 nm，衍射角(2θ)的范围为5-50°，扫描步长为 0. 02°/sec，扫描速度为 2°/min。
(3)结构分析
称取高分子膜/水凝胶膜粉末样品~5.0 mg，用热重/差热联用仪（SDT2960，美国TA公司）进行TG、DTG和DSC分析。参数设定：保护气体为氮气100 mL/min，温度范围25 °C ~ 800 °C，升温速度10 °C/min。使用傅里叶变换红外光谱仪（Nicolet 6700，美国Thermo Fisher）检测高分子膜/水凝胶膜粉末样品的结构特征。取约100 mg溴化钾晶体与1 mg待测样品粉末混合，研磨。取少量混合样品粉末压成薄片后测试。扫描次数16次，分辨率4 cm -1，光谱范围在4000 ~ 1000 cm -1。膜状样品直接用ATR附件测定傅里叶变换红外全反射光谱。粉末状样品用荷兰帕纳科公司X-射线衍射光谱仪（X’Pert-Pro MPD）测试，Cu靶，管压40 kV，管流50 mA，λ= 1.5406 nm，衍射角(2θ)的范围为5-50°，扫描步长为 0. 02°/sec，扫描速度为 2°/min。
(4)扫描电镜观察
先将水中达到溶胀平衡的高分子膜或水凝胶膜置于-197 ℃液氮中冷冻数分钟，取出后用镊子制作样品的断裂面，接着继续置于真空冷冻干燥机中冻干；取薄片样品固定载物台上。表面喷金70 s，在日立S-4700冷场发射扫描电镜（SEM，Regulus 8230）下观察膜样品的表面以及横截面或断裂面表观形态。扫描电镜测试参数设置为：加速电压15 kV。
先将水中达到溶胀平衡的高分子膜或水凝胶膜置于-197 ℃液氮中冷冻数分钟，取出后用镊子制作样品的断裂面，接着继续置于真空冷冻干燥机中冻干；取薄片样品固定载物台上。表面喷金70 s，在日立S-4700冷场发射扫描电镜（SEM，Regulus 8230）下观察膜样品的表面以及横截面或断裂面表观形态。扫描电镜测试参数设置为：加速电压15 kV。
(5)体外酶解
用无菌PBS分别配制一定活性单位的蛋白酶水溶液，酶溶液需过滤除菌。将干态的高分子膜/水凝胶膜或其他实验样品称重记为M 0，并分别浸入一定体积的蛋白酶溶液中，每组重复3个样品，最终将这些样品酶溶液置于25°C、37°C或 50°C恒温摇床中振荡（110 rpm）培养酶解。每天观察降解情况和更换酶液。培养结束后取出一组膜样品用PBS冲洗后，烘干称重记做M n，最后按照下式计算酶解残留率（%）平均值和标准误差（±SD）。
用无菌PBS分别配制一定活性单位的蛋白酶水溶液，酶溶液需过滤除菌。将干态的高分子膜/水凝胶膜或其他实验样品称重记为M 0，并分别浸入一定体积的蛋白酶溶液中，每组重复3个样品，最终将这些样品酶溶液置于25°C、37°C或 50°C恒温摇床中振荡（110 rpm）培养酶解。每天观察降解情况和更换酶液。培养结束后取出一组膜样品用PBS冲洗后，烘干称重记做M n，最后按照下式计算酶解残留率（%）平均值和标准误差（±SD）。
质量剩余率（%）= M
n/M
0×100%
(6)生物相容性试验
本实验选用小鼠成纤维细胞L929为试验对象，观察小鼠成纤细胞在高分子膜/水凝胶膜等膜状样品上着生、增殖与生长以及细胞的形态，评估这些样品的细胞着生与生物相容性。本实验的生物培养基为含有10 %胎牛血清、1 %青链霉素的DMEM高糖培养基。细胞培养过程中的所有操作均在超净工作台中进行。细胞培养箱的环境设置为恒温37° C，二氧化碳浓度为5%。
(6)生物相容性试验
本实验选用小鼠成纤维细胞L929为试验对象，观察小鼠成纤细胞在高分子膜/水凝胶膜等膜状样品上着生、增殖与生长以及细胞的形态，评估这些样品的细胞着生与生物相容性。本实验的生物培养基为含有10 %胎牛血清、1 %青链霉素的DMEM高糖培养基。细胞培养过程中的所有操作均在超净工作台中进行。细胞培养箱的环境设置为恒温37° C，二氧化碳浓度为5%。
先将样品置于25℃的PBS液中保存1 h，然后将其切割成圆形样品，每个样品重复20片分别置于96孔板中，各加入50 µL无菌的PBS溶液，盖上板盖后置于365 nm紫外光下照射5 min灭菌处理，随后用无菌PBS清洗3次。选取对数生长期的L929细胞，向96孔板中接种1.0 mL密度为10000/mL的细胞悬液，采用细胞计数试剂盒(Cell counting Kit-8, CCK-8)每隔24 h测定L929细胞在丝素塑料上的细胞活力。向96孔板中加入10 µL CCK-8溶液，37°C继续培养4 h。为了避免生物塑料对吸光度的影响，将待测细胞培养液转移至新的96孔板中，放入酶标仪中测定细胞培养液在450 nm处的吸光度值，每组20个平行。
本实施例提供一种单向纳米孔脱水制备的具有优良力学性能的丝素膜。
(1) 丝素膜的制备
按附图1提供的模具、实施例1和实施例2提供的单向纳米孔脱水方法，选用截留~ 10 kDa分子量的透析膜作为模具底部进行安装。从模具顶部加样孔加入5 mL的3.0 %再生液体丝素水溶液，加完用橡皮塞堵住加样孔，水平放置在一个模具架上，然后置于室温（25℃）或相对湿度（35%RH）环境下，纳米孔单向脱水10 ~ 16 h后，模具杯中丝素液已经干燥成片状物，浸入水中少许湿润后可从模具上取下纳米孔滤膜与丝素膜，获得力学性能优良的单向纳米孔脱水丝素膜（SFM-U）。
按附图1提供的模具、实施例1和实施例2提供的单向纳米孔脱水方法，选用截留~ 10 kDa分子量的透析膜作为模具底部进行安装。从模具顶部加样孔加入5 mL的3.0 %再生液体丝素水溶液，加完用橡皮塞堵住加样孔，水平放置在一个模具架上，然后置于室温（25℃）或相对湿度（35%RH）环境下，纳米孔单向脱水10 ~ 16 h后，模具杯中丝素液已经干燥成片状物，浸入水中少许湿润后可从模具上取下纳米孔滤膜与丝素膜，获得力学性能优良的单向纳米孔脱水丝素膜（SFM-U）。
(2)不同体积（厚度）丝素膜的力学性能
分别吸取以5.0、7.0、10.0、15.0、18.0 mL丝素溶液(3%)，用直径为30 mm模具通过单向纳米孔脱水制备了五种不同厚度的丝素膜（SFM-U），其厚度也从0.205 mm 到1.028 mm，基本上是丝素膜的厚度随丝素体积或重量的增加而增厚。说明5 mL到18 mL丝素溶液(3%)都是在7 cm 2面积上形成的丝素膜。但从拉伸强度分析可以发现，随着丝素体积的增加，形成的膜厚度也越厚。也就是单向纳米孔脱水形成丝素膜的时间延长，其拉伸强度和断裂伸长率会有明显的下降。从表1中可以观察到在1天内能形成的丝素膜其机械性能最强达到11.8 MPa，断裂伸长率也是最大达到490%。
分别吸取以5.0、7.0、10.0、15.0、18.0 mL丝素溶液(3%)，用直径为30 mm模具通过单向纳米孔脱水制备了五种不同厚度的丝素膜（SFM-U），其厚度也从0.205 mm 到1.028 mm，基本上是丝素膜的厚度随丝素体积或重量的增加而增厚。说明5 mL到18 mL丝素溶液(3%)都是在7 cm 2面积上形成的丝素膜。但从拉伸强度分析可以发现，随着丝素体积的增加，形成的膜厚度也越厚。也就是单向纳米孔脱水形成丝素膜的时间延长，其拉伸强度和断裂伸长率会有明显的下降。从表1中可以观察到在1天内能形成的丝素膜其机械性能最强达到11.8 MPa，断裂伸长率也是最大达到490%。
表1 丝素溶液体积对丝素膜厚度与机械性能的影响
(3)浓度的影响
从表2结果中可以发现使用三种丝素浓度10 mg/mL、30 mg/mL和45 mg/mL，分别取15.0 mL、5.0 mL和3.4 mL而制备的三种丝素膜重量相仿，丝素厚度也相差不大，而它们的拉伸强度和断裂伸长率也几乎相仿。结果说明单向纳米孔脱水制备时丝素溶液的浓度对形成生物膜及其机械性能的影响不大。
从表2结果中可以发现使用三种丝素浓度10 mg/mL、30 mg/mL和45 mg/mL，分别取15.0 mL、5.0 mL和3.4 mL而制备的三种丝素膜重量相仿，丝素厚度也相差不大，而它们的拉伸强度和断裂伸长率也几乎相仿。结果说明单向纳米孔脱水制备时丝素溶液的浓度对形成生物膜及其机械性能的影响不大。
表2 丝素浓度对丝素膜厚度与机械性能的影响
(4)湿度的影响
在25℃温度环境中，分别取5.0 mL丝素液（3.0%）置于35%、50%、85%和95%四种不同的相对湿度下进行纳米孔脱水12 ~ 48 h，制备的四种丝素膜实验结果由表3所示，湿度对形成丝素膜的厚度、拉伸强度和断裂伸长率有影响。
在25℃温度环境中，分别取5.0 mL丝素液（3.0%）置于35%、50%、85%和95%四种不同的相对湿度下进行纳米孔脱水12 ~ 48 h，制备的四种丝素膜实验结果由表3所示，湿度对形成丝素膜的厚度、拉伸强度和断裂伸长率有影响。
表3 湿度对丝素膜厚度与机械性能的影响
(5)温度
在相对湿度50%RH环境和微型风扇通风条件下中，分别在七种不同温度（4 ℃、15℃、25℃、35℃、45℃、50℃、70℃）下，用单向纳米孔脱水12 h ~ 48 h分别将5.0 mL 3%丝素溶液制备了7种丝素膜，实验结果由表4所示。可以发现温度对膜厚度、机械性能和溶胀率都有明显影响。制备时温度越高，脱水形成的丝素膜越薄，拉伸强度越强，断裂伸长率越小，溶胀率也越低。4°C时单向纳米孔脱水形成的丝素膜最厚为0.236 mm，拉伸强度最低为7.91 MPa，几乎是70 °C时的厚度一半；而断裂伸长率在25 ℃时最大。
在相对湿度50%RH环境和微型风扇通风条件下中，分别在七种不同温度（4 ℃、15℃、25℃、35℃、45℃、50℃、70℃）下，用单向纳米孔脱水12 h ~ 48 h分别将5.0 mL 3%丝素溶液制备了7种丝素膜，实验结果由表4所示。可以发现温度对膜厚度、机械性能和溶胀率都有明显影响。制备时温度越高，脱水形成的丝素膜越薄，拉伸强度越强，断裂伸长率越小，溶胀率也越低。4°C时单向纳米孔脱水形成的丝素膜最厚为0.236 mm，拉伸强度最低为7.91 MPa，几乎是70 °C时的厚度一半；而断裂伸长率在25 ℃时最大。
表4 温度对丝素膜厚度、机械性能及溶胀率
(6)纳滤膜孔径的影响
本实施例用五种截留分子量范围不同的透析膜作为纳滤膜进行试验。分别吸取5.0 mL再生液体丝素溶液(30 mg/mL)加入模具中，置于25℃、35%RH和微型风扇通风环境条件下，纳米孔单向脱水12 h ~ 36 h，实验结果由表5表示。可以观察到五种不同孔径的透析膜制备时都能形成相仿的丝素膜，其厚度在0.225 mm和0.271 mm之间，其溶胀率也在56.79 % ~ 63.89 %之间，经过统计分析这二者都没有显著差异。结果表明，制备丝素膜时所用的截留分子量在0.1 KDa ~ 1000 KDa之间透析膜没有影响丝素膜的形成。
本实施例用五种截留分子量范围不同的透析膜作为纳滤膜进行试验。分别吸取5.0 mL再生液体丝素溶液(30 mg/mL)加入模具中，置于25℃、35%RH和微型风扇通风环境条件下，纳米孔单向脱水12 h ~ 36 h，实验结果由表5表示。可以观察到五种不同孔径的透析膜制备时都能形成相仿的丝素膜，其厚度在0.225 mm和0.271 mm之间，其溶胀率也在56.79 % ~ 63.89 %之间，经过统计分析这二者都没有显著差异。结果表明，制备丝素膜时所用的截留分子量在0.1 KDa ~ 1000 KDa之间透析膜没有影响丝素膜的形成。
表5 纳滤膜孔径对丝素膜厚度与溶胀率的影响
(7)厚度与力学性能的关系比较
表6中二种类型的丝素膜是在完全相同的实验环境（25℃、35%RH和微型风扇通风）条件下制备的。制备时分别取浓度为3 %再生丝素溶液35.0 mL、52.5 mL和70.0 mL以单向纳米孔脱水12 h ~ 48 h分别制备面积为50 cm 2的三种不同厚度的脱水型丝素膜（SFM-U）。用同样浓度的35 mL丝素液在面积50 cm 2的聚丙烯圆盒内鼓风制备蒸发型丝素膜（SFM-E），接着用80%甲醇水溶液浸渍1 ~ 2 min，成为b化的丝素膜（SFM-EM）作对照样品。按实施例3方法用拉伸仪拉伸测试机械性能，重复测定15次，计算平均值和标准差（±SD）。
表6中二种类型的丝素膜是在完全相同的实验环境（25℃、35%RH和微型风扇通风）条件下制备的。制备时分别取浓度为3 %再生丝素溶液35.0 mL、52.5 mL和70.0 mL以单向纳米孔脱水12 h ~ 48 h分别制备面积为50 cm 2的三种不同厚度的脱水型丝素膜（SFM-U）。用同样浓度的35 mL丝素液在面积50 cm 2的聚丙烯圆盒内鼓风制备蒸发型丝素膜（SFM-E），接着用80%甲醇水溶液浸渍1 ~ 2 min，成为b化的丝素膜（SFM-EM）作对照样品。按实施例3方法用拉伸仪拉伸测试机械性能，重复测定15次，计算平均值和标准差（±SD）。
实验结果表明，单向纳米孔脱水形成的丝素膜（SFM-U）与对照组蒸发型丝素膜（SFM-EM）在膜厚度、拉伸性能等差异很大（表6）。SFM-U厚度与其丝素液加入量几乎成正比；35 mL 3%丝素溶液制备的丝素膜厚度为0.19 mm，而普通SFM-EM厚度只有0.10 mm，前者是后者的1.9倍。这充分说明单向纳米孔脱水丝素膜的内部结构比蒸发型宽松很多。但是，SFM-U的拉伸力较蒸发膜强1.8倍，达到7.26 N；随着脱水膜厚度的增加，其拉伸力也成正比增强；由于拉伸强度与膜厚度相关，所以三种单向纳米孔脱水丝素膜也与蒸发膜相近，都为13.60 MPa。最后二种膜的断裂伸长性能差异更大，蒸发膜伸长率~100%，而三种SFM-U膜伸长率高4~6倍。
表6 丝素膜的厚度和机械性能比较
(8)应力应变曲线对比
单向纳米孔脱水制备的三种厚度（0.19 mm、0.31 mm和0.43 mm）丝素膜（SFM-U）的3%丝素用量分别是35.0 mL、52.5 mL、70.0 mL（见表6），膜面积均为50 cm 2，对照组SFM-EM的3%丝素用量和膜面积最低浓度的单向纳米孔脱水膜完全相同。参见附图2，是本实施例提供的丝素膜应力与应变曲线对比图；图中，SFM-EM: 蒸发型丝素膜＋甲醇浸渍处理；SFM-U: 单向纳米孔脱水法制备的三种不同厚度丝素膜；从图2中可以观察到，蒸发膜厚度仅为0.10 mm，而相同丝素量和膜面积的脱水丝素膜厚度高达0.19 mm，较前者厚1.9倍，其拉伸力较蒸发膜高1.8倍；而按拉伸强度计算这二种膜均为13.6 MPa上下，而三种单向纳米孔脱水膜的平均断裂伸长率是蒸发膜5倍以上。
单向纳米孔脱水制备的三种厚度（0.19 mm、0.31 mm和0.43 mm）丝素膜（SFM-U）的3%丝素用量分别是35.0 mL、52.5 mL、70.0 mL（见表6），膜面积均为50 cm 2，对照组SFM-EM的3%丝素用量和膜面积最低浓度的单向纳米孔脱水膜完全相同。参见附图2，是本实施例提供的丝素膜应力与应变曲线对比图；图中，SFM-EM: 蒸发型丝素膜＋甲醇浸渍处理；SFM-U: 单向纳米孔脱水法制备的三种不同厚度丝素膜；从图2中可以观察到，蒸发膜厚度仅为0.10 mm，而相同丝素量和膜面积的脱水丝素膜厚度高达0.19 mm，较前者厚1.9倍，其拉伸力较蒸发膜高1.8倍；而按拉伸强度计算这二种膜均为13.6 MPa上下，而三种单向纳米孔脱水膜的平均断裂伸长率是蒸发膜5倍以上。
本实施例提供一种单向纳米孔脱水制备的丝素膜的结构特性与表征。
（1）丝素膜红外光谱
通常蛋白质分子中典型的C–O伸缩振动引起酰胺I带(1638 cm -1) 和N–H振动引起酰胺II带(1545 cm -1)二个主要吸收峰。参见附图3a，是本发明提供的丝素膜红外光谱图；图中，蒸发型丝素膜（SFM-E）经甲醇浸渍处理成为不溶于水的丝素膜（SFM-EM）；单向纳米孔脱水法制备的丝素膜（SFM-U）。从丝素膜的红外光谱图3中，可以观察到普通蒸发型的丝素膜（SFM-E）的酰胺I谱带在1636.3 cm -1处显示一个强峰，主要对应于无规卷曲部分。当置于80%甲醇水溶液中浸渍1~ 2 min后（SFM-EM），酰胺I谱带整体向低波数方向移动到1619.9 cm -1处出现一个非常强的吸收峰，很显然膜的二级结构已经转变成以β-折叠为主导的结构。而单向纳米孔脱水形成的SFM-U明显与上述SFM-E不同，在1640.2 cm -1处出现一个强峰，充分说明经过单向纳米孔脱水形成的丝素膜结构以II型β-转折即所谓的silk I结构为主。在酰胺II，蒸发型SFM-E及其β-化SFM-EM峰值几乎都出现在1515.3 cm -1处，说明都有β-折叠结构存在；而SFM-U显著与上述二者都不同，在1529.3 cm -1处有较强的肩峰，这与Lu等报道的小孔水份缓慢蒸发形成的丝素膜肩峰更显著（Lu et al., Acta Biomaterialia 2010, 6: 1380-1387）。这充分说明通过单向纳米孔脱水引起丝素分子结构重排的情况与上述报道的丝素结构变化有一些区别的。
通常蛋白质分子中典型的C–O伸缩振动引起酰胺I带(1638 cm -1) 和N–H振动引起酰胺II带(1545 cm -1)二个主要吸收峰。参见附图3a，是本发明提供的丝素膜红外光谱图；图中，蒸发型丝素膜（SFM-E）经甲醇浸渍处理成为不溶于水的丝素膜（SFM-EM）；单向纳米孔脱水法制备的丝素膜（SFM-U）。从丝素膜的红外光谱图3中，可以观察到普通蒸发型的丝素膜（SFM-E）的酰胺I谱带在1636.3 cm -1处显示一个强峰，主要对应于无规卷曲部分。当置于80%甲醇水溶液中浸渍1~ 2 min后（SFM-EM），酰胺I谱带整体向低波数方向移动到1619.9 cm -1处出现一个非常强的吸收峰，很显然膜的二级结构已经转变成以β-折叠为主导的结构。而单向纳米孔脱水形成的SFM-U明显与上述SFM-E不同，在1640.2 cm -1处出现一个强峰，充分说明经过单向纳米孔脱水形成的丝素膜结构以II型β-转折即所谓的silk I结构为主。在酰胺II，蒸发型SFM-E及其β-化SFM-EM峰值几乎都出现在1515.3 cm -1处，说明都有β-折叠结构存在；而SFM-U显著与上述二者都不同，在1529.3 cm -1处有较强的肩峰，这与Lu等报道的小孔水份缓慢蒸发形成的丝素膜肩峰更显著（Lu et al., Acta Biomaterialia 2010, 6: 1380-1387）。这充分说明通过单向纳米孔脱水引起丝素分子结构重排的情况与上述报道的丝素结构变化有一些区别的。
（2）X-射线衍射光谱
在温度25℃和湿度35%和微型风扇通风条件下，由5.0 mL 30 mg/mL再生丝素溶液经过单向纳米孔脱水 24 h制备了实验样品丝素膜（SFM-U）。而另二种对照样品是在PP平板上在相同条件下蒸发12 h制备的溶于水的SFM-E及其80%甲醇水溶液中浸渍1 ~ 2 min制备的不溶于水的SFM-EM。参见附图3b，是本发明实施例提供的丝素膜的X-射线衍射光谱图；图中，蒸发型丝素膜（SFM-E）经甲醇浸渍处理成为不溶于水的丝素膜（SFM-EM）；单向纳米孔脱水法制备的丝素膜（SFM-U）。从图3bX-射线衍射光谱中可以观察到，常规蒸发制备的丝素膜（SFM-E）仅在21.84°附近有一个很宽阔峰，说明这是可溶性丝素膜典型的非晶态结构的衍射图。而甲醇处理后引起b化的丝素膜其衍射峰出现在9.11°、20.44°、24.14°位置，很显然丝素膜以b-折叠为主的Silk II结构。而本发明通过单向纳米孔脱水方法制备的丝素膜衍射峰完全不同。分别出现在12.01°、19.87°、24.11°、28.06°、32.40°和36,81°。
在温度25℃和湿度35%和微型风扇通风条件下，由5.0 mL 30 mg/mL再生丝素溶液经过单向纳米孔脱水 24 h制备了实验样品丝素膜（SFM-U）。而另二种对照样品是在PP平板上在相同条件下蒸发12 h制备的溶于水的SFM-E及其80%甲醇水溶液中浸渍1 ~ 2 min制备的不溶于水的SFM-EM。参见附图3b，是本发明实施例提供的丝素膜的X-射线衍射光谱图；图中，蒸发型丝素膜（SFM-E）经甲醇浸渍处理成为不溶于水的丝素膜（SFM-EM）；单向纳米孔脱水法制备的丝素膜（SFM-U）。从图3bX-射线衍射光谱中可以观察到，常规蒸发制备的丝素膜（SFM-E）仅在21.84°附近有一个很宽阔峰，说明这是可溶性丝素膜典型的非晶态结构的衍射图。而甲醇处理后引起b化的丝素膜其衍射峰出现在9.11°、20.44°、24.14°位置，很显然丝素膜以b-折叠为主的Silk II结构。而本发明通过单向纳米孔脱水方法制备的丝素膜衍射峰完全不同。分别出现在12.01°、19.87°、24.11°、28.06°、32.40°和36,81°。
本实施例提供一种单向纳米孔脱水制备的丝素膜酶解特性与生物相容性。
丝素生物材料最终的目的一般是植入体内进行组织或器官的支撑或修复，需要对其生物降解效率进行分析。参见附图4a，是本发明实施例提供的丝素膜在体外的中性蛋白酶降解图；图4中可以看到，单向纳米孔脱水制备的SFM-U在中性蛋白酶液中1d内可以降解20%左右，随着降解天数增加，失重率增加，在降解到第14d时，重量已经剩余不足3%了；上述结果表明了这种SFM-U的水稳定性极好，但其特有的二级结构易被中性蛋白酶降解，本发明制备的材料非常适合于生物降解的体内植入物。
按实施例3所述的鼠成纤细胞L929方法在单向纳米孔脱水丝素膜（SFM-U）上进行培养实验，每天用CCK8定量法测定普通培养皿上和丝素膜上L-929细胞的活力。实验结果参见附图4b，是本发明实施例提供的丝素膜上L929细胞的生长曲线图；从中可以，细胞在单向纳米孔脱水形成的丝素膜上培养的第3天开始，L-929细胞就已经出现贴壁生长，随后细胞的附着逐渐增多，在培养到5d时，细胞仍然正常生长，几乎长满整个丝素膜表面，并呈现了典型的成纤维细胞形态。这些细胞的生长状态几乎与对照组生长在普通培养皿的情况相仿。参见附图5a和图5b，分别是本发明提供的丝素膜（SFM-U直径80 mm）和该丝素膜上L929细胞第4d的生长状态图。
本实施例提供一种单向纳米孔脱水制备的丝素塑化/复合/交联膜的特性。
按实施例1、2和附图1的单向纳米孔脱水方法，将含有5%甘油、25 μL聚乙二醇二缩水甘油醚（PEGDE）或0.1 mg罗丹明101的5 mL的3.0 %丝素水溶液的混合液分别加入到直径为30 mm的模具中，置于25℃和35%RH相对湿度环境下，同时，在模具下方增加风扇使空气快速流动，单向脱水10 ~ 16 h后，分别获得了力学性能优良的多功能丝素-甘油塑化膜（SFM-G）、丝素-PEGDE交联膜（SFM-PEGDE）和丝素-罗丹明101复合膜（SFM-R101）。SFM-G塑化膜较未加塑化剂的SFM-U更柔软，丝素-PEGDE交联膜的力学性能较SFM-U更强，SFM-R101日光照射下呈淡红色，在365 mm紫外光照下呈亮红色，主要是R101发出的荧光色；日光下SFM-G和SFM-PEGDE与SFM-U一样，呈半透明的乳白色，紫外光照射下都呈淡蓝色，都是丝素蛋白呈现的颜色。SFM-G的应力与应变曲线与未塑化的SFM-U相似，但拉伸强度有显著增强（18.36 MPa），断裂伸长率（382%）略低。SFM-PEGDE力学性能明显优于未交联的SFM-U。SFM-R101应力与应变曲线与SFM-U相似，但拉伸强度（13.59 MPa）与断裂伸长率（380%）略低于SFM-U丝素膜。
本实施例提供一种单向纳米孔脱水制备的固定化脲酶丝素膜制备。
按实施例1、2、4和附图1的单向纳米孔脱水方法，选用截留~ 10 kDa分子量的透析膜作为模具底部。从模具顶部加样孔加入含一定活性单位的脲酶的5 mL的3.0 %再生液体丝素水溶液，于25℃和35%RH环境下，鼓风纳米孔脱水10 ~ 16 h后，从模具上取下固定化脲酶丝素膜（SFM-Urease）。脲酶活力测定方法如下，取5ml的尿素（0.3M）溶液于37℃的水浴加热5 min，将一定量脲酶或过夜浸润的固定化脲酶丝素膜（0.2 g）放入尿素溶液中，继续在37℃水浴摇床中以110 rpm转速反应10 min；反应完成后取适量的反应液加双蒸水液稀释至200 μL，随后依次加入10 μL的10%酒石酸钠钾和20 μL的奈氏试剂，充分混匀，静置反应15 min后，在420 nm处测定吸光度值；最后将吸光度值代入铵浓度标准曲线（0、0.1、0.2、0.3、0.4、0.5 mL的0.25 M的硫酸铵溶液）计算出铵离子浓度，将催化生成1 μmol/min铵氮定义为一个酶活单位来计算固定化酶活。
表7所示固定化脲酶丝素膜中九种不同含量脲酶的活性回收率，以游离脲酶的活性测定值为100%，然后通过建立的游离脲酶标准曲线的线性方程计算固定化酶的活性回收率。从表中可以发现，丝素膜中脲酶含量从1.5 U到25 U逐步增加，其固定化酶的活性回收率也随之变化，当丝素/脲酶比达到1:4时，即0.2 g丝素膜中含有0.8 U脲酶时，酶活性回收率可以达到75%。
表7 固定化脲酶丝素膜
本实施例提供一种单向纳米孔脱水制备的阿霉素缓释丝素膜。
按实施例1、2、4和附图1的单向纳米孔脱水方法，选用截留~ 10 kDa分子量的透析膜作为模具底部。从模具顶部加样孔加入含0、50、100、150、200、250 mg 阿霉素（DOX）的5 mL的3.0 %再生液体丝素水溶液，于25℃和35%RH环境下，鼓风单向纳米孔脱水10 ~ 16 h后，从模具上取下阿霉素缓释丝素膜（SFM-DOX）。
A549细胞培养方法：用打孔器将SFM-U、SFM-DOX膜打成一定大小的圆片结构，用PBS冲洗5min，接着浸泡在75%乙醇中10 min，重复上述步骤三次，紫外照射30 min。接着将膜放入12孔板中，加入F12培养基，放入培养箱过夜。用PBS冲洗丝素膜，将待传代的A549细胞和膜一同加入12孔板中，加入含血清的F12培养基，摇匀。将12孔板放入培养箱，后续每天定期观察细胞状态。
参见附图6A，分别是本发明实施例提供的5种浓度的DOX-固定化的丝素膜阿霉素缓释的外观照片，a, b, c, d 和e分别表示0.2 mg、0.4 mg、0.6 mg、0.8 mg和1.0 mg/g DOX。参见附图6B，分别是本发明实施例提供的SFM-DOX（0.6 mg/g）上A549细胞第4d的培养照片。从图6A中，可以发现这五种膜随着DOX浓度的升高而丝素膜颜色加深，从淡粉红到红色。而且，加入DOX后丝素膜的透明度也没有受到任何影响。本实验以人肺泡腺癌基底上皮细胞A549为模型，在体外进行了固定化DOX丝素膜的抗肿瘤活性试验，结果由图6B所示。对照组A549细胞在不含DOX的丝素膜上着生、生长、增殖正常，图6Ba为培养第4天细胞状态。而A549细胞在含DOX的SFM-DOX上第2天就能观察到有细胞死亡，到第4天几乎全部已经死亡（图6Bb）。这一结果表明这种具有广谱抗癌作用的阿霉素制成缓释丝素膜后仍具有抑制肿瘤生长的作用。
本实施例提供一种单向纳米孔脱水制备的导电丝素-聚吡咯修饰膜。
按实施例1、2和附图1的单向纳米孔脱水方法，选用截留~ 10 kDa分子量的透析膜作为模具底部。，5 mL 3.0 %再生液体丝素水溶液在单向纳米孔脱水模具（直径30 mm）中，置于25℃和35% RH环境下，单向纳米孔脱水10 ~ 16 h后从模具上取下丝素膜（SFM-U）。接着，将10 mL 2%吡咯（pyrrole/Py，使用前蒸馏溶解）、0.02 M柠檬酸和0.02 M磺基水杨酸钠的混合水溶液加入SFM-U丝素膜后，磁力搅拌2 h；再加入10 mL的1.0 M FeCl
3溶液在-20 °C下进行原位聚合过夜。反应后丝素膜样品用水和乙醇轮流彻底洗涤数次，主要去除未反应的吡咯单体，由此获得了原位聚合聚吡咯的丝素-聚吡咯修饰膜（图7a和7b）。参见附图7，是本发明提供的导电性的丝素-聚吡咯修饰膜。
本实施例提供一种定向纳米孔脱水制备的酪蛋白交联水凝胶膜。
按实施例2和附图1所述的单向纳米孔脱水方法，选用截留~ 10 kDa分子量的透析膜作为模具底部。从模具顶部加样孔(4)，加入含0.025 mL PEGDE的3%酪蛋白Casein水溶液5 mL于模具（直径30 mm）中，加完用橡皮塞(5)堵住加样孔(4)，水平放置在一个模具架上置于室温（25℃）或相对湿度（35%RH）环境下，同时，在模具下方增加风扇使空气快速流动，然后单向纳米孔脱水10 ~ 16 h后，可从模具上取下纳米孔滤膜与酪蛋白交联膜（CA-PEGDE）。另外，按实施例2所述的流延法制备的对照组样品—蒸发型酪蛋白交联膜（图7c）。
参见附图7，是本发明实施例提供的酪蛋白交联水凝胶膜(c)和普通蒸发形成的酪蛋白交联水凝胶膜(d)外观照片。如图7所示，普通蒸发的酪蛋白-PEGDE交联膜不平整、皱折且厚度很薄(~ 0.15 mm)且不均匀（图7d）；而本发明方法制备的酪蛋白-PEGDE交联膜（图左）平整、非常透明、富弹性、膜厚均匀，厚度为0.55 mm，是前者2~3倍以上（图7c）。
本实施例提供一种单向纳米孔脱水制备的丝素生物塑料。
按实施例2、实施例5~10和附图1制备的各种丝素膜、复合膜、交联膜、荧光膜、缓释膜或导电膜等，浸入25℃水中至少湿润2 h，然后置于在拉伸仪上湿态拉伸4 ~ 5倍，并暂定约10 ~ 20 min，取下拉伸过的丝素膜呈乳白色，称为丝素´5倍拉伸膜（SFM×5）或称丝素生物塑料（SFB）。这种膜干态和湿态相似，像人工高分子塑料，不仅有弹性还有韧性，不易开裂或破碎。再通过机械切割或粉碎加工成具有各种功能的丝素片剂、颗粒或粉末。这种丝素生物塑料具有良好的生物相容性，在体内和体外都不易被蛋白酶降解或胃液消化，适用于医用组织工程材料的场景应用。
参见附图8，是本发明实施例提供的用SFM-U拉伸5倍形成的SFB和对照组样品SFM-EM。如图8所示，普通蒸发形成的并甲醇处理的丝素膜（SFM-EM）为实验对照组，单向纳米孔脱水形成的丝素膜拉伸强度与蒸发膜相似，为13.60 MPa左右。但断裂伸长率是前者的5倍以上。根据上述结果，本实验设计制备了另一种脱水型的丝素拉伸膜，即当脱水膜机械拉伸到500%时（未断裂）就停止拉伸并保持数分钟，取下后这种伸长5倍的纳米孔脱水的丝素膜（SFM´5）呈乳白色，干态不易断裂和破碎，且坚硬似塑料，干态拉伸前后的性能完全不同。所以，在这里又称它为丝素生物塑料（SFB）。将此拉伸的脱水膜（SFM´5）同样湿润后作拉伸实验，结果如图所示，其拉伸强度达到了51.05 MPa，是原来脱水膜拉伸强度的3.3倍，当然，其断裂伸长率相应有大幅下降仅50%。
本实施例提供一种定向纳米孔脱水制备的聚乙烯醇水凝胶膜及其机械性能。
按实施例1、实施例2和附图1所述的单向纳米孔脱水方法，将35.0 mL 10%聚乙烯醇（PVA）水溶液加入到模具内，在25.0℃和35%RH条件下，添加微型风扇加快单向纳米孔脱水，脱水12~ 16 h后即得到面积为50.0 cm
2 聚乙烯醇水凝胶膜（PVAHM）。
表8中PVAHM是按从模具上刚取下时的状态裁切成长方条状膜，按实施例3力学性能的测试方法测定湿态膜的机械性能。PVAHM厚度不同，其拉伸性能明显受到影响，水凝胶膜越厚，拉力越强，范围在1~50 N之间；断裂伸长率越大，范围在350%~800%之间。不同体积的PVA溶液会形成不同厚度的水凝胶膜，随着体积和厚度的增加，PVA水凝胶膜的最大负荷Fmax也逐渐增大。断裂伸长率BE与PVA的体积呈正相关。因为测量拉伸仪器的精度问题，PVA少于2.5 mL、多于12.5 mL的情况无法准确的测量，所以在可测量的范围内，用该种方法制备的PVA水凝胶，体积与厚度越大，其机械性能也会越大。
表8 聚乙烯醇水凝胶膜厚度与其机械性能的关系
不同温度下制膜所消耗的时间不同，随着温度的增加，水分流失的速度加快，所以温度越高，制膜所需的时间就越短。就图9a拉伸结果来看，相对而言，温度的增加能使得PVAHM的最大负荷、断裂伸长率、抗拉强度有明显增加。25°C时PVAHM的拉伸强度已接近3.0 MPa，断裂伸长率也达到400%；当温度从40°C上升到55°C时，定向纳米孔脱水形成的PVAHM拉伸强度和断裂伸长率都同时上升2~3倍；当温度上升到70°C时，这二个指标大到了峰值，分别为10.5 MPa和890%。参见附图9a，是本发明提供的PVAHM机械性能受温度变化的影响。
从图9b中可以发现，在25℃单向纳米孔脱水形成的PVAHM力学性能良好，拉伸强度达到2.95 MPa，而此时断裂伸长率也达到407%；当单向脱水时将温度提高到70℃，脱水时间明显缩短1/3，而不仅拉伸强度大大提高，增强3.6倍；其断裂伸长率竟然还增加了40%。所以，这种水凝胶模的力学性能可以通过制备时的温度加以调节。参见附图9b，是本发明提供的25°C和70°C下单向纳米孔脱水形成的PVAHM应力与应变曲线。
本实施例提供一种定向纳米孔脱水制备的PVA复合/交联水凝胶膜。
(1)PVA复合/交联水凝胶膜制备
按实施例1、实施例2和附图1所述的方法，将35.0 mL 的10%聚乙烯醇（PVA）水溶液与酪蛋白和淀粉水溶液或交联剂MBA（N, N-甲叉双丙烯酰胺）表9、10或表11所示的重量百分比（w%）的混合液分别加入到面积为50.0 cm 2模具内，用橡皮塞(5)堵住加样孔(4)使室内密闭。在25.0℃和35% RH条件下添加风扇使空气流通，单向纳米孔脱水12~ 16 h后，即得到面积为分别为50 cm 2的四种水凝胶膜（PVAHM、PVA-CA、PVA-DOS和PVA-MBA）。
按实施例1、实施例2和附图1所述的方法，将35.0 mL 的10%聚乙烯醇（PVA）水溶液与酪蛋白和淀粉水溶液或交联剂MBA（N, N-甲叉双丙烯酰胺）表9、10或表11所示的重量百分比（w%）的混合液分别加入到面积为50.0 cm 2模具内，用橡皮塞(5)堵住加样孔(4)使室内密闭。在25.0℃和35% RH条件下添加风扇使空气流通，单向纳米孔脱水12~ 16 h后，即得到面积为分别为50 cm 2的四种水凝胶膜（PVAHM、PVA-CA、PVA-DOS和PVA-MBA）。
表9所示为酪蛋白掺入量对PVA-CA复合水凝胶膜机械性能的影响。可以发现酪蛋白掺入伸长率量越多，对复合水凝胶膜拉力的影响就越大；而断裂伸长率相反，随着酪蛋白量增加，其断裂伸长率就越大。从表10中可以观察到，PVA与淀粉（DOS）按不同比例混合后制成了PVA-DOS复合水凝胶膜，其拉伸力随着DOS比例的增加而明显减小，而断裂伸长率倒是略有上升。从表11中可以发现，聚乙烯醇交联水凝胶膜中MBA加入对其机械性能有影响。MBA加入量越多，拉伸力越强，而断裂伸长率有明显增加，而溶胀率略有下降。当含0.5%MBA的PVA交联膜具有拉伸力为14.67 N，断裂伸长率达到460%左右。
表9 酪蛋白含量对PVA-CA复合水凝胶膜机械性能的影响
表10淀粉含量对PVA-DOS复合水凝胶膜机械性能的影响
表11 MBA含量对PVA-MBA水凝胶交联膜机械性能的影响
(2)PVA复合/交联水凝胶膜的机械性能与溶胀率
表12中对PVA水凝胶膜、PVA-10%CA、PVA-10%DOS和PVA-0.2%MBA复合/交联膜的溶胀前后的膜外形尺寸、重量溶胀率及其机械性能等作了比较。从表中结果就可以了解到各种水凝胶膜溶胀前后的变化非常大。刚从直径为80 mm模具上卸下时四种膜都比较柔软，属于不完全干的状态，放一段时间或烘箱中100°C烘干1 h就能获得完全干燥状态的PVA水凝胶膜/复合/交联膜。此时膜非常坚硬，当浸入水中25°C溶胀24 h后，PVA膜、PVA-10%CA、PVA-10%DOS以及PVA-0.2%MBA的直径都分别从80 mm伸展到104 mm、106 mm、105 mm和103 mm。其溶胀率明显增涨。但是，这些水凝胶膜的透明程度、水中稳定性和力学性能等没有明显的影响。
表12中对PVA水凝胶膜、PVA-10%CA、PVA-10%DOS和PVA-0.2%MBA复合/交联膜的溶胀前后的膜外形尺寸、重量溶胀率及其机械性能等作了比较。从表中结果就可以了解到各种水凝胶膜溶胀前后的变化非常大。刚从直径为80 mm模具上卸下时四种膜都比较柔软，属于不完全干的状态，放一段时间或烘箱中100°C烘干1 h就能获得完全干燥状态的PVA水凝胶膜/复合/交联膜。此时膜非常坚硬，当浸入水中25°C溶胀24 h后，PVA膜、PVA-10%CA、PVA-10%DOS以及PVA-0.2%MBA的直径都分别从80 mm伸展到104 mm、106 mm、105 mm和103 mm。其溶胀率明显增涨。但是，这些水凝胶膜的透明程度、水中稳定性和力学性能等没有明显的影响。
表12 PVA水凝胶膜/复合膜/交联膜溶胀前后大小与重量以及拉伸性能
参见附图9b，是本发明提供的四种PVAHM、PVA-10%CA、PVA-10%DOS、PVA-0.2%MBA的应力与应变曲线。从图10中可以看出，与单独PVA水凝胶膜相比（图10a），PVA中掺入无论10%酪蛋白还是淀粉其形成的PVA-复合水凝胶膜，在力学性能上与单独PVA水凝胶相比较除断裂伸长率略有提高外，拉伸强度没有显著的变化。加入交联剂MBA也没有引起大的变化，
(3)PVA水凝胶膜/复合/交联水凝胶膜的热性能、红外光谱和X-射线衍射图谱
参见附图10b，是本发明提供的四种PVAHM、PVA-10%CA、PVA-10%DOS、PVA-0.2%MBA及其市售PVA粉末的热分析DSC图谱。从图10b中可以观察到PVA粉末的DSC图谱在223.9和293.7°C出现了二个熔融峰，而且后一个熔融峰更强。可能说明粉末中PVA链内或链间有强氢键存在。当经过纳米孔单向脱水引起了PVA链间分子重排，出现了更多或更强的链内或链间羟基之间的强氢键，四种水凝胶膜、复合/交联水凝胶膜均出现了第三个熔融峰。与CA和DOS共混制备的水凝胶分别提高到345.9°C和357.6°C。而单独PVA水凝胶膜与仅加少量交联剂MBA水凝胶膜几乎一样，除在290°C附近出现较弱的第2个熔融峰以外，分别出现355.5°C和357.7°C更高温度的熔融峰。
(3)PVA水凝胶膜/复合/交联水凝胶膜的热性能、红外光谱和X-射线衍射图谱
参见附图10b，是本发明提供的四种PVAHM、PVA-10%CA、PVA-10%DOS、PVA-0.2%MBA及其市售PVA粉末的热分析DSC图谱。从图10b中可以观察到PVA粉末的DSC图谱在223.9和293.7°C出现了二个熔融峰，而且后一个熔融峰更强。可能说明粉末中PVA链内或链间有强氢键存在。当经过纳米孔单向脱水引起了PVA链间分子重排，出现了更多或更强的链内或链间羟基之间的强氢键，四种水凝胶膜、复合/交联水凝胶膜均出现了第三个熔融峰。与CA和DOS共混制备的水凝胶分别提高到345.9°C和357.6°C。而单独PVA水凝胶膜与仅加少量交联剂MBA水凝胶膜几乎一样，除在290°C附近出现较弱的第2个熔融峰以外，分别出现355.5°C和357.7°C更高温度的熔融峰。
参见附图10c，是本发明提供的PVAHM、PVA-10%CA、PVA-10%DOS、PVA-0.2%MBA四种样品的红外光谱。从图10c中可以观察到PVA-粉末样品的红外谱图在 3285.6 cm
-1左右有一个较宽的吸收峰, 这是O–H的伸缩振动峰。经过纳米孔单向脱水形成的水凝胶膜这个O–H伸缩振动峰明显增强，吸收峰向高波数漂移在3273.7 cm
-1，说明氢键的结合能力明显增强。当加入10%酪蛋白和淀粉的复合水凝胶膜向低波数较大幅度移动，说明氢键的结合力明显增强。图10d为四种PVA水凝胶膜的XRD图谱。PVA粉末样品在2θ的11.28°，19.52°，22.53°和40.53°处有明显的衍射峰，特别是在19.52°处有最强的衍射峰。当PVA水溶液经过纳米孔单向脱水后，形成的聚乙烯醇水凝胶膜，无论是加入其它蛋白、多糖或交联剂还是没有加入任何物质的单独成份的水凝胶膜，都引起了PVA链内或链间分子重排，在链内或链间羟基之间出现了更多或更强的氢键，在2θ的11.28°和22.53°二个小衍射峰几乎消失，而在19.52°处强衍射峰没有明显变化，但2θ稍有漂移为19.34°。这些结果充分说明纳米孔单向脱水引起了PVA链内或链间分子的重排，晶型发生了明显的变化。参见附图10c，是本发明提供的PVAHM、PVA-10%CA、PVA-10%DOS、PVA-0.2%MBA四种样品的XRD图谱。
(4)PVA水凝胶膜/复合/交联水凝胶膜的电镜观察
参见附图11，是本发明提供的PVAHM(a)、PVA-10%CA(b)、PVA-10%DOS(c)和PVA-0.2%MBA(d)四种水凝胶膜的横断面扫描电镜图。从图11中可以清楚地观察到单独PVAHM的微观结构，呈现典型的海绵状多孔结构。网状分布均匀，孔径0.5~1.5 μm之间。加入10%酪蛋白（CA）后，其网络孔径变大了5~10倍。当加入10%铁棍山药淀粉（DOS）后，膜表面形成的孔非常稀少，孔径又明显变小，膜内部孔的贯通性很差．多为不连贯的海绵状孔，似乎呈发泡状态。而加入0.2%MBA交联剂的聚乙烯醇交联膜也呈网络结构，其孔径要比单独聚乙烯醇水凝胶膜小得多，水凝胶网络更致密。
参见附图11，是本发明提供的PVAHM(a)、PVA-10%CA(b)、PVA-10%DOS(c)和PVA-0.2%MBA(d)四种水凝胶膜的横断面扫描电镜图。从图11中可以清楚地观察到单独PVAHM的微观结构，呈现典型的海绵状多孔结构。网状分布均匀，孔径0.5~1.5 μm之间。加入10%酪蛋白（CA）后，其网络孔径变大了5~10倍。当加入10%铁棍山药淀粉（DOS）后，膜表面形成的孔非常稀少，孔径又明显变小，膜内部孔的贯通性很差．多为不连贯的海绵状孔，似乎呈发泡状态。而加入0.2%MBA交联剂的聚乙烯醇交联膜也呈网络结构，其孔径要比单独聚乙烯醇水凝胶膜小得多，水凝胶网络更致密。
(5)PVA水凝胶膜/复合/交联水凝胶膜的生物相容性
参见附图12，是本发明提供的为L929细胞在四种水凝胶膜上1~5天内每天测定的吸光度值变化曲线。图12可以真实地反应L929细胞在这些膜上着生、增殖与生长情况。在PVA水凝胶膜上培养的L929细胞几乎与对照组相仿，细胞梭形清晰，细胞内核质也清晰可见，整个细胞饱满、立体感强，两端似乎扎根到蛋清膜内（图13）。而加入10%酪蛋白的PVA复合水凝胶膜（PVA-CA）有点呈乳白色，透光性稍差，影响光学显微镜成像，但L929细胞第4天生长密度相仿。PVA-10%DOS透光性优于PVA-10%CA膜，细胞生长状态和密度几乎与对照组一样。PVA-0.2%MBA交联膜细胞生长速度、密度和状态与PVA-CA膜相仿。参见附图13，是本发明提供的聚乙烯醇水凝胶膜上培养第4天的L929细胞生长状态。
参见附图12，是本发明提供的为L929细胞在四种水凝胶膜上1~5天内每天测定的吸光度值变化曲线。图12可以真实地反应L929细胞在这些膜上着生、增殖与生长情况。在PVA水凝胶膜上培养的L929细胞几乎与对照组相仿，细胞梭形清晰，细胞内核质也清晰可见，整个细胞饱满、立体感强，两端似乎扎根到蛋清膜内（图13）。而加入10%酪蛋白的PVA复合水凝胶膜（PVA-CA）有点呈乳白色，透光性稍差，影响光学显微镜成像，但L929细胞第4天生长密度相仿。PVA-10%DOS透光性优于PVA-10%CA膜，细胞生长状态和密度几乎与对照组一样。PVA-0.2%MBA交联膜细胞生长速度、密度和状态与PVA-CA膜相仿。参见附图13，是本发明提供的聚乙烯醇水凝胶膜上培养第4天的L929细胞生长状态。
本实施例提供一种单向纳米孔脱水制备的固定化大肠杆菌聚乙烯醇水凝胶膜与增殖稳定性。
按实施例1、实施例2和附图1所述的单向纳米孔脱水方法，以截留~ 10 kDa分子量的透析膜作为模具底部。将10% PVA水溶液和以1´10
2 ~ 1´10
4数量的大肠杆菌（EC）溶液以不同比例混合均匀，接着取5.0 mL混合液加入到模具内，将模具放置到水平模具架上，在无菌环境、25℃和35%RH条件下，鼓风进行单向纳米孔脱水，大约5 ~ 16 h后模具杯中PVA-大肠杆菌溶液已经脱水形成片状物，浸入无菌水中去除滤膜后即获得水不溶、柔软、弹性和透明的聚乙烯醇-大肠杆菌水凝胶复合膜（PVA-EC）。
为了调查大肠杆菌在PVAHM中固定化是否对大肠杆菌的生存和增殖产生影响，利用12孔培养板进行了PVA-EC重复培养实验，每12 h更换新鲜培养液并测度培养液在600 nm处吸光度值，每天测定2次，进行了20天37℃的LB培养基连续培养与检测，以非固定化的游离大肠杆菌的吸光度值为100%，计算20天共40次的固定化大肠杆菌增殖率的相对值；然以20天为横座标，相对百分值为纵座标作图，实验结果由图14所示。连续20天40次重复换液培养，其增殖吸光度值线性拟合形成一条几乎水平的直线，其相对增殖率均在90%。这结果充分说明聚乙烯醇水凝胶既能有效地固定化大肠杆菌，还能几乎不影响大肠杆菌的活性与增殖速度。所以，这种固定化大肠杆菌PVA水凝胶膜（PVA-EC）可以于工程菌反应器、细菌燃料电池、生物传感器等多种领域得到应用。参见附图14，是本发明提供的PVAHM中固定化大肠杆菌的20天40次重复增殖的稳定性。
Claims (10)
- 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的制备方法，其特征在于：将高分子水溶液或它的混合物溶液从容器顶部的加样孔中加入到模具中，所述模具以纳米孔滤膜为底部的容器，用密封盖将加样孔密闭；溶液中的水分子经滤膜纳米孔向下单向脱水，在纳米孔滤膜上表面得到高分子膜/水凝胶膜。
- 根据权利要求1所述的一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的制备方法，其特征在于：所述的高分子水溶液包括天然高分子、人工合成高分子水溶液，或经物理混合或化学交联而成的50 kDa以上的混合高分子水溶液；按质量百分比，高分子的浓度为1～20 %；所述的混合物包括蛋白质、多糖、酶、交联剂、塑化剂、药物、色素、生长因子、微生物；按质量百分比混合物的浓度为0.1～20 % 。
- 根据权利要求2所述的一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的制备方法，其特征在于：所述的天然高分子包括酪蛋白，家蚕丝、蜘蛛丝、柞蚕丝、蓖麻蚕丝或野蚕丝的再生液体丝素蛋白中的一种；所述的人工合成高分子包括聚乙烯醇。
- 按权利要求1制备方法得到的一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜。
- 一种基于单向纳米孔脱水的导电高分子膜/水凝胶膜的制备方法，其特征在于：将高分子水溶液或它的混合物溶液从容器顶部的加样孔中加入到模具中，所述模具以纳米孔滤膜为容器的底部，用密封盖将加样孔密闭；溶液中的水分子经纳米孔滤膜向下单向脱水，在纳米孔滤膜上表面得到高分子膜/水凝胶膜；将得到的高分子膜/水凝胶膜与吡咯在水溶液中进行原位聚合反应，得到一种聚吡咯修饰的导电高分子膜/水凝胶膜。
- 根据权利要求5所述的一种基于单向纳米孔脱水的导电高分子膜/水凝胶膜的制备方法，其特征在于：所述的高分子包括天然高分子酪蛋白、家蚕丝、蜘蛛丝、柞蚕丝、蓖麻蚕丝或野蚕丝的再生液体丝素蛋白中的一种，或人工合成高分子包聚乙烯醇。
- 按权利要求5制备方法得到的一种基于单向纳米孔脱水的导电高分子膜/水凝胶膜。
- 一种用于制备基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的装置，其特征在于：以纳米孔滤膜为容器的底部，容器的顶部设有加样孔和与其配合的密封盖；所述的纳米孔滤膜的孔径为截留0.1 ~ 1000 kDa分子量的透析膜，或滤孔≤50 nm的高分子合成膜。
- 根据权利要求8所述的一种用于制备基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的装置，其特征在于：包括用于安装所述装置的模具架，在容器底部的纳米孔滤膜下方设置高吸水材料或单向脱水加速器；所述的单向脱水加速器包括流动空气产生装置、恒温恒湿箱、负压腔、渗透压差腔。
- 根据权利要求8所述的一种用于制备基于单向纳米孔脱水的功能性高分子膜/水凝胶膜的装置，其特征在于：所述的纳米孔滤膜包括再生纤维透析膜、平面透析玻璃纸、陶瓷膜、聚丙烯酰胺水凝胶膜、尼龙膜。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18/200,490 US20230295387A1 (en) | 2022-01-29 | 2023-05-22 | Unidirectional nanopore dehydration-based functional polymer membrane or hydrogel membrane, preparation method thereof and device thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210112449.1A CN114702704B (zh) | 2022-01-29 | 2022-01-29 | 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 |
| CN202210112449.1 | 2022-01-29 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US18/200,490 Continuation US20230295387A1 (en) | 2022-01-29 | 2023-05-22 | Unidirectional nanopore dehydration-based functional polymer membrane or hydrogel membrane, preparation method thereof and device thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023143540A1 true WO2023143540A1 (zh) | 2023-08-03 |
Family
ID=82166332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2023/073618 WO2023143540A1 (zh) | 2022-01-29 | 2023-01-28 | 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 |
Country Status (3)
| Country | Link |
|---|---|
| US (1) | US20230295387A1 (zh) |
| CN (1) | CN114702704B (zh) |
| WO (1) | WO2023143540A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114702704B (zh) * | 2022-01-29 | 2023-11-24 | 苏州大学 | 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028536A (zh) * | 2007-03-02 | 2007-09-05 | 上海交通大学 | 聚乙烯醇/丝胶共混凝胶薄膜的制备方法 |
| CN102847194A (zh) * | 2012-09-17 | 2013-01-02 | 浙江星月生物科技股份有限公司 | 一种难溶于水的支架型丝素蛋白膜及其制备与应用 |
| CN109793928A (zh) * | 2017-11-16 | 2019-05-24 | 南杰 | 一种胶原材料制备的脑膜或脊膜生物膜片及其制备方法 |
| CN112401157A (zh) * | 2020-10-27 | 2021-02-26 | 苏州大学 | 一种保鲜蛋清结晶粉、制备及保鲜贮藏方法 |
| CN114702704A (zh) * | 2022-01-29 | 2022-07-05 | 苏州大学 | 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981561B (zh) * | 2014-05-23 | 2016-10-05 | 苏州大学 | 一种电聚丝素水凝胶膜的制备方法、装置及其应用 |
| CN208710260U (zh) * | 2018-03-20 | 2019-04-09 | 珠海水丝新材料有限公司 | 一种单向渗透脱水水凝胶膜 |
| CN110777078B (zh) * | 2019-11-08 | 2021-05-11 | 电子科技大学 | 一种序列化灌注式微流体装置 |
| CN111870740B (zh) * | 2020-08-06 | 2021-12-24 | 苏州大学 | 一种复合蛋白膜及其制备方法 |
| CN112062994B (zh) * | 2020-09-09 | 2022-03-08 | 苏州大学 | 一种蛋清生物塑料、制备方法及其应用 |
-
2022
- 2022-01-29 CN CN202210112449.1A patent/CN114702704B/zh active Active
-
2023
- 2023-01-28 WO PCT/CN2023/073618 patent/WO2023143540A1/zh unknown
- 2023-05-22 US US18/200,490 patent/US20230295387A1/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028536A (zh) * | 2007-03-02 | 2007-09-05 | 上海交通大学 | 聚乙烯醇/丝胶共混凝胶薄膜的制备方法 |
| CN102847194A (zh) * | 2012-09-17 | 2013-01-02 | 浙江星月生物科技股份有限公司 | 一种难溶于水的支架型丝素蛋白膜及其制备与应用 |
| CN109793928A (zh) * | 2017-11-16 | 2019-05-24 | 南杰 | 一种胶原材料制备的脑膜或脊膜生物膜片及其制备方法 |
| CN112401157A (zh) * | 2020-10-27 | 2021-02-26 | 苏州大学 | 一种保鲜蛋清结晶粉、制备及保鲜贮藏方法 |
| CN114702704A (zh) * | 2022-01-29 | 2022-07-05 | 苏州大学 | 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 |
Also Published As
| Publication number | Publication date |
|---|---|
| US20230295387A1 (en) | 2023-09-21 |
| CN114702704A (zh) | 2022-07-05 |
| CN114702704B (zh) | 2023-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Qi et al. | Investigation of Salecan/poly (vinyl alcohol) hydrogels prepared by freeze/thaw method | |
| Li et al. | Muscle-inspired MXene/PVA hydrogel with high toughness and photothermal therapy for promoting bacteria-infected wound healing | |
| Hu et al. | Biocompatible fibroin blended films with recombinant human-like collagen for hepatic tissue engineering | |
| Huang et al. | An injectable nano-hydroxyapatite (n-HA)/glycol chitosan (G-CS)/hyaluronic acid (HyA) composite hydrogel for bone tissue engineering | |
| Tonda-Turo et al. | Comparative analysis of gelatin scaffolds crosslinked by genipin and silane coupling agent | |
| Li et al. | One-pot, self-catalyzed synthesis of self-adherent hydrogels for photo-thermal, antimicrobial wound treatment | |
| Cho et al. | Fabrication and characterization of porous alginate/polyvinyl alcohol hybrid scaffolds for 3D cell culture | |
| Ceylan et al. | Effect of crosslinking methods on the structure and biocompatibility of polyvinyl alcohol/gelatin cryogels | |
| CN108409988B (zh) | 一种海绵状大孔聚乙烯醇水凝胶的制备方法 | |
| Xu et al. | Conductive and antimicrobial macroporous nanocomposite hydrogels generated from air-in-water Pickering emulsions for neural stem cell differentiation and skin wound healing | |
| He et al. | Shape memory composite hydrogel based on sodium alginate dual crosslinked network with carboxymethyl cellulose | |
| Samal et al. | Silk/chitosan biohybrid hydrogels and scaffolds via green technology | |
| Cho et al. | Time-dependent alginate/polyvinyl alcohol hydrogels as injectable cell carriers | |
| WO2023143540A1 (zh) | 一种基于单向纳米孔脱水的功能性高分子膜/水凝胶膜、制备方法及装置 | |
| Foglia et al. | A new method for the preparation of biocompatible silica coated-collagen hydrogels | |
| Chen et al. | Study on the cross-linking effect of a natural derived oxidized chitosan oligosaccharide on the porcine acellular dermal matrix | |
| CN110152055A (zh) | 海藻酸胺化衍生物/细菌纤维素纳米晶复合凝胶构筑的功能性药物缓释医用敷料 | |
| Song et al. | POSS–PU electrospinning nanofibers membrane with enhanced blood compatibility | |
| CN115350334A (zh) | 一种聚酰亚胺基复合气凝胶材料的制备方法、产品及应用 | |
| Cirillo et al. | Adhesion and function of rat liver cells adherent to silk fibroin/collagen blend films | |
| Yang et al. | Fabrication, hydrolysis and cell cultivation of microspheres from cellulose-graft-poly (l-lactide) copolymers | |
| CN113583455B (zh) | 一种胶原蛋白-改性壳聚糖双网络水凝胶、生物墨水、制备方法和应用 | |
| RU2471824C1 (ru) | Биосовместимый биодеградируемый пористый композиционный материал и способ его получения | |
| Zhao et al. | Novel high strength PVA/soy protein isolate composite hydrogels and their properties | |
| CN113248743A (zh) | 一种生物相容的可降解的三维纤维素凝胶及其制备方法和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23746417 Country of ref document: EP Kind code of ref document: A1 |