CN111921000A - 氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法 - Google Patents
氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法 Download PDFInfo
- Publication number
- CN111921000A CN111921000A CN202010680672.7A CN202010680672A CN111921000A CN 111921000 A CN111921000 A CN 111921000A CN 202010680672 A CN202010680672 A CN 202010680672A CN 111921000 A CN111921000 A CN 111921000A
- Authority
- CN
- China
- Prior art keywords
- graphene oxide
- group
- insulin
- growth factor
- wound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 109
- 229910021389 graphene Inorganic materials 0.000 title claims abstract description 106
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 title claims abstract description 40
- 102100037852 Insulin-like growth factor I Human genes 0.000 title claims abstract description 40
- 230000029663 wound healing Effects 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000002131 composite material Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims description 23
- 208000027418 Wounds and injury Diseases 0.000 claims abstract description 51
- 206010052428 Wound Diseases 0.000 claims abstract description 50
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 23
- 230000012010 growth Effects 0.000 claims abstract description 13
- 230000035876 healing Effects 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 24
- 229960003638 dopamine Drugs 0.000 claims description 23
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 22
- 230000001580 bacterial effect Effects 0.000 claims description 19
- 238000011534 incubation Methods 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 230000007547 defect Effects 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- PCTMTFRHKVHKIS-BMFZQQSSSA-N (1s,3r,4e,6e,8e,10e,12e,14e,16e,18s,19r,20r,21s,25r,27r,30r,31r,33s,35r,37s,38r)-3-[(2r,3s,4s,5s,6r)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-19,25,27,30,31,33,35,37-octahydroxy-18,20,21-trimethyl-23-oxo-22,39-dioxabicyclo[33.3.1]nonatriaconta-4,6,8,10 Chemical compound C1C=C2C[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2.O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 PCTMTFRHKVHKIS-BMFZQQSSSA-N 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 241000699670 Mus sp. Species 0.000 claims description 9
- 238000011156 evaluation Methods 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 8
- 241000191967 Staphylococcus aureus Species 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 108050006400 Cyclin Proteins 0.000 claims description 6
- 108010013198 Daptomycin Proteins 0.000 claims description 6
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 6
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 claims description 6
- 229960005484 daptomycin Drugs 0.000 claims description 6
- 238000000556 factor analysis Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000011550 stock solution Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 6
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- 238000002329 infrared spectrum Methods 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 4
- 231100000135 cytotoxicity Toxicity 0.000 claims description 4
- 230000003013 cytotoxicity Effects 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 210000002950 fibroblast Anatomy 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 241000283707 Capra Species 0.000 claims description 3
- 229920002101 Chitin Polymers 0.000 claims description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 206010063560 Excessive granulation tissue Diseases 0.000 claims description 3
- 238000004566 IR spectroscopy Methods 0.000 claims description 3
- 102000018697 Membrane Proteins Human genes 0.000 claims description 3
- 108010052285 Membrane Proteins Proteins 0.000 claims description 3
- 239000006180 TBST buffer Substances 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 238000002038 chemiluminescence detection Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 239000010931 gold Substances 0.000 claims description 3
- 229910052737 gold Inorganic materials 0.000 claims description 3
- 210000001126 granulation tissue Anatomy 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 239000007928 intraperitoneal injection Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229960001412 pentobarbital Drugs 0.000 claims description 3
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000013641 positive control Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000000853 adhesive Substances 0.000 claims description 2
- 230000001070 adhesive effect Effects 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims 2
- 230000008901 benefit Effects 0.000 abstract description 3
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 26
- 230000000694 effects Effects 0.000 description 9
- 239000010410 layer Substances 0.000 description 8
- 210000004379 membrane Anatomy 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 5
- 230000012292 cell migration Effects 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 210000003491 skin Anatomy 0.000 description 5
- 239000003102 growth factor Substances 0.000 description 4
- 239000002114 nanocomposite Substances 0.000 description 4
- 229920001690 polydopamine Polymers 0.000 description 4
- 230000033115 angiogenesis Effects 0.000 description 3
- 239000012620 biological material Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 2
- 102000013275 Somatomedins Human genes 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229960003085 meticillin Drugs 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000237536 Mytilus edulis Species 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 229920000297 Rayon Polymers 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000008952 bacterial invasion Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000020638 mussel Nutrition 0.000 description 1
- 239000002060 nanoflake Substances 0.000 description 1
- 239000002064 nanoplatelet Substances 0.000 description 1
- 239000002135 nanosheet Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- ZRHANBBTXQZFSP-UHFFFAOYSA-M potassium;4-amino-3,5,6-trichloropyridine-2-carboxylate Chemical compound [K+].NC1=C(Cl)C(Cl)=NC(C([O-])=O)=C1Cl ZRHANBBTXQZFSP-UHFFFAOYSA-M 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000006934 radiodermatitis Diseases 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 208000037974 severe injury Diseases 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 230000008591 skin barrier function Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
- A61L2300/104—Silver, e.g. silver sulfadiazine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明公开了氧化石墨烯‑纳米银/胰岛素样生长因子‑1复合敷料制备及创面愈合方法,尤其是氧化石墨烯‑纳米银/胰岛素样生长因子‑1复合敷料制备,包括纯氧化石墨烯膜(A组)、氧化石墨烯膜‑纳米银(B组)、氧化石墨烯膜‑胰岛素样生长因子‑1(C组)、氧化石墨烯膜‑纳米银/胰岛素样生长因子‑1(D组)。本发明在成功制备氧化石墨烯膜‑纳米银/胰岛素样生长因子‑1敷料的基础上,D组样品很好地实现了抗菌作用和促生长性能,对创面愈合有很大的益处。
Description
技术领域
本发明涉及氧化石墨烯技术领域,尤其涉及氧化石墨烯-纳米银/ 胰岛素样生长因子-1复合敷料制备及创面愈合方法。
背景技术
纳米银(nanosilver,NS)是一种对革兰氏阳性菌和革兰氏阴性菌都具有强大的广谱杀菌活性的著名抗菌剂,包括可抑制耐甲氧西林金黄色葡萄球菌(MRSA)等多种耐药菌。有研究认为,纳米银的毒性作用仅在高浓度下才会发生,纳米银掺入材料可以减轻毒性。因此,纳米银被认为是一种理想的生物材料包涵体抗菌剂。据报道,各种生长因子(如表皮生长因子、血小板源生长因子,和纤维母细胞生长因子2) 可以分别在不同动态阶段发挥关键部分,包括迁移,增殖及血管生成。因此,我们用胰岛素样生长因子-1作为模型因子对制备的材料进行修饰,以增强伤口愈合效果。胰岛素样生长因子(IGF)已被证明可在体外刺激角质细胞增殖。有研究表明胰岛素样生长因子-1,可调节组织生长和修复蛋白,尤其是对糖尿病创面患者十分重要。基底层和成纤维细胞中缺乏胰岛素样生长因子-1,可能导致糖尿病患者创面愈合延迟。它应该与具有持续释放特性的药物输送系统相结合,例如水凝胶,以实现更好的利用。
近年来,氧化石墨烯在抗菌方面的优异性能引起了越来越多的关注。已有报道基于氧化石墨烯的纳米复合材料在很大程度上有利于伤口愈合,其抗菌性能的根源在于氧化石墨烯边缘所致的细胞膜紊乱及氧化应激诱导之间的协同作用。此外,氧化石墨烯表面含有丰富的羟基、环氧基团、羧基等表面基团,其亲水性和生物相容性明显增强,同时可以被其他化学物质进一步修饰。因此,我们特意对氧化石墨烯实现功能化,以增强其抗菌作用,同时提高其稳定性。目前,为了防止氧化石墨烯的聚集,已经有几例采用化学材料接枝法,其中交联剂的使用包括聚多巴胺、Fe3O4和银纳米颗(AgNPs),但银纳米颗的细胞毒性值得警惕。
因此,氧化石墨烯膜和纳米银/胰岛素样生长因子-1的综合研究成为热点,不仅具有广泛的应用潜力,而且纳米银抗菌和胰岛素样生长因子-1促生长的联合作用前景广阔。本文成功制备了一系列氧化石墨烯膜-纳米银/胰岛素样生长因子-1敷料,并在体外对其抗菌性能进行了系统研究。此外,还研究了它们对小鼠创伤愈合的实际作用,所得结果对氧化石墨烯敷料的进一步研究具有指导意义。
发明内容
基于背景技术存在的技术问题,本发明提出了氧化石墨烯-纳米银银/胰岛素样生长因子-1复合敷料制备及创面愈合方法。
本发明提出的氧化石墨烯-纳米银复合敷料制备,包括纯氧化石墨烯膜(A组)、氧化石墨烯膜-纳米银(B组)、氧化石墨烯膜-胰岛素样生长因子-1(C组)、氧化石墨烯膜-纳米银/胰岛素样生长因子-1(D 组)。
氧化石墨烯--纳米银/胰岛素样生长因子-1复合敷料制备,其特征在于,包括如下步骤:
S1准备A组氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司(货号:100027,CAS号:7440-44-0,参数:尺寸:9x9cm厚度:约25微米);
S2制备氧化石墨烯-多巴胺膜:
S21在100mL去离子水中加入131.14g tris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris-多巴胺溶液,浓度为2mg/mL,pH值为8.5;
S22将S1中的氧化石墨烯膜浸泡在上述溶液中12h后,将混合物转移到振动筛中,在37℃下以100r/min的速度振动;
S3制备B组氧化石墨烯-纳米银样品:
先将达托霉素粉溶解于去离子水中,制备10mg/mL的纳米银水溶液,再将S1中的氧化石墨烯样品,经去离子水仔细清洗,孵育在达托霉素水溶液中,其中孵育温度37℃;震动速度:100r/min;孵化时间,12h。
S4制备C组氧化石墨烯膜-胰岛素样生长因子-1:将S2中制备好的氧化石墨烯-多巴胺膜,溶解在10μg/ml的胰岛素样生长因子 -1溶液中,孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S5制备D组氧化石墨烯膜-纳米银/胰岛素样生长因子-1:将氧化石墨烯膜-多巴胺加入到S3和S4步骤中相同浓度的纳米银/胰岛素样生长因子-1混合物中,孵育温度37℃;震动速度:100r/min;孵化时间,12h,制备得氧化石墨烯膜-纳米银/胰岛素样生长因子-1。
氧化石墨烯-纳米银复合敷料的创面愈合方法,包括如下步骤:
S1扫描电镜观察材料表面结构:将A组和D组样品进行喷金,并彻底干燥,然后用扫描电镜仪器在真空中观察得到的薄膜,详细地拍摄孔径结构;
S2傅里叶红外光谱观察材料合成成分:将制备的样品在600cm-1 -4000cm-1波数范围内,通过傅里叶红外光谱测量表征其化学结构;
S3接触角测试仪观察材料亲疏水性:ABCD四组样本被放置水平,每个材料表面均滴1μL去离子水,封闭静置12h,用形成的液滴测量接触角,每个样品测试三次,得到平均角度值;
S4评估材料体外抗菌性:将大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体培养、扩增至1×109CFU/mL的密度,再经LB稀释至1×104CFU/mL的密度,提取100mL菌液。采用96孔板,将ABCD 每个样本放置3个孔,每孔滴入200μL菌液,孵化24小时,37℃孵化温度。采用分光光度计评估菌液变化,细菌原液标准OD值设为 0.7,24h后再次行OD值检测,观察不同材料对细菌原液影响。
S5评估材料体外细胞毒性:原代成纤维细胞来源于正常的新生小鼠,并进一步将细胞传代至第二代和第三代。采用96孔板进行细胞计数及培养,2000个细胞/孔,将每组样品与血管内皮细胞共同培养,每组3孔。第1、3、5、7天依次检测,37℃条件下孵化后加上LB培养基溶液中(150μL/孔),上述操作一式三份;
S6评估材料促细胞迁移能力:将血管内皮细胞接种于24孔板(2 ×104/孔)后,使用DMEM培养基培养,使用枪头划出一道划痕,并记录时间为0h,将ABCD四组材料与该细胞进行共培养,用活细胞工作站显微镜进行24小时观察,在单个实验中,每组设置6个重复,使用ImageJ 1.48V软件(美国NIH公司)进行具体测量,上述操作一式三份;
S7小鼠创面模型建立及不同材料对创面愈合影响:小鼠的腹腔内注射戊巴比妥钠(1%,70μL/g)麻醉,然后使用打孔器建立全层皮肤缺损模型,缺损面积直径0.6厘米,每个创面滴加细菌溶液(5μL 108/毫升)培养大肠杆菌和金黄色葡萄球菌,用75%的酒精对材料进行消毒,用磷酸缓冲盐溶液漂洗彻底去除杂质,然后,在伤口上涂上准备好的薄膜,然后用粘胶毛巾固定,伤后1、3、5、7天拍照更换材料,采用山东贝诺医药生物科技有限公司【国家发明专利号 ZL200620082586.1】采购的市售甲壳素敷料(CCD)作为阳性对照;
S8创面愈合计算:比较创面愈合前后创面面积,计算愈合率,采用IPP6.0软件辅助,根据“感兴趣区域”(AOI)功能选择目标创面面积,我们用“大小计数”方法测量像素面积,伤口面积可按伤口愈合率=(创面面积-愈合一定时间后的创面面积)/创面面积×100%的公式计算;
S9创面蛋白表达:创面使用材料敷贴第7天后用Wester Blot 方法检测PCNA及CD31表达。即在小鼠全层创面缺损创面中取样约 10mm×10mm的小方块,包括表皮和肉芽组织,并将其立即置于液氮中冷冻,随后裂解提取蛋白。抗CD31抗体(货号:ab28364,品牌:Abcam,生产地:UK)和抗PCNA抗体(货号:ab15497,品牌:Abcam, 生产地:UK)均按1:1000稀释,抗tubulin抗体(品牌:Sungene,生产地:China)按1:2000稀释,所有抗体在使用前一晚维持在4℃,将HRP(中国中山生物公司)标记的山羊抗兔二抗1:2000稀释,与样品在25℃孵育1h,在TBST中洗涤5次后,将收获的PDVF膜送化学发光检测(美国Thermal Scientific);
S10通过Origin软件,采用单因素方差分析和双因素方差分析分别分析两组之间和两组以上的显著性差异,实验数据以均数±标准差表示,P<0.05被认为有统计学意义。
本发明中,所述氧化石墨烯膜-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法,我们注意到附着复合材料中的离子在氧化石墨烯膜-纳米银/胰岛素样生长因子-1(D组)中沉淀,傅里叶红外光谱分析表明纳米银和胰岛素样生长因子-1可能粘附在氧化石墨烯膜上,接触角试验表明,材料的亲水性逐渐增加,B、D组体外抗菌活性相似,优于A、C组;4个实验组均优于对照组(P>0.05),另一方面,C组和D组在1-7D内均能有效促进细胞增殖(P<0.05),且细胞迁移活性相似,高于A组和B组(彼此接近);实验组均优于对照组(P<0.05,此外,通过监测CD31和PCNA的表达水平,D组标本在覆盖皮肤缺损时可促进血管生成和细胞繁殖(P<0.05)。伤后7 天,A、B、C、D组分别为对照组、试验组,愈合率分别为40.6%、53.0%、 67.1%、61.5%、76.5%。
本发明在成功制备氧化石墨烯膜-纳米银/胰岛素样生长因子-1 敷料的基础上,d组样品很好地实现了抗菌作用和促生长性能,对创面愈合有很大的益处。
附图说明
图1a为氧化石墨烯膜原理图,b为A组和d组具有代表性的扫描电镜图像;
图2a为傅里叶红外光谱图,b为A组(氧化石墨烯膜)、B组(氧化石墨烯膜-纳米银)、C组(氧化石墨烯膜-胰岛素样生长因子-1)、D 组(氧化石墨烯膜-纳米银/胰岛素样生长因子-1)的接触角图;c为接触角动态测量图;
图3为抗菌活性测定图;
图4a为在0~24h内观察不同分组对细胞迁移的影响图,b)为各群体的迁移百分比图;
图5为Western blot检测创面全层组织中CD31和PCNA的表达图;
图6为感染创面愈合的影响a创面愈合图,b为创面愈合3,7天的统计数据图,c为创面完全愈合所需的时间图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
氧化石墨烯--纳米银/胰岛素样生长因子-1复合敷料制备,其特征在于,包括如下步骤:
S1准备A组氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司(货号:100027,CAS号:7440-44-0,参数:尺寸:9x9cm厚度:约25微米);
S2制备氧化石墨烯-多巴胺膜:
S21在100mL去离子水中加入131.14g tris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris-多巴胺溶液,浓度为2mg/mL,pH值为8.5;
S22将S1中的氧化石墨烯膜浸泡在上述溶液中12h后,将混合物转移到振动筛中,在37℃下以100r/min的速度振动;
S3制备B组氧化石墨烯-纳米银样品:
先将达托霉素粉溶解于去离子水中,制备10mg/mL的纳米银水溶液,再将S1中的氧化石墨烯样品,经去离子水仔细清洗,孵育在达托霉素水溶液中,其中孵育温度37℃;震动速度:100r/min;孵化时间,12h。
S4制备C组氧化石墨烯膜-胰岛素样生长因子-1:将S2中制备好的氧化石墨烯-多巴胺膜,溶解在10μg/ml的胰岛素样生长因子 -1溶液中,孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S5制备D组氧化石墨烯膜-纳米银/胰岛素样生长因子-1:将氧化石墨烯膜-多巴胺加入到S3和S4步骤中相同浓度的纳米银/胰岛素样生长因子-1混合物中,孵育温度37℃;震动速度:100r/min;孵化时间,12h,制备得氧化石墨烯膜-纳米银/胰岛素样生长因子-1。
参照图1-6,氧化石墨烯-纳米银复合敷料制备,包括纯氧化石墨烯膜(A组)、氧化石墨烯膜-纳米银(B组)、氧化石墨烯膜-胰岛素样生长因子-1(C组)、氧化石墨烯膜-纳米银/胰岛素样生长因子-1(D 组)。
氧化石墨烯-纳米银复合敷料的创面愈合方法,包括如下步骤:
S1扫描电镜观察材料表面结构:将A组和D组样品进行喷金,并彻底干燥,然后用扫描电镜仪器在真空中观察得到的薄膜,详细地拍摄孔径结构;
S2傅里叶红外光谱观察材料合成成分:将制备的样品在600cm-1 -4000cm-1波数范围内,通过傅里叶红外光谱测量表征其化学结构;
S3接触角测试仪观察材料亲疏水性:ABCD四组样本被放置水平,每个材料表面均滴1μL去离子水,封闭静置12h,用形成的液滴测量接触角,每个样品测试三次,得到平均角度值;
S4评估材料体外抗菌性:将大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体培养、扩增至1×109CFU/mL的密度,再经LB稀释至1×104CFU/mL的密度,提取100mL菌液。采用96孔板,将ABCD 每个样本放置3个孔,每孔滴入200μL菌液,孵化24小时,37℃孵化温度。采用分光光度计评估菌液变化,细菌原液标准OD值设为 0.7,24h后再次行OD值检测,观察不同材料对细菌原液影响。
S5评估材料体外细胞毒性:原代成纤维细胞来源于正常的新生小鼠,并进一步将细胞传代至第二代和第三代。采用96孔板进行细胞计数及培养,2000个细胞/孔,将每组样品与血管内皮细胞共同培养,每组3孔。第1、3、5、7天依次检测,37℃条件下孵化后加上LB培养基溶液中(150μL/孔),上述操作一式三份;
S6评估材料促细胞迁移能力:将血管内皮细胞接种于24孔板(2 ×104/孔)后,使用DMEM培养基培养,使用枪头划出一道划痕,并记录时间为0h,将ABCD四组材料与该细胞进行共培养,用活细胞工作站显微镜进行24小时观察,在单个实验中,每组设置6个重复,使用ImageJ 1.48V软件(美国NIH公司)进行具体测量,上述操作一式三份;
S7小鼠创面模型建立及不同材料对创面愈合影响:小鼠的腹腔内注射戊巴比妥钠(1%,70μL/g)麻醉,然后使用打孔器建立全层皮肤缺损模型,缺损面积直径0.6厘米,每个创面滴加细菌溶液(5μL 108/毫升)培养大肠杆菌和金黄色葡萄球菌,用75%的酒精对材料进行消毒,用磷酸缓冲盐溶液漂洗彻底去除杂质,然后,在伤口上涂上准备好的薄膜,然后用粘胶毛巾固定,伤后1、3、5、7天拍照更换材料,采用山东贝诺医药生物科技有限公司【国家发明专利号 ZL200620082586.1】采购的市售甲壳素敷料(CCD)作为阳性对照;
S8创面愈合计算:比较创面愈合前后创面面积,计算愈合率,采用IPP6.0软件辅助,根据“感兴趣区域”(AOI)功能选择目标创面面积,我们用“大小计数”方法测量像素面积,伤口面积可按伤口愈合率=(创面面积-愈合一定时间后的创面面积)/创面面积×100%的公式计算;
S9创面蛋白表达:创面使用材料敷贴第7天后用Wester Blot 方法检测PCNA及CD31表达。即在小鼠全层创面缺损创面中取样约10mm×10mm的小方块,包括表皮和肉芽组织,并将其立即置于液氮中冷冻,随后裂解提取蛋白。抗CD31抗体(货号:ab28364,品牌:Abcam,生产地:UK)和抗PCNA抗体(货号:ab15497,品牌:Abcam, 生产地:UK)均按1:1000稀释,抗tubulin抗体(品牌:Sungene,生产地:China)按1:2000稀释,所有抗体在使用前一晚维持在4℃,将HRP(中国中山生物公司)标记的山羊抗兔二抗1:2000稀释,与样品在25℃孵育1h,在TBST中洗涤5次后,将收获的PDVF膜送化学发光检测(美国Thermal Scientific);
S10通过Origin软件,采用单因素方差分析和双因素方差分析分别分析两组之间和两组以上的显著性差异,实验数据以均数±标准差表示,P<0.05被认为有统计学意义。
本发明:当严重伤害发生在皮肤上时,一个有效的伤口闭合是至关重要的,因为它有利于防止微生物入侵和能量、电解质或体液的损失。伤口敷料具有广泛的功能,如加速伤口愈合,重建皮肤屏障,保护或准备后续手术的。优良的选矿材料应具有良好的生物相容性,对水蒸气具有足够的渗透性,并具有较强的机械性能。此外,这些材料应该为伤口愈合过程创造无菌、适宜的微环境,以避免炎症或感染带来的不良影响。最近有报道说氧化石墨烯纳米薄片具有抗菌活性。尽管如此,在水溶液中由于强的层间相互作用而引起的逐层聚集阻止了氧化石墨烯纳米片在更广泛的应用上的应用。此外,也有研究报道氧化石墨烯对抗菌性能有微弱甚至不利的影响。为此,本研究通过多巴胺凝胶反应制备氧化石墨烯-纳米银/胰岛素样生长因子-1纳米复合敷料,证实复合材料的抗菌活性。
图1a是氧化石墨烯膜的示意图。可以看出,原始的氧化石墨烯结构在A组氧化石墨烯膜中得到了完整的保存,在高倍镜下观察到清晰的纹理。D组氧化石墨烯膜-纳米银/胰岛素样生长因子-1样品在 D1中仍可见片状结构,D2中规则和纹理发生变化,嵌入的纳米银/多巴胺层在D3中进一步析出。白色箭头表示纳米银层,红色箭头表示多巴胺层。这种现象是由于氧化石墨烯膜结构随反应前的变化而变化的结果,使纳米银层能够有效地附着在膜上。确定了三组(对照组和D组)的红外光谱,结果接近。平均接触角(θ)是A组(91.42±10.87)°,B组(78.80±9.64)°,C组(71.76±8.95)°,D组(56.81± 8.32)°。显然,样品的亲水性逐渐增加,纳米银改善膜可使材料抗菌能力受益。根据早期的研究,氧化石墨烯可以通过破坏细胞膜或诱导氧化应激来达到较高的抗菌活性,而纳米银可以通过扰乱细胞膜来达到同样的效果。上述实验结果显示,用纳米复合材料处理的细菌细胞膜受损。然后,我们想知道氧化石墨烯-纳米银样品是否能达到同等甚至促进的抗菌效果。由于革兰氏阴性菌对氧化石墨烯纳米银的耐药性通常高于革兰氏阳性菌,因此选择大肠杆菌和耐甲氧西林金黄色葡萄球菌进行后续抗菌机制的研究。结果表明,氧化石墨烯纳米片与纳米银配合使用,抗菌效果更佳。具体来说,一方面,氧化石墨烯薄片可以与目标细菌相互作用,包裹住它们的细胞膜;另一方面,氧化石墨烯制得的接枝纳米银与菌膜充分接触,使更多的纳米银能够聚集在目标菌周围。24小时培养金黄色葡萄球菌和大肠杆菌导致测定样品的抗菌活性是按照以下顺序:D组≈B组>C组≈A组(P>0.05,图3)。这项研究证明了复合纳米银膜可增强氧化石墨烯的抗菌活性。尽管合成纳米银颗粒的方法很多,但由于制备过程的复杂性,产生有害的副产品,它们并不受欢迎。近年来,仿生聚多巴胺(PD)因其多用途的表面修饰和还原性能引起了人们对组织工程的极大兴趣。研究人员受蚌粘附现象的启发,发现多巴胺(DA)在碱性环境下进行自聚合形成多巴胺层。一层多聚多巴胺可以涂覆在任何类型的生物材料上,其可通过氧化还原反应材料表面原位形成和整合成金属纳米颗粒。此外,多巴胺是无毒的,在温和的形成过程中不产生有害的副产品。细胞增殖抑制实验(图4)表明,C和D组可以有效地促进1-7天的细胞增殖(P< 0.05),促增值顺序为D组≈C组>B组≈A组>对照组(P<0.05),与促进体外细胞迁移顺序一致。急性放射性皮炎等总是伴随着体表的损伤,即我们的防御屏障被破坏,随之而来的是我们的免疫能力下降。广泛的组织坏死和细菌侵袭会引起创面感染,一直是放疗常见并发症。图6中分别为对照组、A组、B组、C组、D组的7天创面愈合率分别为40.3%、53.0%、67.8%、62.7%、75.4%,D组与对照组比较,差异有统计学意义(P<0.05)。此外,从对照组到D组,这五组的平均完全恢复时间为11.3、10.1、9.4、9.8和9.3天。在伤口愈合的过程中,伤口会与上皮细胞的形成一起收缩。由于人类是皮肤致密的物种,重新上皮化作用是伤口愈合的主要驱动力。为了解决上述缺点,研究人员将重点放在结合生物材料(如纤维蛋白、胶原蛋白、葡聚糖和壳聚糖)的胶囊化生长因子开发。该系统无需多个管理过程,即可针对生长因子进行持续释放。在这些,多巴胺是一个多功能载体,因为它可生物可降解,并可与表皮细胞的已知成分结合,在表皮再生过程中经历重塑。因此,我们也监测了PCNA和CD31的表达水平。结果表明,D组皮肤损伤标本应用能促进血管生成和细胞增殖(P<0.05);因此,伤口愈合的速度可以大大提高。同时,本研究也存在一定的局限性。因此,改进修饰材料的制备方法,提高氧化石墨烯膜的稳定性,提高对革兰氏阴性菌的治疗效果至关重要。此外,下一步我们将把抗菌物质/生长因子与不同类型的氧化石墨烯膜结合起来,从而获得最理想的用于伤口治疗和感染预防的敷料。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (3)
1.氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备,其特征在于,包括纯氧化石墨烯膜(A组)、氧化石墨烯膜-纳米银(B组)、氧化石墨烯膜-胰岛素样生长因子-1(C组)、氧化石墨烯膜-纳米银/胰岛素样生长因子-1(D组)。
2.氧化石墨烯--纳米银/胰岛素样生长因子-1复合敷料制备,其特征在于,包括如下步骤:
S1准备A组氧化石墨烯膜:氧化石墨烯膜购买自南京先锋纳米有限公司(货号:100027,CAS号:7440-44-0,参数:尺寸:9x9cm厚度:约25微米);
S2制备氧化石墨烯-多巴胺膜:
S21在100mL去离子水中加入131.14g tris盐酸溶解,得到的tris溶液中加入多巴胺粉200mg,得到tris-多巴胺溶液,浓度为2mg/mL,pH值为8.5;
S22将S1中的氧化石墨烯膜浸泡在上述溶液中12h后,将混合物转移到振动筛中,在37℃下以100r/min的速度振动;
S3制备B组氧化石墨烯-纳米银样品:
先将达托霉素粉溶解于去离子水中,制备10mg/mL的纳米银水溶液,再将S1中的氧化石墨烯样品,经去离子水仔细清洗,孵育在达托霉素水溶液中,其中孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S4制备C组氧化石墨烯膜-胰岛素样生长因子-1:将S2中制备好的氧化石墨烯-多巴胺膜,溶解在10μg/ml的胰岛素样生长因子-1溶液中,孵育温度37℃;震动速度:100r/min;孵化时间,12h;
S5制备D组氧化石墨烯膜-纳米银/胰岛素样生长因子-1:将氧化石墨烯膜-多巴胺加入到S3和S4步骤中相同浓度的纳米银/胰岛素样生长因子-1混合物中,孵育温度37℃;震动速度:100r/min;孵化时间,12h,制备得氧化石墨烯膜-纳米银/胰岛素样生长因子-1。
3.氧化石墨烯膜-纳米银/胰岛素样生长因子-1复合敷料的创面愈合方法,其特征在于,包括如下步骤:
S1扫描电镜观察材料表面结构:将A组和D组样品进行喷金,并彻底干燥,然后用扫描电镜仪器在真空中观察得到的薄膜,详细地拍摄孔径结构;
S2傅里叶红外光谱观察材料合成成分:将制备的样品在600cm-1-4000cm-1波数范围内,通过傅里叶红外光谱测量表征其化学结构;
S3接触角测试仪观察材料亲疏水性:ABCD四组样本被放置水平,每个材料表面均滴1μL去离子水,封闭静置12h,用形成的液滴测量接触角,每个样品测试三次,得到平均角度值;
S4评估材料体外抗菌性:将大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)菌体培养、扩增至1×109CFU/mL的密度,再经LB稀释至1×104CFU/mL的密度,提取100mL菌液。采用96孔板,将ABCD每个样本放置3个孔,每孔滴入200μL菌液,孵化24小时,37℃孵化温度。采用分光光度计评估菌液变化,细菌原液标准OD值设为0.7,24h后再次行OD值检测,观察不同材料对细菌原液影响;
S5评估材料体外细胞毒性:原代成纤维细胞来源于正常的新生小鼠,并进一步将细胞传代至第二代和第三代。采用96孔板进行细胞计数及培养,2000个细胞/孔,将每组样品与血管内皮细胞共同培养,每组3孔,第1、3、5、7天依次检测,37℃条件下孵化后加上LB培养基溶液中(150μL/孔),上述操作一式三份;
S6评估材料促细胞迁移能力:将血管内皮细胞接种于24孔板(2×104/孔)后,使用DMEM培养基培养,使用枪头划出一道划痕,并记录时间为0h,将ABCD四组材料与该细胞进行共培养,用活细胞工作站显微镜进行24小时观察,在单个实验中,每组设置6个重复,使用ImageJ1.48V软件(美国NIH公司)进行具体测量,上述操作一式三份;
S7小鼠创面模型建立及不同材料对创面愈合影响:小鼠的腹腔内注射戊巴比妥钠(1%,70μL/g)麻醉,然后使用打孔器建立全层皮肤缺损模型,缺损面积直径0.6厘米,每个创面滴加细菌溶液(5μL108/毫升)培养大肠杆菌和金黄色葡萄球菌,用75%的酒精对材料进行消毒,用磷酸缓冲盐溶液漂洗彻底去除杂质,然后,在伤口上涂上准备好的薄膜,然后用粘胶毛巾固定,伤后1、3、5、7天拍照更换材料,采用山东贝诺医药生物科技有限公司【国家发明专利号ZL200620082586.1】采购的市售甲壳素敷料(CCD)作为阳性对照;
S8创面愈合计算:比较创面愈合前后创面面积,计算愈合率,采用IPP6.0软件辅助,根据“感兴趣区域”(AOI)功能选择目标创面面积,我们用“大小计数”方法测量像素面积,伤口面积可按伤口愈合率=(创面面积-愈合一定时间后的创面面积)/创面面积×100%的公式计算;
S9创面蛋白表达:创面使用材料敷贴第7天后用Wester Blot方法检测PCNA及CD31表达。即在小鼠全层创面缺损创面中取样约10mm×10mm的小方块,包括表皮和肉芽组织,并将其立即置于液氮中冷冻,随后裂解提取蛋白。抗CD31抗体(货号:ab28364,品牌:Abcam,生产地:UK)和抗PCNA抗体(货号:ab15497,品牌:Abcam,生产地:UK)均按1:1000稀释,抗tubulin抗体(品牌:Sungene,生产地:China)按1:2000稀释,所有抗体在使用前一晚维持在4℃,将HRP(中国中山生物公司)标记的山羊抗兔二抗1:2000稀释,与样品在25℃孵育1h,在TBST中洗涤5次后,将收获的PDVF膜送化学发光检测(美国Thermal Scientific);
S10通过Origin软件,采用单因素方差分析和双因素方差分析分别分析两组之间和两组以上的显著性差异,实验数据以均数±标准差表示,P<0.05被认为有统计学意义。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010680672.7A CN111921000A (zh) | 2020-07-15 | 2020-07-15 | 氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010680672.7A CN111921000A (zh) | 2020-07-15 | 2020-07-15 | 氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111921000A true CN111921000A (zh) | 2020-11-13 |
Family
ID=73313616
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010680672.7A Pending CN111921000A (zh) | 2020-07-15 | 2020-07-15 | 氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111921000A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106581758A (zh) * | 2017-01-12 | 2017-04-26 | 广东泰宝医疗器械技术研究院有限公司 | 一种具诱导血管化能力的抗菌脱细胞真皮敷料及其制备方法 |
CN109395083A (zh) * | 2018-12-29 | 2019-03-01 | 吉林大学 | 一种具有抗菌活性的载药膜及其制备方法 |
CN110478517A (zh) * | 2019-08-12 | 2019-11-22 | 南昌大学第一附属医院 | 一种负载纳米银和生物活性因子的医用敷料及其制备方法 |
-
2020
- 2020-07-15 CN CN202010680672.7A patent/CN111921000A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106581758A (zh) * | 2017-01-12 | 2017-04-26 | 广东泰宝医疗器械技术研究院有限公司 | 一种具诱导血管化能力的抗菌脱细胞真皮敷料及其制备方法 |
CN109395083A (zh) * | 2018-12-29 | 2019-03-01 | 吉林大学 | 一种具有抗菌活性的载药膜及其制备方法 |
CN110478517A (zh) * | 2019-08-12 | 2019-11-22 | 南昌大学第一附属医院 | 一种负载纳米银和生物活性因子的医用敷料及其制备方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Xu et al. | Bacterial self-defense antibiotics release from organic–inorganic hybrid multilayer films for long-term anti-adhesion and biofilm inhibition properties | |
Yao et al. | Bacterial infection microenvironment-responsive enzymatically degradable multilayer films for multifunctional antibacterial properties | |
Liu et al. | In vivo wound healing and antibacterial performances of electrospun nanofibre membranes | |
Liu et al. | Fabrication of KR-12 peptide-containing hyaluronic acid immobilized fibrous eggshell membrane effectively kills multi-drug-resistant bacteria, promotes angiogenesis and accelerates re-epithelialization | |
Cochis et al. | Silver-doped keratin nanofibers preserve a titanium surface from biofilm contamination and favor soft-tissue healing | |
Fujie et al. | Dual therapeutic action of antibiotic-loaded nanosheets for the treatment of gastrointestinal tissue defects | |
Lu et al. | Novel wound dressing with chitosan gold nanoparticles capped with a small molecule for effective treatment of multiantibiotic-resistant bacterial infections | |
Lu et al. | A ROS-scavenging hydrogel loaded with bacterial quorum sensing inhibitor hyperbranched poly-L-lysine promotes the wound scar-free healing of infected skin in vivo | |
Spasova et al. | Electrospun chitosan‐coated fibers of poly (L‐lactide) and poly (L‐lactide)/poly (ethylene glycol): preparation and characterization | |
El-Newehy et al. | Fabrication of electrospun antimicrobial nanofibers containing metronidazole using nanospider technology | |
Khosravimelal et al. | Fabrication and characterization of an antibacterial chitosan/silk fibroin electrospun nanofiber loaded with a cationic peptide for wound-dressing application | |
Khan et al. | Catechol cross-linked antimicrobial peptide hydrogels prevent multidrug-resistant Acinetobacter baumannii infection in burn wounds | |
Arif et al. | Chitosan-based nanoparticles as delivery-carrier for promising antimicrobial glycolipid biosurfactant to improve the eradication rate of Helicobacter pylori biofilm | |
Barros et al. | Staphylococcus aureus and Escherichia coli dual‐species biofilms on nanohydroxyapatite loaded with CHX or ZnO nanoparticles | |
CN111744051A (zh) | 氧化石墨烯-溶菌酶∕碱性成纤维细胞生长因子复合敷料制备及创面愈合方法 | |
Li et al. | O‐Mannosylation Affords a Glycopeptide Hydrogel with Inherent Antibacterial Activities against E. coli via Multivalent Interactions between Lectins and Supramolecular Assemblies | |
Xie et al. | Nepenthes-inspired multifunctional nanoblades with mechanical bactericidal, self-cleaning and insect anti-adhesive characteristics | |
Lei et al. | Click-crosslinked in-situ hydrogel improves the therapeutic effect in wound infections through antibacterial, antioxidant and anti-inflammatory activities | |
Liu et al. | Biofilm therapy for chronic wounds | |
Wang et al. | Continuous and controllable electro-fabrication of antimicrobial copper-alginate dressing for infected wounds treatment | |
CN111744050A (zh) | 氧化石墨烯-达托霉素∕表皮生长因子复合敷料制备及创面愈合方法 | |
CN111921000A (zh) | 氧化石墨烯-纳米银/胰岛素样生长因子-1复合敷料制备及创面愈合方法 | |
CN107137761B (zh) | 一种甲壳素-两亲离子/季铵盐天然敷料及其制备方法与应用 | |
Dong et al. | Ultrasmall gold Nanoparticles/Carboxymethyl chitosan composite hydrogel: Tough, restorable, biocompatible antimicrobial dressing for wound healing | |
Mothilal et al. | Amikacin sulphate loaded chitosan-diopside nanoparticles composite scaffold for infectious wound healing |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201113 |