CN117257723A - 预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用 - Google Patents
预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用 Download PDFInfo
- Publication number
- CN117257723A CN117257723A CN202311307736.9A CN202311307736A CN117257723A CN 117257723 A CN117257723 A CN 117257723A CN 202311307736 A CN202311307736 A CN 202311307736A CN 117257723 A CN117257723 A CN 117257723A
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- liposome
- protamine
- cell membrane
- tumor recurrence
- 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.)
- Granted
Links
- 239000000017 hydrogel Substances 0.000 title claims abstract description 42
- 206010060932 Postoperative adhesion Diseases 0.000 title claims abstract description 28
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 claims abstract description 46
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 42
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 42
- 239000000499 gel Substances 0.000 claims abstract description 32
- 239000002502 liposome Substances 0.000 claims abstract description 32
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 30
- 239000003814 drug Substances 0.000 claims abstract description 30
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 26
- 108020004459 Small interfering RNA Proteins 0.000 claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 23
- 239000002105 nanoparticle Substances 0.000 claims abstract description 23
- 229960001603 tamoxifen Drugs 0.000 claims abstract description 23
- 239000002245 particle Substances 0.000 claims abstract description 21
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 17
- 239000002114 nanocomposite Substances 0.000 claims abstract description 16
- 108010007568 Protamines Proteins 0.000 claims abstract description 13
- 102000007327 Protamines Human genes 0.000 claims abstract description 13
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 13
- 229940048914 protamine Drugs 0.000 claims abstract description 13
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 12
- 210000000130 stem cell Anatomy 0.000 claims abstract description 11
- 239000004055 small Interfering RNA Substances 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 150000002632 lipids Chemical class 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 239000002246 antineoplastic agent Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 4
- 229940044683 chemotherapy drug Drugs 0.000 claims description 4
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 claims description 3
- 230000003592 biomimetic effect Effects 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 2
- 239000012670 alkaline solution Substances 0.000 claims description 2
- 210000001772 blood platelet Anatomy 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- 229940127089 cytotoxic agent Drugs 0.000 claims description 2
- 210000003617 erythrocyte membrane Anatomy 0.000 claims description 2
- 210000002950 fibroblast Anatomy 0.000 claims description 2
- 210000002865 immune cell Anatomy 0.000 claims description 2
- 210000002540 macrophage Anatomy 0.000 claims description 2
- 239000012567 medical material Substances 0.000 claims description 2
- 210000003954 umbilical cord Anatomy 0.000 claims description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 2
- 229940079593 drug Drugs 0.000 abstract description 23
- 238000011282 treatment Methods 0.000 abstract description 11
- 230000002980 postoperative effect Effects 0.000 abstract description 8
- 230000002829 reductive effect Effects 0.000 abstract description 8
- 230000004888 barrier function Effects 0.000 abstract description 7
- 238000002512 chemotherapy Methods 0.000 abstract description 7
- 238000004393 prognosis Methods 0.000 abstract description 7
- 206010061218 Inflammation Diseases 0.000 abstract description 4
- 230000004054 inflammatory process Effects 0.000 abstract description 4
- 231100000057 systemic toxicity Toxicity 0.000 abstract description 4
- 230000006907 apoptotic process Effects 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 125000002091 cationic group Chemical group 0.000 abstract description 2
- 230000029663 wound healing Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 11
- 206010061535 Ovarian neoplasm Diseases 0.000 description 8
- 206010033128 Ovarian cancer Diseases 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 6
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 239000012620 biological material Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 4
- 238000005538 encapsulation Methods 0.000 description 4
- 238000001794 hormone therapy Methods 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000006399 behavior Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 229940011871 estrogen Drugs 0.000 description 3
- 239000000262 estrogen Substances 0.000 description 3
- 102000015694 estrogen receptors Human genes 0.000 description 3
- 108010038795 estrogen receptors Proteins 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000035876 healing Effects 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 101000876610 Dictyostelium discoideum Extracellular signal-regulated kinase 2 Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 101001052493 Homo sapiens Mitogen-activated protein kinase 1 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 2
- 208000007660 Residual Neoplasm Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 238000001125 extrusion Methods 0.000 description 2
- 239000010408 film Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- SHKUUQIDMUMQQK-UHFFFAOYSA-N 2-[4-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COCCCCOCC1CO1 SHKUUQIDMUMQQK-UHFFFAOYSA-N 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 229920002177 Icodextrin Polymers 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 206010033661 Pancytopenia Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000012084 abdominal surgery Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000000823 artificial membrane Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- VBVAVBCYMYWNOU-UHFFFAOYSA-N coumarin 6 Chemical compound C1=CC=C2SC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 VBVAVBCYMYWNOU-UHFFFAOYSA-N 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 208000024389 cytopenia Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000013020 final formulation Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229940016836 icodextrin Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002357 laparoscopic surgery Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000008384 membrane barrier Effects 0.000 description 1
- 238000011133 mesenchymal stem cell therapy Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000003170 nutritional factors Nutrition 0.000 description 1
- 229960002378 oftasceine Drugs 0.000 description 1
- 230000010494 opalescence Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 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 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002311 subsequent effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 238000011521 systemic chemotherapy Methods 0.000 description 1
- 229940126703 systemic medication Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 230000030968 tissue homeostasis Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/005—Ingredients of undetermined constitution or reaction products 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/042—Polysaccharides
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/043—Proteins; Polypeptides; Degradation products thereof
- A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/145—Hydrogels or hydrocolloids
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
-
- 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
- A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Vascular Medicine (AREA)
- Surgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Inorganic Chemistry (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明属于医药领域，提供了一种预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用。设计了siRNA与他莫昔芬共递送的细胞膜脂质体‑鱼精蛋白‑透明质酸仿生纳米复合颗粒，内部是小干扰RNA与鱼精蛋白压缩成的纳米颗粒，外部为负载他莫昔芬的阳离子脂质体，以实现双药共同递送，减少化疗全身毒性的同时消灭残留细胞，增加对他莫昔芬治疗的敏感性，促进肿瘤细胞凋亡。针对上述所提到的术后粘连等不良预后，拟采用负载间充质干细胞的透明质酸凝胶预防术后粘连，改善术后环境，透明质酸具有良好生物相容性，既作为物理屏障有效预防粘连的形成，其中负载的干细胞还可以促进伤口愈合，改善炎症从而多方面改善预后。
Description
技术领域
本发明属于医药领域，特别涉及预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
卵巢癌是是严重威胁女性健康的恶性肿瘤。多数患者经肿瘤细胞减灭术和化疗药物治疗后仍会复发,最终发展为化疗耐药性疾病。
最新的卵巢癌诊疗指南指出对化疗无效或者不耐受的患者可采用激素治疗。雌激素受体在卵巢肿瘤的生长和转移中占据重要地位，在40％～80％的卵巢癌细胞中表达，当雌激素与之结合后可促进癌细胞的存活和增殖。激素治疗药物有可能成为复发性或耐药性卵巢癌的替代治疗手段，为卵巢癌患者提供化疗以外的其他备选方案，以改善预后。他莫昔芬是目前临床应用的雌激素调节剂，其能与雌二醇竞争雌激素受体，与雌激素受体形成稳定的复合物，并转运入核内，阻止染色体基因开放，从而使癌细胞的生长和发育受到抑制。
然而，即使激素治疗可以大幅度减少全身毒性，但内在或获得性抵抗依然难以避免，相当大比例的患者会出现雌激素非依赖性生长，导致激素治疗耐药。研究证明，MAPK信号传导与他莫昔芬耐药有关，这是一种经典的调节各种细胞功能(包括细胞增殖、存活)的信号通路，该通路激活会降低他莫昔芬治疗的反应和有效性，导致预后不良，反之抑制该通路可以维持细胞对激素治疗的敏感性，有效防止耐药的形成。但当前以siRNA为代表的核酸药物的研发应用面临着重要挑战。核酸药物想要进入体内并发挥药理功能，在递送过程主要面临三类挑战：一是由于核酸酶的存在，此类药物极不稳定；二是潜在的脱靶副作用可能带来不可忽视的毒性和炎症反应；三是进入作用靶点困难，由于siRNA为水溶性负电荷核酸短链，难以穿过脂质双分子层细胞膜，且进入细胞后难以实现溶酶体/内体逃逸。
术后粘连作为腹部和盆腔手术的重要临床并发症，最常见是由腹部外科手术引起，发生率高达90％，粘连的形成涉及缺血、炎症、血管生成和组织修复等多因素。术后粘连也是卵巢癌复发的影响因素之一，粘连的形成涉及缺血、炎症、血管生成和组织修复等多因素.此外，肿瘤切除术后患者较虚弱，一般需在术后2周才能进行化疗或放疗，辅助治疗的延时性可能导致错过杀灭残余肿瘤细胞的最佳时期。因此，采取措施防止粘连产生，及时的消灭残留的肿瘤细胞，是改善预后、防止复发的关键。
目前防术后粘连最常见的非手术方法主要是药物治疗和生物材料屏障给药，但只能在一定程度上减轻或减少粘连，并不能促进腹膜等受损组织的修复。抗炎药和抗凝剂在内的局部或系统药物治疗会在腹膜腔中迅速代谢，这大大降低其预防效果。现有生物材料屏障产品疗效不佳：例如，Interceed和Seprafilm等人工膜屏障产品不能完全覆盖不规则损伤创伤，在实践中不方便；可注射聚合物溶液屏障如Adept(艾考糊精溶液)在局部腹膜创伤中保留时间短。因此，亟待发明一种性能更好、抗粘连效果更好的新型生物材料。
发明内容
为改善临床术后肿瘤细胞残留，化疗全身毒性大、易耐药，导致肿瘤复发率高的临床困境，本发明提供了一种预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用。设计了siRNA与他莫昔芬共递送的细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒，内部是小干扰RNA与鱼精蛋白压缩成的纳米颗粒，外部为负载他莫昔芬的阳离子脂质体，以实现双药共同递送，减少化疗全身毒性的同时消灭残留细胞，增加对他莫昔芬治疗的敏感性，促进肿瘤细胞凋亡。针对上述所提到的术后粘连等不良预后，拟采用负载间充质干细胞的透明质酸凝胶预防术后粘连，改善术后环境，透明质酸具有良好生物相容性，既作为物理屏障有效预防粘连的形成，其中负载的干细胞还可以促进伤口愈合，改善炎症从而多方面改善预后。
为了实现上述目的，本发明采用如下技术方案：
本发明的第一个方面，提供了一种预防术后粘连与肿瘤复发的多功能水凝胶，包括：
细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒；
间充质干细胞；
透明质酸凝胶；
所述透明质酸凝胶中负载有所述细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒和间充质干细胞；
所述水凝胶的分子量为1000-200万；
间充质干细胞的浓度在1000-1000万/ml。
水凝胶制剂作为肿瘤术后局部递药系统已成为近年来抗肿瘤药物递送研究的热门方向，将水凝胶自身特点优势与临床需求结合起来，通过对基质材料的设计来赋予凝胶多种理化性质以改善术后环境，通过对包载治疗剂的搭配组合实现多种途径相结合的抗肿瘤目的。凝胶优势在于能够使药物直接定位于术后创面，填补空腔、预防粘连；提高病灶处的药物浓度，缓慢释药，降低全身给药毒性，并填补手术与全身化疗之间的空白期，有望成为改善患者预后的更理想的术后治疗方案。
目前透明质酸被广泛用作生物材料、药物释放载体及预防黏连物理屏障等是一种天然存在的糖胺聚糖，作为细胞外基质的主要成分，负责维持组织稳态，为细胞提供良好的机械支撑环境，具有优异生物相容性。通过化学改性或结构设计引入其他功能性基团，进行药物释放、细胞培养与递送等，最终实现对受损组织的愈合治疗。
间充质干细胞作为生命的种子细胞，在细胞治疗、系统重建、组织愈合过程中发挥着重大作用。在受伤部位移植MSCs除了分泌多种营养因子和生长因子，改善局部微环境、抑制细胞凋亡、促进残余细胞增殖再生外，并可促使其他内源性干细胞向受损部位归巢，加快内源性修复。尽管间充质干细胞治疗有希望成为新一代治疗药物，但受到递送环境的影响干细胞的存活率不高，极大限制了其治疗效果。本发明将干细胞负载于形态与体积可控的凝胶内，有助于细胞逐步适应宿主微环境，降低引发免疫反应的风险，提高细胞成活率，增强术后恢复的效率。
在一些实施例中，所述细胞膜包括ID8、4T1、B16、LLC、Hepa1-6等所有肿瘤细胞膜，间充质干细胞膜，红细胞、血小板、巨噬细胞膜(RAW 246.7)等。
在一些实施例中，负载药物包括他莫昔芬等激素类药物以及紫杉醇、多西他赛等脂溶性化疗药物。
在一些实施例中，所述细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒包括：脂质体、纳米粒、脂溶性化疗药物；所述脂质体中包载脂溶性化疗药物，所述脂质体内部包载纳米粒。
在一些实施例中，所述纳米粒由鱼精蛋白与透明质酸对核酸进行压缩而得，RNA包括100kd以下的siRNA、circRNA或shRNA。
本发明的第二个方面，提供了上述的预防术后粘连与肿瘤复发的多功能水凝胶的制备方法，包括：
采用脂质体包载他莫昔芬，得到负载他莫昔芬脂质体；
将siRNA与透明质酸、DEPC水、鱼精蛋白涡旋混匀，得到siRNA纳米粒；
将所述siRNA纳米粒与负载他莫昔芬脂质体混合，孵育，得到LPH；
将细胞膜与LPH混合，挤压多次，得到细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒；
将透明质酸与透明质酸-醛基溶解在碱性溶液中，在交联剂存在条件下凝胶化，得到凝胶；
取所述凝胶与细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒凝缩液、间充质干细胞混悬液混合均匀，即得。
在一些实施例中，DOTAP与CHOL脂质摩尔比例在0.5-5：1；(脂质包括dopc\dope\chems\dspc\dppc等脂质)
在一些实施例中，NP与脂质体比例在1：100-2000。
在一些实施例中，HA分子量1000-200万。
在一些实施例中，所述细胞膜与LPH以质量比1:1～20。
在一些实施例中，所述siRNA与透明质酸、鱼精蛋白的摩尔比为1：1-3：1-3。
在一些实施例中，所述透明质酸与透明质酸-醛基的质量比为50-100:0-50。
本发明的第三个方面，提供了上述的预防术后粘连与肿瘤复发的多功能水凝胶在制备预防术后粘连与肿瘤复发医用材料或药物中的应用，负载细胞范围保护骨髓、脂肪、脐带等多种来源干细胞、成纤维细胞、血管内皮细胞、及多种免疫细胞。
本发明的有益效果
(1)针对核酸药物不稳定，难以穿过细胞膜等问题，本发明用鱼精蛋白与透明质酸对核酸进行压缩形成稳定纳米粒，有效防止核酸降解。脂质体中包载他莫昔芬作为抗肿瘤药物，有效增强对于脂溶性药物的摄取，在内部包载纳米粒，与核酸实现共递送，极大增强协同效果，其外部融合肿瘤细胞膜，起到中和电荷的效果，减小制剂毒性的同时还具有同源靶向效作用。
(2)针对目前用于术后粘连产品代谢快，贴合不好等问题，本发明设计透明质酸凝胶可以在术后恢复期缓慢降解，在手术部位持续发挥抗粘附作用。此外透明质酸作为细胞外基质为干细胞提供了适宜的生存环境，大幅提高了干细胞在体内递送的存活率，此外通过HA的醛基和间充质干细胞表面的氨基之间反应形成醛亚胺键，通过可逆的席夫碱形成来实现间充质干细胞在凝胶中的保留与释放。
(3)本发明制备方法简单、实用性强，易于推广。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示例性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1脂质体外观及丁达尔效应；
图2为siRNA NP、lip、LPH、M@LPH透射电镜图；
图3为M@LPH粒径分布图；
图4为M@LPH zeta电位图；
图5为他莫昔芬累积释放曲线；
图6为siRNA NP包封率测定；
图7为siRNA稳定性考察结果图；
图8为ID8细胞膜完整性考察结果图；
图9为细胞摄取结果图；
图10为ERK2基因表达水平测定；
图11为ID8细胞中蛋白表达条带；
图12为透明质酸凝胶外观图；
图13为透明质酸凝胶与所负载干细胞的扫描电镜图；
图14为凝胶的流变图；
图15为凝胶的可注射性考察结果图；
图16为凝胶的自愈合过程；
图17为凝胶中干细胞的分布图；
图18为细胞活力考察结果图；
图19为溶血考察结果图。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
下面结合具体的实施例，对本发明做进一步的详细说明，应该指出，所述具体实施例是对本发明的解释而不是限定。
实施例1
肿瘤细胞膜的提取
为了获得肿瘤细胞膜，用细胞刮刀收集培养在175平方厘米细胞培养瓶中的细胞，1000rpm，离心5分钟。将得到的细胞颗粒加入含有蛋白酶抑制剂苯甲磺酰氟(PMSF)的低渗裂解缓冲液中。在冰浴中处理15分钟后，用超声破碎处理在冰上进一步处理细胞裂解缓冲液40轮(Ton＝3s，Toff＝10s，功率40％)超声处理后，在1000g离心5分钟，仔细收集上清液，在4℃下以12000rpm进一步离心20分钟。重新悬浮在PBS中，以便进一步使用，测定浓度。
实施例2
细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒的制备方法
取DOTAP和CHOL(摩尔比2:1)与他莫昔芬溶于适量CHCl3中，充分混匀后减压旋转蒸发，温度为20℃，转速为80rpm，待溶液蒸干后，形成一层无色透明的薄膜，继续旋蒸10min尽可能减少有机和溶剂残留。向其中加入1mL预热至40℃的DEPC水，水浴超声30s，共三次，置于40℃水浴中以1200rpm的速度搅拌1h，最后用直径为0.22μm的滤膜挤压过滤得负载他莫昔芬脂质体。
取20μM siRNA20μL、1mg/mL HA5μL、30μL DEPC水混合，加入125μg/mL鱼精蛋白40μL涡旋即得siRNA纳米粒。
将siRNA纳米粒与他莫昔芬脂质体以摩尔比1：100的比例进行混合，40℃孵育30min得LPH。将所提细胞膜与LPH以质量比1:10的比例混合，用脂质体挤出机挤压10次，即得细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒(M@LPH)。
将所得M@LPH溶液中加入甘露糖(0.1％w/w)在-80℃冰箱中预冻过夜，在冻干机中冻干24h，用0.9％氯化钠溶液溶解得M@LPH凝缩液，浓度为M@LPH溶液的20-100倍。
实施例3
负载MSCs与M@LPH透明质酸凝胶制备方法
在透明质酸钠溶液中逐滴加入高碘酸钠溶液，避光搅拌，加入乙二醇终止反应，并在纯水中透析48小时，冷冻干燥即得HA-CHO(OHA)。将270mg HA与30mg OHA溶解于3ml0.25M氢氧化钠溶液中，加入50μl 1,4-丁二醇二缩水甘油醚(BDDE)交联剂，40℃水浴搅拌3小时，加入适量0.25M盐酸调节pH至7。取1g所得凝胶，与100μl M@LPH凝缩液混合均匀后，再与100μL间充质干细胞PBS或空白培养基混悬液(500万/ml)充分混合，即得负载间充质干细胞透明质酸凝胶。
实验例
A.如图1所示，实施例2中通过薄膜水化法制备脂质纳米颗粒，脂质成膜透明均匀，所得的脂质纳米粒溶液具有淡蓝色乳光，可见明显的丁达尔效应，证明脂质纳米粒成功制备。
B.如图2所示，在透射电镜中清晰可见纳米粒，脂质体以及脂质纳米粒的微观形态，压缩后的RNA形态圆整，大小均一，脂质体结构明显，粒径较为均一，两者混合后可见其核壳结构，即证明脂质纳米粒制备成功。
C-D.粒径分布与表面电荷通过粒径电位分析仪测定。测定最终制剂粒径结果为245.93nm，电位约33.57mV，如图3、图4所示。
E.如图5所示，通过高效液相色谱测定他莫昔芬的载药量包封率与累积释放率。完成了包括专属性、回收率以及日间、日内精密度在内的方法学验证，最终测得包封率约为93.80％，载药量约为6.33％。pH 7.4与5.0分别模拟体内正常环境与肿瘤细胞溶酶体内环境，对他莫昔芬释放行为进行体外模拟，结果可知在pH 7.4的PBS中，制剂比游离药物释放速率要慢，在72h时累计释放率才为30％左右，明显延长药物释放行为，使脂质体在到达靶组织前不会发生破裂渗漏；而在pH 5.0时药物能较从脂质体中释放，72h释放量可分别达80％，远高于在pH7.4时的释放量，充分表明脂质体在血液中较稳定，而被肿瘤细胞内吞后可快速释放药物，从而发挥后续作用。
F.如图6所示，琼脂糖凝胶电泳测定siRNA的包封率与稳定性。通过琼脂糖凝胶电泳筛选鱼精蛋白与siRNA比例，发现当比例1：1时，游离RNA条带消失，则证明其全部包载于制剂内；
G.如图7所示，降解考察也可以看出，相比于游离siRNA24h内消失，在鱼精蛋白压缩后，可以稳定至48h以上不被降解，具有较好的稳定性。
H.如图8所示，M@LPH在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中保留的蛋白质组分与纯ID8细胞膜的电泳条带一致，脂质体中未检测到蛋白质信号，证明制剂中细胞膜成功融合于脂质体中，且完整性良好。
I.如图9所示，用香豆素6模拟药物在细胞水平的摄取情况，在共聚焦显微镜下明显看出有脂质体包覆后药物摄取显著高于游离药物，且加入siRNA NP与细胞膜未明显观察到细胞摄取减弱。
J.如图10所示，实时定量PCR分析验证制备的细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒在小鼠卵巢癌细胞(ID8)中干扰mRNA合成的能力。结果表明，细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒可以实现较强的mRNA干扰，优于市售转染制剂。
K.如图11所示，通过western blotting实验分析ERK2的相对蛋白表达量，M@LPH组几乎看不见的条带。
L-M.如图12、图13所示，为了表征凝胶的微观结构，将实验例2制备得到的载药水凝胶水凝胶冷冻在-80℃冰箱过夜预冻，冷冻干燥24小时。在扫描电子显微镜下观察其表面形态。从中可以看出，水凝胶呈现出连续的孔状三维结构，这种结构使水凝胶具有了较大的比表面积，更有利于药物的扩散，且较大的孔隙尺寸可以为细胞提供足够的空间及为其传输营养和空气的流通。
N.如图14所示，使用流变仪测定水凝胶的流变学性能，进一步研究了其力学性能。在频率扫描并且在0.1至50Hz的测试频率范围内没有观察到交集，表明水凝胶在低频下均具有稳定的凝胶状状态，G′值高于G″值，表明水凝胶保持稳定的固体(弹性)状态。然后，进行时间扫描测试以确认水凝胶的稳定性。水凝胶在测试时间(0-500s)内可以保持稳定的G′和G″值。
O-P理想的术后抗粘连产品应该是可注射和自愈的，以方便外科医生手术，特别是腹腔镜手术。因此，水凝胶是否可注射是通过进行宏观物理挤出来确定的。简而言之，将制备的水凝胶块使用无菌针头直接挤压。如图15所示，可以看到，所获得的水凝胶可以很容易地通过针头，挤出部分可以快速无缝融合。为了更加具体评估水凝胶的自我愈合行为，进行宏观恢复实验，用来定性观察自愈过程。如图16所示，两个表面粗糙的水凝胶盘被罗丹明B和天青A染色。随后，在室温下沿着切割线保持两个不同颜色的半圆盘紧密接触，无需任何外部干预即可完全融合成一个整体的水凝胶圆盘，且表面也变得光滑。在愈合过程中，两种染料分子不断地在切割表面上扩散，并最终相互渗透，在边界处呈现紫色。
R.如图17所示，共聚焦观察MSCs分布情况，将MSCs用钙黄绿素标记后与凝胶混合，在共聚焦显微镜3D模式下观察，可见MSCs均匀分布在凝胶中。
S.如图18所示，通过细胞活力测定生物材料的细胞相容性用于体内应用。将凝胶用培养基在37℃条件下浸提24小时后，通过CCK8测定评估水凝胶的细胞相容性。MSCs在细胞在不同浓度的水凝胶浸提溶液下均显示出超过90％的细胞活力，表明水凝胶表现出良好的生物相容性。
T另一方面，水凝胶在作为腹膜创伤的屏障应用时直接接触伤口，因此必须考虑血液相容性。选择小鼠红细胞悬浮液来评估血液相容性。用水凝胶处理2h后，未观察到明显的溶血，所有水凝胶提取物的溶血率均低于5％，表明水凝胶具有良好的血液相容性，对术后粘附在体内是安全的，如图19所示。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种预防术后粘连与肿瘤复发的多功能水凝胶，其特征在于，包括：
细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒；
间充质干细胞；
透明质酸凝胶；
所述透明质酸凝胶中负载有所述细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒和间充质干细胞；
所述水凝胶的分子量为1000-200万；
间充质干细胞的浓度在1000-1000万/ml。
2.如权利要求1所述的预防术后粘连与肿瘤复发的多功能水凝胶，其特征在于，所述细胞膜包括：肿瘤细胞膜，间充质干细胞膜，红细胞、血小板或巨噬细胞膜。
3.如权利要求1所述的预防术后粘连与肿瘤复发的多功能水凝胶，其特征在于，所述细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒包括：脂质体、纳米粒、脂溶性化疗药物；所述脂质体中包载脂溶性化疗药物，所述脂质体内部包载纳米粒。
4.如权利要求3所述的预防术后粘连与肿瘤复发的多功能水凝胶，其特征在于，所述纳米粒由鱼精蛋白与透明质酸对核酸进行压缩而得，RNA包括100kd以下的siRNA、circRNA或shRNA。
5.一种权利要求1-4任一项所述的预防术后粘连与肿瘤复发的多功能水凝胶的制备方法，其特征在于，包括：
采用脂质体包载他莫昔芬，得到负载他莫昔芬脂质体；
将siRNA与透明质酸、DEPC水、鱼精蛋白涡旋混匀，得到siRNA纳米粒；
将所述siRNA纳米粒与负载他莫昔芬脂质体混合，孵育，得到LPH；
将细胞膜与LPH混合，挤压多次，得到细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒；
将透明质酸与透明质酸-醛基溶解在碱性溶液中，在交联剂存在条件下凝胶化，得到凝胶；
取所述凝胶与细胞膜脂质体-鱼精蛋白-透明质酸仿生纳米复合颗粒凝缩液、间充质干细胞混悬液混合均匀，即得。
6.如权利要求1所述的预防术后粘连与肿瘤复发的多功能水凝胶的制备方法，其特征在于，DOTAP与CHOL脂质摩尔比例在0.5-5：1；
或，NP与脂质体比例在1：100-2000。
7.如权利要求1所述的预防术后粘连与肿瘤复发的多功能水凝胶的制备方法，其特征在于，所述细胞膜与LPH以质量比1:1～20。
8.如权利要求1所述的预防术后粘连与肿瘤复发的多功能水凝胶的制备方法，其特征在于，所述siRNA与透明质酸、鱼精蛋白的摩尔比为1：1-3：1-3。
9.如权利要求1所述的预防术后粘连与肿瘤复发的多功能水凝胶的制备方法，其特征在于，所述透明质酸与透明质酸-醛基的质量比为50-100:0-50。
10.权利要求1-4任一项所述的预防术后粘连与肿瘤复发的多功能水凝胶在制备预防术后粘连与肿瘤复发医用材料或药物中的应用，其特征在于，负载细胞包括：来源骨髓、脂肪或脐带的干细胞、成纤维细胞、血管内皮细胞、及免疫细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311307736.9A CN117257723B (zh) | 2023-10-10 | 2023-10-10 | 预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311307736.9A CN117257723B (zh) | 2023-10-10 | 2023-10-10 | 预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117257723A true CN117257723A (zh) | 2023-12-22 |
| CN117257723B CN117257723B (zh) | 2024-05-10 |
Family
ID=89217549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311307736.9A Active CN117257723B (zh) | 2023-10-10 | 2023-10-10 | 预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117257723B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050181023A1 (en) * | 2004-02-17 | 2005-08-18 | Young Janel E. | Drug-enhanced adhesion prevention |
| KR20180047442A (ko) * | 2016-10-31 | 2018-05-10 | 케이비바이오메드 주식회사 | 경구투여용 간암표적성 siRNA 나노전달체 및 이의 제조방법 |
| CN110354311A (zh) * | 2019-08-30 | 2019-10-22 | 青岛大学 | 细胞外基质复合透明质酸凝胶及其制备方法、应用和生物材料 |
| US20200163885A1 (en) * | 2017-07-25 | 2020-05-28 | Lemonex Inc. | Composition for delivering physiologically active ingredients into blood vessel |
| US20220273851A1 (en) * | 2019-09-21 | 2022-09-01 | Bioregen Biomedical (Changzhou) Co., Ltd. | Disulfide crosslinked hyaluronic acid gel for postoperative abdominal (pelvic) adhesion prevention and preparation method thereof |
-
2023
- 2023-10-10 CN CN202311307736.9A patent/CN117257723B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050181023A1 (en) * | 2004-02-17 | 2005-08-18 | Young Janel E. | Drug-enhanced adhesion prevention |
| KR20180047442A (ko) * | 2016-10-31 | 2018-05-10 | 케이비바이오메드 주식회사 | 경구투여용 간암표적성 siRNA 나노전달체 및 이의 제조방법 |
| US20200163885A1 (en) * | 2017-07-25 | 2020-05-28 | Lemonex Inc. | Composition for delivering physiologically active ingredients into blood vessel |
| CN110354311A (zh) * | 2019-08-30 | 2019-10-22 | 青岛大学 | 细胞外基质复合透明质酸凝胶及其制备方法、应用和生物材料 |
| US20220273851A1 (en) * | 2019-09-21 | 2022-09-01 | Bioregen Biomedical (Changzhou) Co., Ltd. | Disulfide crosslinked hyaluronic acid gel for postoperative abdominal (pelvic) adhesion prevention and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| ALI FATEHI HASSANABAD等: "Prevention of Post-Operative Adhesions: A Comprehensive Review of Present andEmerging Strategies", 《BIOMOLECULES》, vol. 11, no. 7, 14 July 2021 (2021-07-14), pages 1 - 42 * |
| WEI TANG等: "Macrophage membrane-mediated targeted drug deliveryfor treatment of spinal cord injury regardless of themacrophage polarization states", 《ASIAN JOURNAL OF PHARMACEUTICAL SCIENCES》, vol. 16, no. 4, 31 July 2021 (2021-07-31), pages 459 - 470 * |
| YUESONG WU等: "Macrophage cell membrane-basednanoparticles: a new promisingbiomimetic platform for targeteddelivery and treatment", 《JOURNAL OF NANOBIOTECHNOLOGY》, vol. 20, 31 December 2022 (2022-12-31), pages 1 - 41 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117257723B (zh) | 2024-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yasin et al. | Advances in hyaluronic acid for biomedical applications | |
| Luo et al. | Development and application of hyaluronic acid in tumor targeting drug delivery | |
| ES2895947T3 (es) | Composición y conjuntos para matrices de microgel pseudoplástico | |
| EP3103485A1 (en) | Material comprising a polymer capable of forming a hydrogel and nanoparticles | |
| Chen et al. | Low-molecular-weight heparin-functionalized chitosan-chondroitin sulfate hydrogels for controlled release of TGF-β3 and in vitro neocartilage formation | |
| Mi et al. | Postsurgical wound management and prevention of triple-negative breast cancer recurrence with a pryoptosis-inducing, photopolymerizable hydrogel | |
| Li et al. | Fabrication of a multi-level drug release platform with liposomes, chitooligosaccharides, phospholipids and injectable chitosan hydrogel to enhance anti-tumor effectiveness | |
| Tong et al. | Supramolecular hydrogel-loaded Prussian blue nanoparticles with photothermal and ROS scavenging ability for tumor postoperative treatments | |
| Shokrani et al. | Polysaccharide-based nanocomposites for biomedical applications: a critical review | |
| Chang et al. | Self-healing pectin/cellulose hydrogel loaded with limonin as TMEM16A inhibitor for lung adenocarcinoma treatment | |
| CN110755365A (zh) | 基于间充质干细胞外泌体的水凝胶及其喷雾剂的制备方法 | |
| Li et al. | Temperature-and pH-responsive injectable chitosan hydrogels loaded with doxorubicin and curcumin as long-lasting release platforms for the treatment of solid tumors | |
| CN111097070B (zh) | 一种用于抑制肿瘤和促进修复的可注射生物活性水凝胶 | |
| Vegad et al. | pH stimuli-responsive hydrogels from non-cellulosic biopolymers for drug delivery | |
| Ilomuanya | Hydrogels as biodegradable biopolymer formulations | |
| Zhao et al. | Bioabsorbable nano-micelle hybridized hydrogel scaffold prevents postoperative melanoma recurrence | |
| Fan et al. | Hydrogel-exosome system in tissue engineering: A promising therapeutic strategy | |
| Zhao et al. | Preparation and evaluation of oxidized-dextran based on antibacterial hydrogel for synergistic photodynamic therapy | |
| US12048715B2 (en) | Cis-platinum cross-linked protein hydrogel and preparation method thereof | |
| CN117257723B (zh) | 预防术后粘连与肿瘤复发的多功能水凝胶制备方法与应用 | |
| Lugasi et al. | Targeted delivery of CBD-loaded poly (RGD) proteinoid nanoparticles for antitumor therapy | |
| CN116370402A (zh) | 一种药物可控释放的原位纳米复合水凝胶的制备方法 | |
| Yang et al. | Evaluation of cytotoxicity and anti-angiogenesis induced by crocetin-delivering alginate hydrogel/chitosan nanoparticle carrier system on human ovarian cancer cell line (A2780): an in vitro evaluation | |
| Moscovici et al. | Bacterial polysaccharides versatile medical uses | |
| Wang et al. | Preparation of genetically or chemically engineered exosomes and their therapeutic effects in bone regeneration and anti-inflammation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |