Vývoj nanovlákenných nosičů pro biomedicínské aplikace
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Disertační práce je věnována vývoji a testování materiálu pro enzymatický debridement. Konkrétně se jedná o optimalizaci postupu imobilizace proteolytických enzymů na křemičitá nanovlákna. Tímto způsobem upravená nanovlákna mají sloužit k bezbolestnému odstranění nekrotické tkáně díky proteolytické aktivitě enzymů, která katalyzuje odštěpení nekrózy od zdravé spodiny rány. Hlavním úkolem je na základě rozsáhlé rešerše, dostupných technik a analyzačních přístrojů vytvořit vhodný postup kovalentní imobilizace enzymů na nanovlákna a vybrat nejlepší proteolytický enzym. Zvolený postup byl vždy porovnáván s již známým, ale pro medicínu nevyhovujícím postupem, který využívá (3-Aminopropyl)triethoxysilan a glutaraldehyd. Z hlediska techniky imobilizace byl nejpodrobněji testován postup s využitím (3-Aminopropyl)triethoxysilanu, sukcinanhydridu a následně esteru vytvořeného z N-hydroxysukcinimidu a 1-Ethyl-3-(3-dimethylaminopropyl)karbodiimidu. Protože výsledky testů cytotoxicity prováděných na myších 3T3 fibroblastech nebyly v případě výše uvedeného postupu uspokojivé, byl vyvinut ještě další postup imobilizace, ve kterém byl 1-Ethyl-3-(3-dimethylaminopropyl)karbodiimid nahrazen N,N-Disuccinimidyl karbonátem.V druhé části výzkumu jsem imobilizovala na nanovlákna postupně 7 proteolytických enzymů. Jejich proteolytická aktivita byla měřena pomocí spektrofotometrických metod a byla testována za různých podmínek - změny hodnoty pH, teploty, vlivu teploty na stabilitu enzymů apod. Jako nejvhodnější enzym byl označen trypsin z hovězího pankreatu, výborných výsledků bylo dosaženo také s proteázou z Aspergillus oryzae.Účinnost vytvořených nanovláken s imobilizovaným enzymem byla ověřena pomocí in vitro testů na prasečí kůži. Úspěšná imobilizace byla potvrzena v rámci testů stability vzorků - při dodržení navrhovaných podmínek skladování, trypsin i po 180 dnech vykazoval více jak 90% proteolytickou aktivitu.
The dissertation is devoted to the development and testing of the material for an enzymatic debridement. Specifically, it is about optimizing the process of proteolytic enzymes immobilization onto silica nanofibers. The nanofibers treated in this way are intended to painlessly removal of the necrotic tissue due to the proteolytic activity of the enzymes, which catalyze the cleavage of necrosis from the healthy base of the wound.The main aim is to optimaze a suitable procedure for the covalent immobilization of enzymes onto nanofibers and to choose the best proteolytic enzyme on the basis of an extensive testing, available techniques and analysis devices. The chosen procedure was always compared with the already known but unsuitable procedure for medicine, which uses (3-Aminopropyl) triethoxysilane and glutaraldehyde. In terms of the immobilization proces, the procedure using (3-Aminopropyl) triethoxysilane, succinic anhydride and subsequently an ester formed from N-hydroxysuccinimide and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide was tested in the most detail. Because the results of the cytotoxicity assays performed on mouse 3T3 fibroblasts were not satisfactory, another immobilization procedure was developed in which 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide was replaced by N,N'-Disuccinimidyl carbonate.In the second part of the research, I immobilized 7 proteolytic enzymes onto nanofibers surface. By spectrophotometric methods was measured their proteolytic activity and was tested it under various conditions - changes of pH value and temperature, the effect of temperature on the enzymes stability, etc. Trypsin from bovine pancreas was identified as the most suitable enzyme, excellent results were also obtained with the protease from Aspergillus oryzae. The efficacy of the created nanofibers with immobilized enzyme was verified by in vitro tests on pig skin. Successful immobilization process was confirmed in sample stability tests - under the proposed storage conditions, trypsin showed more than 90% proteolytic activity even after 180 days from immobilization.
The dissertation is devoted to the development and testing of the material for an enzymatic debridement. Specifically, it is about optimizing the process of proteolytic enzymes immobilization onto silica nanofibers. The nanofibers treated in this way are intended to painlessly removal of the necrotic tissue due to the proteolytic activity of the enzymes, which catalyze the cleavage of necrosis from the healthy base of the wound.The main aim is to optimaze a suitable procedure for the covalent immobilization of enzymes onto nanofibers and to choose the best proteolytic enzyme on the basis of an extensive testing, available techniques and analysis devices. The chosen procedure was always compared with the already known but unsuitable procedure for medicine, which uses (3-Aminopropyl) triethoxysilane and glutaraldehyde. In terms of the immobilization proces, the procedure using (3-Aminopropyl) triethoxysilane, succinic anhydride and subsequently an ester formed from N-hydroxysuccinimide and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide was tested in the most detail. Because the results of the cytotoxicity assays performed on mouse 3T3 fibroblasts were not satisfactory, another immobilization procedure was developed in which 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide was replaced by N,N'-Disuccinimidyl carbonate.In the second part of the research, I immobilized 7 proteolytic enzymes onto nanofibers surface. By spectrophotometric methods was measured their proteolytic activity and was tested it under various conditions - changes of pH value and temperature, the effect of temperature on the enzymes stability, etc. Trypsin from bovine pancreas was identified as the most suitable enzyme, excellent results were also obtained with the protease from Aspergillus oryzae. The efficacy of the created nanofibers with immobilized enzyme was verified by in vitro tests on pig skin. Successful immobilization process was confirmed in sample stability tests - under the proposed storage conditions, trypsin showed more than 90% proteolytic activity even after 180 days from immobilization.
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Debridement; Imobilizace enzymů; Proteolytický enzym; Křemičitá nanovlákna; Odstranění nekrózy, Debridement; Immobilization of enzymes; Proteolytic enzyme; Silica nanofibers, Removing of necrosis