Influence of physical properties of polycaprolactone nanofibers on their cytotoxicity

Nanotehnologija predstavlja enega izmed najbolj zanimivih področij raziskav v začetku 21. stoletja. Pri tem imajo nanovlakna vedno večjo vlogo na področju naprednih dostavnih sistemov, tkivnega inženirstva in obnavljanja tkiv. Nanovlakna so trdna vlakna s premerom v nanometrskem območju in teoretično neomejeno dolžino. V literaturi najdemo veliko podatkov o nanovlaknih, a je njihova varnost nanovlaken doslej malo raziskana, zato je to vsebina moje magistrske naloge. Cilj raziskav je bil opredeliti fizikalne lastnosti nanovlaken, ki bi lahko citotoksično vplivale na celice v stiku z njimi. Za izdelavo nanovlaken, ki smo jih pripravili z elektrostatskim sukanjem, smo uporabili polimer polikaprolakton. Najprej smo v preliminarnih raziskavah definirali vpliv lastnosti polimerne raztopine, procesnih in okoljskih parametrov na proces elektrostatskega sukanja in morfologijo nanovlaken. Z načrtovanjem eksperimentov smo izdelali model, s katerim smo ugotovili, pri katerih parametrih lahko pripravimo nanovlakna določenih premerov in velikosti por med njimi. Lastnosti nanovlaken smo ovrednotili na več različnih načinov: z optičnim in vrstičnim elektronskim mikroskopom smo določili premer nanovlaken, površino por med nanovlakni ter pridobili natančne informacije o izgledu membrane nanovlaken na nanomerilu. Z metodo merjenja stičnega kota smo preučili hidrofilne in hidrofobne lastnosti membrane nanovlaken ter na koncu izbrana nanovlakna ovrednotili še z dinamično diferenčno kalorimetrijo in termogravimetrično analizo. Nanovlakna smo testirali na celicah limfocitov in preučevali metabolno aktivnost limfocitov s testom metabolne aktivnosti ter citotoksičnost nanovlaken s testom laktat dehidrogenaze. Pri raziskavah na limfocitih smo testirali vpliv premera nanovlaken, velikosti por, prisotnosti vozlov, vpliv urejenosti nanovlaken, različne debeline in različne poroznosti membrane. Ugotovili smo, da velikost por in debelina membrane nanovlaken iz polikaprolaktona vplivata na metabolno aktivnost limfocitov, zaradi mehanske imobilizacije celic, kar lahko privede do citotoksičnosti ali manjše metabolne aktivnosti. Rezultati, ki smo jih dobili, prispevajo k poglobljenemu razumevanju vplivov nanovlaken na limfocite in njihove citotoksičnosti.Nanotechnology is one of the most interesting research area of the early 21st century and nanofibers are playing an increasingly important role in advanced delivery systems, tissue engineering, and tissue regeneration. Nanofibers are solid fibers with a diameter in the nanometer range and a theoretically unlimited length. Literature data about nanofiber are plentiful, however, a few research has been done on their safety, therefore, this is the subject of my master\u27s thesis. The aim of the study was to determine the influence of physical properties of nanofibres that could have a cytotoxic effect on the cells present. Polycaprolactone polymer was used to produce nanofibers, which were made by electrospinning. Firstly, we conducted preliminary studies to determine the key factors of the polymer solution, process and environmental parameters on the electrospinning and the morphology of the nanofibers. Secondly, using Design of experiments method, we established a model to define parameters at which nanofibers with specific diameters and pore sizes between them can be produced. The properties of the nanofibers were evaluated in several ways: The diameter of the nanofibers, the surface area of the pores between the nanofibers and accurate information about the nanofibres morphology were obtained using optical microscopy and scanning electron microscopy. The hydrophilic and hydrophobic properties of the nanofiber scaffold were investigated by the contact angle measurement method and finally, the selected nanofibers were evaluated by thermal analysis. The nanofibers were tested on lymphocyte cellstheir metabolic activity was investigated by MTS assay and their cytotoxicity was investigated using LDH assay. In studies on lymphocytes, we tested the influence of nanofiber diameter, pore size, different thicknesses and different membrane porosity, influence of nanofiber allignment and presence of nodules. We discovered that the pore size and membrane thickness of polycaprolactone nanofibers affect the metabolic activity of lymphocytes, due to the mechanical immobilization of cells, which leads to cytotoxic effect or decreased metabolic activity. The results obtained with this study will help to better understand effect of nanofibers on lymphocytes and their cytotoxicity