Development of combination products prepared with different nanoparticular systems and polymeric nanofibers

In this thesis, melatonin loaded multilayer nanoparticles were prepared by using layer-by-layer coating technology of nanoparticles and nanofibers were prepared by electrospinning method for modular wound healing applications. For this purpose, nanofibers were modified and functionalized with the FNIII9*10 recombinant peptide sequence, consisting of 9th and 10th type III repeats of fibronectin. Combined wound care products were prepared by combining multilayer nanoparticles with FNIII9*10 functionalized nanofibers. Multilayer nanoparticles were prepared by layer coating technology of nanoparticles with three different core materials using two different methods. To achieve that, silver nanoparticles and poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles were studied. Melatonin loading into both the core and the layers has been tried separately. Maxiumum melatonin encapsulation was achieved with PLGAMEL1 coded nanoparticles with a 11.44% encapsulation value and then this nanoparticles were coated with chitosan. Chitosan coated and melatonin loaded PLGA multilayerd nanoparticles (PLGAMEL1/CHI) were preapared as proposed formulation with a narrow particle dispertion value of 0.15, particle size of 280 nm and a zeta potential of +56 mV in phosphate buffer. In vitro dissolution studies revealed that all melatonin content in nanoparticles were released in 3 hours. Polymers, such as polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polycaprolactone (PCL) were used to obtain nanofibers via electrospinning method. PCL nanofibers were functionalized by FNIII9*10 recombinant peptide and protein quantification studies revealed that 53.9% of the peptide was succesfully attached to the nanofibers. Melatonin loaded PCL nanofibers were also prepared and an in vitro release study was performed for 5 days. Melatonin loaded PCL nanofibers showed a slower melatonin release than melatonin loaded PLGA nanoparticles. No significant changes were observed in drug content of melatonin loaded PCL nanofibers during 2 months stability testing in 4°C, 25°C 60% and 40°C 75% relative humidity conditions. Multilayer nanoparticles were combined with FNIII9*10 functionalized PCL nanofibers using lyophilization technique as one of the proposed final formulation. For this purpose, PLGAMEL1/CHI multilayered nanoparticle dispersion was added and mixed with FNIII9*10 functionalized PCL nanofibers in multiwell plates seperately and then the water content was lyophilized for two days to achieve the final wound healing scaffold. Melatonin loaded PCL nanofibers and FNIII9*10 functionalized PCL nanofibers were also evaluated as final formulations and cell culture studies were performed with these 3 final formulations. MRC-5 human lung fibroblast cell line was used to determine the proliferative dose of melatonin. It was observed that significant cell proliferation starts from 125 µM melatonin application in this cell line. Final formulations consisting of 250 µM melatonin was prepared and biocompatibility studies were performed in human skin fibroblast, BJ and immortalized human keratinocyte, HACAT cell lines. No cytotoxicity effect was observed with the proposed final formulations. In fact, all formulations significantly improved cell proliferation values compared to the control. The adhesion of the cells to the nanofibers was demonstrated by fluorescence and electron microscopy. In vitro wound closure assay was performed using 2-well silicone inserts using solutions of FNIII9*10, melatonin and their mixture on cell culture plates. It was observed with optical imaging studies that, FNIII9*10 only and FNIII9*10 and melatonin mixture applications fully closed the wound area in 24 hours. Finally, wound dressings that mimic ESM both physically and biochemically are successfully prepared and their activity and efficiency were demonstrated using in vitro cell culture studies.