Collagen/keratin/alginate based hydrogel delivering micro-RNA to modulate the angiogenic properties on human umbilical vein endothelial cells (HUVECs)

In the last years, the integration of genome editors and regulatory ribonucleic acids (RNAs) with biomaterials has gained attention, especially in the field of therapeutics and personalized medicine. This thesis study aimed to develop a functional, biocompatible, and micro-RNA carrying hydrogel based on collagen, keratin, and alginate for the modulation of angiogenesis. For this purpose, firstly keratin and collagen were isolated from waste human hair and bovine Achilles tendon, respectively. For both isolated proteins, all the characteristic peaks of keratin and collagen were observed by Fourier transform infrared spectroscopy (FT-IR). Two major bands were observed in human hair keratin samples at approximately 45 kDa and 63 kDa, while collagen samples gave four bands at around 300 kDa, 245 kDa, 135 kDa, and 100 kDa on SDS-PAGE. Col/Ker/Alg hydrogels with different ratios were prepared and their chemical, microstructural properties, percent porosity, in vitro hydrolytic degradation behavior, water uptake properties, in vitro protein release behaviors, and biocompatibility were characterized to optimize the hydrogels for the preparation of PEI:miR-21 plasmid complex loaded hydrogels for subsequent investigations. The hydrogel group consisting of 1% w/v of each component showed the highest swelling rate and porosity percentage (80.89%), the lowest degradation rate (only 15.19% at the end of 21 days), and better biocompatibility. The miR-21 expression vector was loaded onto this hydrogel group to be used as a gene-activated matrix (GAM) to observe the effects on HUVECs. To observe the effects of the complex and hydrogels on HUVECs, fluorescent microscopy, confocal laser scanning microscopy (CSLM), and RT-qPCR for gene expression analysis were conducted. miR-21, pro-angiogenic factor VEGFA, direct targets of miR-21 and PTEN, SPRY1, and SPRY2 were examined. In vitro studies revealed that hydrogels and over-expression of miR-21 were not toxic on cells and provided a successful transient transfection of HUVECs by observing green fluorescent protein (GFP) reporter. The migration of HUVECs into the hydrogel and the transfection was observed by CSLM and the GFP fluorescence signal increased from day 1 to day 4. Gene expression results for miR-21 were higher in the transfected groups (TCP control and PEI: plasmid loaded hydrogels) as expected. PTEN, SPRY1, and SPRY2 gene expression decreased in transfected groups since they are the direct targets of miR-21, yet VEGFA gene expression increased in those groups compared to the negative controls. In addition, regular tubular sprouting as seen in angiogenesis was not observed in any experimental groups.