Development of gene silencing method with RNA i̇nterference in target cells usi̇ng poly lactic-co-glycolic acid nanoparti̇cles

Pancreatic cancer is considered among the highest cancer incidence and mortality in the world and in Turkey. These data indicate that existing treatment strategies are not sufficiently effective. In spite of developed modern methods, pancreatic cancer, can be treated with surgical methods only. In addition, chemotherapy drugs used for therapeutic purposes can not target the tumor and have a harmful effect on healthy tissues; these methods have several disadvantages, such as radiotherapy used for therapeutic purposes, resulting in loss of function in healthy tissue exposed to radiation. Traditional treatment methods for cancer treatment are replaced by new generation therapies specific to the tumor. Targeted therapies are of great interest of scientific authorities as they do not harm healthy cells and have high selectivity to the tumor tissue. The RNA interference (RNAI) technique is a natural, biological process in which gene expression is inhibited as a result of the destruction of specific mRNA molecules that are complementary to the cytoplasm of the single strand of double stranded miRNA encoded by DNA in the nucleus. The binding of antisense-acting molecules to the target mRNA inhibits the expression of the gene. In the RNAi mechanism, a variety of molecules such as microRNA (miRNA) and small interfering RNA (siRNA) are used which exhibit an antisense effect. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles; used for diagnosis and treatment of cancer; shows superior properties such as characteristic biodegradability, high biocompatibility, low toxicity compared to other nano drug delivery systems. It is possible to silence the gene at the level of mRNA with the siRNA loaded into the PLGA nanoparticles. The GPR87 (G protein-coupled receptor 87) gene is the gene encoding the cell surface receptor included in the GPCR (G protein-coupled receptors) family. The GPR87 gene, which is overexpressed in pancreatic cancer, plays an important role in maintaining the viability of cells. Silencing of the gene using the GPR87 gene, siRNA-loaded PLGA nanoparticles, significantly affects the survival of the cells. In this study, the GPR87 gene was targeted by PLGA nanoparticles in different cells, and the gene was aimed to be post transcribed by the RNAi mechanism. For this purpose, the GPR87 gene was isolated from the HEK293T cell line, and the gene isolated polymerase chain reaction (PCR) amplification was performed with the gene-specific primers. The PCR product and the psiCHECK2 vector were ligated by XhoI. The GPR87- psiCHECK ™ -2 recombinant vector was transformed into E. coli One Shot® Mach1 ™ -T1R cells. Transformation control was controlled by PCR, agarose gel electrophoresis and bioinformatics. Then, the generation of siRNA specific to the GPR87 gene sequence was performed. The first generation of siRNA production was carried out by the oligonucleotide template design. After hybridization with this template T7 promoter primer was achieved, polymerization with Klenow DNA polymerase was performed to obtain dsRNA. In the second stage of the thesis, the synthesis, characterization, optimum release time and toxicity of PLGA nanoparticles were determined. For this purpose, PLGA nanoparticles were first prepared by water-oil-water (W1 / O / W2) double emulsion solvent evaporation method. Using different techniques, nanoparticles were prepared in three different formulations and siRNA was loaded into these nanoparticles. Characterization of the obtained siRNA-PLGA nanoparticles were performed, and the efficiency of the Encapsulation efficiency (% EE) and loading efficiency (LE) were calculated. In addition, the in vitro release process of the generated nanoparticles was optimized and cytotoxicity was measured. In the final stage, the gene silencing caused by nanoparticles at human embryonic kidney cell line HEK293T and pancreatic cell line 1.1B4 were determined by luciferase activity. The gene silencing results obtained from siRNA-PLGA nanoparticles produced using three different protocols were not equal. It was determined that the nanoparticles had high encapsulation and loading efficiency, the in vitro release time was optimum, and the prepared siRNA-PLGA nanoparticles caused a complete post-transcriptional silencing of the gene which consequently caused %15 cell death.