Kanser Tedavisinde Gen Susturulması İçin Hyalüronik Asit-Polietilenimin Taşıyıcı Sisteminin Geliştirilmesi

Abstract

Target-specific delivery of small interfering siRNAs in cancer treatment is one of today's leading research topics. Within the scope of the thesis, effective transport of siRNAs via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low molecular weight polyethyleneimine (PEI) based transport systems. Within the thesis study, gold nanoparticles which are inorganic based nanoparticles capable of giving photothermal response and their conjugates with PEI and HA are added to the structure, which is first in the literature. As a consequence, a combination of gene silencing, photothermal therapy and chemotherapy, which is one of the most important features of this thesis, has been provided. In the first part of the thesis study, PEI and HA-based siRNA delivery systems were synthesized and characterized. The dimensions of the PEI-HA, PEI-SS-HA, AuNP, AuPEI NP, AuPEI-HA and AuPEI-HA-DOX particles were measured as, 330 nm, 203 nm, 91 nm, 25 nm, 366 nm and 692 nm, respectively. The formation of conjugates was confirmed by 1H-NMR, FT-IR and elemental analysis methods. The electrostatic interaction of the synthesized conjugates with siRNA was determined by the gel retardation analysis and the change in zeta potentials. In the second part of the study, effects of the conjugates on the viability of target cells (L929 mouse fibroblast cell line, MDA-MB-231 breast cancer cell line and CAPAN-1 pancreatic cancer cell line) were determined by the WST-1 cell viability test; in general (except AuPEI NP) the cell viability was above 50% when the synthesized particles (100 µg/mL) were applied. In the application of radiation following the conjugate/siRNA complex (especially those containing Au), application increased the cytotoxic effect on the MDA-MB-231 cell line. Apoptotic-necrotic effects of conjugates were investigated by double staining method, and overall apoptotic effect was low in all three cell lines. However, the necrotic effect was highest in both MDA-MB-231 and CAPAN-1 cell lines, after administration of the AuPEI-HA-DOX conjugates (50% and 25%, respectively). The effects of the complexes of the synthesized conjugates and siRNA on the cell proliferation were also investigated by RTCA real-time cell proliferation analysis. Antiproliferative effect was not observed in L929 cell line in accordance with cytotoxicity results, whereas the highest antiproliferative effects in MDA-MB-231 and CAPAN-1 cell lines were observed after administration of PEI-HA/siRNA, PEI-SS-HA/siRNA, AuPEI NP/siRNA and AuPEI-HA-DOX/siRNA complexes. The effects of suppression of target gene expression in cancer cell lines were investigated by Real Time-PCR method. All conjugates or complexes in MDA-MB-231 cell line were able to suppress target gene expression of AuPEI NP/siRNA complex in CAPAN-1 cell line. It was found that all conjugates or complexes in the MDA-MB-231 cell line, AuPEI NP/siRNA complex in the CAPAN-1 cell line could suppress the target CXCR4 gene expression. All these data show that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.