Development of A Gene-Knockdown Approach Based on Sirna/Gold Nanopartıcles for Breast Cancer Therapy

Abstract

Breast cancer is considered to be one of the leading causes of death in the 21st century and has claimed the lives of many people. As one of its subtypes, triple-negative breast cancer (TNBC) is a devastating disease, owing to the aggressive course, early metastasis, drug resistance, and poor clinical outcome and patient survival. Recently, different gene therapy approaches such as the use of siRNAs have brought hope for the treatment of TNBC. Although siRNA-based therapeutics offer tremendous potential as targeted therapies, their low transfection efficiency, and in vivo degradation by serum endo and exo-nucleases have prevented their delivery into tumors and made that an important obstacle for their translation into the clinic. So, the development of safe and effective delivery systems with robust target knockdown is of paramount importance. To address this problem, in this thesis it was aimed to develop a novel gold nanoparticle/siRNA based nanotherapeutic for gene knockdown approach in triple negative breast cancer. In the first and second sections of this thesis, it was aimed to develop gold nanoparticle based delivery vehicles and evaluate their cellular biodistribution. In this regard, highly stable and monodisperse polyethylenimine (PEI) functionalized gold nanoparticles were synthesized by a new method of surface modification. For the first time in literature, it was found that PEI functionalized gold nanoparticles in the size range of 45-82 nm, can enter to the cell nucleolus even in postmitotic dorsal root ganglion (DRG) neurons which are known to be very difficult cell types to transfect. It was shown that PEI functionalization increased the uptake rate of gold nanoparticles and delayed the fast excretion rate. Also, it was observed that PEI functionalization increased the emission signal intensity of gold nanoparticles. With a great potential for theranostic applications, the cellular biodistribution of nanoparticles was clearly visualized under both confocal and two-photon microscopes. In the third section, it was aimed to develop a siRNA therapeutic for TNBC therapy based on PEI-functionalized gold nanoparticles. With this purpose, eukaryotic elongation factor 2 kinase (eEF-2K) was chosen as the therapeutic target and conjugated with nanoparticles. Prepared siRNA therapeutic was highly monodisperse (PDI of 0.1) and stable. It was shown that this siRNA therapeutic was highly effective for eEF-2K gene down-regulation in vitro and in vivo and showed remarkable antitumor efficacy that was associated with eEF-2K knockdown, inhibition of Src and MAPK-ERK signaling pathways in a TNBC orthotopic tumor model. A significant tumor inhibition (90%) was achieved by only one intravenous injection of siRNA therapeutic per week at a relatively low dose of 8 µg/mouse (0.3 mg/kg), which is below the limit of any potential toxicity. Finally, siRNA therapeutic conjugated with doxorubicin was developed for combinational therapy approach which decreased the cell viability significantly in compare to sole doxorubicin chemotherapeutic drug. In conclusion, a potent theranostic gold nanoparticle based gene delivery vehicle was developed and used in the preparation of eEF-2K siRNA based therapeutics. It was concluded that prepared eEF-2K siRNA therapeutics are promising for TNBC treatment and imaging.