Development of Jak1 Sirna Medıated Nanotherapeutıcs for Non-Melanoma Skın Cancer Therapy

Abstract

Non-melanoma skin cancer is one of the most common cancers with still no effective treatment. Due to the side effects observed after long and high dose treatments and acquired resistance to the chemotherapy drugs, different attempts have been made to overcome these problems and to provide more effective treatment with low dose usage. With successful results and advances in gene therapy, it has been observed that concurrent gene silencing and drug administration may be a better approach in cancer treatment compared to drug/drug combination therapies. The main purpose of this study was to investigate the therapeutic efficiency of liposomal formulation for delivery of both JAK1 siRNA and 5-Fluorouracil (5-FU) in non-melanoma skin cancer. For this purpose, combined nanoliposome formulations containing both 5-fluorouracil (5-FU), a cytotoxic chemotherapeutic agent, or JAK1 (Janus kinase 1) siRNA have been prepared and characterized in terms of size, polydispersity, zeta potential, morphology, encapsulation efficiency, cytotoxicity, and stability. Prepared liposomal nanoformulations were uniform and had a size of 80-120 nm, the PDI below 0.3, and remained stable for a period of 3 months. Positive zeta potential of liposomal nanoformulations successfully internalized across cell membrane, and delivered 6-FAM or cy5 labeled siRNA to the cells even within 30 minutes of incubation, and could remain in the A-431 cells as observed under fluorescence or confocal microscope. Encapsulation or hydrophilic 5-FU in the nanoliposomes was achieved at different drug/lipid ratios, and showed the highest encapsulation efficiency at the 0.2 drug/lipid ratio, and had the 1st order release kinetics. In the evaluation of nanoliposome formulations toxicities, cells express viability over 80 % for the treatment concentrations up to 5x103 µg/mL after 24 hours of incubation. Prolonged incubation reduced cell viability to 70 % at the concentration of 5x103 µg/mL. Delivery of 5-FU using nanoliposome formulations resulted in a significant reduction of cell viability after 24 hours incubation at the 5-FU concentration of 5 µM and above (p<0.05). Evaluation of JAK1 mRNA expression was done using RT-qPCR, proceeding with relative protein expression using Western Blot analysis. Nanoliposome formulations containing JAK1 siRNA were effective for in vitro gene silencing, by inhibition of mRNA expression by 50 %. Similarly, JAK1 protein level was also reduced after treatment with JAK1 siRNA nanoliposomes. After finalizing in vitro gene knockdown studies, in vivo studies have been initiated. Treatment efficacy with the most optimal formulations was evaluated in nude male mice. First of all, the tumor model was formed with inoculation of 1X106 to the right flank region of mice and after tumor volume reached approximately 50-100 mm3, intratumoral administration of nanoliposome formulations containing 5-FU or JAK1 have been initiated. Nanoliposomes formulations containing either 5-FU, JAK1 siRNA or both, significantly suppressed the tumor volume increase compared to the control group (p<0.05), showing the tumor inhibitory rate of approximately 80 % after 28 days of treatment. With relatively less tumor areas observed, 5-FU or siRNA containing groups also reduced the expression of JAK1, ERK, AKT, pAKT, pERK proteins. In addition, JAK1-siRNA loaded nanotherapeutics strongly induced apoptosis in vivo. Based on these findings, the delivery of JAK1 siRNA and 5-FU by developed nanoliposomes appears to be a promising treatment strategy for non-melanoma skin cancer.