Theranostic use of K-RAS siRNA-loaded magnetic nanoparticles in colon cancer

Cancer is a disease that is frequently seen today and reduces the quality of human life. KRAS is knowing GTPase protein that can activate many proteins such as c-Raf and PI3K in the EGFR signaling pathways. Active mutation of KRAS causes cancer and KRAS mutations are frequently seen in colon cancer. Nanobiotechnology or Nanomedicine is the most developed technology in the field of health today due to the transport of drugs and sending them to the target region. Strategies for silencing mutated genes hold some therapeutic hopes. In this study, it was aimed to integrate new synthesis super magnetic iron oxide nanoparticles (SPION) into liposome nanoparticles as part of the gene transport system for KRAS gene silencing. Firstly, supermagnetic iron oxide nanoparticles were synthesized by co-precipitation method and KRAS siRNA was encapsulated together with, supermagnetic iron oxide nanoparticles by a cationic liposome prepared as DSPC:DC-Cholesterol:PEG. The synthesized nanoparticle was characterized by TEM, FTIR, XRD,VSM and Zeta Potential. Silencing of KRAS gene, BCL-2 and BAX gene expression, cytotoxicity, wound healing and apoptosis tests were performed in Caco-2 and HT29 colon cancer cell lines with different dose rates of siRNA loaded magnetoliposomes. The Mission siRNA transfection agent used as positive control for both Caco-2 and HT29, for Caco-2 cells Mission siRNA TA was the highest rate of silencing of the KRAS gene was 65%. Following the Mission siRNA TA gene silencing was observed as %58 in sample No:4 compared to the control. The lowest silencing rate of 39% was observed in sample No:3. For HT29 cells, the highest rate of suppression of the KRAS gene is 72% by Mission siRNA TA. It was observed that the gene suppression was 64% by No:1 sample and other samples closed to the No:1 sample. Considering the BCL-2 and BAX gene expresion, it was found that Caco-2 cells lead to apoptosis while HT29 cells which was observed that cancer specifically escaped apoptosis. Considering the cytotoxic effect of MTT on Caco-2 cells that cells death is observed at all doses at the 48th hour and this decrease is followed by the 72nd hour values. Cytotoxic effect of MTT for HT29 cells observed that all doses showed cytotoxicity at 72 hours. Caco-2 wound healing values that all doses showed a small effect while most affect in samples No:1 and No:4 to compare with other samples. Wound healing for HT29 cells was observed to close at the latest in samples No: 3 and No: 4. In the apoptosis values determined by fluorescent microscope with Annexin v, it was observed that there was more necrosis in the positive control and sample No: 4 for Caco-2 cells and the rate of late apoptosis was higher in sample No:4 for HT29 cells.