Uzma Azeem Awan
Gene silencing activity of a specific oncogene transcript to reduce expression of encoded protein by complementary base-pairing mechanism is a potential molecular therapeutic approach. Systemic delivery of therapeutic oligonucleotides (oligos) is one of the major limitations due to lack of stability, poor cellular uptake, degradation and reduced efficacy associated with RNA interference (RNAi) technology. Gene interference therapeutic strategies seek safe and efficient delivery vehicles. Hepatocellular carcinoma (HCC) is the 6th most common cancer worldwide. Abnormally elevated expression of cyclooxygenase-2 (COX-2) has been frequently observed to regulate tumor growth, invasion and metastasis in HCC. Here we have designed, a nano-therapeutic plasmonic carriers comprising gold nanorods (GNRs) which incorporate and release COX-2 interfering conjugated oligos upon illumination with near infrared (NIR) continuous wave (CW) laser (800 nm) at specific intracellular location. HepG2 cells were used to determine cellular uptake and gene silencing activity by GNRs conjugated fluorescence labeled single and double stranded oligos irradiated with NIR laser. Gene knock down was demonstrated and validated by fluorescence repression and western blot analysis. Effect of COX-2 knockdown on downstream molecular pathway was determined. Applying the optimized parameters transfection efficiencies of 78% were achieved in cells using a fluorescent labeled single and double stranded oligos while maintaining a high cell viability of >81%. Dark field microscopy, confocal microscopy and fluorescence microscopy showed significant preferential uptake of nanobiocomposite by the cells. In vitro laser triggered delivery of nanobiocomposite resulted in 81% down regulation of targeted COX-2 protein. Specific inhibition of COX-2 at translation level significantly affects downstream molecular pathway involved in cancer. Our findings emphasize that gold nanoparticle mediated laser transfection provides a potential gene interfering technique with spatial and temporal control, a novel molecular therapeutic approach for cancer treatment.
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