COX-2 RNA INTERFERENCE BY OLIGONUCLEOTIDES ON A NANOPLASMONIC CARRIER-BASED OPTICAL SWITCH (ONCOS) IN HEPATOCELLULAR CARCINOMA

Abstract

In this dissertation, nanoplasmonic optical antennae (gold nano particles) are utilized as nanoplasmonic gene switches for on-demand and systematic gene regulation in living systems. The plasmon resonance of nanoplasmonic gene switches (gold nanorods) is specifically tuned to the near infrared spectral region where cells and tissues are essentially transparent. Cyclooxygenase-2 (COX-2) is an immediate early response gene in various cancers and is one of the most critical enzymes in tumor metastasis. COX-2 non-selective inhibition as well as inhibition of the constitutive isozyme COX-1 by selective and non selective Non-steroidal anti-inflammatory drugs (NSAIDs), causes several adverse effects like renal, cardiac and gastrointestinal toxicity. Therefore COX-2 inhibition should be localized and very specific without inhibiting COX-1. Conventional gene-interfering techniques lack spatial and temporal control. This study employed RNAi to specifically knockdown endogenous COX-2 expression in the HepG2 (hepatocellular carcinoma) and MDA-MB-231 (breast cancer) cell lines using nano plasmonic optical switches and NIR laser. Nanoparticles, gold nanorods (GNRs) are successfully prepared with tunable longitudinal plasmon resonance (LSPR) wavelengths between 682-906 nm. The hydrodynamic diameter of the GNRs was estimated to be 20.90±2.15 to 50.5±2.13 nm. The zeta potential of the prepared GNRs was measured as +33.3± 0.34 -40.1±0.71 mV. We tailored the aspect ratios of GNRs between 2.5 to 4.6 by optimizing growth conditions that include concentrations of reactants continuous stirring at acidic pH. Fine tuning of LSPR is achieved across a broad range by varying silver ion and seed concentration. Colloidal stability of GNRs was studied through UV-vis spectroscopy based particle instability parameter (PIP < 0.1). GNRs xxvii are stable at 28-60°C; however, prolonged high temperature (>60°C) and alkaline pH can trigger colloidal instability. GNRs remain stable at higher salt concentration, physiological and slightly acidic pH. GNRs can be stored in 0.001 M CTAB for 03 months without compromising their stability. PEG-GNRs are quite stable in cellular media solution (PIP < 0.1). In vitro toxicological assays of CTAB-GNRs and PEG-GNRs solutions with (human breast adenocarcinoma cell (MCF-7), human Rhabdomyosarcoma cell line (RD) and non-tumorigenic epithelial mammary gland cell line (MCF12-F) had confirmed that the toxicity is caused by free CTAB in solution. PEGylation of GNRs substantially had reduced the cellular toxicity as higher survival rates were observed in both normal and cancerous cell after incubation with PEG-GNRs solution. Gold nanorods, with aspect ratio 3.4 (λmax = 785 nm) and gold nanospheres (GNS), with diameters of ~30 nm (λmax = 530 nm) were selected for gene knockdown. Fluorescence labeled thiol reduced 21 mer DNA oligos directed to the 5′ end of COX-2 mRNA were conjugated with GNS and GNRs. UV-Vis spectroscopy calculations reveal 225 DNA Strands per particle in case of GNS and 385 DNA Strands per particle in case of GNRs. The flow cytometric based biocompatibility analysis for conjugated optical switches revealed neither significant (p<0.05) level of apoptotic cells nor necrotic (approximately 95% healthy population). The percentage cellular uptake of GNS with transfection agent is about 38% whereas, with ssDNA conjugated GNS the uptake increased to 56%, and it increased more to 67% when dsDNA conjugated GNS were used. The percentage cellular uptake of GNRs with transfection agent is about 26% whereas, with ssDNA conjugated GNRs the uptake increased to 50.3%, and it increased more to 60.1% xxviii when dsDNA conjugated GNRs were used. Dark field microscopy and confocal microscopy results for cellular uptake are consistent with UV-Vis spectroscopy. After conventional RNAi technique using nanocarriers the COX-2 protein expression level was reduced by 38% by gold nanospheres conjugated with COX-2 antisense oligos compared to their untreated counterpart after 48 h in MDA-MB-231cells. Comparatively, the silencing efficacy of conjugated nanocarriers after 72 h was significantly (p<0.05) increased to 78%. Average COX-2 protein level was reduced with silencing efficacy of 98% after ONCOS activation in MDA-MB-231 cells. The silencing efficacy after ONCOS activation in HepG2 cells is 96% compare to untreated cells. The decrease in protein level in the ONCOS-activated cells versus the control sample indicates that ONCOS has effectively interfered with COX-2 protein expression. We may conclude optical switches can be conjugated with oligos which can be released by remote optical extinction at desired intracellular location ad at specific time to block the expression of target gene. We demonstrate a novel gene-interfering technique presenting spatial and temporal control.