In Vivo Monitoring of Liposomal Encapsulated siRNA Delivery

Abstract

RNA interference (RNAi) is being widely explored as a means of tumour therapy due to the specific and potent silencing of targeted genes. However, in vivo delivery of RNAi effectors, such as small interfering RNA (siRNA) and detection of delivery is fraught with problems. In this thesis a novel theranostic PEGylated siRNA nanoparticle, termed liposome encapsulated siRNA (LEsiRNA) was developed and tested in vitro and in vivo. The LEsiRNA nanoparticles were formulated to be MR sensitive and contain labels for fluorescence microscopy/histology. The nanoparticles were shown to be an ideal size for in vivo delivery, under 100 nm, stable over 4 days and have a MR relaxivity better than the clinically used contrast agent Dotarem. The LEsiRNA nanoparticles were tested in vitro to assess their suitability for use in vivo. It was found that incubation of the particles with cells led to a decrease in protein level of the targeted cancer gene, Survivin, an inhibitor of apoptosis. In vivo assessment of the LEsiRNA nanoparticles demonstrated their potential as a theranostic agent, delivering therapy, while simultaneously allowing monitoring of tumour delivery. The LEsiRNA nanoparticles, administered by intravenous injection, were shown to accumulate in xenograft tumours by MRI at 16 and 24 hours post administration. Fluorescence microscopy of ex vivo tumour samples was used to corroborate the MR results and simultaneously demonstrate co-localisation of nanoparticles and siRNA within the tumours. The LEsiRNA nanoparticle-mediated delivery of the anti-cancer Survivin siRNA caused a significant reduction in both Survivin expression and tumour growth when compared to controls. These results suggest that LEsiRNA nanoparticles can be valuable as an in vivo delivery agent for siRNA therapy to tumours. As a way to non-invasively assess the tumour therapy effect caused by the Survivin LEsiRNA nanoparticles, an MR contrast agent targeted to apoptotic cells was investigated. This agent was tested in vitro and found, by MRI, to selectively bind to apoptotic cells. This suggests that the agent could be used in vivo to give an early prognosis, after a therapeutic intervention, without the need for an invasive biopsy. The favourable biodistribution of the LEsiRNA nanoparticles to tumours and the subsequent successful functional delivery of Survivin siRNA, with simultaneous monitoring by MRI, suggests that these LEsiRNA nanoparticles have real potential for the future treatment of cancer. The ability to non-invasively detect the effect of the LEsiRNA nanoparticles on tumours in vivo, afforded by the apoptotic targeted contrast agent, is also paramount for future clinical applications. Therefore, these two agents have great potential in the treatment and prognosis of cancer.