Development of sesbania mosaic virus nanoparticles for imaging

Abstract

AbstractThe capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to displaymany imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carriedout bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactivelysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelledSeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preferencefor entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescenceof SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivitythan that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled withCy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. Theparticles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blotanalysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, whichwere identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexinA2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. Theresults presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop invivo imaging and targeted drug delivery agents for cancer diagnosis and therapy