Interface of Physics and Biology: Engineering Virus-Based Nanoparticles for Biophotonics

Abstract

Virus-based nanoparticles (VNPs) have been used for a wide range of applications, spanning basic materials science and translational medicine. Their propensity to self-assemble into precise structures that offer a three-dimensional scaffold for functionalization has led to their use as optical contrast agents and related biophotonics applications. A number of fluorescently labeled platforms have been developed and their utility in optical imaging demonstrated, yet their optical properties have not been investigated in detail. In this study, two VNPs of varying architectures were compared side-by-side to determine the impact of dye density, dye localization, conjugation chemistry, and microenvironment on the optical properties of the probes. Dyes were attached to icosahedral cowpea mosaic virus (CPMV) and rod-shaped tobacco mosaic virus (TMV) through a range of chemistries to target particular side chains displayed at specific locations around the virus. The fluorescence intensity and lifetime of the particles were determined, first using photochemical experiments on the benchtop, and second in imaging experiments using tissue culture experiments. The virus-based optical probes were found to be extraordinarily robust under ultrashort, pulsed laser light conditions with a significant amount of excitation energy, maintaining structural and chemical stability. The most effective fluorescence output was achieved through dye placement at optimized densities coupled to the exterior surface avoiding conjugated ring systems. Lifetime measurements indicate that fluorescence output depends not only on spacing the fluorophores, but also on dimer stacking and configurational changes leading to radiationless relaxation—and these processes are related to the conjugation chemistry and nanoparticle shape. For biological applications, the particles were also examined in tissue culture, from which it was found that the optical properties differed from those found on the benchtop due to effects from cellular processes and uptake kinetics. Data indicate that fluorescent cargos are released in the endolysosomal compartment of the cell targeted by the virus-based optical probes. These studies provide insight into the optical properties and fates of fluorescent proteinaceous imaging probes. The cellular release of cargo has implications not only for virus-based optical probes, but also for drug delivery and release systems.

Tobacco mosaic virus-based protein nanoparticles and nanorods for chemotherapy delivery targeting breast cancer

“…Effective cell killing of the covalently-delivered DOX for the DOX TMV and DOX SNP formulations can be explained by the intracellular fates of the protein-based nanocarrier: in previous cellular trafficking experiments we have shown that TMV is internalized by cancer cells through endocytosis and targeted to the endolysosome, where the free drug is released following degradation of the proteinaceous carrier [56,57]. …”

“…Based on our previous studies on cell trafficking of TMV [56, 57], it is expected that upon cell targeting and internalization, the protein-based carriers are targeted to the endolysosomal compartment, where the proteinaceous plant virus-based carriers will be metabolically degraded through hydrolase and protease activity, therefore yielding efficient drug release inducing effective cell killing. Indeed our data confirmed that drug efficacy was maintained upon conjugation to or encapsulation into the TMV and SNP-based carriers (Fig.…”

Molecular Interactions in Organic Nanoparticles for Phototheranostic Applications

“…66 A second investigation into CPMV as a fluorescence agent for biological imaging suggests that surface dye labeling density can affect the observed fluorescence intensity as well as the cell−virus interactions. 73 CPMV nanoparticles were modified with Cy5 dyes at various ratios to elucidate the optimal amount of dye for loading. They found that 27 dyes per CPMV particle yielded maximal fluorescence, after which quenching occurs.…”

Featured Article: Delivery of chemotherapeutic vcMMAE using tobacco mosaic virus nanoparticles

“…These results are as expected, as we and others have previously shown that TMV is internalized by cancer cells through a combination of endocytosis and macropinocytosis and that the particles then co-localize with endolysosomal markers. [28][29][30] We hypothesize that within the protease-rich environment of the endolyosome, the prodrug TMV-vcMMAE is cleaved at the protease-cleavable vc linker, resulting in release of the active MMAE component. In fact, in our previous studies, we have shown that TMV-conjugated cargos are cleaved and released in endolysosomal extracts even when conjugated via amide bonds.…”