Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models

Abstract

The application of plant virus-derived nanostructures in materials science, biomedical research and engineering has recently been promoted by the development of fluorescence-labeled viruses for optical imaging in tissue culture and preclinical animal models. Most studies reported thus far have focused on the application of viruses that have been chemically modified with organic dyes. In this investigation, we sought to develop and study genetically-engineered virus-based biomaterials that incorporate green or red fluorescent proteins. The genetic introduction of such imaging moieties is advantageous because post-harvest modifications are not required, thus minimizing the number of manufacturing steps and maximizing the yields of each fluorescent probe. Specifically, we engineered the filamentous plant virus Potato virus X (PVX) to display green fluorescent protein (GFP) or mCherry as N-terminal coat protein (CP) fusions, producing a 1 : 3 fusion protein to CP ratio. The infection of Nicotiana benthamiana plants with the recombinant GFP-PVX and mCherry-PVX particles was documented by fluorescence imaging, structural analysis and genetic characterization to determine the stability of the chimeras and optimize the molecular farming protocols. We also demonstrated the application of fluorescent mCherry-PVX filaments as probes for optical imaging in human cancer cells and a preclinical mouse model. Cell viability assays and histological analysis following the administration of mCherry-PVX indicated the biocompatibility and rapid tissue clearance of the particles. Such particles could therefore be functionalized with additional cancer-specific detection ligands to provide tools for molecular imaging, allowing the investigation of molecular signatures, disease progression/recurrence and the efficacy of novel therapies.

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…Using both mouse and chicken chorioallantoic membrane (CAM) models with tumor xenografts, the passive partitioning of particles to the tumor can be observed ( Figure 7 ). 84, 143 As discussed in the previous section, evaluation of localization of particles inside the tumor revealed enhanced accumulation and penetration of rod-shaped particles. 143 …”

Stealth filaments: Polymer chain length and conformation affect the in vivo fate of PEGylated potato virus X

“…For example, we recently demonstrated expression of green fluorescent protein (GFP) and other fluorescent proteins as genetic coat protein fusions. [18] Furthermore, solvent-exposed lysine side chains offer a convenient means of modification with non-peptide-based ligands (e.g. therapeutics or contrast agents) via chemical bioconjugation.…”

Virus-Based Nanoparticles as Versatile Nanomachines

“…In order to complete these genetic manipulations, viral genomes often must be refactored to eliminate overlaps in the protein-coding sequences that are to be modified (128). The genetic addition of GFP or the red fluorescent protein mCherry to the potato virus X (PVX) coat protein allowed for in vivo imaging of human tumor xenographs in mice (129). Similarly, bacteriophage λ has been coated with fluorescent molecules via bioconjugation to addressable lysine side chains (130) and also by the genetic engineering of the coat protein gpD to decorate the particle with GFP (131).…”

“…Not only can small chemical modifiers such as contrast agents, drugs, and peptides be displayed, but there is also an opportunity to present entire proteins as genetic fusions. Examples include the display of full-length fluorescent proteins for optical imaging (129); the genetic fusion of functional enzymes to the internal (154) or external (155) surfaces of VNPs, allowing them to be used as nanoreactors; and the presentation of complex protein structures such as the I-domain of the anthrax toxin cellular receptor in a complex with the anthrax protective antigen as a vaccination platform (110). Another interesting avenue is the use of subviral structures such as the tail, which functions as a molecular motor (156).…”