Formulation and Characterization of Plant Virus Nanoparticles that Target Tumor Cells

Nanotechnology has been extensively applied in tumor cell targeting and drug delivery. Plant viruses represent a class of nanoparticles that has unique advantages of low toxicity, uniform shapes and robust capsid proteins (CP). This research is mainly focused on constructing Plant Virus Nanoparticles (PVN) based on Red Clover Necrotic Mosaic Virus (RCNMV). RCNMV has T=3 icosahedral symmetry and is composed of 180 subunits. One distinctive characteristic of RCNMV is that under the removal of divalent cations, the CP subunits are rotated, which leads to the formation of pores throughout the CP. The pore formation is reversible and thus can be taken advantage of for drug infusion.;The CP of RCNMV can be conjugated with peptides for the purpose of targeting cancer cells. The conventional method of conjugation is to use the bifunctional linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) to link lysines on the CP and cysteines on the targeting peptide. A detailed mass spectrometry (MS) analysis was conducted on these peptide conjugated PVN. The results indicate extensive peptide modifications on the R domain, and various unwanted modification of the linker, hydrolyzed linker and intra-peptide crosslinks. In view of the issues brought by the sulfo- SMCC linker, an alternative strategy of using maleimide peptide was proposed. The maleimide peptide directly conjugates to the cysteines on the CP and reduced redundant modifications. MS analysis on the new PVN revealed that one out of the five cysteines was modified with the maleimide peptide.;Besides the conventional chemical modification, other attempts with innovative approaches to functionalize the CP of RCNMV were carried out. One approach was to genetically insert a targeting peptide sequence to have it displayed on the loops or C-terminus of the CP. A total of 20 mutants were designed and tested, and only one of them successfully produced intact virion in plants. This particular mutant was with insertion of an RGD sequence at C-terminus. By comparing it to the native RCNMV, it was concluded that the mutant RCNMV was a T=1 virus, and consequently incapable of being used for infusion. Unfortunately, the mutant RCNMV did not show increased signal over the control in a cell delivery experiment.;A second approach consisted of using a scaffold protein specified as RCNMV binding protein (RBP). RBP was screened from a library of Sso7d mutants by our collaborator, Dr. Balaji Rao. The RBP has a moderate affinity to RCNMV. Because of the need for higher affinity in cell delivery experiments, two tandem RBPs were constructed and denoted as RBP-eGFP-RBP and RBP-RBPe-GFP. However, the binding affinities of the tandem RBPs were even weaker than those of singular RBP. In order to determine the reason for the decreased affinity, the binders were built virtually and subjected to molecular dynamics (MD) simulations. While the binding seemed geometrically feasible, a hypothesis was suggested that the two RBPs tend to form intra-molecular dimers given their proximity in the tandem binder constructs. The hypothesis was later confirmed by MD simulation. Further, the simulation tool was applied to a well-characterized system: the Fc part of human immunoglobulin and its binders in order to validate the method. A possible solution to the dimerization problem is to introduce partially rigid linkers, as to keep the binders apart while offering sufficient flexibility to permit both binding proteins to bind to their targets.;In addition to RCNMV, studies were also conducted on two other plant viruses. Under certain circumstances, Turnip Yellow Mosaic Virus (TYMV) can form an empty shell with a large opening, named artificial top component (ATC). Infusion and capping of the opening on ATC were attempted. Geminiviruses were used to construct vectors for the purpose of gene delivery and silencing. Unfortunately, the goals of formulating PVN based on these viruses were not achieved.