| Red clover necrotic mosaic virus (RCNMV) is a highly robust (Tm=60 °C), 36 nm icosahedral plant virus. The capsid of RCNMV is assembled from 180 chemically equivalent coat proteins (CPs). The CPs arrange in a T=3 symmetry, in 1 of 3 conformations forming the asymmetric subunit (ASU). There are two Ca(II) binding sites per CP; the removal of divalent cations causes the CP subunits of the ASU to rotate away from each other forming a ∼13 Å channel. These channels lead to the highly organized bipartite genome of RCNMV and can be closed by adding back Ca(II). Titrimetric analysis and tryptophan fluorescence was used to determine the affinity of RCNMV for Ca(II) to be ∼Kd < 300 nM.;It has been shown that doxorubicin (Dox) can be infused into the capsid at a mole ratio of ∼1000:1, Dox-to-virus, and unlike other nanoparticles, there is no detectable leakage. The high loading of Dox is most likely due to intercalation into the genome and significant intercalation or exposure to denaturants was observed to cause loss of capsid stability. To better understand the limitations of cargo loading, Dox and other intercalating molecules (rhodamine 800, ethidium bromide, and propidium iodide) were assayed to determine optimum infusion conditions. Dox was observed to have a propensity to aggregate. In order to manage the Dox aggregation, the infusion buffer was changed from 50 mM Tris-HCl/50 mM NaOAc/50 mM EDTA or 200 mM EDTA at pH 8.0 to 5 mM HEPES/5 mM Na4EDTA/10 mM NaCl pH 7.8. The Dox:RCNMV infusion mole ratio was also lowered from 5000:1 to 500:1 and the incubation temperature was changed from 4 °C to 22 °C for <12 hours, opposed to 24 hours.;To impart targeting functionality to RCNMV, biomimetic peptides were conjugated to either the surface capsid lysines or cysteines using standard bioconjugation methods. For all of the biomimetic peptides screened, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) was used to orthogonally attach the cysteineterminated peptides to the surface lysines of RCNMV. The efficacy of a plant virus nanoparticle (PVN), loaded with a cargo (Dox or propidium iodide) and armed with a targeting peptide, was tested in vitro against several cell line using cell viability assays.;ADH304, an N-cadherin targeting peptide, was synthesized to contain a lys[maleimide]. Dox infused RCNMV was armed with ADH304maleimide by conjugating the peptide to the endogenous C267 located in the P-domain. The use of a cysteine reactive peptide increased the PVN yield from ∼30% to ∼70% by eliminating several synthetic steps. The efficacy of this PVN formulation was tested on a human melanoma tumor in a xenograft mouse model. This results of this study showed that a) a PVN could suppress tumor growth at 4 × the MTD (maximum tolerated dose) without acute toxicity and b) Dox, otherwise not effective against melanoma, was able to suppress tumor growth when formulated in a PVN. This is most likely due to the PVN entering the tumor cells through an endocytic pathway versus the simple diffusion of Dox which is subject to cellular efflux pumps. |
