Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting

Abstract

Multivalent nanoparticle platforms are attractive for biomedical applications because of their improved target specificity, sensitivity, and solubility. However, their controlled assembly remains a considerable challenge. An efficient hydrazone ligation chemistry was applied to the assembly of Cowpea mosaic virus (CPMV) nanoparticles with individually tunable levels of a VEGFR-1 ligand and a fluorescent PEGylated peptide. The nanoparticles recognized VEGFR-1 on endothelial cell lines and VEGFR1-expressing tumor xenografts in mice, validating targeted CPMV as a nanoparticle platform in vivo.

Figure 1. CPMV: Strategy for labeling the virus capsid

A. Surface exposed lysines on CPMV are covalently modified with a benzaldehyde group for ligation (red). B. The hydrazide group (purple) on the ligand F56f (blue) reacts specifically with some of the benzaldehydes on CPMV. C. A second ligation with hydrazide-PEG500f (black) labels remaining benzaldehyde sites on CPMV, yielding a doubly-modified nanoparticle. D. Three-dimensional structure of CPMV particle showing large subunits (light and dark green), and small subunits (medium green). Lysines available for conjugation are shown in red (image created in RasMol and DeepView using coordinates generated in VIPER from 1NY7.pdb).

Figure 2. CPMV conjugation with F56 peptide-fluorescein and PEG-fluorescein

a. Relative fluorescence of PEG-conjugated CPMVs; P1, 10-fold excess; P2, 20-fold excess; P3, 50-fold excess; P4, 100-fold excess of PEG500f over benzaldehyde groups. b. Relative fluorescence of F56f and F56f/PEG500f mixed conjugated CPMVs; F1, 100-fold excess F56f; FP1, 100-fold excess F56f / 20fold excess PEG500f; FP2, 100- fold excess F56f / 50-fold excess PEG500f; FP3, 100-fold excess F56f / 100-fold excess PEG500f over benzaldehyde groups. c. SDS-PAGE of CPMV conjugates stained with Coomassie showing large (L) and small (S) subunits. d. SDS-PAGE of CPMV conjugates by UV transillumination. Lane labels in (b) correspond to those in (a) and (c).

Figure 3. Binding of CPMV conjugates to endothelial cellsin vitro

a. Cell adhesion assay with human endothelial cells (AE.hy926). Results are normalized to PBS control (y axis, Binding Efficiency: relative binding, normalized to PBS). Assay was performed in duplicate. Positive control: fibronectin (FN); negative control: native CPMV (CPMV); see Table 1 for an explanation of CPMV conjugates. b. Fluorescence visualization of CPMV conjugates FP3 (loaded with PEG500f and F56f), P4 (loaded with PEG500f only) and F1 (loaded with F55f only) bound to endothelial cells (EA.hy926) and fibroblasts (MEF). CPMV particles are shown in green. Nuclei were stained with DAPI (in blue).

Figure 4. Targeting CPMV to tumors in vivo

a,b. Confocal images showing that FP3 (a), but not P4 nanoparticles (b), bind to tumor tissue. CPMV was stained with fluorescently-labeled secondary antibodies. c. Expression of VEGFR-1 on HT-29 cells in vitro studied by flow cytometry. Red histogram = anti-VEGFR-1 and secondary antibody. Black histogram = secondary antibody only. d,e. HT-29 tumors stained ex vivo with anti-VEGFR-1 antibodies (d) or secondary antibody only (e). f-i. FP3 or P4 particles localize within HT-29 tumor xenografts following intravenous administration in nude/WEHI mice. CPMV was stained using specific antibodies. j,k. Intra-tumoral localization of particles (green) and endothelial cells (red) following intravenous administration of FP3 (j) or PBS (k). CPMV was stained using specific antibodies, and endothelial cells were stained using anti-CD31 antibody. Blue = DAPI stain to indicate cell nuclei.