Folic acid-mediated targeting of cowpea mosaic virus particles to tumor cells

Abstract

Cowpea mosaic virus (CPMV) is a well-characterized nanoparticle that has been used for a variety of nanobiotechnology applications. CPMV interacts with several mammalian cell lines and tissues in vivo. To overcome natural CPMV targeting and redirect CPMV particles to cells of interest, we attached a folic acid-PEG conjugate by using the copper-catalyzed azide-alkyne cycloaddition reaction. PEGylation of CPMV completely eliminated background binding of the virus to tumor cells. The PEG-folate moiety allowed CPMV-specific recognition of tumor cells bearing the folate receptor. In addition, by testing CPMV formulations with different amounts of the PEG-FA moiety displayed on the surface, we show that higher-density loading of targeting ligands on CPMV may not be necessary for efficient targeting to tumor cells. These studies help to define the requirements for efficiently targeting nanoparticles and protein cages to tumors.

Figure 1. Synthesis of the PEG-folic acid conjugates

Figure 2. Virus integrity assessed by size exclusion chromatography and TEM, and Western blot

A. Size exclusion FPLC analysis of CPMV-PEG-FA and WT-CPMV. B. Coomassie stain of WT-CPMV (1) and CPMV-(PEG-FA)₆₀ (2), showing small (S) and large (L) capsid subunit proteins. C–D. Western blots of WT-CPMV (1) and CPMV-(PEG-FA)₆₀ at higher concentration (2). Left panel, detection using rabbit anti-CPMV antibody. Right panel, detection using mouse anti-folic acid antibody. L: large subunit, and S: small subunit. E–F. Typical TEM image of WT-CPMV (e) and CPMV-(PEG-FA)₆₀ (f), showing intact particles. The bar represents 200 nm.

Figure 3. Fluorescent microscopy of monolayers of HeLa (top) and KB (bottom) cells

Cells were incubated with the indicated virus particles (37°C, 2 h), followed by permeabilization and treatment with anti-CPMV antibody and then with a green fluorescent secondary antibody conjugate. Nuclei are stained with DAPI (blue). Scale bar, 10µm.

Figure 4. Measurement of virus binding to tumor cells using flow cytometry

A. Binding of CPMVs to HeLa tumor cells. B. Binding of CPMV to KB tumor cells. The peaks from left to right are cells only (dotted histogram), CPMV-PEG (short dashed lines), WT-CPMV (long dashed lines), and CPMV-(PEG-FA)₆₀ (filled histograms), respectively.

Figure 5. Competition of CPMV-PEG-FA using free FA

A. CPMV-PEG-FA bearing 60 FA/particle (1 µM FA) was incubated with KB cells in the presence of increasing amounts of FA as indicated. Error bars represent the mean +/− S.D. of triplicate experiments. B. Competition of CPMV-(PEG-FA)₂₀ (grey), CPMV-(PEG-FA)₃₅ (white bars) or CPMV-(PEG-FA)₆₀ was performed using increasing amounts of FA as indicated. In each case, the concentration of virus particles was 17 nM, giving rise to the following concentrations of displayed FA: CPMV-(PEG-FA)₂₀ = 0.33 µM FA; CPMV-(PEG-FA)₃₅ = 0.6 µM FA; CPMV-(PEG-FA)₆₀ = 1µM FA.