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. 2013 Dec 10;172(2):568-78.
doi: 10.1016/j.jconrel.2013.04.023. Epub 2013 May 9.

Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery

Ibrahim Yildiz  1 , Karin L Lee, Kevin Chen, Sourabh Shukla, Nicole F Steinmetz
Affiliations

Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery

Ibrahim Yildiz et al. J Control Release. .

Abstract

This work is focused on the development of a plant virus-based carrier system for cargo delivery, specifically 30nm-sized cowpea mosaic virus (CPMV). Whereas previous reports described the engineering of CPMV through genetic or chemical modification, we report a non-covalent infusion technique that facilitates efficient cargo loading. Infusion and retention of 130-155 fluorescent dye molecules per CPMV using DAPI (4',6-diamidino-2-phenylindole dihydrochloride), propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide), and acridine orange (3,6-bis(dimethylamino)acridinium chloride), as well as 140 copies of therapeutic payload proflavine (PF, acridine-3,6-diamine hydrochloride), is reported. Loading is achieved through interaction of the cargo with the CPMV's encapsidated RNA molecules. The loading mechanism is specific; empty RNA-free eCPMV nanoparticles could not be loaded. Cargo-infused CPMV nanoparticles remain chemically active, and surface lysine residues were covalent modified with dyes leading to the development of dual-functional CPMV carrier systems. We demonstrate cargo-delivery to a panel of cancer cells (cervical, breast, and colon): CPMV nanoparticles enter cells via the surface marker vimentin, the nanoparticles target the endolysosome, where the carrier is degraded and the cargo is released allowing imaging and/or cell killing. In conclusion, we demonstrate cargo-infusion and delivery to cells; the methods discussed provide a useful means for functionalization of CPMV toward its application as drug and/or contrast agent delivery vehicle.

Keywords: Cowpea mosaic virus; Drug delivery; Infusion; Viral nanoparticle.

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Figures

Figure 1
Figure 1
A) Structure of DAPI (4’,6-diamidino-2-phenylindole dihydrochloride), propidium iodide (PI, 3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide), and acridine orange (AO, 3,6-bis(dimethylamino)acridinium chloride). B) Size exclusion chromatography of CPMV-DAPI, CPMV-PI, and CPMV-AO shows the typical elution profile of intact CPMV, co-elution of the dyes indicates loading. C) UV/visible spectra of CPMV-DAPI, CPMV-PI, and CPMV-AO showing the CPMV typical peak at 260 nm and the dye-specific absorbance peak at 358, 493, and 470 nm, respectively. D) Native agarose gel electrophoresis of CPMV and eCPMV after incubation with DAPI, PI, AO. The gels were visualized and documented under UV light and then stained with Coomassie blue and photographed under white light.
Figure 2
Figure 2
Electrophoretic separation of CPMV-DAPI and their coat proteins. A) Denaturing gel electrophoresis using a NuPAGE gel and B) native gel using an agarose gel. 1 = CPMV, 2 = CPMV-DAPI, 3 = A555-CPMV, 4 = A555-CPMV-DAPI, M = molecular weight standard; the bands are labeled in the center of the gels (in kDa). Gels were visualized under UV light and under white light after Coomassie blue (CB) staining.
Figure 3
Figure 3
The fate of CPMV-DAPI in HeLa cells. Time and temperature-dependency: CPMV-DAPI was incubated with HeLa cells at 4°C versus 37°C for 10 min versus 60 min. A, C, E, G = CPMV channel (in green), B, D, F, H = overlay of CPMV channel (in green), cell membrane stain (WGA, in red), and DAPI in blue. Concentration dependence: I-L showing cells after incubation with different concentrations of free DAPI versus CPMV-DAPI, cell membrane in red, DAPI in blue (no CPMV staining). Colocalization: M = overlay of DAPI in blue (note in this experiment cells were stained using free DAPI, and not DAPI delivered through CPMV), endolysosomes (Lamp-1 marker) in red, and CPMV in green, N = Lamp-1 only in red, O = CPMV only in green, P = colocalization analysis using ImageJ and colocalization highlighter plug-in, colocalized signals are shown in white. The scale bars are 50 μm in length.
Figure 4
Figure 4
Characterization of drug-loaded CPMV. A) UV/visible spectroscopy of CPMV-PF showing the CPMV and proflavine-specific absorbance maxima at 260 nm and 450 nm. B) Native gel electrophoresis of CPMV and eCPMV with and without proflavine (PF) (it should be noted that non-purified samples were analyzed on the gel to show the migration pattern of free PF versus (e)CPMV); gels were documented under UV light, and then stained with Coomassie blue and photographed under white light. The bright bands in proflavine-positive samples indicate free dye that migrates towards the cathode in the electrophoretic field (on top).
Figure 5
Figure 5
Cell viability assays. HeLa (in black) were exposed to proflavine and CPMV-PF at 0.3 μM, 0.6 μM, 1.8 μM, and 2.9 μM concentration of proflavine (equates to a CPMV concentration of 0.002 μM, 0.004 μM, 0.012 μM, and 0.02 μM) for 24 h, and washed, and incubated for further 24 h in tissue culture medium, prior to analysis of cell viability using XTT assay. C = untreated control cells. HT-29 (in white) and PC-3 (in grey) were exposed to proflavine and CPMV-PF at 1.46 μM, 3.07 μM, 6.13 μM, and 16.06 μM concentration of proflavine (equates to a CPMV concentration of 0.010 μM, 0.021 μM, 0.042 μM, and 0.11 μM) for 24 h, and washed, and incubated for further 24 h in tissue culture medium, prior to analysis of cell viability using XTT assay. C = untreated control cells.
Figure 6
Figure 6
A) Cell binding of A555-CPMV-PF to HeLa and HT-29 cells after 60 min exposure. For these experiments cells were collected using non-enzymatic cell dissociation buffers to avoid the natural CPMV receptor being cleaved off the cell surface; we have not achieved collection of PC-3 cells using this method. B) Confocal microscopy images of HeLa, HT-29, and PC-3 cells after incubation with A555-CPMV-PF. Red = cell membrane (WGA staining), blue = nuclei (DAPI staining), and green = CPMV (from A555 dye). The scale bar is 20 μm.
Scheme 1
Scheme 1
Cartoon of CPMV with its single stranded RNA molecule (in red), CPMV is exposed to a bathing solution containing the cargo of interest (in blue), washing and dialysis is used to remove excess cargo yielding intact CPMV with infused cargo.
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