Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X

Abstract

Nanomaterials with elongated architectures have been shown to possess differential tumor homing properties compared to their spherical counterparts. Here, we investigate whether this phenomenon is mirrored by plant viral nanoparticles that are filamentous (Potato virus X) or spherical (Cowpea mosaic virus). Our studies demonstrate that Potato virus X (PVX) and Cowpea mosaic virus (CPMV) show distinct biodistribution profiles and differ in their tumor homing and penetration efficiency. Analogous to what is seen with inorganic nanomaterials, PVX shows enhanced tumor homing and tissue penetration. Human tumor xenografts exhibit higher uptake of PEGylated filamentous PVX compared to CPMV, particularly in the core of the tumor. This is supported by immunohistochemical analysis of the tumor sections, which indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity make it a potentially superior platform for applications in cancer drug delivery and imaging applications.

Figure 1

A) Bioconjugation scheme showing A647 and PEG5000 conjugation to solvent-exposed Lys side chains on PVX and CPMV. B) TEM of negatively-stained (2% w/v UAc) A647-CPMV-PEG (left) and A647-PVX-PEG5000 (right). C) SDS gel after Coomassie staining of separated coat proteins. M = SeeBlue Plus2 protein marker, numbers indicate molecular weight standards in kDa. 1 = CPMV, 2 = A647-CPMV-PEG, 3 = PVX, and 4 = A647-PVX-PEG5000. CPMV consists of S and L protein; PVX consists of a single coat protein. Lower mobility bands indicate PEGylation. Band density analysis was performed using band analysis tool and ImageJ software. D) Zeta potential of A647-labeled and PEGylated VNPs.

Figure 2. Intravital imaging of VNP uptake in human tumor xenografts in the CAM

A. Avian embryos bearing vascularized GFP-expressing human fibrosarcoma HT1080 or human epithelial carcinoma HEp3 tumors (magenta) were co-injected with 120 μg of PVX-PEG-A555 (red) and 20 μg of CPMV-PEG-647 (green) and visualized 4 h after injection. Scale bar = 190 μm. B. The analysis of whole tumor uptake of CPMV and PVX nanoparticles compared to uptake only in the tumor core was assessed using distinct ROIs (left panel) in HT1080 (middle panel) and HEp3 (right panel) tumors. While whole tumor localization of CPMV and PVX were comparable, PVX accumulated in the core of tumors to a significantly higher degree than CPMV (unpaired t test). C. The localization of nanoparticles was assessed in 8 micron sections of the tumor core using fluorescence microscopy. CPMV (green) and PVX (red) are visualized in the tumor (magenta). While CPMV was visualized in punctate foci, PVX was distributed throughout the tumor in areas devoid of CPMV (white arrowheads).

Figure 3. Tumor homing and biodistribution of VNPs measured using Maestro^™ Imager

A) A647-labeled and PEGylated CPMV and PVX were administered intravenously into nude mice bearing HT-29 xenografts. 24-hours post-injection tissues were collected and imaged; tissues are shown under white light and fluorescence (A647 signal). B) Tumors (2 per animal) were exercised from 3 animals and imaged; qualitative data (left) and quantitative data (right) are presented. C) Intra-tumoral localization of CPMV (pseudo-colored in yellow) and PVX (pseudo-colored in red). Endothelium was immunostained using a FITC-labeled CD31 antibody (pseudocolored in pink). Nuclei were stained with DAPI (blue). Scale bars are 30 microns.

Figure 4. Plasma clearance of A647-labeled, PEGylated PVX and CPMV

Pharmacokinetics were evaluated using healthy Balb/c mice. Blood was collected over a 60-min time period, plasma extracted and the fluorescence intensity measured.

Figure 5

A) Biodistribution of A647-labeled and PEGylated CPMV and PVX nanoparticles and filaments. Three nude mice with human HT-29 tumor xenografts each were injected with PBS (control group), CPMV, and PVX. Tissues were collected and analyzed 24 h post administration. Fluorescence intensity normalized per gram of tissue weight is plotted for all major organs and tumors for each mouse. B) Average biodistribution normalized against PBS samples. The percentage of VNPs detected in each tissue is shown for each organ and tumors analyzed.