To Target or Not to Target: Active vs. Passive Tumor Homing of Filamentous Nanoparticles Based on Potato virus X - PubMed (original) (raw)

To Target or Not to Target: Active vs. Passive Tumor Homing of Filamentous Nanoparticles Based on Potato virus X

Sourabh Shukla et al. Cell Mol Bioeng. 2015.

Abstract

Nanoparticles are promising platforms for the diagnosis and treatment of cancer. Diverse classes and shapes of materials have been investigated to establish design principles that achieve the effective partitioning of medical cargos between tumors and healthy tissues. Molecular targeting strategies combined with specific nanoparticle shapes confer tissue-specificity on the carriers, allowing the cell-specific delivery of cargos. We recently developed a filamentous platform technology in which the plant virus Potato virus X (PVX) was used as a scaffold. These particles are flexible 515 × 13 nm filaments that encourage passive tumor homing. Here we sought to advance the PVX platform by including a molecular targeting strategy based on cyclic RGD peptides, which specifically bind to integrins upregulated on tumor cells, neovasculature, and metastatic sites. Although the RGD-targeted filaments outperformed the PEGylated stealth filaments in vitro, enhanced tumor cell targeting did not translate into improved tumor homing in vivo in mouse tumor models. The RGD-PVX and PEG-PVX filaments showed contrasting biodistribution profiles. Both formulations were cleared by the liver and spleen, but only the stealth filaments accumulated in tumors, whereas the RGD-targeted filaments were sequestered in the lungs. These results provide insight into the design principles for virus-based nanoparticles that promote the delivery of medical cargos to the appropriate cell types.

Keywords: Biodistribution; Cancer; Integrins; Nanoparticle shape; Tumor targeting.

PubMed Disclaimer

Figures

Figure 1

Figure 1

(a) Nicotiana benthamiana plants infected with Potato virus X (PVX) and (b) a negatively-stained transmission electron micrograph of PVX.

Figure 2

Figure 2

Modification of PVX with PEG and RGD ligands. _N_-hydroxysuccinimide (NHS) chemistry was used to conjugate A647 and SM(PEG)4 bifunctional linkers to PVX lysine residues. The cRGDfC peptide (with terminal cysteine) was then conjugated using the maleimide handle to produce RGD-PVX. Similarly, A647 and PEG (5-kDa MW) were conjugated to PVX by NHS chemistry to yield PEG-PVX filaments.

Figure 3

Figure 3

Characterization of PEG-PVX and RGD-PVX particles: (a) UV/Vis spectroscopy was used to determine number of dye molecules attached and to determine the concentration of PEG-PVX and RGD-PVX particles. (b) SDS-PAGE was used to confirm the covalent conjugation of PEG and RGD peptides to the PVX coat proteins (CPs). ImageJ software was used to determine the percentage of modified coat proteins. (c) Quantification of dye molecules and PEG/RGD ligands per PVX filament based on UV/Vis and SDS-PAGE. (d) Transmission electron microscopy was used to confirm the structural stability of PVX particles after modification (left to right: PVX, PEG-PVX and RGD-PVX). Scale bar = 100 nm.

Figure 4

Figure 4

Comparative biodistribution of PEG-PVX and RGD-PVX particles injected intravenously into NCR-nu/nu mice with subcutaneous HT-29 tumor xenografts. Ex vivo Maestro analysis was carried out using dissected tissues 24 h post-administration. (a) Maestro images (yellow boxes show enlarged view of the lungs from animals treated with RGD-PVX, showing hot spots for PVX sequestration). (b) Quantitative analysis.

Figure 5

Figure 5

Immunofluorescence analysis of tumor and lung sections. The tumor sections were stained with antibodies specific for the endothelial marker CD31 (red, a + b), integrin α v [red, c + d (inset: higher magnification)], the tumor macrophage marker CD68 (pink, e + f) and DAPI (blue) to determine the localization of PEG-PVX and RGD-PVX within the tumor. (g–i) Whole lung (g) and lung sections (h, i) from mice treated with RGD-PVX were stained for α v integrin (red) and CD68+ macrophages (pink). Co-localization analysis was used to highlight the hotspots of RGD-PVX accumulation with integrins and CD68 macrophages (i). Scale bars = 50 _μ_m.

Figure 6

Figure 6

Biodistribution of PEG-PVX and RGD-PVX particles in healthy Balb/c mice. Tissues were collected for ex vivo Maestro™ fluorescence analysis 24 h after the administration of PEG-PVX and RGD-PVX. (a) Maestro images (yellow boxes show enlarged view of the lungs from animals treated with RGD-PVX, showing hot spots for PVX sequestration). (b) Quantitative analysis.

Figure 7

Figure 7

The analysis of PEG-PVX and RGD-PVX interactions with and uptake into cells using flow cytometry and confocal microscopy. (a + d) Flow cytometry was used to determine the interactions between PVX particles and HT-29 cancer cells (a, b) or RAW264.7 macrophages (d, e). The mean fluorescence intensity (MFI) is plotted and error bars indicate the standard deviation (n = 3). (b + e) Competition binding assays using RGD-PVX and free RGD peptides. (c + f) Confocal microscopy shows that RGD-PVX is taken up more efficiently than PEG-PVX by HT-29 cells and RAW264.7 macrophages. Cells were stained with DAPI (blue) to show the nucleus and with the endosomal marker LAMP-1 (pink; in HT-29 cells) or WGA (red; RAW cells). Colocalization of the particles and the endosomal marker indicates cellular uptake. Scale bars = 10 _μ_m.

References

    1. An L, Hu H, Du J, Wei J, Wang L, Yang H, Wu D, Shi H, Li F, Yang S. Paramagnetic hollow silica nanospheres for in vivo targeted ultrasound and magnetic resonance imaging. Biomaterials. 2014;35:5381–5392. doi: 10.1016/j.biomaterials.2014.03.030. - DOI - PubMed
    1. Antonov AS, Antonova GN, Munn DH, Mivechi N, Lucas R, Catravas JD, Verin AD. alphaVbeta3 integrin regulates macrophage inflammatory responses via PI3 kinase/Akt-dependent NF-kappaB activation. J. Cell Physiol. 2011;226:469–476. doi: 10.1002/jcp.22356. - DOI - PMC - PubMed
    1. Arnida, Janát-Amsbury MM, Ray A, Peterson CM, Ghandehari H. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur. J. Pharm. Biopharm. 2011;77:417–423. doi: 10.1016/j.ejpb.2010.11.010. - DOI - PMC - PubMed
    1. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 1994;79:1157–1164. doi: 10.1016/0092-8674(94)90007-8. - DOI - PubMed
    1. Bruckman MA, Randolph LN, VanMeter A, Hern S, Shoffstall AJ, Taurog RE, Steinmetz NF. Biodistribution, pharmacokinetics, and blood compatibility of native and PEGylated tobacco mosaic virus nano-rods and -spheres in mice. Virology. 2014;449:163–173. doi: 10.1016/j.virol.2013.10.035. - DOI - PMC - PubMed

LinkOut - more resources