Amy M. Wen

Increased Tumor Homing and Tissue Penetration of the Filamentous Plant Viral Nanoparticle Potato virus X

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.

In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer

Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The “in situ vaccination” immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic anti-tumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from Cowpea Mosaic Virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic anti-tumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity, and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the anti-tumour immune response. CPMV also exhibited clear treatment efficacy and systemic anti-tumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer.

The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft-Matter Nanorods

The size and shape of nanocarriers can affect their fate in vivo, but little is known about the effect of nanocarrier aspect ratio on biodistribution in the setting of cancer imaging and drug delivery. The production of nanoscale anisotropic materials is a technical challenge. A unique biotemplating approach based on of rod-shaped nucleoprotein nanoparticles with predetermined aspect ratios (AR 3.5, 7, and 16.5) is used. These rigid, soft-matter nanoassemblies are derived from tobacco mosaic virus (TMV) components. The role of nanoparticle aspect ratio is investigated, while keeping the surface chemistries constant, using either PEGylated stealth nanoparticles or receptor-targeted RGD-displaying formulations. Aspect ratio has a profound impact on the behavior of the nanoparticles in vivo and in vitro. PEGylated nanorods with the lowest aspect ratio (AR 3.5) achieve the most efficient passive tumor-homing behavior because they can diffuse most easily, whereas RGD-labeled particles with a medium aspect ratio (AR 7) are more efficient at tumor targeting because this requires a balance between infusibility and ligand-receptor interactions. The in vivo behavior of nanoparticles can therefore be tailored to control biodistribution, longevity, and tumor penetration by modulating a single parameter: the aspect ratio of the nanocarrier.

Fluorescent Nanodiamonds Embedded in Biocompatible Translucent Shells

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 10-20-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells.

Interior Engineering of a Viral Nanoparticle and Its Tumor Homing Properties

The development of multifunctional nanoparticles for medical applications is of growing technological interest. A single formulation containing imaging and/or drug moieties that is also capable of preferential uptake in specific cells would greatly enhance diagnostics and treatments. There is growing interest in plant-derived viral nanoparticles (VNPs) and establishing new platform technologies based on these nanoparticles inspired by nature. Cowpea mosaic virus (CPMV) serves as the standard model for VNPs. Although exterior surface modification is well-known and has been comprehensively studied, little is known of interior modification. Additional functionality conferred by the capability for interior engineering would be of great benefit toward the ultimate goal of targeted drug delivery. Here, we examined the capacity of empty CPMV (eCPMV) particles devoid of RNA to encapsulate a wide variety of molecules. We systematically investigated the conjugation of fluorophores, biotin affinity tags, large molecular weight polymers such as poly(ethylene glycol) (PEG), and various peptides through targeting reactive cysteines displayed selectively on the interior surface. Several methods are described that mutually confirm specific functionalization of the interior. Finally, CPMV and eCPMV were labeled with near-infrared fluorophores and studied side-by-side in vitro and in vivo. Passive tumor targeting via the enhanced permeability and retention effect and optical imaging were confirmed using a preclinical mouse model of colon cancer. The results of our studies lay the foundation for the development of the eCPMV platform in a range of biomedical applications.

Stealth filaments: Polymer chain length and conformation affect the in vivo fate of PEGylated potato virus X.

Nanoparticles hold great promise for delivering medical cargos to cancerous tissues to enhance contrast and sensitivity of imaging agents or to increase specificity and efficacy of therapeutics. A growing body of data suggests that nanoparticle shape, in combination with surface chemistry, affects their in vivo fates, with elongated filaments showing enhanced tumor targeting and tissue penetration, while promoting immune evasion. The synthesis of high aspect ratio filamentous materials at the nanoscale remains challenging using synthetic routes; therefore we turned toward natures materials, developing and studying the filamentous structures formed by the plant virus potato virus X (PVX). We recently demonstrated that PVX shows enhanced tumor homing in various preclinical models. Like other nanoparticle systems, the proteinaceous platform is cleared from circulation and tissues by the mononuclear phagocyte system (MPS). To increase bioavailability we set out to develop PEGylated stealth filaments and evaluate the effects of PEG chain length and conformation on pharmacokinetics, biodistribution, as well as potential immune and inflammatory responses. We demonstrate that PEGylation effectively reduces immune recognition while increasing pharmacokinetic profiles. Stealth filaments show reduced interaction with cells of the MPS; the protein:polymer hybrids are cleared from the body tissues within hours to days indicating biodegradability and biocompatibility. Tissue compatibility is indicated with no apparent inflammatory signaling in vivo. Tailoring PEG chain length and conformation (brush vs. mushroom) allows tuning of the pharmacokinetics, yielding long-circulating stealth filaments for applications in nanomedicine.

Biodistribution and clearance of a filamentous plant virus in healthy and tumor-bearing mice

AIM Nanoparticles based on plant viruses are emerging biomaterials for medical applications such as drug delivery and imaging. Their regular structures can undergo genetic and chemical modifications to carry large payloads of cargos, as well as targeting ligands. Of several such platforms under development, only few have been characterized in vivo. We recently introduced the filamentous plant virus, potato virus X (PVX), as a new platform. PVX presents with a unique nanoarchitecture and is difficult to synthesize chemically. METHODS Here, we present a detailed analysis of PVX biodistribution and clearance in healthy mice and mouse tumor xenograft models using a combination of ex vivo whole-organ imaging, quantitative fluorescence assays and immunofluorescence microscopy. RESULTS & CONCLUSION While up to 30% of the PVX signal was from the colon, mammary and brain tumor tissues, remaining particles were cleared by the reticuloendothelial system organs (the spleen and liver), followed by slower processing and clearance through the kidneys and bile.

Design rules for nanomedical engineering: from physical virology to the applications of virus-based materials in medicine

Physical virology seeks to define the principles of physics underlying viral infections, traditionally focusing on the fundamental processes governing virus assembly, maturation, and disassembly. A detailed understanding of virus structure and assembly has facilitated the development and analysis of virus-based materials for medical applications. In this Physical Virology review article, we discuss the recent developments in nanomedicine that help us to understand how physical properties affect the in vivo fate and clinical impact of (virus-based) nanoparticles. We summarize and discuss the design rules that need to be considered for the successful development and translation of virus-based nanomaterials from bench to bedside.

Interface of Physics and Biology: Engineering Virus-Based Nanoparticles for Biophotonics

Virus-based nanoparticles (VNPs) have been used for a wide range of applications, spanning basic materials science and translational medicine. Their propensity to self-assemble into precise structures that offer a three-dimensional scaffold for functionalization has led to their use as optical contrast agents and related biophotonics applications. A number of fluorescently labeled platforms have been developed and their utility in optical imaging demonstrated, yet their optical properties have not been investigated in detail. In this study, two VNPs of varying architectures were compared side-by-side to determine the impact of dye density, dye localization, conjugation chemistry, and microenvironment on the optical properties of the probes. Dyes were attached to icosahedral cowpea mosaic virus (CPMV) and rod-shaped tobacco mosaic virus (TMV) through a range of chemistries to target particular side chains displayed at specific locations around the virus. The fluorescence intensity and lifetime of the particles were determined, first using photochemical experiments on the benchtop, and second in imaging experiments using tissue culture experiments. The virus-based optical probes were found to be extraordinarily robust under ultrashort, pulsed laser light conditions with a significant amount of excitation energy, maintaining structural and chemical stability. The most effective fluorescence output was achieved through dye placement at optimized densities coupled to the exterior surface avoiding conjugated ring systems. Lifetime measurements indicate that fluorescence output depends not only on spacing the fluorophores, but also on dimer stacking and configurational changes leading to radiationless relaxation—and these processes are related to the conjugation chemistry and nanoparticle shape. For biological applications, the particles were also examined in tissue culture, from which it was found that the optical properties differed from those found on the benchtop due to effects from cellular processes and uptake kinetics. Data indicate that fluorescent cargos are released in the endolysosomal compartment of the cell targeted by the virus-based optical probes. These studies provide insight into the optical properties and fates of fluorescent proteinaceous imaging probes. The cellular release of cargo has implications not only for virus-based optical probes, but also for drug delivery and release systems.

Detection and Imaging of Aggressive Cancer Cells Using an Epidermal Growth Factor Receptor (EGFR)-Targeted Filamentous Plant Virus-Based Nanoparticle

Molecular imaging approaches and targeted drug delivery hold promise for earlier detection of diseases and treatment with higher efficacy while reducing side effects, therefore increasing survival rates and quality of life. Virus-based nanoparticles are a promising platform because their scaffold can be manipulated both genetically and chemically to simultaneously display targeting ligands while carrying payloads for diagnosis or therapeutic intervention. Here, we displayed a 12-amino-acid peptide ligand, GE11 (YHWYGYTPQNVI), on nanoscale filaments formed by the plant virus potato virus X (PVX). Bioconjugation was used to produce fluorescently labeled PVX-GE11 filaments targeted toward the epidermal growth factor receptor (EGFR). Cell detection and imaging was demonstrated using human skin epidermoid carcinoma, colorectal adenocarcinoma, and triple negative breast cancer cell lines (A-431, HT-29, MDA-MB-231), all of which upregulate EGFR to various degrees. Nonspecific uptake in ductal breast carcinoma (BT-474) cells was not observed. Furthermore, co-culture experiments with EGFR(+) cancer cells and macrophages indicate successful targeting and partitioning toward the cancer cells. This study lays a foundation for the development of EGFR-targeted filaments delivering contrast agents for imaging and diagnosis, and/or toxic payloads for targeted drug delivery.