Karin L. Lee

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.

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.

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.

Virus‐based nanoparticles as platform technologies for modern vaccines

Nanoscale engineering is revolutionizing the development of vaccines and immunotherapies. Viruses have played a key role in this field because they can function as prefabricated nanoscaffolds with unique properties that are easy to modify. Viruses are immunogenic via multiple pathways, and antigens displayed naturally or by engineering on the surface can be used to create vaccines against the cognate virus, other pathogens, specific molecules or cellular targets such as tumors. This review focuses on the development of virus-based nanoparticle systems as vaccines indicated for the prevention or treatment of infectious diseases, chronic diseases, cancer, and addiction. WIREs Nanomed Nanobiotechnol 2016, 8:554-578. doi: 10.1002/wnan.1383 For further resources related to this article, please visit the WIREs website.

Shaping bio-inspired nanotechnologies to target thrombosis for dual optical-magnetic resonance imaging

Arterial and venous thrombosis are among the most common causes of death and hospitalization worldwide. Nanotechnology approaches hold great promise for molecular imaging and diagnosis as well as tissue-targeted delivery of therapeutics. In this study, we developed and investigated bioengineered nanoprobes for identifying thrombus formation; the design parameters of nanoparticle shape and surface chemistry, i.e. incorporation of fibrin-binding peptides CREKA and GPRPP, were investigated. Two nanoparticle platforms based on plant viruses were studied - icosahedral cowpea mosaic virus (CPMV) and elongated rod-shaped tobacco mosaic virus (TMV). These particles were loaded to carry contrast agents for dual-modality magnetic resonance (MR) and optical imaging, and both modalities demonstrated specificity of fibrin binding in vitro with the presence of targeting peptides. Preclinical studies in a carotid artery photochemical injury model of thrombosis confirmed thrombus homing of the nanoprobes, with the elongated TMV rods exhibiting significantly greater attachment to thrombi than icosahedral (sphere-like) CPMV. While in vitro studies confirmed fibrin-specificity conferred by the peptide ligands, in vivo studies indicated the nanoparticle shape had the greatest contribution toward thrombus targeting, with no significant contribution from either targeting ligand. These results demonstrate that nanoparticle shape plays a critical role in particle deposition at the site of vascular injury. Shaping nanotechnologies opens the door for the development of novel targeted diagnostic and therapeutic strategies (i.e., theranostics) for arterial and venous thrombosis.

High Aspect Ratio Nanotubes Formed by Tobacco Mosaic Virus for Delivery of Photodynamic Agents Targeting Melanoma

Melanoma is a highly aggressive cancer that is unresponsive to many traditional therapies. Recently, photodynamic therapy has shown promise in its treatment as an adjuvant therapy. However, conventional photosensitizers are limited by poor solubility and limited accumulation within target tissue. Here, we report the delivery of a porphyrin-based photosensitizer encapsulated within a plant viral nanoparticle. Specifically, we make use of the hollow, high aspect ratio nanotubes formed by the nucleoprotein components of tobacco mosaic virus (TMV) to encapsulate the drug for delivery and targeting of cancer cells. The cationic photosensitizer was successfully and stably loaded into the interior channel of TMV via electrostatic interactions. Cell uptake and efficacy were evaluated using a model of melanoma. The resulting TMV-photosensitizer exhibited improved cell uptake and efficacy when compared to free photosensitizer, making it a promising platform for improved therapy of melanoma.

Shape matters: the diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct

Nanomaterial-based carrier systems hold great promise to deliver therapies with increased efficacy and reduced side effects. While the state-of-the-art carrier system is a sphere, recent data indicate that elongated rods and filaments have advantageous flow and margination properties, resulting in enhanced vascular targeting and tumor homing. Here, we report on the distinct diffusion rates of two bio-inspired carrier systems: 30 nm-sized spherical cowpea mosaic virus (CPMV) and 300×18 nm-sized tobacco mosaic virus (TMV) with a tubular structure, using a spheroid model of the tumor microenvironment and fluorescent imaging.

Genetic Engineering and Chemical Conjugation of Potato Virus X

Here we report the genetic engineering and chemical modification of potato virus X (PVX) for the presentation of various peptides, proteins, and fluorescent dyes, or other chemical modifiers. Three different ways of genetic engineering are described and by these means, peptides are successfully expressed not only when the foot and mouth disease virus (FMDV) 2A sequence or a flexible glycine-serine linker is included, but also when the peptide is fused directly to the PVX coat protein. When larger proteins or unfavorable peptide sequences are presented, a partial fusion via the FMDV 2A sequence is preferable. When these PVX chimeras retain the ability to assemble into viral particles and are thus able to infect plants systemically, they can be utilized to inoculate susceptible plants for isolation of sufficient amounts of virus particles for subsequent chemical modification. Chemical modification is required for the display of nonbiological ligands such as fluorophores, polymers, and small drug compounds. We present three methods of chemical bioconjugation. For direct conjugation of small chemical modifiers to solvent exposed lysines, N-hydroxysuccinimide chemistry can be applied. Bio-orthogonal reactions such as copper-catalyzed azide-alkyne cycloaddition or hydrazone ligation are alternatives to achieve more efficient conjugation (e.g., when working with high molecular weight or insoluble ligands). Furthermore, hydrazone ligation offers an attractive route for the introduction of pH-cleavable cargos (e.g., therapeutic molecules).

Plasmonic Nanodiamonds – Targeted Core-shell Type Nanoparticles for Cancer Cell Thermoablation

Targeted biocompatible nanostructures with controlled plasmonic and morphological parameters are promising materials for cancer treatment based on selective thermal ablation of cells. Here, core-shell plasmonic nanodiamonds consisting of a silica-encapsulated diamond nanocrystal coated in a gold shell are designed and synthesized. The architecture of particles is analyzed and confirmed in detail using electron tomography. The particles are biocompatibilized using a PEG polymer terminated with bioorthogonally reactive alkyne groups. Azide-modified transferrin is attached to these particles, and their high colloidal stability and successful targeting to cancer cells overexpressing the transferrin receptor are demonstrated. The particles are nontoxic to the cells and they are readily internalized upon binding to the transferrin receptor. The high plasmonic cross section of the particles in the near-infrared region is utilized to quantitatively ablate the cancer cells with a short, one-minute irradiation by a pulse 750-nm laser.

Utilizing Viral Nanoparticle/Dendron Hybrid Conjugates in Photodynamic Therapy for Dual Delivery to Macrophages and Cancer Cells.

Photodynamic therapy (PDT) is a promising avenue for greater treatment efficacy of highly resistant and aggressive melanoma. Through photosensitizer attachment to nanoparticles, specificity of delivery can be conferred to further reduce potential side effects. While the main focus of PDT is the destruction of cancer cells, additional targeting of tumor-associated macrophages also present in the tumor microenvironment could further enhance treatment by eliminating their role in processes such as invasion, metastasis, and immunosuppression. In this study, we investigated PDT of macrophages and tumor cells through delivery using the natural noninfectious nanoparticle cowpea mosaic virus (CPMV), which has been shown to have specificity for the immunosuppressive subpopulation of macrophages and also targets cancer cells. We further explored conjugation of CPMV/dendron hybrids in order to improve the drug loading capacity of the nanocarrier. Overall, we demonstrated effective elimination of both macrophage and tumor cells at low micromolar concentrations of the photosensitizer when delivered with the CPMV bioconjugate, thereby potentially improving melanoma treatment.