Neetu M. Gulati

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.

Silica-coated Gd(DOTA)-loaded protein nanoparticles enable magnetic resonance imaging of macrophages.

The molecular imaging of in vivo targets allows non-invasive disease diagnosis. Nanoparticles offer a promising platform for molecular imaging because they can deliver large payloads of imaging reagents to the site of disease. Magnetic resonance imaging (MRI) is often preferred for clinical diagnosis because it uses non-ionizing radiation and offers both high spatial resolution and excellent penetration. We have explored the use of plant viruses as the basis of for MRI contrast reagents, specifically Tobacco mosaic virus (TMV), which can assemble to form either stiff rods or spheres. We loaded TMV particles with paramagnetic Gd ions, increasing the ionic relaxivity compared to free Gd ions. The loaded TMV particles were then coated with silica maintaining high relaxivities. Interestingly, we found that when Gd(DOTA) was loaded into the interior channel of TMV and the exterior was coated with silica, the T1 relaxivities increased by three-fold from 10.9 mM-1 s-1 to 29.7 mM-1s-1 at 60 MHz compared to uncoated Gd-loaded TMV. To test the performance of the contrast agents in a biological setting, we focused on interactions with macrophages because the active or passive targeting of immune cells is a popular strategy to investigate the cellular components involved in disease progression associated with inflammation. In vitro assays and phantom MRI experiments indicate efficient targeting and imaging of macrophages, enhanced contrast-to-noise ratio was observed by shape-engineering (SNP > TMV) and silica-coating (Si-TMV/SNP > TMV/SNP). Because plant viruses are in the food chain, antibodies may be prevalent in the population. Therefore we investigated whether the silica-coating could prevent antibody recognition; indeed our data indicate that mineralization can be used as a stealth coating option to reduce clearance. Therefore, we conclude that the silica-coated protein-based contrast agent may provide an interesting candidate material for further investigation for in vivo delineation of disease through macrophage imaging.

Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays

The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe.

Multiple Administrations of Viral Nanoparticles Alter in Vivo Behavior—Insights from Intravital Microscopy

Multiple administrations of nanoparticle-based formulations are often a clinical requirement for drug delivery and diagnostic imaging applications. Steady pharmacokinetics of nanoparticles is desirable to achieve efficient therapeutic or diagnostic outcomes over such repeat administrations. While clearance through mononuclear phagocytic system is a key determinant of nanoparticle persistence in vivo, multiple administrations could potentially result in altered pharmacokinetics by evoking innate or adaptive immune responses. Plant viral nanoparticles (VNPs) represent an emerging class of programmable nanoparticle platform technologies that offer a highly organized proteinaceous architecture and multivalency for delivery of large payloads of drugs and molecular contrast agents. These very structural features also render them susceptible to immune recognition and subsequent accelerated systemic clearance that could potentially affect overall efficiency. While the biodistribution and pharmacokinetics of VNPs have been reported, the biological response following repeat administrations remains an understudied area of investigation. Here, we demonstrate that weekly administration of filamentous plant viruses results in the generation of increasing levels of circulating, carrier-specific IgM and IgG antibodies. Furthermore, PVX specific immunoglobulins from the serum of immunized animals quickly form aggregates when incubated with PVX in vitro. Such aggregates of VNP-immune complexes are also observed in the mouse vasculature in vivo following repeat injections when imaged in real time using intravital two-photon laser scanning microscopy (2P-LSM). The size of aggregates diminishes at later time points, coinciding with antibody class switching from IgM to IgG. Together, our results highlight the need for careful in vivo assessment of (viral) nanoparticle-based platform technologies, especially in studying their performance after repeat administration. We also demonstrate the utility of intravital microscopy to aid in this evaluation.

Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs

Platform technologies based on plant virus nanoparticles (VNPs) and virus-like particles (VLPs) are attracting the attention of researchers and clinicians because the particles are biocompatible, biodegradable, noninfectious in mammals, and can readily be chemically and genetically engineered to carry imaging agents and drugs. When the Physalis mottle virus (PhMV) coat protein is expressed in Escherichia coli, the resulting VLPs are nearly identical to the viruses formed in vivo. Here, we isolated PhMV-derived VLPs from ClearColi cells and carried out external and internal surface modification with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries, respectively. The uptake of dye-labeled particles was tested in a range of cancer cells and monitored by confocal microscopy and flow cytometry. VLPs labeled internally on cysteine residues were taken up with high efficiency by several cancer cell lines and were colocalized with the endolysosomal marker LAMP-1 within 6 h, whereas VLPs labeled externally on lysine residues were taken up with lower efficiency, probably reflecting differences in surface charge and the propensity to bind to the cell surface. The infusion of dye and drug molecules into the cavity of the VLPs revealed that the photosensitizer (PS), Zn-EpPor, and the drugs crystal violet, mitoxantrone (MTX), and doxorubicin (DOX) associated stably with the carrier via noncovalent interactions. We confirmed the cytotoxicity of the PS-PhMV and DOX-PhMV particles against prostate cancer, ovarian and breast cancer cell lines, respectively. Our results show that PhMV-derived VLPs provide a new platform technology for the delivery of imaging agents and drugs, with preferential uptake into cancer cells. These particles could therefore be developed as multifunctional tools for cancer diagnosis and therapy.

Bioinspired Shielding Strategies for Nanoparticle Drug Delivery Applications

Nanoparticle delivery systems offer advantages over free drugs, in that they increase solubility and biocompatibility. Nanoparticles can deliver a high payload of therapeutic molecules while limiting off-target side effects. Therefore, delivery of an existing drug with a nanoparticle frequently results in an increased therapeutic index. Whether of synthetic or biologic origin, nanoparticle surface coatings are often required to reduce immune clearance and thereby increase circulation times allowing the carriers to reach their target site. To this end, polyethylene glycol (PEG) has long been used, with several PEGylated products reaching clinical use. Unfortunately, the growing use of PEG in consumer products has led to an increasing prevalence of PEG-specific antibodies in the human population, which in turn has fueled the search for alternative coating strategies. This review highlights alternative bioinspired nanoparticle shielding strategies, which may be more beneficial moving forward than PEG and other synthetic polymer coatings.

13. Functional Role of Adenovirus Penton in Modulating In Vivo Properties of Liver-Targeted and Liver-Detargeted Adenovirus Variants

Pharmacokinetic studies of adenovirus (Ad) vectors after intravascular delivery demonstrate that the majority of an administered virus dose is rapidly sequestered from the circulation by the liver. The molecular determinants that target Ad to the liver have been found on each of the virus capsid proteins, including hexon, penton, and fiber. The penton protein in the majority of Ad serotypes comprises a hypervariable loop containing an RGD motif that has been shown to interact with a number of cellular integrins upon virus entry into the cell. The binding of penton to integrins triggers virus internalization and relaxes the virus structure for subsequent endosomal disassembly. However, RGD motif interaction with integrins on tissue macrophages leads to production of inflammatory cytokines and significantly contributes to systemic Ad toxicity. Despite its importance for the virus entry into the cell, the penton is not well employed in the development of therapeutic Ad vectors. Therefore, in this study we evaluated strategies for modulating Ad penton interactions with cellular integrins. Ad vectors were created bearing substitutions of the RGD motif with mimetic non-RGD-containing peptides derived from extracellular proteins laminin 1 and laminin 3. These peptides are interacting with a different subset of integrins compared to those binging penton RGD motif. Unlike wild type (WT) Ad5, these penton-modified vectors were able to utilize β4 integrin for cell entry. The in vivo studies demonstrated that penton-modified Ads cannot activate a full inflammatory cascade and their in vivo toxicity, therefore, is dramatically reduced. Although the penton-mutated vectors were infecting a set of cancer cell lines in vitro and hepatocytes in vivo with a similar efficacy as the WT Ad5, interactions of virus with Kupffer cells and virus accumulation in the liver after intravascular vector delivery remain similar for penton-mutated and WT Ad vector. We also developed Ad vectors containing mutations abrogating virus interactions with all types of liver cells. Additionally to penton modifications, we introduced a T425A mutation in the hexon hyper variable loop HVR7 to completely abrogate virus interaction with blood coagulation FX and prevent transduction of hepatocytes. Furthermore, a set of mutations in the hexon HVR1 region were introduced to prevent Ad uptake by Kupffer cells. Due to penton modifications these vectors had very low toxicity and combinations of three mutations diverted the vector away from the liver. These data demonstrate that RGD loop modification represents a useful approach for introducing beneficial properties to the Ad vectors for greatly reducing their systemic toxicity and enable liver de-targeting. CryoEM analyses are currently underway to identify the structural differences in the capsid between the penton mutants and the wild type Ad.

Characterization of the Shielding Properties of Serum Albumin on a Plant Viral Nanoparticle

1. Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, Ohio USA. 2. Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio USA. 3. Departments of Radiology, Macromolecular Science and Engineering, Materials Science and Engineering, and Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio USA.

CryoEM based models for adenovirus neutralization by human alpha-defensin 5

Defensins are peptides of the innate immune system with potent antimicrobial activity. Despite strong interest in defensins as potential pharmaceutical compounds, commercial development of these peptides has been challenging. New generations of modified antimicrobial peptides are being pursued to improve their potential as pharmaceuticals. Strategies include peptide mimetics, hybrid peptides, peptide congeners, stabilized peptides, peptide conjugates and immobilized peptides [1]. Our laboratories have been working on understanding the molecular mechanisms of defensins against adenovirus to characterize key interaction sites. There are six human alpha-defensins, including HD5, and multiple beta-defensins. Defensin action against enveloped viruses includes membrane disruption, as well as interference with viral membrane fusion. Alpha-defensins also neutralize viruses that lack envelopes including human adenovirus (HAdV), human papillomavirus (HPV), and polyomaviruses.