Andrzej S. Pitek

Virus-Based Nanoparticles as Versatile Nanomachines

Nanoscale engineering is revolutionizing the way we prevent, detect, and treat diseases. Viruses have played a special role in these developments because they can function as prefabricated nanoscaffolds that have unique properties and are easily modified. The interiors of virus particles can encapsulate and protect sensitive compounds, while the exteriors can be altered to display large and small molecules in precisely defined arrays. These properties of viruses, along with their innate biocompatibility, have led to their development as actively targeted drug delivery systems that expand on and improve current pharmaceutical options. Viruses are naturally immunogenic, and antigens displayed on their surface have been used to create vaccines against pathogens and to break self-tolerance to initiate an immune response to dysfunctional proteins. Densely and specifically aligned imaging agents on viruses have allowed for high-resolution and noninvasive visualization tools to detect and treat diseases earlier than previously possible. These and future applications of viruses have created an exciting new field within the disciplines of both nanotechnology and medicine.

The Protein Corona of Plant Virus Nanoparticles Influences their Dispersion Properties, Cellular Interactions, and In Vivo Fates

Biomolecules in bodily fluids such as plasma can adsorb to the surface of nanoparticles and influence their biological properties. This phenomenon, known as the protein corona, is well established in the field of synthetic nanotechnology but has not been described in the context of plant virus nanoparticles (VNPs). The interaction between VNPs derived from Tobacco mosaic virus (TMV) and plasma proteins is investigated, and it is found that the VNP protein corona is significantly less abundant compared to the corona of synthetic particles. The formed corona is dominated by complement proteins and immunoglobulins, the binding of which can be reduced by PEGylating the VNP surface. The impact of the VNP protein corona on molecular recognition and cell targeting in the context of cancer and thrombosis is investigated. A library of functionalized TMV rods with polyethylene glycol (PEG) and peptide ligands targeting integrins or fibrin(ogen) show different dispersion properties, cellular interactions, and in vivo fates depending on the properties of the protein corona, influencing target specificity, and non-specific scavenging by macrophages. Our results provide insight into the in vivo properties of VNPs and suggest that the protein corona effect should be considered during the development of efficacious, targeted VNP formulations.

POxylation as an alternative stealth coating for biomedical applications

Polyethylene glycol (PEG) polymers are currently used in a variety of medical formulations to reduce toxicity, minimize immune interactions and improve pharmacokinetics. Despite its widespread use however, the presence of anti-PEG antibodies indicates that this polymer has the potential to be immunogenic and antigenic. Here we present an alternative polymer, poly(2-oxazoline) (POx) for stealth applications, specifically shielding of a proteinaceous nanoparticle from recognition by the immune system. Tobacco mosaic virus (TMV) was used as our testbed due to its potential for use as a nanocarrier for drug delivery and molecular imaging applications.

Elongated Plant Virus-Based Nanoparticles for Enhanced Delivery of Thrombolytic Therapies

Thrombotic cardiovascular disease, including acute myocardial infarction, ischemic stroke, and venous thromboembolic disease, is the leading cause of morbidity and mortality worldwide. While reperfusion therapy with thrombolytic agents reduces mortality from acute myocardial infarction and disability from stroke, thrombolysis is generally less effective than mechanical reperfusion and is associated with fatal intracerebral hemorrhage in up to 2-5% of patients. To address these limitations, we propose the tobacco mosaic virus (TMV)-based platform technology for targeted delivery of thrombolytic therapies. TMV is a plant virus-based nanoparticle with a high aspect ratio shape measuring 300 × 18 nm. These soft matter nanorods have favorable flow and margination properties allowing the targeting of the diseased vessel wall. We have previously shown that TMV homes to thrombi in a photochemical mouse model of arterial thrombosis. Here we report the synthesis of TMV conjugates loaded with streptokinase (STK). Various TMV-STK formulations were produced through bioconjugation of STK to TMV via intervening PEG linkers. TMV-STK was characterized using SDS-PAGE and Western blot, transmission electron microscopy, cryo-electron microscopy, and cryo-electron tomography. We investigated the thrombolytic activity of TMV-STK in vitro using static phantom clots, and in a physiologically relevant hydrodynamic model of shear-induced thrombosis. Our findings demonstrate that conjugation of STK to the TMV surface does not compromise the activity of STK. Moreover, the nanoparticle conjugate significantly enhances thrombolysis under flow conditions, which can likely be attributed to TMVs shape-mediated flow properties resulting in enhanced thrombus accumulation and dissolution. Together, these data suggest TMV to be a promising platform for the delivery of thrombolytics to enhance clot localization and potentially minimize bleeding risk.

Featured Article: Delivery of chemotherapeutic vcMMAE using tobacco mosaic virus nanoparticles

The first-line treatment for non-Hodgkin’s lymphoma is chemotherapy. While generally well tolerated, off-target effects and chemotherapy-associated complications are still of concern. To overcome the challenges associated with systemic chemotherapy, we developed a biology-inspired, nanoparticle drug delivery system (nanoDDS) making use of the nucleoprotein components of the tobacco mosaic virus (TMV). Virus-based nanoparticles, including the high-aspect ratio soft nanorods formed by TMV, are growing in popularity as nanoDDS due to their simple genetic and chemical engineerability, size and shape tunability, and biocompatibility. In this study, we used bioconjugation to modify TMV as a multivalent carrier for delivery of the antimitotic drug valine-citrulline monomethyl auristatin E (vcMMAE) targeting non-Hodgkin’s lymphoma. We demonstrate successful synthesis of the TMV-vcMMAE; data indicate that the TMV-vcMMAE particles remained structurally sound with all of the 2130 identical TMV coat proteins modified to carry the therapeutic payload vcMMAE. Cell uptake using Karpas 299 cells was confirmed with TMV particles trafficking to the endolysosomal compartment, likely allowing for protease-mediated cleavage of the valine-citrulline linker for the release of the active monomethyl auristatin E component. Indeed, effective cell killing of non-Hodgkin’s lymphoma in vitro was demonstrated; TMV-vcMMAE was shown to exhibit an IC50 of ∼250 nM. This study contributes to the development of viral nanoDDS. Impact statement Due to side effects associated with systemic chemotherapy, there is an urgent need for the development of novel drug delivery systems. We focus on the high-aspect ratio nanotubes formed by tobacco mosaic virus (TMV) to deliver antimitotic drugs targeted to non-Hodgkin’s lymphoma. Many synthetic and biologic nanocarriers are in the development pipeline; the majority of systems are spherical in shape. This may not be optimal, because high-aspect ratio filaments exhibit enhanced tumor homing, increased target cell interactions and decreased immune cell uptake, and therefore have favorable properties for drug delivery compared to their spherical counterparts. Nevertheless, the synthesis of high-aspect ratio materials at the nanoscale remains challenging; therefore, we turned toward the nucleoprotein components of TMV as a biologic nanodrug delivery system. This work presents groundwork for the development of plant virus-based vehicles for use in cancer treatment.

Nanomedicine: The Protein Corona of Plant Virus Nanoparticles Influences their Dispersion Properties, Cellular Interactions, and In Vivo Fates (Small 13/2016).

The interactions between targeted plant virus-based nanoparticles and the proteins they encounter when injected into the bloodstream are investigated by N.F. Steinmetz and co-workers, on page 1758. The identification of which plasma proteins bind to the surface of plant viral nanoparticles and how that affects their dispersion, targeting, and clearance by the mononuclear phagocytic system, is demonstrated. The consequences of these interactions on biodistribution are also shown.

Interactions Between Plant Viral Nanoparticles (VNPs) and Blood Plasma Proteins, and Their Impact on the VNP In Vivo Fates

Plant viral nanoparticles (VNPs) are currently being developed as novel vessels for delivery of diagnostic and therapeutic cargos to sites of disease. With a rapid increase in the number of VNP variants and their potential applications in nanomedicine, the properties they acquire in the bloodstream need to be investigated. Biomolecules present in plasma are known to adsorb onto the surface of nanomaterials (including VNPs), forming a biointerface called the protein corona, which is capable of reprogramming the properties of VNPs. Here we describe a few general methods to isolate and study the VNP-protein corona complexes, in order to evaluate the impact of protein corona on molecular recognition of VNPs by target cells, and clearance by phagocytes. We outline procedures for in vivo screening of VNP fates in a mouse model, which may be useful for evaluation of efficacy and biocompatibility of different VNP based formulations.

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.