Alaa A. A. Aljabali

CPMV-DOX delivers

The plant virus, Cowpea mosaic virus (CPMV), is developed as a carrier of the chemotherapeutic drug doxorubicin (DOX). CPMV-DOX conjugate, in which eighty DOX molecules are covalently bound to external surface carboxylates of the viral nanoparticle (VNP), shows greater cytotoxicity than free DOX toward HeLa cells when administered at low dosage. At higher concentrations, CPMV-DOX cytotoxicity is time-delayed. The CPMV conjugate is targeted to the endolysosomal compartment of the cells, in which the proteinaceous drug carrier is degraded and the drug released. This study is the first demonstrating the utility of CPMV as a drug delivery vehicle.

Cowpea mosaic virus unmodified empty viruslike particles loaded with metal and metal oxide.

The development of methods for the production of empty Cowpea mosaic virus (CPMV) virus-like particles (VLPs) that are devoid of RNA, eVLPs, has renewed promise in CPMV capsid technologies. The recombinant nature of CPMV eVLP production means that the extent and variety of genetic modifications that may be incorporated into the particles is theoretically much greater than those that can be made to infectious CPMV virions due to restrictions on viral propagation of the latter. Free of the infectious agent, the genomic RNA, these particles are now finding potential uses in vaccine development, in vivo imaging, drug delivery, and other nanotechnology applications that make use of internal loading of the empty particles. Here we describe methods for the genetic modification and production of CPMV eVLPs and describe techniques useful for their characterization.

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.

Environmentally benign synthesis of virus-templated, monodisperse, iron-platinum nanoparticles

The use of an engineered variant of a plant virus, Cowpea mosaic virus (CPMV), as a template for directed mineralization provides an environmentally benign route to monodisperse iron-platinum nanoparticles of approximately 30 nm diameter.

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.

Peptide-Controlled Access to the Interior Surface of Empty Virus Nanoparticles

The structure of Cowpea mosaic virus (CPMV) is known to high resolution, thereby enabling the rational use of the particles in diverse applications, from vaccine design to nanotechnology. A recently devised method for the production of empty virus‐like particles (eVLPs) has opened up new possibilities for CPMV capsid‐based technologies, such as internal mineralisation of the particle. We have investigated the role of the carboxyl (C) terminus of the small coat (S) protein in controlling access to the interior of CPMV eVLPs by determining the efficiency of internal mineralisation. The presence of the C‐terminal 24‐amino acid peptide of the S protein was found to inhibit internal mineralisation, an effect that could be eliminated by enzymatic removal of this region. We have also demonstrated the amenability of the C terminus to genetic modification. Substitution with six histidine residues generated stable particles and facilitated external mineralisation by cobalt. These findings demonstrate consistent internal and external mineralisation of CPMV, and will aid the further exploration and development of the use of eVLPs for bionanotechnological and medical applications.

CPMV-polyelectrolyte-templated gold nanoparticles.

The use of polyelectrolyte surface-modified Cowpea mosaic virus (CPMV) for the templated synthesis of narrowly dispersed gold nanoparticles is described. The cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), is electrostatically bound to the external surface of the virus capsid; the polyelectrolyte promotes the adsorption of anionic gold complexes, which are then easily reduced, under mild conditions, to form a metallic gold coating. As expected, the templated gold nanoparticles can be further modified with thiol reagents. In contrast, reaction of polyelectrolyte-modified CPMV (CPMV-PA) with preformed gold nanoparticles results in the self-assembly of large, hexagonally packed, tessellated-spheres.

Genetic engineering and characterization of Cowpea mosaic virus empty virus-like particles.

The development of methods for the production of empty Cowpea mosaic virus (CPMV) virus-like particles (VLPs) that are devoid of RNA, eVLPs, has renewed promise in CPMV capsid technologies. The recombinant nature of CPMV eVLP production means that the extent and variety of genetic modifications that may be incorporated into the particles is theoretically much greater than those that can be made to infectious CPMV virions due to restrictions on viral propagation of the latter. Free of the infectious agent, the genomic RNA, these particles are now finding potential uses in vaccine development, in vivo imaging, drug delivery, and other nanotechnology applications that make use of internal loading of the empty particles. Here we describe methods for the genetic modification and production of CPMV eVLPs and describe techniques useful for their characterization.

Structure-based design and experimental engineering of a plant virus nanoparticle for the presentation of immunogenic epitopes and as a drug carrier

Biomaterials research for the discovery of new generation nanoparticles is one of the most active areas of nanotechnoloy. In the search of nature-made nanometer-sized objects, plant virus particles appear as symmetrically defined entities that can be formed by protein self-assembly. In particular, in the field of plant virology, there is plenty of literature available describing the exploitation of plant viral cages to produce safe vaccine vehicles and nanoparticles for drug delivery. In this context, we have investigated on the use of the artichoke mottled crinkle virus (AMCV) capsid both as a carrier of immunogenic epitopes and for the delivery of anticancer molecules. A dual approach that combines both in silico tools and experimental virology was applied for the rational design of immunologically active chimeric virus-like particles (VLPs) carrying immunogenic peptides. The atomic structures of wild type (wt) and chimeric VLPs were obtained by homology modeling. The effects of insertion of the HIV-1 2F5 neutralizing epitope on the structural stability of chimeric VLPs were predicted and assessed by detailed inspection of the nanoparticle intersubunit interactions at atomic level. Wt and chimeric VLPs, exposing on their surface the 2F5 epitope, were successfully produced in plants. In addition, we demonstrated that AMCV capsids could also function as drug delivery vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first systematic predictive and empirical research addressing the question of how this icosahedral virus can be used for the production of both VLPs and viral nanoparticles for biomedical applications.

Structural insights into magnetic clusters grown inside virus capsids.

Magnetic nanoparticles have multiple applications in materials science. In particular, virus capsids have been suggested as promising templates for building up nanometric-sized magnetic clusters by taking advantage of their inner cavity as a nanoreactor. In this study we investigate the magnetization of individual cobalt-filled cowpea mosaic virus empty virus-like particles using atomic force microscopy. We also combine the analysis of the effects of dehydration on the structure of virus particles with a comparison of their magnetic signal to that provided by commercially available magnetic nanoparticles of similar size. These two approaches allow the evaluation of the structure of the metallic cluster grown inside the virus capsid. We conclude that, rather than forming solid clusters, cobalt inside viruses forms a discontinuous structure that does not completely fill the virus cavity and reaches about 10% of its volume.