Pratik Singh

Canine parvovirus-like particles, a novel nanomaterial for tumor targeting.

Specific targeting of tumor cells is an important goal for the design of nanotherapeutics for the treatment of cancer. Recently, viruses have been explored as nano-containers for specific targeting applications, however these systems typically require modification of the virus surface using chemical or genetic means to achieve tumor-specific delivery. Interestingly, there exists a subset of viruses with natural affinity for receptors on tumor cells that could be exploited for nanotechnology applications. For example, the canine parvovirus (CPV) utilizes transferrin receptors (TfRs) for binding and cell entry into canine as well as human cells. TfRs are over-expressed by a variety of tumor cells and are widely being investigated for tumor-targeted drug delivery. We explored whether the natural tropism of CPV to TfRs could be harnessed for targeting tumor cells. Towards this goal, CPV virus-like particles (VLPs) produced by expression of the CPV-VP2 capsid protein in a baculovirus expression system were examined for attachment of small molecules and delivery to tumor cells. Structural modeling suggested that six lysines per VP2 subunit are presumably addressable for bioconjugation on the CPV capsid exterior. Between 45 and 100 of the possible 360 lysines/particle could be routinely derivatized with dye molecules depending on the conjugation conditions. Dye conjugation also demonstrated that the CPV-VLPs could withstand conditions for chemical modification on lysines. Attachment of fluorescent dyes neither impaired binding to the TfRs nor affected internalization of the 26 nm-sized VLPs into several human tumor cell lines. CPV-VLPs therefore exhibit highly favorable characteristics for development as a novel nanomaterial for tumor targeting.

Plasma Clearance of Bacteriophage Qβ Particles as a Function of Surface Charge

Self-assembled protein capsids have gained attention as a promising class of nanoparticles for biomedical applications due to their monodisperse nature and versatile genetic and chemical tailorability. To determine the plasma clearance and tissue distribution in mice of the versatile capsid of bacteriophage Qbeta, the particles were decorated with gadolinium complexes using the CuI-mediated azide-alkyne cycloaddition reaction. Interior surface labeling was engineered by the introduction of an azide-containing unnatural amino acid into the coat protein for the first time. Clearance rates were conveniently monitored by quantitative detection of Gd using inductively coupled plasma optical emission spectroscopy and were found to be inversely proportional to the number of complexes attached to the exterior surface of the particle. This phenomenon was correlated to changes in exterior surface charge brought about by acylation of surface-exposed amine groups in the initial step of the bioconjugation protocol. When primary amine groups were reintroduced by azide-alkyne coupling, the circulation time increased accordingly. These results show that nanoparticle trafficking may be tailored in predictable ways by chemical and genetic modifications that modulate surface charge.

Tomato Bushy Stunt Virus (TBSV), A Versatile Platform for Polyvalent Display of Antigenic Epitopes and Vaccine Design

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDelta52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T=1, T=3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.