Structurally uniform protein nanoparticles derived from the self-assembly of viral capsid proteins are attractive platforms for the multivalent display of cell-targeting motifs for use in nanomedicine. Virus-based nanoparticles are of particular interest because the scaffold can be manipulated both genetically and chemically to simultaneously display targeting groups and carry a functional payload. Here, we displayed the human epidermal growth factor (EGF) on the exterior surface of bacteriophage Qβ as a C-terminal genetic fusion to the Qβ capsid protein. The co-assembly of wild-type Qβ and EGF-modified subunits resulted in structurally homogeneous nanoparticles displaying between 5 and 12 copies of EGF on their exterior surface. The particles were found to be amenable to bioconjugation via standard methods as well as the high-fidelity copper catalyzed azide-alkyne cycloaddition reaction (CuAAC). Such chemical derivatization did not impair the ability of the particles to specifically interact with the EGF receptor. Additionally, the particle-displayed EGF remained biologically active promoting auto-phosphorylation of the EGF receptor and apoptosis of A431 cells. These results suggest that hybrid Qβ-EGF nanoparticles could be useful vehicles for targeted delivery of imaging and/or therapeutic agents.

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qβ VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA.

The single-coat protein (CP) of bacteriophage Qβ self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.

Qβ virus-like particles encapsulating multiple copies of fluorescent proteins were generated in high yields using a modular system enhanced by specific engineered RNA-protein interactions. The resulting particles were structurally indistinguishable from recombinant Qβ alone. The encapsidated proteins were nearly identical in photochemical properties to monomeric analogues, were more stable toward thermal degradation, and were protected from proteolytic cleavage. Residues on the outer capsid surface were chemically derivatized by acylation and azide-alkyne cycloaddition without affecting the fluorescence properties of the packaged proteins. A high affinity carbohydrate-based ligand of the CD22 receptor was thereby attached, and specific cell labeling by the particles was successfully detected by flow cytometry and confocal laser microscopy.

Phenotypic high throughput screens are a valuable tool for identifying new chemical compounds with antimalarial activity. Traditionally, these screens have focused solely on the symptomatic asexual blood stage of the parasite's lifecycle; however, in order to discover new medicines for malaria's treatment and prevention, robust screening technologies against other parasite lifecycle stages are required. This review highlights recent advances and progress toward phenotypic screening methodologies over the past several years, with a focus on exoerythrocytic stage screens.