Virus-like particles (VLPs) as iron oxide contrast agent carriers for enhanced prostate tumor imaging [electronic resource]
Keywords: Chemical Engineering.

Virus-like particles (VLPs)—non-infectious nanoparticles composed of viral capsid proteins—hold enormous potential as molecular delivery agents. They are small and uniform in size and possess an ordered surface, which provides multiple attachment points for molecules such as antibodies. VLPs are thus an ideal scaffold for specifically targeting prostate cancer, a disease in which the tumor cells possess well-characterized surface markers. For diagnostic purposes, however, we need to couple the VLP with an entity that can detect even the smallest prostate tumor cell populations. This sensitivity can be imparted by magnetic resonance imaging (MRI) contrast agents known as superparamagnetic iron oxide nanoparticles (SPIONs), which have been shown to enable detection down to the single cell level. Here, we explore the engineering of a novel SPION-loaded VLP construct which exhibits both the sensitivity and specificity necessary for early and accurate detection of prostate cancer. First, we developed a novel biphasic method for stabilizing organic-soluble SPIONs of diameters up to 20 nm in an aqueous environment and concomitantly functionalizing their surface such that they are suitable for loading inside VLPs. We studied various parameters to optimize phase transfer of monodisperse, water-soluble SPIONs while maximizing relaxivities (r2 ≥ 250 mMFe^-1s^-1). This functionalization technique enables us to produce SPIONs with fine control over surface chemistry while minimizing coating thickness. We then evaluated various strategies for loading the functionalized SPIONs into VLPs. First, we examined using the MS2 bacteriophage VLP, which natively self-assembles via an interaction with an RNA stem loop sequence. We attempted to load the SPIONs either as the VLP capsid monomers were being produced in a cell-free protein synthesis system (co-translationally), or after VLP production and disassembly (post-translationally). Stability issues arising from SPION-RNA interactions precluded VLP assembly attempts in the former case, while minimal VLP assembly around the SPIONs was observed in the latter. We then shifted our efforts to using the hepatitis B core protein (HBc) VLP, which is slightly larger in diameter than MS2 and assembles predominantly via hydrophobic interactions. We focused on a posttranslational loading strategy in which the HBc VLP component dimers are first produced and purified before introducing the SPIONs. We developed an effective method for affinity-purifying the HBc subunit dimers by adding a hexahistidine tag at the C-terminus of the protein. The C-terminus was also mutated to include a series of charged amino acids with the purpose of enabling templated assembly of the VLP around the functionalized SPIONs via electrostatic interactions between the dimers and the SPIONs. Initial transmission electron microscopy (TEM) and dynamic light scattering (DLS) data suggest that this assembly approach is capable of producing SPIONs encapsidated within VLPs. We are optimistic that the research presented here lays the groundwork for utilizing VLPs as molecular carriers for enhanced MRI of prostate cancer, as well as for other diagnostic and therapeutic applications through modification of VLP surface ligands and payloads.