The research program described herein involves the construction and evaluation of versatile carriers that can image the various stages of atherosclerosis using PET. The basic platform for these applications is the hollow protein shell of a virus that has been modified on its exterior and interior surfaces with synthetic molecules. These studies will yield a well-understood platform for the study of cardiovascular disease, as well as key design information for the construction of future nanoscale delivery vehicles.
The availability of well-defined nanoscale materials offers exciting new approaches for the diagnosis and treatment of disease. In contrast to small molecule drugs, the relatively large dimensions of these structures al- low many different components to be combined into a complex, multifunctional device. However, these materials have been difficult to produce in practice due to the lack of suitable scaffolds that offer low toxicity, high stability in biological settings, and strategic sites for the attachment of building blocks for biomedical applications. To fulfill this need, the research program described herein will convert the hollow protein shell of a virus into a series of targeted imaging agents for the study and diagnosis of cardiovascular disease. This will be accomplished by combining the extensive protein modification experience of the Francis group (UC Berkeley Chemistry Department) with the cardiovascular imaging and microPET expertise of the UCSF Center for Molecular and Functional Imaging. Specifically, we will install groups on the outside of genome-free MS2 viral capsids to cause them to localize at the early (targeting VCAM-1), middle (targeting macrophages), and late (targeting fibrin clots) stages of atherosclerosis. The inside surfaces of these carriers will be doubly functionalized with fluorescent chromophores and chelators for long-lived radionuclides (such as 64Cu). The radiotracers will allow the capsids to be imaged using PET, and will assist in quantitative biodistribution studies. The fluorophores will allow the detection and differentiation of the capsid agents during histopathological staining. Preliminary studies have already characterized the baseline biodistribution properties of untargeted capsids, indicating that they display unexpectedly long blood circulation times. In the proposed studies, the biodistribution of each targeted agent will first be characterized in healthy mice in order to determine the effects of external functionalization on clearance time. They will then be imaged in ApoE(-/-) mice that have been fed a high fat diet for varying lengths of time. Sites of in vivo accumulation will be verified using contrast-enhanced CT scans, as well as histopathology of excised regions. In addition to verifying their ability to localize with the appropriate bimolecular targets, we will determine whether the agents are capable of distinguishing the different stages of atherosclerotic plaque formation.