Detection of targeted perfluorocarbon nanoparticle binding using 19F diffusion weighted MR spectroscopy.

A principal objective in cancer research and diagnosis is the noninvasive detection of small preclinical lesions, which afford opportunities for early intervention. Molecular imaging with targeted MRI contrast agents has emerged as a promising diagnostic approach offering high resolution depiction of pathological anatomy and detection of associated disease biomarkers (1,2). However, molecular MRI with contrast agents (lanthanide- or iron oxide-based) entails indirect assessment of the effect of targeted agents on the relaxation of surrounding water molecules (3), achieving only relative tumor contrast enhancement above the background proton signal. Additionally, intravenous injection of iron oxide compounds for molecular MRI require a delay of 24 h or more for blood clearance to avoid detection of an overwhelming blood pool signal (4). As lengthy delays pose a problem for efficient clinical imaging of patients, it would be preferable to use an agent with a unique MR signature that could be detected shortly after injection and was devoid of background signal or susceptibility artifact. Our laboratory has developed targeted liquid perfluorocarbon (PFC) nanoparticles for molecular imaging that are operational at both clinical (1.5 Tesla [T]) and research (11.7T) field strengths (5). Nanoparticles have been targeted to angiogenesis by incorporating high-affinity peptidomimetic binding ligands specific for αvβ3-integrin, which is highly up-regulated on activated malignant neovasculature endothelium (6). These 200-nm PFC particles contain very high concentrations of 19F (~100M), a high-signal nucleus for MRI. Because the low physiological concentration of 19F in biological tissues is essentially undetectable by MRI (7), PFC nanoparticles offer a unique MR signature with no inherent tissue background signal. To date, most cancer-related molecular MRI efforts have focused on detecting relatively large and readily apparent xenograft tumors, while studies on early detection of precancerous lesions remain lacking. Genetically engineered mouse models exhibiting well-defined multi-stage carcinogenesis offer opportunities for using molecular imaging to track stage-specific molecular events that drive lesion growth. The K14-HPV16 mouse is a model of squamous cell cancer, derived by incorporating human papilloma virus (HPV) transgenic segments E6 and E7 into the mouse genome under control of the K14 keratin promoter (8). At 5–8 months of age, dysplastic lesions in the ear epidermis are associated with expansive dermal papillation and a dense supporting network of neovasculature. In 18% of mice, these lesions contain foci of microinvasive cancer, which appear as grossly visible malignancies by 12 months of age (9). We propose that the early angiogenic substrate in this model might serve as an image-based biomarker of precancerous lesions with an appropriate targeted molecular contrast agent. In this work, we used a targeted MRI contrast agent with a unique 19F-based signature in conjunction with a novel application of 19F diffusion sensitive MRI techniques similar to those used by Neil and Ackerman to study the pseudo-diffusion of intravascular perfluorocarbon emulsions (10). We used these techniques to selectively suppress the unbound and flowing 19F nanoparticle signal in the blood pool. This strategy allowed for specific and rapid detection of the 19F signal derived only from targeted nanoparticles bound to lesion neovasculature. We demonstrated that 19F diffusion sensitive MRS directly reports nanoparticle binding to the up-regulated microvasculature of precancerous squamous lesions by comparing the decay of diffusion sensitive 19F signal in the epidermis of the ear in K14-HPV16 mice and controls.