Targeted delivery of nitroxide spin probes, using immunoliposomes, for electron paramagnetic resonance imaging of tumors in vivo

Abstract

Metastasis is the cause of approximately 90% of cancer-associated deaths. Detection of metastatic lesions is a major clinical challenge and current imaging methods frequently fail to detect micrometastases (0.2 - 2.0 mm). Electron Paramagnetic Resonance Imaging (EPRI) is an emergent in vivo imaging modality. In an analogous manner to MRI, EPRI detects magnetic-field-induced resonant absorption of radio-frequency light. The key difference is that EPRI involves resonant absorption by the unpaired electrons in paramagnetic free radicals, whereas MRI involves resonant absorption by hydrogen nuclei (protons). Because the free radical concentration in the body is negligible, EPRI requires the use of exogenous stable free radicals - known as spin probes. The use of exogenous spin probes in EPRI is a key advantage that allows high signal-to-background contrast generation. Therefore, EPRI represents an ideal in vivo imaging technique for identifying and locating metastatic lesions in cancer. Liposomes are lipid bilayer vesicles that can encapsulate a wide variety of molecules in their aqueous lumen. Antibody conjugation to the liposomal surface allows targeting to specific tissues - such liposomes are called immunoliposomes. Analogously to fluorophores, high concentrations of spin probes exhibit "self-quenching", a phenomenon where spectral signals are greatly attenuated at high concentration. Thus, spin probes encapsulated in immunoliposomes - at high concentration - are spectroscopically "dark". Immunoliposome endocytosis by tumor cells leads to liposomal degradation, releasing the spin probes, which become diluted in the intracellular volume. This dilution relieves self-quenching, restoring the spin probe's spectral signal, making the tumor appear "bright," and thus highlighted in EPRI. This thesis details the development of an immunoliposome delivery system for targeting spin probes to HER2-overexpresing xenograft tumors in mice. Specifically, this research demonstrates: 1) the development of an anti-HER2 single-chain antibody (scFv) for streamlined production of immunoliposomes; 2) the efficacy of fusogenic INF7 peptide for mediating endosome-to-cytosol delivery of immunoliposome-encapsulated imaging probes, which minimizes tumor cell probe extrusion; 3) the efficacy of adding multiple ionic charges to imaging probes to prolong cell retention. As a result of these enhancements, we hope to produce better signal buildup and retention in the tumor, thus improving the signal-to-noise ratio in imaging.