Cellular uptake of electron paramagnetic resonance imaging probes through endocytosis of liposomes

Abstract

Electron paramagnetic resonance imaging (EPRI) allows detection and localization of paramagnetic spin probes in vivo and in real time. We have shown that nitroxide spin probes entrapped in the intracellular milieu can be imaged by EPRI. Therefore, with the development of a tumor-targetable vehicle that can efficiently deliver nitroxides into cells, it should be possible to use nitroxide spin probes to label and image cells in a tumor. In this study, we assess the potential of liposomes as a delivery vehicle for imaging probes. We demonstrate that liposomes can stably encapsulate nitroxides at very high concentrations (> 100 mM), at which nitroxides exhibit concentration-dependent quenching of their EPR signal—a process analogous to the quenching of fluorescent molecules. The encapsulating liposomes thus appear spectroscopically “dark”. When the liposomes are endocytosed and degraded by cells, the encapsulated nitroxides are liberated and diluted into the much larger intracellular volume. The consequent relief of quenching generates a robust intracellular nitroxide signal that can be imaged. We show that through endocytosis of nitroxide-loaded liposomes, CV1 cells can achieve intracellular nitroxide concentrations of ~1 mM. By using tissue phantom models, we verify that this concentration is more than sufficient for in vivo EPR imaging.

Clearance and Biodistribution of Liposomally Encapsulated Nitroxides: A Model for Targeted Delivery of Electron Paramagnetic Resonance Imaging Probes to Tumors

“…Administration through immunoliposomes results in relatively more nitroxide than 67 Ga in the kidneys. One potential explanation is partial leakage of nitroxides from circulating liposomes, which previous studies have suggested is a possibility (Burks et al, 2009). Such leaked nitroxides, being small anions, would be filtered by, and thus FIG.…”

“…Importantly, the total amount of nitroxide detected in tissues by 3 h postinjection (Ͻ1% total nitroxide dose) is only a small fraction of what has been cleared from the circulation at that time (ϳ20% total dose). The fact that these nitroxides are exceptionally resistant to bioreduction in the blood/plasma (Burks et al, 2009;Miyake et al, 2010) and are further protected from metabolic degradation by being encapsulated in liposomes implies that most cleared nitroxides are subjected to one of two fates: metabolic degradation by the liver and kidney (Fig. 7) or possibly excretion by the kidneys into the urine.…”

“…The amount of nitroxide observed in tumors after 3 h is ϳ5 nmol/g, which corresponds to a concentration of ϳ5 M. Our preliminary imaging studies of Hc7 tumors and previous studies (Burks et al, 2009(Burks et al, , 2010b suggest this concentration should be just visible in reconstructed images, which have the benefit of signal averaging to increase signal-to-noise ratio (SNR). Several recent advances in EPR imaging technology should markedly improve the ease of tumor imaging with nitroxides delivered through immunoliposomes.…”

“…Classical, sterically stabilized immunoliposomes were prepared as described previously (Burks et al, 2009(Burks et al, , 2010b. In brief, all liposomes were primarily composed of 1,2-distearoylphosphatidylcholine and cholesterol at a molar ratio of 3 distearoylphosphatidylcholine to 2 cholesterol.…”

“…We have further demonstrated that these nitroxides can be encapsulated in liposomes at high concentrations (Ͼ100 mM) and delivered to cells through endocytosis of liposomes (Burks et al, 2009). Liposomes encapsulating high concentrations of nitroxides are especially attractive carriers because nitroxides exhibit the phenomenon of "self-quenching," analogous to that of fluorophores, where spectral signals become greatly attenuated at high concentration.…”