Annexin A5–Conjugated Polymeric Micelles for Dual SPECT and Optical Detection of Apoptosis

Abstract

Imaging of apoptosis can allow noninvasive assessment of disease states and response to therapeutic intervention for a variety of diseases. The purpose of this study was to develop and evaluate a multimodal nanoplatform for the detection of apoptosis. Methods To modulate the pharmacokinetics of annexin A5, a 36-kDa protein that binds specifically with phosphatidylserine, annexin A5 was conjugated to polyethylene glycol-coated, core-crosslinked polymeric micelles (CCPM) dually labeled with near-infrared fluorescence fluorophores and a radioisotope (indium 111). To evaluate the specificity of the binding of annexin A5-CCPM to apoptotic cells, both fluorescence microscopy and cell binding studies were performed in vitro. Pharmacokinetics, biodistribution, dual nuclear and optical imaging, and immunohistochemical studies were carried out in 2 xenografted tumor models to evaluate the potential applications of annexin A5-CCPM. Results In cell-based studies, annexin A5-CCPM exhibited strongly specific binding to apoptotic tumor cells. This binding could be efficiently blocked by annexin A5. In mice, annexin A5-CCPM displayed a mean elimination half-life of 12.5 h. The mean initial concentration in blood was predicted to be 22.4% of the injected dose/mL, and annexin A5-CCPM was mainly distributed in the central blood compartment. In mice bearing EL4 lymphoma treated with cyclophosphamide and etoposide and in mice bearing MDA-MB-468 breast tumors treated with poly(L-glutamic acid)-paclitaxel and cetuximab (IMC-C225) anti-EGFR antibody, the tumor apoptosis was clearly visualized by both single photon emission computed tomography and fluorescence molecular tomography. In contrast, there was little accumulation of this nanoradiotracer in the tumors of untreated mice. The biodistribution data were consistent with the imaging data, with tumor-to-muscle and tumor-to-blood ratios of 38.8 and 4.1, respectively, in treated mice, and 14.8 and 2.2, respectively, in untreated mice bearing EL4 lymphoma. Moreover, further studies demonstrated that the conventional Tc-99m-labeled HYNIC-annexin A5 and the plain CCPM control exhibited significantly lower uptake in the tumors of the treated mice than annexin A5-CCPM. Immunohistochemistry staining study showed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of tumor apoptosis. Conclusions Annexin A5-CCPM allowed visualization of tumor apoptosis by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in assessing and validating early response to therapy.

Apoptosis Imaging Probe Predicts Early Chemotherapy Response in Preclinical Models: A Comparative Study with 18F-FDG PET

“…Noninvasive imaging with 18 F-FDG PET enables the monitoring of changes in metabolic activity and has become a gold standard for the assessment of early treatment response (1)(2)(3)(4). 18 F-FDG is a glucose analog that reflects glucose metabolic activity in tumor cells; an effective therapy often leads to a significant decrease in 18 F-FDG uptake.…”

“…18 F-FDG is a glucose analog that reflects glucose metabolic activity in tumor cells; an effective therapy often leads to a significant decrease in 18 F-FDG uptake. However, 18 F-FDG is not a tumor-specific agent.…”

“…18 F-FDG is a glucose analog that reflects glucose metabolic activity in tumor cells; an effective therapy often leads to a significant decrease in 18 F-FDG uptake. However, 18 F-FDG is not a tumor-specific agent. Inflammatory cells, which infiltrate tumors as a result of chemotherapy, also have a high intracellular accumulation of 18 F-FDG, perhaps even higher than that of the viable tumor cells, leading to an underestimation of the therapeutic effect (5-7).…”

“…However, 18 F-FDG is not a tumor-specific agent. Inflammatory cells, which infiltrate tumors as a result of chemotherapy, also have a high intracellular accumulation of 18 F-FDG, perhaps even higher than that of the viable tumor cells, leading to an underestimation of the therapeutic effect (5-7). Moreover, some molecularly targeted drugs are cytostatic-that is, they seem not to alter glucose metabolism enough for 18 F-FDG PET to have significant predictive value (8).…”

“…Inflammatory cells, which infiltrate tumors as a result of chemotherapy, also have a high intracellular accumulation of 18 F-FDG, perhaps even higher than that of the viable tumor cells, leading to an underestimation of the therapeutic effect (5-7). Moreover, some molecularly targeted drugs are cytostatic-that is, they seem not to alter glucose metabolism enough for 18 F-FDG PET to have significant predictive value (8). Therefore, the development of an imaging technique that is capable of more accurately predicting early treatment response would benefit cancer patients undergoing anticancer molecular therapy and chemotherapy.…”