Investigating the potential of Cu-64-annexin V as an imaging agent for cell death

Objectives Annexin V is a calcium-dependent glycoprotein, that has been shown to detect apoptosis by binding to the membrane-bound phosphatidylserine (PS), an anionic phospholipid, with nanomolar affinity (Kd = 7 nmol). PS is normally confined to the inner leaflet of the plasma membrane and the exposure of PS on the cell’s outer surface occurs in the early stages of apoptosis. The ability to monitor apoptosis in association with disease progression or regression would be clinically relevant. Thus, we plan to optimize the synthesis of the positron emission tomography (PET) imaging agent, copper-64-DOTA-annexin V, investigate structural changes, and determine affinity and binding to PS in vitro.

Methods We synthesized, purified, and characterized copper-64-DOTA-annexin V. We studied the chemical purity by high pressure liquid chromatography, characterized by MALDI-TOF, and investigated structural changes and thermal denaturing via fluorescence and circular dichorism studies respectively. Lastly we explored the potential of copper-64-DOTA-annexin V to detect treatment-induced apoptosis in colorectal carcinoma cells, HCT-116.

Results The imaging agent, copper-64-DOTA-annexin V was successfully synthesized and purified. Structural changes to Annexin V were observed upon inclusion of the chelating agent, DOTA. Copper-64-DOTA-annexin V was shown to recognize PS and detect treatment-induced apoptosis in HCT 116 cells.

Conclusions The inclusion DOTA to annexin V resulted in structural changes to the protein and potentially results in the exposure of tryptophan residues that were previously embedded. Despite these changes, copper-64-DOTA-annexin V was shown to bind to PS with high affinity, and was sensitive to treatment-induced apoptosis