Targeting Phosphatidylserine with a ⁶⁴Cu-Labeled Peptide for Molecular Imaging of Apoptosis

Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 (⁶⁴Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with ⁶⁴Cu to prepare radiotracer ⁶⁴Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, and ^natCu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer ⁶⁴Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide ⁶⁴Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC₅₀ values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, and ^natCu-NOTA-PSBP-6 were 600 μM, 30 μM, and 23 μM, respectively. A competitive radiometric cell binding assay confirmed binding of ⁶⁴Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca²⁺-independent manner. PET imaging studies demonstrated significantly higher uptake of ⁶⁴Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV_5min 0.95 ± 0.04) compared to control tumors (SUV_5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer ⁶⁴Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional "fine-tuning" of ⁶⁴Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.