Radiopharmacological evaluation of a novel caspase responsive probe for PET imaging of tumor apoptosis

Objectives: Early evaluation of tumor response to therapy is very important for promoting the development of anti-tumor drugs and optimizing the treatment method. A ¹⁸F-labeling caspase-responsive probe was designed and synthesized based on the peptide sequence DEVD as a recognition group and the peptide sequence HEHEHE as a tag for reducing the liver uptake of the probe as well as cysteine and 2-cyanobenzothiazole (CBT) for triggering the intermolecular click condensation reaction and self-assembly of the probe, which could enhance and prolong the radioactive signal in the tumor site. This study aimed to develop a novel smart probe with optimized pharmacokinetics for PET imaging of tumor apoptosis.

Methods: The probe ¹⁸F-1 was radiosynthesized using a convenient 'one-step' ¹⁸F-labeling method with the ammoniomethyl-trifluoroborate moiety. Its octanol/water partition coefficient, stability in PBS and FBS as well as biocompatibility were studied systematically. Enzyme kinetics studies were performed to test the sensitivity and specificity of the probe towards caspase-3. The sensitivity and specificity of ¹⁸F-1 for detecting the drug-induced apoptosis was fully evaluated in vitro and in vivo. The effect of cold precursor 1 on the cell uptake and tumor imaging of ¹⁸F-1 was also assessed. The level of activated caspase-3 in A549 cells and tumors with or without apoptosis induction was analyzed and compared by western blotting and histological staining analysis.

Results: The probe ¹⁸F-1 was successfully prepared with radiolabeling yield of 83% and radiochemical purity of 99%. Stability and MTT assay showed that ¹⁸F-1 was stable and biocompatible, and the partition coefficient revealed it was highly hydrophilic (logP = -1.95 ± 0.03). It can be activated and cleaved by caspase-3 sensitively and specifically (K_m=345 μM, k_cat = 10.89 s^-1) and then underwent an intermolecular cyclization to form nanosized particles (138 nm). Cell uptake studies showed that the retained ¹⁸F-1 in DOX-treated A549 cells was 3.8 folds of that in untreated cells at 4 h, and microPET imaging showed the retained ¹⁸F-1 signal in DOX-treated tumor was 6.25 folds of that in untreated tumor with low liver uptake. Immunostaining analysis showed that PET imaging results correlated well with the expression level of activated caspase-3.

Conclusions: A novel smart caspase-responsive probe with low liver uptake and high signal-to-noise ratio was designed and evaluated. It could serve as a promising PET imaging probe for timely and noninvasive evaluation of tumor therapy. Financial Supports: Natural Science Foundation of Jiangsu Province (BK20181128) and Major Scientific Research Project of Wuxi Commission of Health (Z201913).