Rational design of caspase-responsive smart molecular probe for positron emission tomography imaging of drug-induced apoptosis

Purpose: Positron emission tomography (PET) imaging of apoptosis is very important for early evaluation of tumor therapeutic efficacy. A stimuli-responsive probe based on the peptide sequence Asp-Glu-Val-Asp (DEVD), [(18)F]DEVD-Cys(StBu)-PPG(CBT)-AmBF(3) ([(18)F]1), for PET imaging of tumor apoptosis was designed and prepared. This study aimed to develop a novel smart probe using a convenient radiosynthesis method and to fully examine the sensitivity and specificity of the probe response to the tumor treatment. Methods: The radiolabelling precursor DEVD-Cys(StBu)-PPG(CBT)-AmBF(3) (1) was synthesized through multistep reactions. The reduction together with caspase-controlled macrocyclization and self-assembly of 1 was characterized and validated in vitro. After [(18)F]fluorination in the buffer (pH= 2.5), the radiolabelling yield (RLY), radiochemical purity (RCP) and stability of the probe [(18)F]1 in PBS and mouse serum were investigated by radio-HPLC. The sensitivity and specificity of [(18)F]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 [(18)F]1 was also assessed. The level of activated caspase-3 in Hela cells and tumors with or without apoptosis induction was analyzed and compared by western blotting and histological staining. Results: The whole radiosynthesis process of [(18)F]1 was around 25 min with RLY of 50%, RCP of over 99% and specific activity of 1.45 ± 0.4 Ci/µmol. The probe was very stable in both PBS and mouse serum within 4 h. It can be activated by caspase-3 and then undergo an intermolecular cyclization to form nanosized particles. The retained [(18)F]1 in DOX-treated HeLa cells was 2.2 folds of that in untreated cells. Within 1 h microPET imaging of the untreated Hela-bearing mice, the injection of [(18)F]1 resulted in the increase of the uptake ratio of tumor to muscle (T/M) only from 1.74 to 2.18, while in the DOX-treated Hela-bearing mice T/M increased from 1.88 to 10.52 and the co-injection of [(18)F]1 and 1 even led to the increase of T/M from 3.08 to 14.81. Conclusions: A caspase-responsive smart PET probe [(18)F]1 was designed and prepared in a kit-like manner. Co-injection of [(18)F]1 and 1 generated remarkably enhanced tumor uptake and signal-to-noise ratio in the tumor-bearing mice with drug-induced apoptosis, which correlated well with the expression level of activated caspase-3. This early readout of treatment response ensured that the probe [(18)F]1 could serve as a promising PET imaging probe for timely and noninvasive evaluation of tumor therapy.