Urokinase-plasminogen-activator sensitive polymeric photosensitizer prodrugs: design, synthesis and in vitro evaluation

Polymeric photosensitizer prodrugs (PPPs) to be selectively activated by urokinase plasminogen activator (uPA) have been developed. They are composed of uPA-cleavable photosensitizer-peptide-conjugates covalently attached to a poly-lysine backbone (25 kDa). Phototoxicity of PPPs is efficiently reduced by energy transfer between closely positioned photosensitizers (PSs) on the polymeric backbone. Enzymatic cleavage by uPA, a serine protease that is up-regulated in certain types of cancer, leads to the release of PS-peptidyl-fragments and restoration of phototoxicity. In the current study, conjugates with different ε-lysine side chain modifications of the polymer-backbone were synthesized and characterized with respect to in vitro properties such as fluorescence and reactive oxygen species (ROS) quenching, as well as enzymatic cleavage by uPA. Based on these in vitro results and a cell screening experiment for photo- and dark toxicity, two compounds, PEG20-N-uPA-PPP and N-uPA-PPP, were selected as the most promising candidates for further evaluation in two uPA-expressing cell lines, DU-145 and PC-3. Here, the pegylated compound PEG20-N-uPA-PPP showed enhanced selective prodrug activation by uPA in comparison to N-uPA-PPP, pinpointing the need for prolonged extracellular residence time of uPA-sensitive prodrugs. In preliminary PDT-experiments, a dose-dependent phototoxic effect of this compound was found in both tested cell lines. In conclusion, this study highlights the fact that backbone substitution units may impact not only photochemical and physicochemical properties of the PPPs, but play an important role for cellular prodrug localization and thus site selective enzymatic activation.