A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(L)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme.

We have developed novel polymeric photosensitizer prodrugs (PPPs) for improved photodynamic therapy. In PPPs, multiple photosensitizer units are covalently coupled to a polymeric backbone via protease-cleavable peptide linkers. These initially non-photoactive compounds become fluorescent and phototoxic after specific enzymatic cleavage of the peptide linkers and subsequent release of the photosensitizer moieties. Tethering the photosensitizer via a short and easily modified amino acid sequence to the polymeric backbone allows for the targeting of a wide variety of proteases. Model compounds, sensitive to trypsin-mediated cleavage, with different pheophorbide a-peptide loading ratios and backbone net charges were evaluated with respect to their solubility, "self-quenching" capacity of fluorescence emission, and reactive oxygen species (ROS) generation. In addition, linker sequence impaired selectivity toward enzymatic cleavage was demonstrated either by incubating PPPs with different enzymes having trypsin-like activity or by introducing a single d-arginine mutant in the peptide sequence. In vitro cell culture tests confirmed dose-dependent higher phototoxicity of enzymatically activated PPPs compared to the nonactivated conjugate after irradiation with white light. These data suggest that similar compounds adapted to disease-associated proteases can be used for selective photodynamic therapy.

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