Two dual stimuli-activated photosensitizers were developed, in which two or three glutathione (GSH)-responsive 2,4dinitrobenzenesulfonate (DNBS)-substituted zinc(II) phthalocyanine units were connected via one or two cathepsin B-cleavable Gly-Phe-Leu-Gly peptide linker(s). These dimeric and trimeric phthalocyanines were fully quenched in the native form due to the photoinduced electron transfer to the DNBS substituents and the self-quenching of the phthalocyanine units. In the presence of GSH and cathepsin B, or upon internalization into A549 and HepG2 cancer cells, these probes were activated through the release of free phthalocyanine units. The intracellular fluorescence intensity was increased upon post-incubation with GSH ester or reduced upon pre-treatment with a cathepsin B inhibitor. Upon light irradiation, these photosensitizers became highly cytotoxic with IC 50 values of 0.21−0.39 μM. The photocytotoxicity was also dependent on the intracellular GSH and cathepsin B levels. The results showed that these conjugates could serve as smart photosensitizers for targeted photodynamic therapy.

An advanced photodynamic molecular beacon (PMB) was designed and synthesized, in which a distyryl boron dipyrromethene (DSBDP)-based photosensitizer and a Black Hole Quencher 3 moiety were connected via two peptide segments containing the sequences PLGVR and GFLG, respectively, of a cyclic peptide. These two short peptide sequences are well-known substrates of matrix metalloproteinase-2 (MMP-2) and cathepsin B, respectively, both of which are overexpressed in a wide range of cancer cells either extracellularly (for MMP-2) or intracellularly (for cathepsin B). Owing to the efficient Förster resonance energy transfer between the two components, this PMB was fully quenched in the native form. Only upon interaction with both MMP-2 and cathepsin B, either in a buffer solution or in cancer cells, both of the segments were cleaved specifically, and the two components could be completely separated, thereby restoring the photodynamic activities of the DSBDP moiety. This PMB could also be activated in tumors, and it effectively suppressed the tumor growth in A549 tumor-bearing nude mice upon laser irradiation without causing notable side effects. In particular, it did not cause skin photosensitivity, which is a very common side effect of photodynamic therapy (PDT) using conventional “always-on” photosensitizers. The overall results showed that this “double-locked” PMB functioned as a biological AND logic gate that could only be unlocked by the coexistence of two tumor-associated enzymes, which could greatly enhance the tumor specificity in PDT.

The synthesis and use of oxetane modified dipeptide building blocks in solution and solid-phase peptide synthesis (SPPS) is reported.

A multifunctional photodynamic molecular beacon (PMB) has been designed and synthesized which contains an epidermal growth factor receptor (EGFR)‐targeting cyclic peptide and a trimeric phthalocyanine skeleton in which the three zinc(II) phthalocyanine units are each substituted with a glutathione (GSH)‐responsive 2,4‐dinitrobenzenesulfonate (DNBS) quencher and are linked via two cathepsin B‐cleavable GFLG peptide chains. This tailor‐made conjugate is fully quenched in the native form due to the photoinduced electron transfer effect of the DNBS moieties and the self‐quenching of the phthalocyanine units. It can target the EGFR overexpressed in cancer cells, and after receptor‐mediated endocytosis, it can be activated selectively by the co‐existence of intracellular GSH and cathepsin B, both of which are also overproduced in cancer cells, in terms of fluorescence emission and singlet oxygen generation. The cell‐selective behavior of this PMB has been demonstrated using a range of cancer cells with different expression levels of EGFR, while the stimuli‐responsive properties have been studied both in vitro and in various aqueous media. The overall results show that this advanced PMB, which exhibits several levels of control of the tumor specificity, is a promising photosensitizer for precise antitumoral photodynamic therapy.