13,15-N-Cycloimide derivatives of chlorin p6 with isonicotinyl substituent are photosensitizers targeted to lysosomes

Four monocationic cycloimide derivatives of chlorin p₆ (CICD) were studied as photosensitizers and compared to a structurally similar neutral derivative. Cationic CICD are highly photostable (quantum yield of photobleaching is about 1 × 10^-5, generate singlet oxygen under irradiation (quantum yields are 0.3-0.45), can be involved in a photo-induced substrate-dependent generation of superoxide radicals, but do not produce OH*. 17,18-δ-lacton 13²-(N-methylisonicotinylamido)-13, 15-cycloimide mesochlorin p₆ (2) and 13²-(N- methylisonicotinylamido)-13,15-cycloimide mesochlorin p₆ methyl ester (3) possess high cancer cell killing photodynamic activity, but they provide no photoinduced bactericidal effect. Substitution of an ethyl group with a hydroxyethyl or acetyl group at position 3 of the macrocycle results in a decrease in extinction and intracellular accumulation that finally leads to the reduced photocytotoxicity. Cationic CICD are targeted to lysosomes, and their intracellular penetration occurs most probably via caveolae-dependent endocytosis. Photodynamic treatment with cationic CICD results in the cell death via necrosis at both sub-phototoxic (40-70% of dead cells) and phototoxic (90-100% of dead cells) regimes of cell treatment. Irradiation induces lysosome damage, leakage of CICD from lysosomes and development of protease activity in cytoplasm, whereas mitochondria are not affected with irradiation. A positive charge of cationic CICD modified drastically an internalization pathway, sites of intracellular localization and mechanisms of photoinduced cytotoxicity as compared to previously studied neutral and anionic CICD. Our experiments with different CICD show that varying charge and structure of substituents it is possible to modulate many cellular properties of CICD in order to find the best molecular template of the advanced near-IR photosensitizer for photodynamic therapy.