Unravelling the modes of phototoxicity of NIR absorbing chlorophyll derivative in cancer cells under normoxic and hypoxic conditions.

The efficacy of photodynamic treatment (PDT) against deep-seated tumor is hindered by low penetration depth of light as well as hypoxic conditions which prevails in tumor. To overcome this limitation, Near-infrared (NIR) absorbing photosensitizers have been investigated actively. In the present study we evaluated the PDT efficacy of an NIR absorbing chlorophyll derivative 'Cycloimide Purpurin-18 (CIPp-18)' in Human Breast carcinoma (MCF-7) and cervical adenocarcinoma (Hela) cells under normoxic and hypoxic conditions. PDT with CIPp-18 (2.0 µM, 3 h) and NIR light (700 ± 25 nm, 0.36-1.4 J /cm²) induced potent phototoxicity in both the cell lines. Under hypoxic conditions, PDT induced ~ 32% and 42% phototoxicity at LD₅₀ and LD₇₀ light dose, respectively, which corresponds to phototoxic dose under normoxia. CIPp-18 in neat buffer (pH 7.4) showed generation of singlet oxygen (¹O₂) as well as superoxide (O₂^·-) radicals. Studies on ROS generation in cells using fluorescence probes and the effect of mechanistic probes of ¹O₂ (Sodium Azide, Histidine, D₂O) and free radicals (DMSO, Mannitol, Cyanocobalamin, SOD-PEG) on phototoxicity show that ¹O₂ plays major role in phototoxicity under normoxia. Whereas, under hypoxic conditions, PDT led to no significant generation of ROS and phototoxicity remained unaffected by cyanocobalamin, a quencher of O₂^·-. Moreover, CIPp-18 showed localization in cell membrane and PDT led to more pronounced loss of membrane permeability in cells under hypoxia than for normoxia. These results demonstrate that CIPp-18 is suitable for PDT of cancer cells under hypoxia and also suggest that phototoxicity under hypoxia is mediated via ROS-independent contact-dependent mechanism.