Effects of hexyl aminolevulinate and Amphinex based photodynamic therapy and photochemical internalization - in vitro studies using rat bladder cancer cells
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Photodynamic therapy (PDT) is a treatment modality which involves application of photosensitizers and their ability to accumulate selectively and damage cancer cells in presence of light. Light induced damage to cancer tissues is irreversible which mediates the formation of reactive oxygen species. Photochemical internalization (PCI) is a novel technology which is based on principles of PDT and is a site specific technology which releases entrapped drugs in light exposed cells. The present study aimed to assess the effect of hexyl aminolevulinate (HAL) and the new photosensitizer Amphinex in rat bladder cancer cells (AY-27) using blue and red light. The focus of the thesis was also to examine the cytotoxicity effect of Bleomycin in cancer cells with Amphinex using PCI technique. Furthermore, bleaching effect of Amphinex in bladder cancer cells was also studied using different light doses. In this project, dose response curves were established for HAL (10 μM, 3.5 h) and Amphinex (0.2 μg/ml, 18 h) treated AY-27 cells 24 h post illumination with blue light (435 nm). Similarly, cell survival with Amphinex based PDT was also done 48 h post illumination with red light (652 nm). The LD 50dose from Amphinex based PDT was further used for PCI in combination with Bleomycin to see cytotoxic effects of photochemical delivery of Bleomycin in bladder cancer cells. All cell viability results were confirmed by measurements of mitochondrial activity as found by MTT assay. Finally, fluorescence spectroscopy was used to investigate the bleaching effect of Amphinex in cancer cells after delivery of different red light doses. LumiSource® (PCI Biotech, Oslo) and Quanta System (Solbiate Ololna, Italy) were the two light sources used for blue and red light illumination of AY-27 cells. A decrease in cell viability was observed in both HAL and Amphinex treated cells after PDT. Similarly, reduction in the cell survival was assessed in PCI of Amphinex with Bleomycin and both photosensitizers were found to be effective in inducing death in AY- 27 cells after PDT treatment with blue and red light. Likewise, there has been marked reduction in cell survival of cancer cells with Amphinex based PCI in combination with Bleomycin presenting the potential cytotoxic effect of Bleomycin. A continuous reduction in fluorescence intensity was also observed with increasing light dose in Amphinex treated cells using red light. The decline in fluorescence was due to continuous photobleaching effect of Amphinex in presence of the light.