Effect of photodynamic therapy using hexyl aminolevulinate and Amphinex – in vitro and in vivo studies
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Photodynamic therapy (PDT) is based on administration of photosensitizers (PS) which preferentially accumulates in tumor cells, followed by exposure of light that excites PS molecules and generation of singlet oxygen or other reactive oxygen species. All together these reactions destroy the tumor cells. Photochemical internalization (PCI) is a novel technology based on the same principle as PDT. By using PCI-techniques and sub-lethal PDT doses for modifying intracellular distribution of actual drugs, it is possible to deliver endocytoced macromolecules into the cytosol. The aim of this thesis was to characterize the effects of hexyl aminolevulinate (HAL), an indirect precursor of protoporphyrin IX (PpIX), and the novel sensitizer Amphinex (PCI Biotech AS, Oslo) in both PDT- and PCI experiments. The human colon carcinoma cell line (WiDr) and the rat bladder transitional carcinoma cell line (AY-27) were used as models for studying cell destruction induced by PDT. Both red (652 nm) and blue light (435 nm) were used in combination with HAL or Amphinex, resulting in characteristic LD50 doses for both cell lines. After HAL based PDT (using blue light) the relative cell survival percent decreases in AY-27 cells (from 78.5% ± SD to 1% ± SD) and in WiDr cells (from 95%±SD to 1%±SD). In both cell lines the light alone do not have much effect in the viability of cell but in case of AY-27 cells HAL exhibit some cytotoxic effect. The LD50 light dose for AY-27 and WiDr cells using same concentration of HAL (10μM, 3.5 h, dark) is 0.296 Jcm-2 and 0.116 Jcm-2. The result shows that HAL based PDT using blue light is more effective for WiDr cells in vitro compared to AY-27 cells. Similarly, Amphinex based PDT, (blue light), in WiDr cells resulted in the LD50 of 12.284 Jcm-2, 1.702 Jcm-2 and 1.624 Jcm-2 when cells were incubated with 0.2 μg/ml, 0.4 μg/ml and 0.6 μg/ml of Amphinex (18 h, dark). This shows that HAL is a more effective photosensitizer when blue light is used. Amphinex based PDT (red light) resulted into LD50 of 3.75 Jcm-2 when cells were incubated with Amphinex (0.4 μg/ml, 18h, dark). It can be concluded that red light is more effective when Amphinex is used as the photosensitizer. The LD50 dose resulted in small difference during PCI (Amphinex, red light, bleomycin) using lower concentration of bleomycin (0.05-5 μM). But the LD50 light dose decreases drastically when higher concentration of bleomycin (10-50 μM) was used. The effects of light on the bleaching of the photosensitizer were also monitored by measuring the fluorescence of Amphinex in WiDr cells incubated with Amphinex and illuminated with different light doses. The bleaching effects of Amphinex on WiDr cells were considerable increased during higher and higher red light (652 nm) doses. A result in this thesis has demonstrated that HAL and Amphinex based PDT using both blue and red light is effective against AY-27 cells and WiDr cells. Amphinex based PCI using red light was also effective in killing the WiDr cells. The bleaching of the Amphinex is very less at the LD50 used in these experiments. The mechanism behind these result were not studied so further experiments have to be done to understand the mechanism of the working of PDT and PCI using HAL and Amphinex. Some in vivo experiments were already done and presented in this thesis by inducing tumor growth after installation of AY-27 cells in rat bladders (Fisher female rats). The histology of the rat bladder walls were monitored for describing the efficacy of PDT. Only some preliminary data from experiments done by the supervisor in collaboration with PCI Biotech, AS, were present in this study.