الفهرس 
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Abstract
Using Nanoplatforms as carriers for photosensitizers has been found to enhance the efficacy of photodynamic therapy by improving bioavailability, preventing drug aggregation, and enhancing photosensitizer solubility to obtain a high therapeutic outcome. In this study, the photodynamic cytotoxicity of methylene blue (MB) loaded on hydroxyapatite nanoparticles (HA) was tested against osteosarcoma cancer cell line, Saos-2. HA and HA loaded with methylene blue (HA-NPs-MB) were prepared by chemical precipitation method. The prepared HA and HA-NPs-MB were characterized by TEM, Zeta potential, FTIR, and XRD. TEM images revealed that HA nanoparticles have a rod shape with a diameter around 14 to 17 nm and length around 46 to 64 nm. FTIR and Zeta potential confirmed the adsorption of cationic MB on HA. XRD pattern was identical to the standard XRD pattern of hydroxyapatite nanoparticles. Incubation of Saos-2 cells for 24 hours with of HA-NPs-MB and then irradiation of cells for 5 minutes with laser emitting diode at 800 nm, causes a reduction of cell viability (determined by MTT assay) more than the free methylene blue. The LC50 was 57.53 μg/ml and 86.99 μg/ml for HA-NPs-MB and free MB, respectively. Thus, nanoformulation of MB greatly reduced the dose of methylene blue required for effective photodynamic therapy. In the absence of laser, no cytotoxicity was observed for either HA-NPs-MB or free MB. No cytotoxicity was detected after incubation of cells with HA in the presence or the absence of laser. This study also investigated the mode of cell death after incubation of cells with free MB and HA-NPs-MB then exposure to laser radiation. The results revealed that the majority of cells died by apoptosis while a minor fraction of cells died by necrosis, especially in the case of HA-NPs-MB. Levels of caspase-3 and death receptor-4 (DR-4) were more elevated in case of HA-NPs-MB than free MB. The effect of prepared nanoparticles and free MB on Raw murine macrophage (RAW 264.7) viability was also examined using MTT assay. The results indicated that HA-NPs-MB in the presence of laser has a great cytotoxic effect on macrophage cells compared to other treatments. This may present an advantage through decreasing macrophage that promotes tumor growth. A slight elevation of nitric oxide (NO) produced by murine macrophages (RAW 264.7) was noticed after treatment with HA-NPs-MB followed by laser irradiation compared to free MB or HA in the presence or absence of laser, indicating that photodynamic therapy with HA-NPs-MB may result in promoting the arrival of more oxygen and drugs to tumor tissues through vasodilation moreover, it may prime the immune system to kill tumor cells in the long term after PDT treatment. This is the first study to assess the effect of HA, free MB and HA-NPs-MB on the osteosarcoma cancer cell line (Saos-2). In conclusion, HA-NPs-MB was more efficient in destroying macrophage cells and Saos-2 cells through apoptosis in the presence of laser irradiation than either MB or HA.