Study Of PDT-induced Tumor Damages With Phosphorescent Oxygen Nanosensors Based On PtTFPP

Photodynamic therapy (PDT) has been known as a well-recognized alternative modality by virtue of high selectivity, minimum invasiveness and simple performance. In terms of in vitro cytotoxicity of PDT, it is usually assessed by MTT assay. In this work a phosphorescent nanosensors specifically for intracellular oxygen was utilized to assess PDT-induced cell damages. The result of cytotoxicity from this measurement was in agreement with cell obtained from MTT assay. Moreover, the direct damage of PDT on cell mitochondria was successfully detected by monitoring respiration instantly after PDT treatment, which is actually beyond the scope of MTT assay.The main research of this thesis is as follows:(1) Firstly, a series of in vitro photodynamic experiments have been explored from the concentration of protoporphyrin sodium salt and irradiation dose based on MTT assays. Then, the toxicity effect of different PDT conditions on HepG2 cells were mastered and the administrated PDT-dosage was screened in advance for the future study.(2) Phosphorescent nanoparticles (NPs) are prepared by a one-step reprecipitation-encapsulation method for sensing ec-O2 and ic-O2 in HepG2 cells, which are based on polystyrene and silicone hybrid material as a substrate doped with the oxygen probe Pt(II)-meso-tetra (pentafluoropheny) porphine (PtTFPP) and the reference dye coumarin 6 (C6). The surface of the NPs is modified with poly-L-lysine (PLL), which exhibit good biocompatibility and efficient cellular uptake by MTT assay and confocal microscopy images for sensing ic-O2. Then, the cellular respiration of HepG2 cells after PDT is monitored using the time-resolved fluorescence microplate reader. It turned out that ic-O2 was demonstrated to be consumed much faster than extracellular O2 with respective O2 nanosensors and its measurement is more accurate. Using the ic-O2 nanosensors, PDT-treated cells with different degree of impairments were the resolved according to oxygen consumption rate (OCR). The evolving trend of cytotoxicity derived from OCRs was also in agreement with cell viability obtained from MTT assay. Moreover, the direct damage of PDT on cell mitochondria was successfully detected by monitoring respiration instantly after PDT treatment, which is actually beyond the scope of MTT assay.