Photodynamic Therapy For Pancreatic Carcinoma With Photosensitizer-loaded TiO₂Nanoparticles:an In Vitro And In Vivo Study

PART I Tumoricidal effects of photosan-loaded TiO2nanospheres induced PDT on Panc-1cellsObjectiveTo explore the tumoricidal effects of Photodynamic Therapy (PDT) with free Photosan and Photosan-loaded hollow Titanium dioxide (TiO2) nanoparticles (abbreviated to Nano-Photosan) on pancreatic carcinoma cells (Panc-1), and evaluate the therapeutic effects of nano-Photosan induced PDT with optimal parameters in vitro.Methods1. Tumoricidal effects of free Photosan and Nano-Photosan on H6C7cells and Panc-1cells were determined by MTT assay.2. The apoptosis and necrocytosis of H6C7and Panc-1cells caused by free photosan and Nano-Photosan induced PDT respectively were detected by Annexin V-FITC/PI flowcytometry.Results1. MTT assay demostrated that neither photosensitizer nor light alone inhibits the growth of Panc-1and H6C7cells. The photodynamic effects were positively related to the concentration of photosensitizers, light dose, and incubating time. With the same parameters, the Photosan-loaded nanoparticles induced PDT has a more severe inhibition effect on cancer cell growth than the free photosan induced PDT. With the optimal parameters regarding to free photosan and nano-photosan respectively, nano-photosan induced PDT has more significantly growth inhibition on Panc-1cell than the free Photosan induced PDT. Moreover nano-photosan groups take effects within a shorter time than free Photosan. Neither Photosan-loaded nanoparticles nor free photosan has effects on H6C7cells cultured from normal pancreas.2. Annexin V-FITC/PI flowcytometry revealed that, PDT induced by both the free Photosan and Photosan-loaded nanoparticles inhibit Panc-1cells by apoptosis passway mainly. With the optimal parameters regarding to free photosan and nano-photosan respectively, nano-photosan can cause a higher rate of apoptosis and necrocytosis in Panc-1cell than free photosan. Conclusion1. Irradiation with corresponding wave length is initially required by both free Photosan and nano-Photosan to induce photodynamic effect.2. Nano-Photosan exhibits faster, stronger and more effective tumoricidal effects than free Photosan on growth inhibition of Panc-1cells, and both of the two photosensitizers may are biologicaly safe to normal pancreas cells.3. Mechanisms of the tumoricidal effects on Panc-1cell caused by both the free Photosan and nano-Photosan are mainly related to apoptosis. PART II Tumoricidal effects of photodynamic therapy on pancreatic carcinoma xenografs with nano-Photosan in vivoObjectiveTo establish the nude mice xenografts model with Panc-1cells and investigate the effects on tumor growth inhibition of both free Photosan’ and Nano-Photosan induced PDT in vivo.Methods1. The pancreatic carcinaoma xenografts model of nude mice is established.2. To compare the theraputic effect as well as side effects of free Photosan and Nano-Photosan induced PDT on human pancreatic carcinoma xenografts with nude mice model.Results1. The success rate of xenografts model establishment is almost 100%. It takes7±2days on average for tumors reached the volume of about0.2cm3.2. The photodynamic effects in therapeutic groups (i.e. free Photosan group and Nano-Photosan group) are significantly better than the control group (both P<0.05). The two therapeutic groups did not display any statistical difference until6day post-irradiated (P<0.05). Tumor weight and volume of the two therapeutical groups was significanty less than that of the control group. Furthemore the tumor weight and volume of nano-Photosan group are significantly smaller than that of the free photosan group (P<0.05).3.14days after treatment, HE stain microscopy reveals that the apoptosis and necrocytosis of Nano-Photosan group are significantly higher than those of the free Photosan group. The panc-1cells alive can be seen in free Photosan group not in nano-Photosan group.4. There were no obvious side effects found in both free Photosan and nano-Photosan groups. Conclusion1. Both free Photosan and Nano-Photosan have significant tumoricidal effects on tumor growth of Panc-1xenograft tumors in nude mice model. And the nano-Photosan induced PDT is significantly better than the free Photosan with less time consumption.2. Both free Photosan and Nano-Photosan indued PDT may are biologically safe in vivo. PART III Photodynamic effect of nano-Photosan induced PDT on Panc-1cell and its mechanismObjectiveTo investigate the impacts of Nano-Photosan induced PDT on cell cycle and autophage of Panc-1cell and its related mechanism. Methods1. To perform the PDT induced by both photosensitizers on Panc-1cells with optimal parameters obtained from the above experiments. And cell cycle of Panc-1treated with both photosensitizers is detected by PI simple staining combined with flow cytometry.2. The mRNA expression level of TGF-β1is determined by real-time fluorescent quantitative PCR and the proteins expression differences of TGF-β1, LC3B, and Beclinl in Panc-1cell are determined by western-blot.3. The autophage of Panc-1cells treated with both free Photosan and Nano-photosan are evaluated by transmission electron microscope (TEM). Results1. Cell cycle is blocked at G1phase in nano-Photosan treated group not in free Photosan group.2. The expression of TGF-β1in Panc-1cells from PDT groups are higher than that from the control group. And both free Photosan and Nano-Photosan can increase the expression level of TGF-β1after Photodynamic irradiation. Compare to the free Photosan, nano-Photosan decreased the expression level of LC3B and Beclinl in Panc-1cells dramatically10hours after PDT.3. Some autophagosomes and autophagic vacuoles were found in both groups, but the number of autophagosomes and autophagic vacuoles of Panc-1cells in nano-photosan group was significantly less than that in photosan group.Conclusion1. Nano-Photosan induced PDT could block the cell cycle of Panc-1cells at G1phase.2. PDT increased mRNA expressions of TGF-β1in Panc-1cells restrain the cell proliferation and induced autophagy, Photosan-loaded nanospheres-PDT reversed the increasing tendency of LC3B and Beclinl induced by TGF-β1.3. Autophagy decreased after Photosan-loaded nanospheres-PDT compared to it with free Photosan-PDT, the autophagy could also be affected by the cell phase which the cells are currently at.