The Mechanism And Killing Effects Of Photosensitizer-loaded Hollow Silica Nanoparticles On Photodynamic Therapy For Cholangiocarcinoma

Aims:1. Preparation Photosan-loaded hollow silica nanoparticles (Photosan-loaded HSNPs), and study the drug safety of Photosan-loaded HSNPs.2. Study and compare the effect of photodynamic therapy (PDT) with Photosan and Photosan-loaded HSNPs on cholangiocarcinoma in vitro.3. Study and compare the effect of PDT with Photosan and Photosan-loaded HSNPs on cholangiocarcinoma in vivo.4. Study and compare the possible ways of PDT with Photosan and Photosan-laoded HSNPs inhibiting the growth of cholangiocarcinoma.Methods:1. Prepare the Photosan-loaded hollow silica nanoparticles.2. In vitro experiments, test the killing effect of PDT with Photosan or Photosan-loaded HSNPs for human cholangiocarcinoma QBC939cells using MTT assay. Observe the influence of PDT effects with different photosensitizer incubation times, different concentrations of photosensitizers and different light dose, and study the dark toxicity of the2photosensitizers as well as compare the differences of their effects. Flow cytometry analysis further investigate the PDT effect of2photosensitizers, and compare the difference between them. Using an optical microscope to observe the morphological changes of the cells before and after PDT.3. Establish the human cholangiocarcinoma in nude mice xenograft model, and observe the differences of inhibiting effect of PDT with Photosan of Photosan-loaded HSNPs. Observe whether the photosensitizers have toxic or side effects on animals. Observe the morphological changes of tumor cells before and after PDT by biopsy. 4. Detect the impacts of PDT for mice’s tumors on vascular generation, invasion capacity and cell apoptosis, and compare the differences between the two photosensitizers. The methods were detecting the differences in the level of mRNA and protein expression of related genes using PCR, Western-blot, and immunohistochemistry.Results:1. Prepare the Photosan-loaded hollow silica nanoparticles successfully.2. MTT assay shows that the PDT both with Photosan and Photosan-loaded HSNPs have obvious killing effect on cholangiocarcinoma cells (P<0.05), and the latter one has even better killing effect (P<0.05). Within a certain range, the effect of PDT shows a positive relationship with the photosensitizer incubation time, the concentration of photosensitizer and light dose. There is no obvious dark toxicity on both two kinds of photosensitizers. But these two kinds of photosensitizers have different optimal interaction parameters. The optimal interaction parameter of Photosan-loaded HSNPs has lower photosensitizer concentration and shorter incubation time. Flow cytometry assay with Photosan or Photosan-loaded HSNPs shows that the latter has obviously stronger cell killing effect to the former in the condition of they both using their own optimal interaction parameters (P<0.05). Both of them kill the cells through the way of apoptosis. But the latter has higher proportion of cell necrosis to the former. With an optical microscope we can also find that the killing effect on tumor cells of Photosan-loaded HSNPs is stronger, and the percentage of necrotic cells is higher.3. Animal experiments show that PDT with Photosan or Photosan-loaded HSNPs both have significantly inhibition effect to cholangiocarcinoma by using their own optimal interaction parameters (P<0.05), and the latter one is even stronger than the former (P<0.05). Biopsy findings also show that the latter has more obvious anti-tumor effect.4. The genes expression at mRNA and protein levels relating to tumor angiogenesis, invasion in cholangiocarcinoma were reduced (P<0.05), and the apoptosis-related genes were up-regulated after PDT with Photosan or Photosan-loaded HSNPs (P<0.05). Compared to the former, the latter has more strongly influence on the tumor invasion ability and tumor cell apoptosis (P<0.05)Conclusions:1. Photosan-loaded HSNPs can be prepared by one-step wet chemical-based synthetic route. HSNPs have no significant toxicity.2. Photosan and Photosan-loaded HSNPs both have obvious killing effect on cholangiocarcinoma cells, while the latter’s killing effect is stronger. In a certain range, the effect of PDT shows a positive relationship with the photosensitizer incubation time, the concentration of photosensitizer and light dose. Comparing to Photosan, Photosa-loaded HSNPs bring stronger PDT effect, faster rate to achieve effective therapeutic concentration.3. PDT with Photosan and Photosan-loaded HSNPs both proved to have a significant therapeutic effect on nude mice xenograft model of human cholangiocarcinoma.4. The possible approachs of PDT with Photosan and Photosan-loaded HSNPs in inhibiting the growth of cholangiocarcinoma are:inhibition of tumor angiogenesis, reduction of tumor invasion ability, as well as induction of tumor cells apoptosis, while the latter reduce tumor invasion ability and induce apoptosis of tumor cells more significantly.