Doping And Modification Of Nanometer TiO 2 And Its Mechanisms For Photodynamic Therapy

Photodynamic therapy (PDT), as a new cancer therapy, has attracted wide attention. Semiconductor titanium dioxide (TiO2) was noticed as a potential photosensitizer due to its low toxicity, high chemical stability, good photoreactivity, and excellent biocompatibility. However, TiO2can only be activated by UV light and cannot resolve in water, which hinder its application and development in PDT. Improvement of the optical absorption and dispersibility of TiO2, will facilitate its application in the field of PDT.In this work, the nitrogen-doped (N-TiO2) nanoparticles were prepared by calcination, whose absorption was expanded from UV to the visible region. We focused on the killing effects and mechanism of N-TiO2on cancer cells with visible light irradiation. Furthermore, surface modification of TiO2and N-TiO2nanoparticles was conducted to enhance their dispersibility in aqueous solutions. The conjugates of surface-modified TiO2(or N-TiO2) and AlPcS4molecules were prepared. The photokilling effect of the conjugates on cancer cells with visible light irradiation was studied. The main results are as follows:1. N-TiO2nanoparticles were prepared by calcining the anatase TiO2nanoparticles under ammonia atmosphere. The N-TiO2showed higher absorbance in the visible region than the pure TiO2. The cytotoxicity and visible-light-induced phototoxicity of the pure-and N-TiO2were examined for HeLa, QGY and KB cancer cell lines. No significant cytotoxicity was detected. However, under visible-light irradiation, the cancer cells incubated with N-TiO2were killed more effectively than that with the pure TiO2. The survival fraction of the cells decreased with the increased incubation concentration of the nanoparticles. Furthermore, the intracellular distributions of N-TiO2nanoparticles were examined by laser scanning confocal microscopy (LSCM). The co-localization of N-TiO2nanoparticles with nuclei or Golgi complexes was observed. After visible-light irradiation, some micronuclei were detected as a sign of the nucleus aberration. This is an evidence of the direct damage to the nuclei resulted from the photoexcited N-TiO2nanoparticles.2. A comparison of the killing effects between N-TiO2and TiO2on HeLa cells with visible light irradiation was conducted. Under visible-light irradiation,N-TiO2 produced more reactive oxygen species (ROS) and specifically more O2/H2O2, while less ROS but more OH·were produced by TiO2. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after visible light PDT were measured and compared for N-TiO2-and TiO2-treated cells by LSCM. The N-TiO2resulted in more loss of MMP and higher increase of Ca2+and NO in cells than pure TiO2. The cell morphology changes with time were also examined by the confocal microscope. Compared to the cells incubated with TiO2, the cells incubated with N-TiO2exhibited more serious distortion and membrane breakage at60min after the PDT.3. To improve the solubility of TiO2in aqueous solutions, TiO2-NH2nanoparticles were prepared by the surface modification of TiO2. The surface of TiO2-NH2was positively charged and its dispersibility in water was greatly improved. The uptake of TiO2-NH2into living cells was more effectively than that of bare TiO2according to the observation by scanning transmission X-ray microscopy. For the possibility to use red light (its wavelength is within the "optical window" of human body) in PDT, the Pc-TiO2composite was prepared by mixing the negatively charged sulfonated aluminum phthalocynaine (AIPCS4) molecules with the positively charged TiO2-NH2in solution, which can be effectively bound due to the electrostatic force. It was observed using the LSCM that Pc-TiO2can enter the cells more effectively than free AIPcS4. The Pc-TiO2conjugates showed no significant toxicity even after24hours incubation with cells. The cells were killed by the conjugates more effectively than the free AlPcS4irradiated by a red light source, as well as a broad-band visible light source. Moreover, the Pc-N-TiO2was prepared by the same method using AlPcS4and N-TiO2as the reactants. Under broad-band visible light irradiation, the photokilling effect of the Pc-N-TiO2was greatly enhanced than that of Pc-TiO2.