Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".

We recently clarified that the side-scatter(ed) light (SSC) of flow cytometry (FCM) could be used as a guide to measure the uptake potential of nanoparticles [ Suzuki et al. Environ. Sci. Technol. 2007 , 41 , 3018 - 3024 ]. In this paper, the method was improved so as to be able to determine simultaneously the uptake potential of nanoparticles and the production of reactive oxygen species (ROS), and correlations with genotoxicity were evaluated. In the FCM analysis, SSC and fluorescence of 6-carboxy-2,7'-diclorodihydrofluorescein diacetate, di(acetoxy ester) based on ROS production were concurrently detected after treatments with ZnO, CuO, Fe(3)O(4), TiO(2), and Ag nanoparticles. The ZnO and CuO nanoparticles caused high ROS production, which was more significant in the cells with higher SSC intensity. The increase of SSC intensity was more significant for TiO(2) than ZnO and CuO, whereas ROS production was higher for ZnO and CuO than TiO(2), suggesting that the extent of ROS production based on the uptake of nanoparticles differed with each kind of nanoparticle. ROS production was correlated with generation of the phosphorylated histone H2AX (γ-H2AX), a marker of DNA damage, and an antioxidant, n-acetylcysteine, could partially suppress the γ-H2AX. This method makes it possible to predict not only uptake potential but also genotoxicity.

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Photodynamic therapy (PDT) is a less-invasive treatment for cancer through the administration of less-toxic porphyrins and visible-light irradiation. Photosensitized damage of biomacromolecules through singlet oxygen (O) generation induces cancer cell death. However, a large quantity of porphyrin photosensitizer is required, and the treatment effect is restricted under a hypoxic cellular condition. Here we report the phototoxic activity of P(V)porphyrins: dichloroP(V)tetrakis(4-methoxyphenyl)porphyrin (CLP(V)TMPP), dimethoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (MEP(V)TMPP), and diethyleneglycoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (EGP(V)TMPP). These P(V)porphyrins damaged the tryptophan residue of human serum albumin (HSA) under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was barely inhibited by sodium azide, a quencher of O. Fluorescence lifetimes of P(V)porphyrins with or without HSA and their redox potentials supported the electron-transfer-mediated oxidation of protein. The photocytotoxicity of these P(V)porphyrins to HeLa cells was also demonstrated. CLP(V)TMPP did not exhibit photocytotoxicity to HaCaT, a cultured human skin cell, and MEP(V)TMPP and EGP(V)TMPP did; however, cellular DNA damage was barely observed. In addition, a significant PDT effect of these P(V) porphyrins on a mouse tumor model comparable with the traditional photosensitizer was also demonstrated. These findings suggest the cancer selectivity of these P(V)porphyrins and lower carcinogenic risk to normal cells. Electron-transfer-mediated oxidation of biomacromolecules by P(V)porphyrins using long-wavelength visible light should be advantageous for PDT of hypoxic tumor.