TiO₂ colloid solutions were prepared[13,14] by hydrolysis of titanium isopropoxide, Ti [OCH (CH₃) ₂]₄ (97%, Aldrich Chemical Co). In brief, 12.5 mL of Ti [OCH (CH₃) ₂]₄ was added to 2 mL isopropanol, then the mixture was added to 150 mL of distilled deionized water containing 2 mL of 700 mL/L nitric acid and vigorously stirred for 6 h at 75 °C. Approximately 150 mL of TiO₂ colloid solution being stable for several months at 4 °C was obtained after the organic layer was removed. The average diameter determined by Zetasizer 3000HS_A (USA) was 21.2 nm.

The pH value of TiO₂ colloid solutions used in the subsequent experiment had to be adjusted from 1.8 to 5.5-6.5 in order not to damage the normal growth of cells. Therefore, 1 mol/L NaOH aqueous solution and 1.5 mL/L polyvinyl alcohol were added to the colloid solutions before the pH adjustment to prevent the TiO₂ from precipitation. The final TiO₂ colloid solutions were sterilized by autoclaving and then diluted to the required concentration. Other chemical reagents used were all of analytical purity from commercial sources.

Human colon carcinoma cell lines Ls-174-t were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. The cell lines were cultured in RPMI 1640 (Gibco) medium supplemented with calf serum 100 ml/L, penicillin (100 × 10 ³μ/L) and streptomycin (100 mg/L). pH was maintained at 7.2-7.4 by equilibration with 50 mL/L CO₂ . Temperature was maintained at 37 °C. Cells were sub-cultured with a mixture of ethylenedinitrile tetraacetic acid (EDTA) and trypsin. All experiments were performed using cells during the exponential growth phase. Cell concentration was determined by using a hemocytometer and the cell density was adjusted to the required final concentration.

Ls-174-t cells were treated with TiO₂ diluted in RPMI 1640 medium for 2 h at 37 °C. Then the solutions were irradiated with a GGZ-300W high pressure Hg lamp (E_max = 365 nm) at room temperature. A UV pass filter was used to obtain a light wavelength between 300-400 nm. The light intensity at the liquid surface was measured by a VLX-3W radiometer-photometer (USA). The incident light intensity was 3.7 mW/cm². In our study, three groups of tests were carried out. One group was treated in the absence of TiO₂. Another group was treated in the absence of UVA. The third group was treated with different TiO₂ concentrations and irradiated by UVA.

Viable cells in the samples were measured by using the MTT staining method[15]. MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide] was dissolved in phosphate-buffered saline ( PBS, pH7.4) at 5 mg/mL and filtered to be sterilized. Twenty microliters of stock MTT solution were added to all wells for an assay, and plates were incubated at 37 °C for 4 h. One hundred and fifty microliters of DMSO were added to all wells and mixed thoroughly to dissolve the blue-violet crystals. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read on a Bio-Rad Novapath^tm microplate reader (Japan), using a test wavelength of 595 nm, taking the solution without MTT as control. Then optical absorptions [A] were obtained. Plates were normally read within 1 h after DMSO was added. The survival rate could be calculated according to [A]_t/[A]_i, where [A]_i is the optical absorption of untreated cells.

Zetasizer 3000HSA (USA) was used to determine the average diameter of TiO₂ nanoparticles. The result is shown in Figure 1. The average volume size of TiO₂ nanoparticles was 21.2 nm.

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Figure 1 Volume size distribution of TiO₂.

The cytotoxicity of TiO₂ nanoparticles (without UVA irradiation) was determined by exposing cells to various concentrations of TiO₂ in RPMI 1640 (Gibco) medium for 24 h. The surviving fraction of the cells was greater than 90% when the concentration of TiO₂ was in the range of 1 000 μg/mL, as shown in Figure 2. The result confirmed that nonirradiated TiO₂ nanoparticles were not toxic to the Ls-174-t cells. It was consistent with those in literatures[16-17].

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Figure 2 After Ls-174-t cells were incubated in RPMI 1640 medium for 24 h without irradiation, the survival of Ls-174-t cells was shown.

The fact that the surviving fraction was greater than 90% after 30 min as shown in Figure 3 (A) indicated that TiO₂ nanoparticles without UVA irradiation showed little toxicity to living cells. The killing effect of UVA without TiO₂ is shown in (B) with the surviving fraction of Ls-174-t cells given as a function of the UVA light irradiation time. About 20% cells were killed after 30 min exposure, whereas after 20 min exposure, more than 90% of the cells survived. Once TiO₂ was added, the Ls-174-t cells were killed at a much higher rate as shown in (C). For example, in the presence of 1 000 μg/mL of TiO₂, 44% of the cells were killed after 10 min of UVA irradiation, and after 30 min irradiation, 80 % of the cells were killed. Therefore it was concluded that photoexcited TiO₂ nanoparticles had an active killing effect on Ls-174-cells.

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Figure 3 Effect of light and TiO₂ on viability of Ls-174-t cells. (A) TiO₂ (1 000 μg/mL) in the dark; (B) no TiO₂ in the light; (C) TiO₂ (1 000 μg/mL) in the light. Initial cell concentration: 5 × 10⁵ cell/mL, light intensity: 3.7 mW/cm².

The effect of the TiO₂ concentration ranging from 200 to 1 000 μg/mL on the rate of cell killing by UVA light is shown in Figure 4. The light intensity was 3.7 mW/cm² and kept constant. The experimental results demonstrated that cell viability decreased monotonically as TiO₂ concentration increased and cell viability decreased with time.

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Figure 4 Influence of TiO₂ concentration on Ls-174-t cells activity. (A) 204 μg/mL, (B) 524 μg/mL, (C) 804 μg/mL, (D) 1 029 μg/mL.

Although a higher concentration of TiO₂ could achieve a higher reaction rate, the difficulties in separation and measurement had to be considered. So the effect of a higher TiO₂ concentration (C > 1 029 μg/mL) on Ls-174-t cell activity was not investigated. The concentration of TiO₂ was set at < 1 029 μg/mL with which the dark cytotoxicity was considered to be negligible. The range of TiO₂ concentrations was close to that used previously[18].

Untreated and TiO₂-treated cells were collected by centrifugation and resuspended in RPMI 1640 medium. The samples were pipetted into a 24-well plate, which was directly observed with an inverted phase-contrast microscope. When treated by photoexcited TiO₂, the cellular shape was condensed and nuclei were dispersed in fragments.