Study on the visible-light-induced photokilling effect of nitrogen-doped TiO2 nanoparticles on cancer cells
© Li et al; licensee Springer. 2011
Received: 19 January 2011
Accepted: 21 April 2011
Published: 21 April 2011
Nitrogen-doped TiO2 (N-TiO2) nanoparticles were prepared by calcining the anatase TiO2 nanoparticles under ammonia atmosphere. The N-TiO2 showed higher absorbance in the visible region than the pure TiO2. The cytotoxicity and visible-light-induced phototoxicity of the pure- and N-TiO2 were examined for three types of cancer cell lines. No significant cytotoxicity was detected. However, the visible-light-induced photokilling effects on cells were observed. The survival fraction of the cells decreased with the increased incubation concentration of the nanoparticles. The cancer cells incubated with N-TiO2 were killed more effectively than that with the pure TiO2. The reactive oxygen species was found to play an important role on the photokilling effect for cells. Furthermore, the intracellular distributions of N-TiO2 nanoparticles were examined by laser scanning confocal microscopy. The co-localization of N-TiO2 nanoparticles with nuclei or Golgi complexes was observed. The aberrant nuclear morphologies such as micronuclei were detected after the N-TiO2-treated cells were irradiated by the visible light.
Semiconductor titanium dioxide (TiO2) has been widely studied as a photocatalyst for its high chemical stability, excellent oxidation capability, good photocatalytic activity, and low toxicity [1–4]. Under the irradiation of ultraviolet (UV) light with the wavelength shorter than 387 nm (corresponding to 3.2 eV for the band gap of anatase TiO2), the electrons in the valence band of TiO2 can be excited to the conduction band, thus creating the pairs of photo-induced electron and hole. Then, the photo-induced electrons and holes can lead to the formation of various reactive oxygen species (ROS), which could kill bacteria, viruses, and cancer cells [5–10].
In recent years, TiO2 attracted more attention as a photosensitizer in the field of photodynamic therapy (PDT) due to its low toxicity and high photostability [2, 3]. However, TiO2 can be activated by UV light only, which hinders its applications. Improvement of the optical absorption of TiO2 in the visible region by dye-adsorbed [11, 12] or doping [13, 14] methods will facilitate the practical application of TiO2 as a photosensitizer for PDT. When using dye-adsorbed method, the dyes such as hypocrellin B  and chlorine e6  themselves are well-known PDT sensitizers and will have influence on the PDT efficiency of TiO2. For doping method, anionic species are preferred for the doping rather than cationic metals which have a thermal instability and an increase of the recombination centers of carriers . In addition, cationic metals themselves always present cytotoxicity. Therefore, anionic species doping, especially nitrogen doping, is mostly adopted to improve the absorption of TiO2 in the visible region.
In the present work, the nitrogen-doped TiO2 (N-TiO2) nanoparticles were used as the photosensitizer to test its photokilling efficiency for three types of cancer cell lines. The N-TiO2 nanoparticles were prepared by calcining pure anatase TiO2 nanoparticles under ammonia atmosphere, which was an inexpensive method and easy to operate. The produced N-TiO2 nanoparticles have high stability and effective photocatalytic activity. Their absorption in the visible region was improved and their photokilling efficiency of cells under visible-light irradiation was compared with that of the pure TiO2. The intracellular distributions of these nanoparticles were measured by the laser scanning confocal microscopy (LSCM). The mechanisms of the photokilling effect were discussed.
Preparation and characterization of N-TiO2 nanoparticles
Calcination parameters and the resulted crystalline phases of the TiO2 nanoparticles
Ammonia gas flow rate (L/min)
Rutile and anatase
Pure- and N-TiO2 nanoparticles were dispersed in Dulbecco's modified Eagle's medium with high glucose (DMEM-H), respectively, at various concentrations between 50 and 200 μg/mL. To avoid aggregation, these suspensions were ultrasonically processed for 15 min before using.
The human cervical carcinoma cells (HeLa), human hepatocellular carcinoma cells (QGY), or human nasopharyngeal carcinoma cells (KB) procured from the Cell Bank of Shanghai Science Academy (Shanghai, China) were grown in 96-well plates or Petri dishes in DMEM-H solution supplemented with 10% fetal calf serum in a fully humidified incubator at 37°C with 5% CO2 for 24 h. Then, the culture medium was replaced by TiO2-containing medium and the cells were incubated for 2 h in the dark. After the TiO2 nanoparticles deposited and adhered to the cells, the medium was changed to the TiO2-free DMEM-H solution supplemented with 10% fetal calf serum for further study.
Measurements of photokilling effect and cytotoxicity
To examine the photokilling effect, the cells were irradiated with the visible light from a 150-W Xe lamp (Shanghai Aojia Electronics Co. Ltd., Shanghai, China). Two pieces of quartz lens were used to obtain a concentrated parallel light beam. An IR cutoff filter was set in the light path to avoid the hyperthermia effect. A 400-nm longpass filter was used to cut off the UV light. The visible-light power density at the liquid surface in cell wells was 12 mW/cm2 as measured by a power meter (PM10V1; Coherent, Santa Clara, CA, USA). After irradiation with this visible light for 4 h, cells were incubated in the dark for another 24 h until further analysis were conducted. The cytotoxicity examinations were carried out with the same procedure as the photokilling effect examinations but without the light irradiation, i.e., the TiO2-treated cells were incubated in the dark for 28 h.
The cell viability assays were conducted by a modified MTT method using WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] (Beyotime, Jiangsu, China). Each well containing 100 μL culture medium was added with 10 μL of the WST-8 reagent solution, and the cells were then incubated at 37°C with 5% CO2 for 2 h. Subsequently, the absorbance was measured at 450 nm using a microplate reader (Bio-Tek Synergy™ HT; Bio-Tek® Instruments, Inc., Winooski, VT, USA). The untreated cells were used as the control groups. The surviving fraction represents the ratio of the viable TiO2-treated cells relative to that of the control groups. It should be noted that the TiO2-containing DMEM-H solution will affect the absorbance value at 450 nm. Therefore, when measuring the cell viability, the absorbance values were measured as a reference before the WST-8 dyes were added. Each experiment was performed in triplicate and repeated three times.
Confocal laser scanning microscopy
The cells grown in Petri dishes were incubated with 50 μg/mL TiO2 in DMEM-H for 10 h before the LSCM observation (Olympus, FV-300, IX71; Olympus, Tokyo, Japan). Hoechst 33342 (Beyotime) and BODIPY FL C5-ceramide complexed to BSA (Molecular Probes; Invitrogen Corporation, Eugene, OR, USA) were used as the indicators for nucleus and Golgi complex, respectively. Hoechst 33342 (0.5 μg/mL) or Golgi complex marker (5 μM) was added into the growth medium for 15 to 30 min to stain the nuclei or Golgi complexes, respectively.
The reflection images of the intracellular TiO2 nanoparticles and the fluorescence images of nuclei (or Golgi complexes) were simultaneously obtained by the LSCM in two channels with no filter for the reflecting light and a 585 to 640-nm bandpass filter for the fluorescence. A 488-nm continuous-wave (CW) Ar+ laser (Melles Griot, Carlsbad, CA, USA) or a 405-nm CW semiconductor laser (Coherent) was used as the excitation source. A 60 × water objective was used to focus the laser beam to a spot of about 1 μm in diameter. The differential interference contrast (DIC) micrographs to exhibit the cell morphology were acquired in a transmission channel simultaneously. The three-dimensional (3D) distributions of TiO2 nanoparticles and nuclei (or Golgi complexes) were obtained using the z-scan mode of the microscope.
Results and discussion
Raman spectra of TiO2 nanoparticles
Absorption spectra of TiO2 nanoparticles
Figure 1b shows the absorption spectra of the samples N-550-1 and N-550-2 and pure TiO2. Compared to the pure TiO2, the absorbances of N-550-1 and N-550-2 are higher in the visible region. However, the sample N-550-2 has the higher absorbance than N-550-1 in the region of 400 to 500 nm. Since N-550-1 and N-550-2 were calcinated at the same temperature and with the same amount of ammonia (flow rate times time), it seems that higher ammonia flow rate (N-550-2) could cause more absorption in the visible, which was expected to have higher photokilling efficiency of cells.
Cytotoxicity and photokilling effect
The photokilling effects were measured as described in the experimental section. The surviving fractions of HeLa cells under visible-light irradiations for 4 h in dependence on the concentrations of pure- and N-TiO2 nanoparticles were shown in Figure 2b. As demonstrated in Figure 2b, the visible light showed very little photokilling effect on HeLa cells in the absence of any TiO2 (pure or N-doped) (at the 0 concentration). The surviving fractions (compared to the control cells without irradiation) were around 93%, which might be caused by the light irradiation, the fluctuant temperature during irradiation, and the experimental procedures. The spectrum of the light irradiated on cells (with filters) is also shown in the figure as an inset. It should be noted according to the spectrum in Figure 1b that the pure TiO2 nanoparticles still has some absorption around 400 nm though the band gap of TiO2 was reported to be 3.2 eV (corresponding to a wavelength of 387 nm). Therefore, pure TiO2 exhibited some photokilling effect under visible-light irradiation as shown in Figure 2b. However, the cells treated with N-TiO2 were killed more effectively than that with pure TiO2. The photokilling effects of samples N-550-1 and N-550-2 were quite similar although their absorption spectra showed some difference. It is also demonstrated in Figure 2b that the survival fractions decreased with the increasing concentrations of the TiO2 samples. It decreased to 40% for the cells treated with sample N-550-2 at a concentration of 200 μg/mL.
The photokilling effects of sample N-550-2 at a concentration of 200 μg/mL on QGY and KB cells were also measured as shown in Figure 3. Similar with the photokilling effect on HeLa cells, the QGY and KB cells treated with N-550-2 were also killed more effectively than that with pure TiO2 under the visible-light irradiation. The results revealed that the N-TiO2 might be applied to different cancers as a photosensitizer for PDT.
ROS influence on the photokilling effect
Distribution of TiO2 in cells
In the present work, N-TiO2 nanoparticles were prepared by calcination under ammonia atmosphere, which is an easily operative method and can achieve the product fruitfully. All the cytotoxicities of the pure- or N-TiO2 nanoparticles were quite low. The N-TiO2 samples showed higher absorbance and better photokilling effect than the pure TiO2 in the visible region. Therefore, the N-TiO2 has a higher potential as a photosensitizer for PDT of cancers.
TiO2 is nonfluorescent and cannot be detected by fluorescence imaging. However, it can be monitored by the reflection imaging, which makes it convenient to record simultaneously with the fluorescence image using a LSCM. Co-localization of N-TiO2 nanoparticles with nuclei was observed. After visible-light irradiation, some micronuclei were detected as a sign of the nucleus aberration. Furthermore, ROS was found to play an important role on the photokilling effect for cells. However, the mechanisms for the photokilling effect on cancer cells should be investigated in details further.
This work is supported by the National Natural Science Foundation of China (61008055, 11074053), the Ph.D. Programs Foundation of Ministry of Education of China (20100071120029), and the Shanghai Educational Development Foundation (2008CG03).
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