Introduction
Titanium dioxide (TiO2) is an important biomaterial with its advantages in chemical stability, high refractive index [1,2] and photocatalytic activity [3-5]. Several crystal forms of TiO2 including anatase, brookite, and rutile are known to exist with different physiochemical properties. The anatase and brookite are less stable than the rutile and transform to the rutile by heat or other environmental conditions [6]. Rutile phase TiO2 (R-TiO2) was widely used in pigment and cosmetic industry [7]. R-TiO2 provides the white opacity with high refractive index and protects paints from chalking due to low photocatalytic activity [8]. Similarly R-TiO2 protects the skin from ultraviolet light and reduce the potential risk effect of free radicals on skin aging, which makes R-TiO2 useful for the sunscreens [9,10]. For therapeutic point, TiO2 was reported to have anti-bacteria, anti-fungal and anti-cancer effects [5,11,12]. In dental field, TiO2 was used for oral hygiene as an ingredient of toothpaste or mouthwash [13,14] and for polymeric resin-based composites [15]. Since R-TiO2 has beneficial advantages in chemical stability, low photocatalytic activity and anti-infection activity, we proposed an idea that TiO2 NPs in rutile phase might be suitable for the aesthetic white pigmentation at the site of tooth decay. To pursuit this idea further, we initiated the synthetic process of R-TiO2 NPs and attempted to characterize its properties by various methods. Here, we report the preparation of TiO2 NPs in rutile phase by the fast and simple method: boiling TiCl4 solution in the air, adjusting pH with NH4OH and calcinating at 900℃. The physiochemical properties of the prepared R-TiO2 NPs were examined by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDS), transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurement. Finally R-TiO2 NPs were tested on two oral cancer cell lines, FaDu and YD-15, for the evaluation of cytocompatibility.
Materials and Methods
1. Preparation of rutile phase titanium dioxide nanoparticles
10 mL of TiCl4 (99.9%; Sigma-Aldrich, St. Lous, MO, USA) was slowly added to 100 mL of cold water, stirred for 2 hours at room temperature and then refluxed at 100℃ for 1 hour to form a milky slurry suspension. After cooling down the suspension, 10 mL of (NH4)2SO4 was added and the pH was adjusted to 7.0 with NH4OH solution. Excess of chlorine and sulfate was removed by the centrifugation with distilled water before heating at 100℃ for 12 hours. The dried white cake of TiO2 was grinded and calcinated at 900℃ for 2 hours.
2. Fourier transform infrared spectroscopy
FT-IR spectrum was recorded in the range of 400–4000 cm –1 by using a Nicolet 6700 FT-IR spectrometer (ThermoFisher, Waltham, MA, USA) with 32 scans for each sample.
3. X-ray diffraction
XRD from 20° to 80° was carried out by an X’Pert PRO Alpha- 1 instrument (Malvern Panalytical, Almelo, Netherlands) with the diffractometer using Cu Kα radiation (wavelength λ = 1.54056 Å). The measurement was done with the step-size of 0.02° and scan step-time of 1 second. Data was collected based on angle (2θ ) between diffracted X-ray with incident X-ray beam. The crystallinity of TiO2 was estimated from the specific peak intensity. The particle size and specific surface area of the TiO2 were assessed by using following equations:
D is the crystal size (nm). K is Scherrer’s constant (0.89). λ is the X-ray wavelength of Cu Kα radiation (λ = 1.54 Å). β is the line width of half maximum (radian). θ is Bragg’s diffraction angle. S is specific surface area. d is the theoretical density of TiO2 particles (for rutile, d = 4.25 g/cm3).
4. Scanning electron microscopy and energy dispersive X-ray spectroscopy
SEM-EDS analysis was carried out by using Hitachi S-4800 SEM (Hitachi, Tokyo, Japan) equipped with X-ray microanalysis to determine the surface topography and analyze the element of the prepared TiO2.
5. Transmission electron microscopy and selected area electron diffraction
TEM were recorded from a Hitachi H-9500 high resolution 300 kV analytical TEM system (Hitachi). The sample for TEM was placed on carbon-coated copper grids and dispersed into ethanol.
6. Dynamic light scattering and zeta potential
DLS and zeta potential measurement were performed by using the high performance particle size analyzer Malvern Zetasizer (Malvern Panalytical), dispersing 0.01 mg of TiO2 in 1 mL of solvent (dimethyl sulfoxide [DMSO]: H2O, 1:9 (v/v)).
7. Cell culture
FaDu human hypopharyngeal adenocarcinoma and YD-15 human tongue cancer cells, supplied by the Korean Cell Line Bank, were cultured in MEM and RPMI-1640 media (WELGENE, Gyeongsan, Korea), supplemented with 10 % fetal bovine serum and 100 units/mL penicillin/streptomycin, respectively. The cells were seeded to the density of 2 × 105 cells/mL and incubated for 24 hours in an incubator with 5 % CO2.
8. Cytotoxicity assay
The cytotoxicity of TiO2 NPs in rutile phase was evaluated by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Briefly, cells were seeded at the density of 4 × 104 cells per well in 96-well plates and incubated overnight. Cells were treated with various concentrations of TiO2 NPs in suspension and incubated for 24 or 48 hours. After incubation, the media was removed and 50 μL of MTT solution (0.5 mg/mL) was added and kept in dark for 4 hours at 37℃. MTT solution in each well was replaced by 100 μL of DMSO to dissolve crystal violet formazan. The formazan solutions, after centrifugation, was used to measure the absorbance at 570 nm on MultiskanTM FC Microplate Photometer (ThermoFisher).
Results
1. Preparation of rutile phase titanium dioxide nanoparticles
TiO2 was prepared by the hydrolysis reaction of TiCl4 in strong acidic environment (pH ＜ 1) as described in the reaction scheme below.
The nucleated TiO2 were initially amorphous but, after adjusting pH to 7 with NH4OH solution, converted into the mixed composition of rutile and anatase phase (named as raw TiO2). The transformation of raw TiO2 into phase-pure rutile TiO2 was achieved by the calcination at 900℃ for 2 hours (Fig. 1).
2. Phase composition analysis of rutile phase titanium dioxide nanoparticles
The calcination at 900℃ for 2 hours caused dramatic phase transformation in crystal structure, converting raw TiO2 into highly pure rutile form of R-TiO2 as shown in the XRD pattern (Fig. 1). The characteristic XRD peaks of R-TiO2 NPs were consistent with the ones of standard rutile phases from the database of the Joint Committee on Powder Diffraction Standards [JCPDS 21-1276: 2θ = 27.47°(110), 36.11°(101), 39.21° (200), 41.27°(111), 44.11°(210), 54.33°(211), 56.63°(220), 62.79°(002), 64.08°(310), 69.05°(301), 69.87°(112), 72.83° (320), and 76.87°(202)]. The appearance of high and sharp diffraction peaks after calcination at 900℃ indicated the pure crystalline of R-TiO2 NPs. Average crystal size of the R-TiO2 NPs, after calcination at 900℃, increased from 27 to 76 nm while specific surface area was reduced from 57 to 19 m2/g (Table 1).
3. Surface function analysis of R-TiO2 NPs
FT-IR spectrum of R-TiO2 NPs showed a dramatic change in the relative intensity and the number of bands after calcination (Fig. 2). The characteristic broad bands at 400 to 1000 cm –1, corresponding to Ti-O-Ti vibration of the mixed polymorphic phases of TiO2, and bands at 3,600–3,100 cm –1 and 1,660– 1,640 cm –1, due to the hydrated water, were appeared in raw TiO2. However, TiO2 after calcination at 900℃ for 2 hours, revealed a simple broad band only at 400 to 1000 cm –1, supporting the phase transformation into the rutile phase.
4. Morpholoical feature, element analysis and crystallinity of rutile phase titanium dioxide nanoparticles
The shape of R-TiO2 NPs was spherical as shown in SEM image (Fig. 3A). The elemental composition of R-TiO2 NPs, which was analyzed by EDS, showed two characteristic peaks at 0.2 keV and 4.5 keV corresponding to Ti-O and Ti with the weight percent of Ti and O at 12.4 and 87.6%, respectively (Fig. 3B). In addition, the morphology and inset image in TEMSAED showed partly nonhomogeneous agglomeration with single rutile crystalline of TiO2 NPs (Fig. 3C).
5. Particle size and zeta potential rutile phase titanium dioxide nanoparticles
DLS and zeta potential was measured to estimate the nature of hydrodynamic size and the suspension stability. The R-TiO2 NPs formed a stable dispersion in aqueous suspension with no precipitation. The average hydrodynamic diameter was 550 nm (Fig. 4A) and the zeta potential at –20.8 mV with the conductivity of 0.00816 mS/cm (Fig. 4B).
6. Cytotoxicity of rutile phase titanium dioxide nanoparticles
The cell morphology of FaDu and YD-15 cells, observed at 24 and 48 hours after treating with 100 μg/mL of R-TiO2 NPs, showed no significant change (Fig. 5). Also cytotoxic effect of R-TiO2 NPs on oral cancer cells was not found in an MTT assay which was carried out by treating FaDu and YD-15 cells with various concentrations of R-TiO2 NPs. Both cell growth and proliferation were not affected by R-TiO2 NPs (Fig. 6).
Discussion
In this study, we prepared and characterized R-TiO2 NPs and evaluated its cytocompatibility on oral cancer cells. In the preparation of TiO2, the hydrolysis reaction of TiCl4 was kept at cold condition to avoid the formation of orthotitanic acid Ti(OH)4, which disturbs the homogenous precipitation of TiO2 [16]. Adjusting pH to 7 was an important factor for the formation of titanium dioxide hydrate, TiO(OH)2 or TiO2•nH2O [17,18]. The hydrated chemical structure of raw TiO2, shown at 3,600–3,100 cm –1 and 1,640–1,620 cm –1 in FT-IR bands, were disappeared after calcination at 900℃, due to the transformation into the chemically stable R-TiO2. Average size of R-TiO2 NPs was 76 nm with specific surface area of 19 m2/g, zeta potential of –20.8 mV and average hydrodynamic diameter of 550 nm in DMSO-H2O suspension. R-TiO2 NPs formed a stable dispersion in aqueous solution without precipitation suggesting that the negative zeta potential value of R-TiO2 NPs maintained the nanoparticles in suspension, not forming aggregation.
International agency for research on cancer (IARC) classifies TiO2 into the human carcinogen group 2B based on sufficient evidence in experimental animals and epidemiological studies. However, the effect of oral exposure to TiO2 NPs was debated by IARC and inconclusive due to the absence of standardized procedures for the risk assessment [19]. Cellular effects of TiO2 on toxicity and carcinogenicity were also unclear with conflicting results on different cell lines such as lung cells, nerve cells, cardiovascular cells, dermal and mucosal cells, and reproductive and renal cells [20]. The TiO2 of anatase phase has been reported to have high photocatalytic activity [3,5], supporting its anti-cancer or anti-bacterial activity. However, as far as authors know, there has been no systemic investigation on the toxic effects of R-TiO2 NPs on oral cells or tissues. Our results on cell growth and cell proliferation support that RTiO 2 NPs are not toxic to oral cancer cells of FaDu and YD-15, and also do not have anti-cancer activity, probably due to the low catalytic activity of the rutile TiO2 NPs. The negative zeta potential value of R-TiO2 NPs might also contribute to relieving the cytoxicity of R-TiO2 NPs since electrostatic repulsion between R-TiO2 NPs and negatively charged phospholipids on cell membrane make cells less sensitive to R-TiO2 NPs. Although we demonstrated that R-TiO2 NPs are cytocompatible to oral cancer cells, further investigation with normal oral cells would be necessary to convince that R-TiO2 NPs is safe and suitable for the application of aesthetic tooth pigmentation.
In conclusion, R-TiO2 NPs were prepared by the hydrolysis reaction of TiCl4 and calcination at 900℃. The composition of R-TiO2 NPs, surface functional group, particle size, shape, surface topography, and morphological behavior were analyzed. R-TiO2 NPs were cytocompatible to FaDu and YD-15 oral cancer cells with no cytotoxicity.
