The effect of synthesis conditions on the photokilling activity of TiO2 nanostructures

In this study, Titanium dioxide nanostructures were hydrothermally synthesized at different temperatures 130, 170 and 200 °C. The structural properties and crystallite size of TiO2 nanostructures were confirmed through XRD analyses. Moreover, the morphologies were confirmed using TEM analyses. These analyses confirmed the formation of single phase TiO2 nanostructures. The average crystallite size for the TiO2 nanostructures synthesized at different hydrothermal temperatures of 130, 170, and 200 °C was found to be 11.5 nm, 5.3 nm, and 5 nm, respectively. The impact of changes in these morphologies on the physical characteristics and photokilling activity of cancer cells has been studied. The results showed that the photokilling activity of TiO2 nanostructures is morphology dependent, with TiO2 nanowires having the highest activity and nanosheets having the lowest under our experimental conditions.


Introduction
TiO 2 nanostructures have excellent physical and chemical properties [1]. These properties make it a valuable candidate for a wide range of everyday applications [2]. TiO 2 nanostructures (i.e., nanoparticles, nanorods, nanowires, nanoribbons, etc,) have good photocatalytic properties and can be used as a photocatalyst in antiseptics, antibacterial compositions, and the degradation of organic contaminants and germs [3,4]. TiO 2 nanostructures are a nontoxic material, so they're widely used in paints, cosmetics, and environmental purification [5]. TiO 2 nanostructures are suitable for solar cell applications because of their wide band gap [2]. TiO 2 nanostructures generate reactive oxygen species (ROS) upon irradiation with ultraviolet (UV) light, which makes them a good candidate for photosensitization and radiosensitization applications [6,7]. Several parameters are used for the determination of TiO 2 nanostructures performance in a specific application, such as particle size, crystallinity, phase composition, morphology, and surface chemistry [8]. The change of the morphology plays an important role in the photocatalytic properties of nanostructures, as morphology modification can change the surface reactivity and surface area [9]. Recently, TiO 2 nanostructures with different morphologies have been investigated [10][11][12][13]. The large specific surface area of these morphologies offers a direct pathway for photo-induced electron transfer and improves the photocatalytic properties of TiO 2 nanostructures in different applications [14,15].
Different methods have been reported to synthesize TiO 2 nanostructures with different morphologies; the three main methods are the template-assisted method [16], electrochemical anodization method [17] and the hydrothermal method [18,19]. The hydrothermal method has the advantages of being low-cost and eco-friendly for large-scale production. It also offers access to uniform and different morphologies with excellent reliability, selectivity, and efficiency. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
To change the morphology of TiO 2 nanostructures hydrothermally, we need to control many parameters, including: 1-Starting material: the variety of titania powders such as, commercial, crystalline, amorphous, anatase, rutile, or brookite can produce different morphologies of TiO 2 nanostructures. For example, when the starting material has a relatively large particle size such as Degussa TiO 2 (P25) and rutile TiO 2 , the product is TiO 2 nanotubes with outer diameters of 10 and 20 nm [20,21]. When anatase 8 nm is used, the produced nanotubes have an average diameter in the range of several hundred nanometres [17].
2-Alkaline solution: the rate of titanite nanosheet crystallization is determined by the concentration of dissolved Ti +4 in a solution such as sodium hydroxide (NaOH) or potassium hydroxide (KOH). The reported concentration for hydrothermally prepared TiO 2 nanotubes is 10 M NaOH [16].
3-Pretreatment: the importance of the sonication process is to aid the migration of the OH − and Na + ions across cluster gaps of titania precursors, which accelerate the change of the nanosheet to different morphologies [18].
4-Hydrothermal temperature and time: the morphology and the crystallinity, as well as the length of TiO 2 nanostructures, were found to be affected by the hydrothermal temperature. The amount of TiO 2 nanostructures produced increases with increasing hydrothermal temperature [19]. For example, a nanotube with high purity and a large surface area was obtained at temperatures between 130 and 150°C, while with a temperature increment up to 180°C the main products were nanoribbons or nanowires like morphologies. The reaction time also has an observed effect on the morphology of the produced nanostructure; increasing the reaction time from 72 h to 1 week might cause the transformation of nanotubes into nanofibers or nanowires [18,19].

5-Acid washing:
The importance of acid washing is to remove the free Na + ions to convert Ti-Na to Ti-OH, which assists the transformation of nanosheets into different morphologies. The concentration of HCl destroys the nanotubular structures. These occur because Na + ions in the TiO 2 nanotube (TNT) decrease with the increase of HCl in the solution, and hydrogen displaces sodium [12,13] 6-Calcination alters the structure of nanostructures because it can advance the phase transformation of TiO 2 to an anatase phase, and it's also conductive to the elimination of Na + ions [13,22] In our work, we synthesized TiO 2 nanostructures with different morphologies by changing the hydrothermal temperature and we studied the impact of the change in these morphologies on the physical characteristics and the photokilling activity of TiO 2 nanostructures.

Synthesis of TiO 2 with different morphologies
For changing the morphology of TiO 2 nanostructures, we kept all parameters constant and changed the hydrothermal temperature to three different temperatures: 130, 170, and 200°C. The preparation procedure followed four steps, as shown in figure (1).
First step (Mixing): 2g of commercial TiO 2 was mixed with 80 ml of 10M NaOH under stirring for 10 min the mixture was then transferred to a Teflon lined autoclave and heated at 130, 170 and C°for 24 h.
Second step (Acid washing): the autoclave was allowed to cool naturally to room temperature. The obtained products were collected and washed with diluted HCl (pH 1.6) for 24 h.
Third step (Washing): The acid washed product was washed several times with distilled water until the pH arrived 7.
Fourth step (Annealing): The final product was annealed at 400°C for 2 h.

Characterization of TiO 2 nanostructures
X-ray diffraction (XRD) measurements were taken with an X'Pert PRO-PAN, Cu-K radiation (λ = 1.54056 a) at 40 kV and 30 mA. The prepared samples were examined by infrared spectral analysis by using the KBr disk technique. FTIR Model 6100, Jasco-Japan, has a resolution of 4.00 cm −1 and covers the wave number range of 4000-400 cm −1 . The external features and morphology of the samples were detected by a high-resolution transmission electron microscope (JEOL, JEM 2100, Japan). A UV-visible spectrophotometer (SPECORD).

Cell viability (trypan blue Exclusion test)
Trypan blue Exclusion tests were used to examine the viability of Caco2 cells after exposure to TiO 2 nanostructures. The Trypan Blue Exclusion test was done according to Tennant Protocol [21]. Caco2 cells were seeded in 24-well plates at 1 × 10 5 cells per well and incubated at 37°C with 5% CO 2 . After 24 h of incubation, the cells were washed with phosphate buffered saline (PBS) once and exposed to the three samples of TiO 2 nanostructures with different concentrations (1000, 500, 250, 125, 62.5 and 31.25 μM) and incubated at 37°C with 5% CO 2 . After 24 h of exposure, cells were collected and stained with 0.5% trypan blue (Biowest, MO, USA). Dead and live cells were counted using a hemocytometer (Funakoshi, Tokyo, Japan). Cell viability was calculated by dividing the number of viable cells by the total number of cells. All experiments were carried out in triplicate and all calculations of cell viability were normalized to their corresponding controls.

Result and discussion
3.1. X-ray diffraction (XRD) Figure (2) shows the XRD pattern results for single phase TiO 2 nanostructures (standard card (JCPDS No. 78-2486) [23][24][25][26]. Changing the hydrothermal temperature leads to different growth directions of the obtained TiO 2 nanostructures. As shown in figure 2, the optimum growth direction for samples prepared at 130°C and 170°C is (101), which is consistent with the standard growth direction for anatase TiO 2 nanostructures, whereas as the temperature increased to 200°C, the growth direction changed to (200). This result can be attributed to the change in the morphology of the obtained TiO 2 nanostructures [22,24]. The average crystallite size was found for the TiO 2 nanostructures synthesized at different hydrothermal temperatures (130, 170 and 200°C) to be 11.5 nm, 5.3 nm and 5 nm, respectively. The dislocation density of TiO 2 nanostructures (δ) represents the amount of crystallographic defect or irregularity within an anatase TiO 2 nanostructures and is defined as the number of dislocations per unit area. The dislocation density (δ) of the (111) preferential orientation for TiO 2 nanostructures was determined according to the following expression:  The Microstrain (ε) is one of the most important parameters used to characterize powder samples, which describes the change in the microstructure, size, and shape of the nanostructures and is given by: The calculated structural parameters are presented in table 1. The small values of δ and ε obtained in the present study confirm the good crystallinity of TiO 2 nanostructures synthesized by the hydrothermal route. Figure (3) represents the EDX of the prepared samples at different temperatures, reflecting the purity of the samples. The TiO 2 200 samples showed small traces of sodium ions, which may be the result from the washing step. Figure 4 shows that there are three bands in the samples. The first broad band between 3800 and 3000 cm −1 belongs to stretching hydroxyl (O-H), which represents water as moisture. The second peak was observed between 1626 and 1638 cm −1 , which belongs to the stretching of titanium carboxilate. The third peak between 800 and 450 cm −1 was assigned to the Ti-O stretching bands. The effect of hydrothermal temperature on the morphology of TiO 2 was observed clearly in the strong absorption between 800 and 450 cm −1 , as the temperature increased, the peak intensity increased, which was attributed to the formation of TiO 2 nanostructures [22].

High resolution transmission electron microscope (HR-TEM)
The morphology of samples was imaged using HR-TEM, as shown in figure (5). The formation of TiO 2 nanostructures with different morphologies was observed at the three hydrothermal temperatures, and its tubular structure is quite well preserved. The formed TiO 2 nanostructures at 170°C showed smaller tube diameters (width 5.622 ± 1.44 nm and length 217.828 ± 55.86 nm) and thinner tube walls than those prepared at 130°C (width 8.64 ± 1.43 nm and length 103.64 ± 35.98 nm). While TiO 2 nanostructures formed at 200°C have the largest tube diameters (width 26.28 ± 9.34 nm and length 225.52 ± 81.19 nm), which means that at  130°C TiO 2 nanorods have been formed, and with the increase of the temperature to 170°C like nanowires with a decrease in width and an increase in length were obtained, and at 200°C nanoribbons were formed with a larger width and length [25,26].

UV-Vis spectroscopy
The absorption spectra of TiO 2 nanostructures with different morphologies are given in figure 6. The influence of hydrothermal temperatures was proved by the rise in absorbance with increasing hydrothermal temperature. The direct band gap (E g ) of the TiO 2 nanostructures is calculated (24): The band gap of TiO 2 nanostructures was measured by plotting (αhν) 2 as a function of photon energy. The calculated band gap is 3.33 ev, 3.14 ev and 3.07 ev for TiO 2 130, TiO 2 170 and TiO 2 200 respectively, (figure 7). We found the energy gap (Eg) values of TiO 2 nanostructures in the range of 2.92 to 3.32 eV as reported in table 1. The change in E g values may be related to the quantum confinement effect of TiO 2 nanostructures, which could modify the ability of nanostructures to absorb more light. According to the literature, the change in the structure of nanostructures has a great effect on the optical band gap. The structural change can produce oxygen vacancies, which in turn can act as donor states within the band gap [24,25].

Photokilling activity of TiO 2 nanostructures
Under UV light irradiation, electrons (−) in nanostructures absorb enough energy to transport from the valence band to the conduction band, leaving behind a hole (+). These electron-hole pairs can migrate to the surface of nanostructures and interact with the reactants through reduction and oxidation mechanisms. The final product of this reaction is the formation of reactive oxygen species (ROS) such as singlet oxygen, hydroxyl, superoxide, and hydrogen peroxide. The photokilling activity of nanostructures is determined by the number and type of ROS [27,28]. The photocatalysis and photokilling activities of nanostructures have a direct relationship with size, shape, and surface reactivity. With the change of nanostructures morphology, the structural, electrical, and optical properties will be different. The optical band gap is considered a key parameter, and a good photocatalysis should have a suitable band gap (less than 3.0 eV) that facilitates the mobility of free charges between the conduction and valence bands as well as adopts the potential of the conduction and valence bands [26]. For TiO 2 nanostructures, the optical band gap is 3.2 and 3.0 eV for the anatase and rutile phases, respectively (27). To obtain efficient photokilling activity, the optical band gap should be tuned to be less than 3.0 eV. This can be achieved by modifying the structure through different strategies, including the synthesis conditions or depositing or incorporating metal ions or non-metal dopants into the TiO 2 .

Cell morphology
Normal Caco 2 cells are adherent cells that grow as a confluent monolayer 40 to 70 μm thick with a spindle or polygon shape [27]. Figure 8 represents some microscopic images of Caco2 cells under different conditions of culturing. The morphology of the control is clearly visible in the dark with its normal shape, whereas after 60 min of UV light irradiation, the cells have a slight shrinkage in shape. This indicates that cell apoptosis might start to occur due to UV irradiation [28][29][30][31].
After 24 h of exposure to TiO 2 nanostructures in the dark with low and high concentrations, there is no change in the morphology, which is due to the low toxicity of TiO 2 nanostructures in the dark [28]. When TiO 2 nanostructures were activated with UV light, cell adherence and growth density changed shrinking in the cell shape, contracting into a round shape, and some cellular debris were generated and increased with the increase of TiO 2 nanostructures concentrations. This indicates that the irradiation with UV light activates TiO 2 nanostructures and accelerates the apoptosis process [29].

The effect of concentration
The effect of TiO 2 nanostructures on the viability of Caco2 cells was examined by the Trypan Blue Exclusion Test. There is no effect of all TiO 2 nanostructures concentrations on the viability of CaCo2 cells in the dark (i.e., figure (9)).

The effect of irradiation time
With the increase in irradiation time, the viability of Caco2 cells decreased dramatically. Cell viability was found to decrease with increasing irradiation time at low concentrations (31.25 M), from approximately 91% for TiO 2 200 to 40% for TiO 2 170. TiO 2 170 possesses the highest photokilling effect while TiO 2 200 possesses the lowest photokilling effect, which is due to the change in the size and morphology of TiO 2 nanostructures, figure (10). Similar behavior was observed by Tong et al on bacterial cells. They concluded that the phototoxicity of TiO 2 nanostructures is morphology dependent and the phototoxicity of nanospheres > nanoroads > nanosheets > nanotubes [30]. It's reported that the increase of the surface area the rate of ROS production will increase. However, when dealing with non-spherical TiO 2 nanostructures, the relation between the ROS production is not the only parameter that drive the phototoxicity. There is a complex relationship between the photoactivity, rate of aggregation and interaction surface of nanostructures/cell or bacteria [30,31].
Moreover, the presence of sodium ions may be contributing as a recombination of centers of photogenerated electron-hole pairs. The EDX analysis reflected a trace amount of sodium in TiO 2 −200 sample, figure 3. Figure 11 illustrates that as the irradiation time increases, the cell viability decreases from approximately (92.1% for TiO 2 200) to (30% for TiO 2 170). Similar to the low concentration, TiO 2 170 possesses the highest photokilling effect, while TiO 2 200 possesses the lowest photokilling effect. According to the calculated optical band gap of the three samples, TiO 2 200 has the smallest value (3.07 eV), and highest surface area, and should have the largest photokilling activity. This behavior can be explained as the change of TiO 2 nanostructures morphology changing the surface area, which in turn affects the cellular uptake of nanostructures [32][33][34][35][36][37]. The relationship between ROS production and phototoxicity is not the only factor influencing it. There is a complex relationship between the photoactivity, rate of aggregation, and interaction surface of nanostructures/cell or bacteria. Due to diameter anisotropy, the alignment of TiO 2 nanostructures on the cell surface will differ for different shapes; in the case of nanotubes and nanowires, they preferred parallel alignment, which results in smaller center-to-center distances between nanotubes and is favored by stronger van der Walls attractions TiO 2 nanosheets with larger diameters, on the other hand, tend to overlap and stack each other, significantly reducing  their exposed surface area with cells and bacteria Moreover, with the higher concentration, the exposed surface area will be reduced due to the orientation of non-spherical TiO 2 nanostructures at different angles. These results demonstrate that the photokilling activity of TiO 2 nanostructures is dependent on the exposure time and the morphology of the nanostructures. Even though the effect of the concentration of nanostructures doesn't show up in the dark, it is clear that it does after being exposed to UV light. Figure 12 compares the cell viability of Caco2 cells after 24 h of exposure to TiO2 170 with the lowest concentration (31.25 μM) and the highest concentration (1 mM). With the increase in concentration, the cell viability decreases, which indicates that the toxicity of TiO2 is also dose dependent.

Conclusion
The TiO 2 nanostructures were successfully synthesized using a hydrothermal method at different hydrothermal temperatures (130, 170, and 200°C). The effect of hydrothermal temperature on the structure, morphology, optical properties, and photokilling activity of TiO 2 nanostructures has been discussed. With the increase in the hydrothermal temperature, the formed phase (anatase) did not change, but there was an increase and a decrease in some peak intensities, which was due to the change in the growth direction of TiO 2 nanostructures. The formation of TiO 2 nanostructures was observed at the three hydrothermal temperatures with HE-TEM, and its  tubular structure is quite good. nanostructures prepared at 170°C had smaller tube diameters and thinner tube walls than those prepared at 130°C, whereas TiO 2 nanosprepared at 200°C had larger tube diameters. The band gap decreased with the increase in the hydrothermal temperature. There is no effect of all TiO 2 nanostructures on the viability of CaCo2 cells in the dark, while after irradiation with UV, the photokilling activity of TiO 2 nanostructures was found to be dependent on the exposure time and the morphology. TiO 2 170 possesses the highest photokilling effect, while TiO 2 200 possesses the lowest photokilling effect in both high and low concentrations, which is due to the change in the size and morphology of TiO 2 nanostructures.

Data availability statement
The data that support the findings of this study are available upon reasonable request from the authors.