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{101}-{001} Surface Heterojunction-Enhanced Antibacterial Activity of Titanium Dioxide Nanocrystals Under Sunlight Irradiation Ning Liu, Yun Chang, Yanlin Feng, Yan Cheng, Xiujuan Sun, Hui Jian, Yuqing Feng, Xi Li, and Haiyuan Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16373 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 29, 2017

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ACS Applied Materials & Interfaces

{101}-{001} Surface Heterojunction-Enhanced Antibacterial Activity of Titanium Dioxide Nanocrystals Under Sunlight Irradiation

Ning Liu,1 Yun Chang,1, 2 Yanlin Feng,1, 3 Yan Cheng,1 Xiujuan Sun,1 Hui Jian,1 Yuqing Feng,4 Xi Li,4* and Haiyuan Zhang1, 2, 3 * 1

Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022 (China); 2University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China); 3University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026 (China); 4School of Chemistry and Life Science, Changchun University of Technology, 2055 Yan’an Street, Changchun, Jilin 130012 (China);

* Corresponding Author: Haiyuan Zhang, Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, 5625 Renmin Street, Changchun, Jilin, 130022 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026 (China); Email: [email protected]. Xi Li, Ph.D, School of Chemistry and Life Science, Changchun University of Technology, 2055 Yan’an Street, Changchun, Jilin 130012 (P.R. China); E-mail: [email protected]. Keywords: Titanium dioxide; Nanocrystals; ROS production; Antibacterial activity; Surface heterojunction. 1

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Abstract: The {101}-{001} surface heterojunction constructed on polyhedral titanium dioxide (TiO2) nanocrystals has recently been proposed to be favorable for the efficient electron-hole spatial separation due to the preferential accumulation of electron and hole on {101} and {001} facets, respectively. The formed free electron and hole can promote reactive oxygen species (ROS) production, which potentially can be used for inactivation of bacteria. In the present study, a series of truncated octahedral bipyramid TiO2 nanocrystals (T1, T2, T3 and T4) coexposed with {101} and {001} facets were prepared to form various ratios of {101} to {001} facet for optimization of electron-hole spatial separation efficiency. All these polyhedral TiO2 nanocrystals could more significantly produce ROS than spherical TiO2 nanocrystals (Ts), exhibiting the higher antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria under simulated sunlight irradiation. Among these polyhedral TiO2 nanocrystals, T3 with a {101}/{001} ratio of 1.78 was found to be the best one showing the highest ROS and the most potent antibacterial performance. Scanning electron microscope images of bacteria displayed that the surface membrane structure of both E. coli and S. aureus bacteria was influenced to different extents by these TiO2 nanocrystals, where T3 caused the most severe membrane damage. The molecular mechanism underlying the high antibacterial activity of TiO2 nanocrystals was ascribed to activation of oxidative stress as evidenced by intracellular ROS production, glutathione depletion and membrane lipid peroxidation in bacteria. The surface heterojunction as a completely new strategy holds great promise to develop effective antibacterial nanomaterials. 2


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1. INTRODUCTION Infectious diseases induced by bacteria have always been harmful to human health since the existence of human society.1,2 Many kinds of bacteria can lead to people’s serious illness and even death.3 Therefore, the design of efficient and eco-friendly antibacterial nanomaterials has attracted great attention.4 Photoactive semiconductor nanomaterials are effective and inexpensive materials to solve infections by bacteria.5,6 More importantly, the key advantages of semiconductor nanomaterials compared to organic antibacterial agents are their stability, robustness and long shelf life.7-9 Since the photo-based disinfection method by platinum-doped titanium dioxide (TiO2) mediated photocatalysis was first reported in 1985, TiO2 nanoparticles have attracted significant interest and used for self-cleaning antibacterial applications.10 With the activation of ultraviolet (UV) light irradiation, energy-rich electron and hole are formed on TiO2 nanoparticles, which can react with oxygen and water molecules to form superoxide and hydroxyl radicals, respectively. These reactive oxygen species (ROS) are strong oxidants and can cause oxidative damage to cell membranes and inactivate bacteria.11,12 However, the high recombination rate of photogenerated electrons and holes dramatically hamper its further applications. To this end, various methods have been applied to modify electronic property aiming to improve the electron-hole separation efficiency, such as doping TiO2 with noble metals, non-noble metal or semiconductors to improve the electron-hole separation efficiency.13,14 However, the antibacterial performance is not satisfactory, and it is of great concern that the release of dopants may cause adverse environmental and human health 3



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effects.15,16 Thus, it is necessary to develop a new strategy on TiO2 nanomaterials for their efficient electron-hole separations and ROS production. Crystallographic facet-constructed surface heterojunction is emerging as a new approach for spatial separation of photogenerated electrons and holes on TiO2 nanoparticles.17-20 This surface heterojunction is functionally similar with the known phase-heterojunctions built by two semiconductors with different energy band structures, and the electron-hole pair probably can be separated at the edge, however, the exact mechanism needs a deeper investigation. Since TiO2 {101} and {001} facets have staggered conduction band and valence band edges, the formation of {101}-{001} surface heterojunction can prompt photogenerated electron to preferentially transfer from {001} to {101} facets while the holes move in an opposite direction, leading to electron-hole spatial separation (Figure 1). This means that simultaneously exposing {101} and {001} facets on polyhedral TiO2 nanoparticle surface is beneficial for electron and hole accumulation on {101} and {001} facets, respectively, ultimately producing more ROS and offering stronger antibacterial activity than spherical TiO2 nanoparticles (Figure 1). Moreover, since electron-rich {101} and hole-rich {001} facets are responsible for superoxide and hydroxyl radical generation, respectively, an appropriate relative ratio of {101} to {001} facet is bound to play the key role in the cooperative processing of electron- and hole-based half reactions. Thus, the ratio of {101} to {001} facet probably can be optimized to maximize the electron-hole separation and radical production, beneficial for achieving a higher antibacterial activity even under natural sunlight exposure. 4


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In the present study, a series of truncated octahedral bipyramid TiO2 nanocrystals, composed of eight {101} facets on the sides and two {001} facets on the top and bottom truncation facets, were prepared to form {101}-{001} surface heterojunction and tune the percentages of {101} and {001} facets on TiO2 nanocrystals by a deliberate control of hydrogen fluoride concentration, aiming to achieve an optimal {101}/{001} ratio for efficient electron-hole separation, abundant ROS production and potent antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria under simulated sunlight radiation. The underlying mechanism was further clarified to intensely correlate to activation of oxidative stress. This study will provide a promising new strategy for development of effective antibacterial nanomaterials based on surface heterojunction construction.

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Figure 1. {101}-{001} surface heterojunction-promoted electron-hole spatial separation. The conduction band and valence band edges of {001} facet are higher than those of {101} facets, facilitating electrons to transfer from {001} to {101} facets and holes from {101} to {001} facets under sunlight irradiation. The {101}/{001} ratio can be adjusted to optimize the electron-hole separation efficiency, maximizing the electron and hole accumulation on {101} and {001} facets, respectively.

2. EXPERIMENTAL SECTION Materials. All chemicals were analytical grade and used without any further purification. Titanium (IV) butoxide was purchased from Sigma-Aldrich (St. Louis, MO). Water was purified from a Milli-Q water purification system (Millipore, Bedford, MA).

Synthesis of polyhedral and spherical TiO2 nanocrystals. For the synthesis of anatase TiO2 nanocrystals, titanium (IV) butoxide (TB) were used as a precursor. Typically, 2 mmol of titanium (IV) butoxide (TB) was added to a mixture of hydrogen fluoride (HF) and water (H2O), and the total molar amounts of HF and H2O were kept at 15 mmol. The HF/H2O ratios were set at 0.5, 1, 2, 3, and 4 to achieve spheres and four types of truncated octahedral bipyramid with decreased {101}/{001} facet ratios. The obtained mixture was stirred at ambient temperature for 30 min. Finally, the solvothermal precursor was transferred into a 25 mL Teflon-lined stainless steel autoclave and then held at 180 oC for 18 h. After the solvothermal crystallization, the prepared anatase TiO2 nanocrystals were thus obtained. The produced white 6


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precipitates were washed with ethanol and water, and then dried at room temperature. For all the TiO2 nanocrystals, 5 mg/mL TiO2 suspensions were prepared in Milli-Q water (Millipore, 18.2 MΩ·cm) as the stock solution, and 1 h-sonication in ultrasonic bath (Health-Sonics, 110 W, 42 kHz) was employed for the stock solution before diluting to the desired concentrations for a series of antibacterial assessments.

Physicochemical characterization. The surface morphology and primary sizes of as-prepared anatase TiO2 nanocrystals were analyzed by transmission electron microscopy (TEM, FEI Tecnai F20) and scanning electron microscopy (SEM, Hitachi S-4800-Ⅱ). The structure property of the samples was measured with X-ray diffraction (XRD) patterns which were recorded on a Bruker D8 Focus diffractometer with Cu Kα radiation and a Lynx Eye detector at a scanning rate of 0.02o min-1 over a range of 15−80°.

Abiotic ROS assessment. Total ROS was determined by rhodamine 123 (R123) fluorescence.21 80 µL of R123 (10 µM) in phosphate buffered saline (PBS, pH 7.4) was added to each well of 96 multiwell black plate (Costar, Corning, NY), followed by addition of 20 µL of 250 µg/mL nanocrystal suspension. The resulting solution was irradiated with simulated sunlight (Sun 2000 Solar Simulator, Abet Technologies) for 15 minutes at the density of 0.1 W/cm2, followed by 2 h incubation at room temperature. R123 fluorescence emission spectra excited at 485 nm were recorded from 505 nm to 600 nm using a SpectraMax M3 microplate reader.

Antibacterial assessment. Monocolonies of E. coli and S. aureus on solid Luria-Bertani (LB) agar plates were transferred to 20 mL of liquid LB broth 7



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containing (yeast extract 5 g, tryptone 10 g and NaCl 5 g/L at a pH of 7.0) and grown at 37oC for 12 h under 180 rpm rotation. The bacteria suspension was then diluted with broth to 1×106 colony forming units per mL (cfu/mL). In all experiments, the concentrations of bacteria were determined by measuring the optical density at 600 nm (OD600 nm). Typically, 50 µL of nanocrystal suspension in LB was mixed with 150 µL of bacteria suspension, leading to various concentrations of nanocrystals including 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 µg/mL. The mixed suspension was irradiated with simulated sunlight for 2 h, followed by incubation at 37 °C on a rotary platform at a 180 rpm for the desired time. Growth was monitored spectrophotometrically by periodic measuring of the absorbance at 600 nm. In addition, colony forming capability was evaluated on agar LB plates. 50 µL of 1.6 mg/mL of nanocrystals in LB was mixed with 150 µL of bacteria solution (1×106 cfu/mL), and the resulting suspension was irradiated with simulated sunlight for 2 h, and diluted 100-fold prior to spreading 100 µL on a LB agar plate. The number of the colonies was counted after agar plates were incubated at 37 °C for 24 h under simulated sunlight irradiation. All the above experiments were repeated three times and the average values were given.

Scanning electron microscopy (SEM) observation of bacteria. The morphological and structural properties of E. coli and S. aureus after exposure to TiO2 nanocrystals were analyzed by SEM.22 150 µL of bacteria suspension (1×106 cfu/mL) was mixed with 50 µL of 800 µg/mL TiO2 nanocrystal suspension. The resulting suspension was irradiated with 0.1 W/cm2 simulated sunlight for 2 h, followed by 24 h incubation at 37 °C on a rotary platform at a 180 rpm. After centrifugation, the bacteria was 8


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harvested and fixed on a silicon pellet with 2.5% glutaraldehyde solution at 4oC overnight. Then, the samples were sequentially dehydrated with 30, 50, 70, 90, and 100% ethanol for 20 min, respectively. Before the SEM examination the samples were coated with platinum in a sputter coater SCD050 (BAL-TEC).

Intracellular ROS assessment. Intracellular ROS production in the bacterial cells was determined based on green fluorescence emitted by 2’, 7’-dichloroflorofluorescin diacetate (DCFH-DA).23 150 µL of bacteria suspension (1×106 cfu/mL) was mixed with 50 µL of 800 µg/mL TiO2 nanocrystal suspension, and the resulting suspension was irradiated with 0.1 W/cm2 simulated sunlight for 2 h, followed by 24 h incubation at 37 oC at 180 rpm rotation. After treatment, bacteria suspension was washed and stained with 5 µM DCFH-DA for 20 minutes in the dark at room temperature. Green fluorescence-ROS producing bacteria were visualized by Nikon Ti-S fluorescence microscope (Tokyo, Japan) with 40×objective.

Glutathione (GSH) assessment. Assessment of the GSH content was carried out based on DTNB methods.24 GSH can react with DTNB (5,5’-dithiobis-2-nitrobenzoic acid) to yield a yellow colored 5-thio-2-nitrobenzoic acid (TNB) that absorbs at 414 nm. 150 µL of bacteria suspension (1×106 cfu/mL) was mixed with 50 µL of 800 µg/mL TiO2 nanocrystal suspension, and the resulting suspension was irradiated with 0.1 W/cm2 simulated sunlight for 2 h, followed by 24 h incubation at 37 oC at 180 rpm rotation. After treatment, the bacteria were washed with PBS three times, and lysed by 100 µL of lysis buffer (KeyGEN BioTECH). 30 µL of cell extracts were mixed with 150 µL of 30 µg/mL DTNB for 25 min, and the absorbance at 414 nm that 9



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is proportional to GSH levels was measured using a SpectraMax M3 microplate reader.

Lipid peroxidation assessment. The extent of lipid peroxidation of cell membrane was estimated by Lipid Peroxidation MDA Assay Kit (Beyotime, China). The principle of the assay was that during per-oxidation of lipids, the oxidation of polyunsaturated fatty acids produce malonaldehyde (MDA), which reacts with thiobarbituric acid (TBA) to produce a chromophore with absorption maximum at 532 nm.25 750 µL of bacteria suspension (1×106 cfu/mL) was mixed with 250 µL of 800 µg/mL TiO2 nanocrystal suspension, and the resulting suspension was irradiated with 0.1 W/cm2 simulated sunlight for 2 h, followed by 24 h incubation at 37 oC at 180 rpm rotation. The resulting suspension was centrifuged, and the cell pellet was suspended in 100 µL of SDBME buffer (4.5 mg Tris-HCl, 2.66 mg Tris base, 30 µL 10% SDS, 200 µL 1M DTT and 770 µL H2O) and heated for 5 min in a boiling water bath for cell lysis. An aliquot of 100 µL of cell extract was mixed with 200 µL of TBA-trichloroacetic acid (TCA)-hydrochloric acid (HCl) reagent (15% TCA and 0.375% TBA dissolved in 0.25 mol/L HCl) and heated for another 15 min in boiling water bath. After cooling, the flocculent precipitates were removed by centrifugation at 12000 rpm for 10 min, and then the absorbance of the supernatant was measured at 532 nm.

3. RESULTS AND DISCUSION Preparation and characterization of truncated octahedral bipyramid TiO2 10


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nanocrystals with various ratios of {101} to {001} facet. Anatase truncated octahedral bipyramid TiO2 nanocrystals were solvothermally synthesized in a mixed solution system consisting of titanium (IV) butoxide, water, and hydrofluoric acid (HF). The morphology could be precisely tailored to achieve different percentages of {101} or {001} facets by using different ratios of water (H2O)/hydrofluoric acid (HF). As a control, spherical TiO2 nanocrystals were also prepared following the same methods. Scanning electron microscopy (SEM) images and transmission electron microscopy (TEM) images (Figure 2) confirmed that with the increase of HF/H2O ratios, four types of truncated octahedral bipyramid nanocrystals (T1, T2, T3, and T4) were formed, which had similar side edge lengths of 51±6, 50±5, 53±10 nm and 51±3 nm for T1, T2, T3 and T4, respectively, but gradually shortened lateral edge lengths of 49±5, 41±9, 33±8 nm and 5±2 nm. The {001} facet percentages of T1, T2, T3 and T4 were 2% , 8%, 36% and 92%, respectively, while those of {101} were 98%, 92%, 64% and 8%, respectively. The {101}/{001} ratios were 49, 11.5, 1.78 and 0.087. The spherical TiO2 nanocrystals (Ts) had a primary size of 59±8 nm. All these TiO2 nanocrystals had similar X-ray diffraction (XRD) patterns (Supporting Information

Figure S1) that could be indexed to the anatase crystalline phase according to the standard card (JCPDS No. 21-1272). These nanocrystals could be well-dispersed in water, showing hydrodynamic sizes at the range of 217.5±12.3 to 268.7±16.6 nm (Supporting Information Figure S2).

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Figure 2. SEM and TEM images of spherical (Ts) and polyhedral (T1, T2, T3, and T4) TiO2 nanocrystals. The ratios of {101} to {001} facet on T1, T2, T3, and T4 were 49, 11.5, 1.78 and 0.087, respectively.

Reactive oxygen species (ROS) generation due to efficient electron-hole spatial separation on {101} and {001} facets. Each facet owns its own surface atomic arrangement, which can result in different electronic configuration from the other facet. So far, the surface band gap and surface energy band of facets have been extensively demonstrated based on theoretical calculation.17,26,27,28 It has also been experimentally verified that the oxidation site of anatase TiO2 is mainly on the {001} face and the reduction site is mainly on the {011} face.29,30 Neighboring {101} and {001} facets of truncated octahedral bipyramid TiO2 nanocrystals can form {101}-{001} surface heterojunction, which is functionally similar with the known phase-heterojunctions built by two semiconductors with different energy band structures. The staggered conduction band and valence band edges of {101} and {001} 12


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facets can prompt electron to transfer from {001} to {101} facet through surface heterojunction interface while hole move from {101} to {001} facets. This spatial separation ultimately leads to electron and hole accumulation on {101} and {001} facets, respectively, capable of efficiently producing abundant ROS. Rhodamine 123 (R123) is a useful fluorescent reagent to detect the ROS generation on the surface of nanomaterials. Fluorescence enhancement of R123 at 500 nm is proportional to ROS amounts on TiO2 nanocrystals. Under simulated sunlight irradiation at 0.1W/cm2 for 15 minutes, T1, T2, and T3 could progressively enhance the R123 fluorescence with decremental {101}/{001} ratios from 49 to 1.78 (Figure 3A), while T4 with the further decreased {101}/{001} ratio of 0.087 could not further enhance the fluorescence intensity, in contrast, T4 showed weaker fluorescence intensity than T3 and T2. Moreover, Ts as spherical nanocrystals only induced weak fluorescence enhancement. The ROS production trend could be depicted as T3>T2>T4>T1>Ts. However, without sunlight irradiation, all these TiO2 nanocrystals only generated weak fluorescence enhancements on R123 that was much lower than those under sunlight irradiation (Figure 3B). Obviously, truncated octahedral bipyramid TiO2 nanocrystals (T1-T4) with {101}-{001} surface heterojunction can produce much more pronounced ROS than spherical nanocrystals (Ts), and tuning the {101}/{001} facet ratio from T1 to T4 can significantly affect the ROS production because it potentially adjusts the electron-hole spatial separation efficiency. Theoretically, this ratio can be optimized to achieve the maximal separation efficiency. Actually, in the present study, T3 among the tested TiO2 nanocrystals was found to show the highest 13



ROS production, meaning the {101}/{001} facet ratio of 1.78 is the optimal one to promote electron-hole spatial separation and ROS production. Facet energy-based electron-hole separation are also reported for other materials, such as Cu2O microcrystals, where the valence band energies of Cu2O {100} and {110} facets are higher than that of Cu2O {111} facet, meaning photogenerated holes on {111} facets will transfer to {100} and {110} facets while the electrons on {100} and {110} facets can migrate to {111} facets.31 A

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Figure 3. R123 fluorescence emission spectra in the presence of Ts, T1, T2, T3, and T4 with (A) or without (B) simulated sunlight irradiation. The PBS solution containing 10 µM R123 and 50 µg/mL TiO2 nanocrystals was irradiated with 0.1 W/cm2 simulated sunlight for 15 min or kept in dark, and the emission spectra were recorded from 505 to 600 nm with excitation at 485 nm. The PBS only containing 10 µM R123 was used as control.

Antibacterial activity of TiO2 nanocrystals. ROS can damage a series of cell organelles and biomacromolecule, such as cell membrane, lipids, protein and nucleic acids.32 Nanomaterials with abundant ROS can result in inactivation of bacteria.33 To 14


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examine the antibacterial activity of TiO2 nanocrystals in terms of their ROS production under simulated sunlight irradiation, the 0-6 h growth kinetics of Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria were assessed after exposure to TiO2 nanocrystals in a Luria-Bertani (LB) culture medium. These nanocrystals could also be well dispersed in LB medium, showing homogeneous hydrodynamic sizes from 319.2±13.7 to 381.1±17.3 nm (Supporting Information Figure S3A) and similar zeta potentials from -16.7±0.36 to -18.5±0.37 mV (Supporting Information Figure S3B). Bacteria suspensions were incubated with 400 µg/mL TiO2 nanocrystals (T1-T4 and Ts) for 2 h under the irradiation with 0.1 W/cm2 simulated sunlight, and the following bacteria growth of E. coli and S. aureus were monitored over 6 h by measurement of the optical density of bacteria at 600 nm. Figure 4 shows all these TiO2 nanocrystals could significantly suppress the growth rates of both bacterial strains following a trend of T3>T2>T4>T1>Ts. This trend is in a close agreement with the trend toward ROS production (Figure 2A), showing the higher ROS production is associated with the stronger antibacterial performance. Since these TiO2 nanocrystals showed similar hydrodynamic sizes and negative charges, the potent and different antibacterial activity was not ascribed to sizes and charges.

E. coli was more susceptible to the antibacterial effects of these TiO2

nanocrystals than S. aureus, which is probably because of the different cell wall between E. coli and S. aureus. The cell wall of Gram-positive bacteria (S. aureus) are usually thick, consisting of a large amount of mucopeptides and surface components of lipoteichoic acids, while Gram-negative bacteria (E. coli) have a relatively thin cell 15



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wall.34 In comparison, without simulated sunlight irradiation, the bacteria growth rates were rarely influenced by these TiO2 nanocrystals (Figure S4A and S4B). Moreover, the bacterial viability was further evaluated after exposure to a range of concentrations (0-400 µg/mL) of TiO2 nanocrystals for 6 h. Figure 5 indicates that the bacterial viability was gradually reduced with the increasing concentration of TiO2 nanocrystals, and the similar inactivation effect was observed as T3>T2>T4>T1>Ts. Similarly, without simulated sunlight irradiation, no reduction in bacterial viability was observed in E. coli and S. aureus bacteria after exposure to these TiO2 nanocrystals (Figure S5A and S5B). Furthermore, the influence of light intensity on bacteria viability was also investigated. Bacteria suspensions were incubated with 400 µg/mL TiO2 nanocrystals (T1-T4 and Ts) under the irradiation with 0.025, 0.05, 0.1, and 0.2 W/cm2 simulated sunlight, respectively, and the bacteria viability was evaluated after 6 h exposure (Figure 6A and 6B). The results indicate that the light itself at the density of 0.025 to 0.2 W/cm2 could not affect the bacteria viability, but after exposure to TiO2 nanocrystals, the bacteria viability was significantly reduced in a dose-dependent manner. Under 0.025 W/cm2 sunlight irradiation, the bacteria viability was less influenced.

Obviously, the similar trends were observed in ROS

production and antibacterial performance of TiO2 nanocrystals, suggesting there exists a strong dependence of antibacterial performance towards ROS production.

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Figure 4. The 0-6 h growth curves of E. coli (A) and S. aureus (B) exposed to 400 µg/mL TiO2 nanocrystals under 0.1 W/cm2 simulated sunlight irradiation for 2 h. The bacteria growth was monitored by measuring the optical density at 600 nm.

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Figure 5. The bacterial viability of E. coli (A) and S. aureus (B) exposed to different concentrations (0-400 µg/mL) of TiO2 nanocrystals for 6 h under simulated sunlight irradiation at 0.1 W/cm2 for 2 h. The bacteria viability was determined by comparing the optical density (at 600 nm) of nanoparticle-treated bacteria with that of untreated bacteria.

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Figure 6. The bacterial viability of E. coli (A) and S. aureus (B) exposed to 400 µg/mL of TiO2 nanocrystals for 6 h under 2 h sunlight irradiation at the density of 0.025, 0.05, 0.1, and 0.2 W/cm2, respectively. The bacteria viability was determined by comparing the optical density (at 600 nm) of nanoparticle-treated bacteria with that of untreated bacteria.

Bacteria growth inhibition assessments were also conducted by observing the number of colony-forming units on LB agar plates. Figure 7 shows the formation of bacterial colonies treated with TiO2 nanocrystals (400 µg/mL) for 2 h under simulated sunlight irradiation at 0.1 W/cm2, followed by 24 h cultivation. Compared to the untreated control, TiO2 nanocrystal-treated bacteria showed the significantly reduced colony number, where T3 showed the most remarkable inhibition on the colony formation. As a control, sunlight alone could not cause the photolysis of bacterial cells. Taken all together, the antibacterial activity of Ts, T1, T2, T3 and T4 is proportional to their ROS production, where T3 shows the most potent antibacterial performance against both E. coli and S. aureus bacteria. This result is consistent to above antibacterial activity assessment on liquid LB medium, consolidating the intense relationship between ROS production and antibacterial activity of TiO2 nanocrystals. 18


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Figure 7. Optical images of bacterial colonies formed by E. coli (A) and S. aureus (B) treated or untreated with 400 µg/mL of various TiO2 nanocrystals under simulated sunlight irradiation. Bacterial suspensions (1×106 cfu/mL) were incubated with 400 µg/mL of TiO2 nanocrystals for 2 h under simulated sunlight irradiation, and 100-fold diluted suspension was spread on a LB agar plate, followed by 24 h incubation at 37 °C on a rotary platform at a 180 rpm. Untreated bacteria were used as control.

Morphological changes in bacteria induced by TiO2 nanocrystals. Interaction between TiO2 nanocrystals and bacteria was also investigated by the standpoint of morphological changes induced on bacteria upon exposure to TiO2 nanocrystals. The surface membrane structures of E. coli and S. aureus were closely examined by SEM following the treatment with 200 µg/mL Ts, T1, T2, T3, and T4 for 2 h under 0.1 W/cm2 sunlight irradiation and another 6 h cultivation (Figure 8). Untreated E. coli bacteria were 1-2 µm long rods with a rough appearance of the surface, distinctive for the Gram-negative bacteria (Figure 8A). Compared with these, the morphology of the bacteria exposed to TiO2 nanocrystals was significantly changed, with a heavily damaged, even ruptured cell surface and a collapsed structure of the cell, where T3 caused the most significant damage on the membrane (Figure 8A). In the case of S. 19



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aureus, untreated bacteria were spheres, 300-500 nm in diameter, with relatively smooth surface characteristic for the peptidoglycanic cell wall of Gram-positive bacteria (Figure 8B). After exposure to TiO2 nanocrystals, S. aureus bacteria showed wrinkled and punctured cell walls occasionally in the inhibition zone, and T3 still showed the most potent adverse influence (Figure 8B). Moreover, accumulated nanocrystals on membrane could be clearly discerned in SEM images, and the accumulation was evident in both bacterial strains.

Figure 8. SEM images of E. coli (A) and S. aureus (B) treated or untreated with various TiO2 nanocrytals under simulated sunlight irradiation. Bacterial suspensions (1×106 cfu/mL) were incubated with 200 µg/mL of TiO2 nanocrystals for 2 h under simulated sunlight irradiation, followed by 24 h incubation at 37 °C on a rotary platform at a 180 rpm. Untreated bacteria were used as control.

Molecular mechanism underlying the antibacterial activity of TiO2 nanocrystals. The close relationship between ROS production and antibacterial activity suggests that abiotic ROS production on nanocrystals could give rise to induction of cellular ROS generation in bacteria, and the molecular mechanism underlying the antibacterial activity of TiO2 nanocrystals is probably related to activation of oxidative stress. To 20


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identify the existence of ROS inside the bacteria, 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) is usually used as a visual indicator of the overall oxidative status of biologic cell.15 DCFH-DA can cross the cell membrane into the cell and be hydrolyzed by intracellular esterase to form non-fluorescent DCFH. In the presence of intracellular ROS, DCFH can be oxidized to highly fluorescent dichlorofluorescein (DCF). Therefore, the ROS concentration in the cell is directly proportional to the fluorescent intensity of DCF. Figure 9A illustrates TiO2 nanocrystals (T1, T2, T3, T4 and Ts) could significantly enhance DCF fluorescence in both E. coli and S. aureus bacteria under simulated sunlight irradiation, meaning the increased intracellular ROS level is triggered by these nanocrystals. T3 induced the highest DCF fluorescence enhancement, indicative of the strongest ROS production in T3-treated bacteria. Moreover, with the decrease of particle doses (Figure S6), DCF fluorescence was gradually decreased. At 25 µg/mL, TiO2 nanocrystal–induced DCF fluorescence was not noticeable. When ROS production exceeds the capacity of the antioxidant defense and the redox equilibrium fails to be restored, the higher levels of oxidative stress can trigger GSH depletion, resulting in cellular toxicity. The GSH levels in bacteria were assessed by the GSH-Glo assay. Six hour exposure of both bacterial strains (E. coli and S. aureus) to 200 µg/mL TiO2 nanocrystals (Ts, T1, T2, T3, and T4) under simulated sunlight irradiation could induce the remarkable decline in cellular GSH levels, proportional to their ROS generation (Figure 9B and 9C). Cellular GSH depletion showed a dose-dependent manner to particle doses from 200 to 50 µg/mL, and at 25 µg/mL, 21



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particles could not induce obvious decline in GSH levels compared to that of untreated bacteria. Furthermore, lipid peroxidation, as another signature of ROS damage, can be detected by assaying for malondialdehyde (MDA) that is an oxidized product of polyunsaturated fatty acids. MDA forms an adduct with thiobarbituric acid (TBA), resulting in a pink product with increased absorbance at 532 nm.20 The MDA assessment was carried out to determine the effect of TiO2 nanocrystals on membrane lipid peroxidation under simulated sunlight irradiation. Figure 9D and 9E show that 200 µg/mL TiO2 nanocrystals could significantly increase the concentration of MDA in both E. coli and S. aureus bacteria, and T3 was still the most potent one. The decreased particle doses could induce less MDA generation. At 25 µg/mL, the MDA level was not significantly increased by any particle. Taken all together, intracellular ROS production, GSH depletion and lipid peroxidation arise from TiO2 nanocrystal-treated bacteria, suggesting the bacterial defense system is overwhelmed by TiO2 nanocrystal-induced ROS and the oxidative stress is stimulated. The antibacterial activity of TiO2 nanocrystals could be ascribed to activation of oxidative stress signaling pathway.

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Figure 9. Oxidative stress responses of E. coli and S. aureus bacteria treated or untreated with various TiO2 nanocrytals under simulated sunlight irradiation. (A) Fluorescence images of DCF-stained bacteria showing intracellular ROS production; Cellular GSH levels in E. coli (B) or S. aureus (C) determined by the GSH-Glo assay; Lipid peroxidation assessment of E. coli (D) or S. aureus (E) based on a MDA method. Bacterial suspensions (1×106 cfu/mL) were incubated with 200 µg/mL of TiO2 nanocrystals for 2 h under simulated sunlight irradiation, followed by 6 h incubation at 37 °C on a rotary platform at a 180 rpm.

4. CONCLUSIONS 23



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Polyhedral TiO2 nanocrystals (T1, T2, T3 and T4) coexposed with {101} and {001} facets could produce more ROS than spherical TiO2 nanocrystals (Ts), exhibiting the more potent antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria under simulated sunlight irradiation. The ROS production and antibacterial performance of polyhedral TiO2 nanocrystals could be optimized through tuning the ratio of {101} to {001} facet, where T3 with a ratio of 1.78 was found capable of showing the highest ROS production and the strongest antibacterial activity among these TiO2 nanocrystals. The potent antibacterial activity was ascribed to activation of oxidative stress, which was supported by intracellular ROS production, glutathione depletion and membrane lipid peroxidation in bacteria. These results imply that TiO2 nanocrystals with an optimal ratio of {101} to {001} facet have the potential to be formulated as new antibacterial nanomaterials.

ACKNOWLEDGEMENTS: This work was primarily supported by National Natural Science Foundation of China (21573216, 21501170), Hundred Talent Program of Chinese Academy of Sciences, Science and Technology Development Project Foundation of Jilin Province (20160101304JC, 20160520134JH).

Supporting Information: Detailed information regarding XRD of TiO2 nanocrystals, hydrodynamic sizes and zeta potentials in water and LB, and bacterial growth curves and viability assessments of E. coli and S. aureus under dark condition are provided in the Supporting Information. This material is available free of charge via the Internet at 24


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http://pubs.acs.org.

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