Crystallographic Facet-Induced Toxicological Responses by Faceted

Publication Date (Web): May 13, 2016. Copyright © 2016 American ... Citation data is made available by participants in Crossref's Cited-by Linking se...
0 downloads 0 Views 5MB Size
Crystallographic Facet-Induced Toxicological Responses by Faceted Titanium Dioxide Nanocrystals Ning Liu,† Kai Li,‡ Xi Li,§ Yun Chang,†,∥ Yanlin Feng,†,∥ Xiujuan Sun,† Yan Cheng,† Zhijian Wu,*,‡,∥ and Haiyuan Zhang*,†,∥ †

Laboratory of Chemical Biology and ‡State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, China § School of Chemistry and Life Science, Changchun University of Technology, 2055 Yan’an Street, Changchun, Jilin 130012, China ∥ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Toxicological responses of nanomaterials have been closely correlated to their physicochemical properties, and establishment of a property−activity relationship of nanomaterials is favorable for a deep understanding of the nanomaterials’ toxicity mechanism, prospectively predicting nanomaterials’ potential hazards and rationally designing safer nanomaterials. Faceted nanomaterials usually exhibit more versatile and effective performance than spherical nanomaterials due to their selectively exposed crystallographic facets with high densities of unsaturated atoms. These facets have high surface reactivity, capable of eliciting strong interactions with biological systems. Few studies paid attention to the toxic behaviors of faceted nanomaterials in terms of their distinctive facets. In the present study, the toxicological role of the crystallographic facets of TiO2 nanomaterials was investigated, and the precise property−activity relationship was exploited to clearly understand the toxicity of faceted nanomaterials. A series of faceted TiO2 nanocrystals with the morphology of truncated octahedral bipyramids were prepared to expose different percentages of {101} and {001} facets on the surface. Density functional theory calculation revealed that {101} facets could only molecularly absorb water molecules while {001} facets due to their surfaceunsaturated Ti atoms could dissociate the absorbed water molecules to generate hydroxyl radicals. Biophysical assessments corroborated the increased production of hydroxyl radicals on the {001} facets compared to {101} facets, which endowed {001} facets with strong hemolytic activity and elicited severe toxicities. A series of increased oxidative stress toxicological responses, including cellular ROS production, heme oxygenase-1 expression, cellular GSH depletion, and mitochondrial dysfunctions, were triggered by faceted TiO2 nanocrystals with progressively increased {001} percentages, demonstrating the toxicological roles of {001} facets. KEYWORDS: TiO2 nanocrystals, facets, oxidative stress, surface reactivity, toxicity ENMs.14−17 In the past decade, a series of physicochemical properties of ENMs, such as chemical composition, sizes, shapes, crytallinities, charges, aggregation, and dissolution, etc., have been specialized to correlate with their induced hazardous responses, and the corresponding property−activity relationships were established to understand the toxicity mechanism of nanomaterials.6,18 However, with the rapid development of

A

breakthrough application of engineered nanomaterials (ENMs) in new consumer products will eventually increase the possibility of ENMs coming into contact with humans and other living organisms, giving rise to potential biological injuries.1−4 Understanding the interaction between nanomaterials and biological systems thus is becoming vitally important because such interactions could be biologically inert or beneficial for biological function but could also pose a biological hazard to humans.5−9 Establishment of the property−activity relationship of ENMs at the nano-bio interface would provide deep understanding of the toxicities, prospective toxicity prediction,10−13 and the safe design of © 2016 American Chemical Society

Received: March 8, 2016 Accepted: May 13, 2016 Published: May 13, 2016 6062

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

www.acsnano.org

Article

ACS Nano Scheme 1. Tuning Facet Percentages in TiO2 Nanocrystals

Figure 1. Physicochemical properties of spherical and faceted TiO2 nanocrystals. (A) TEM images. (B) SEM images. (C) XRD spectra. (D) Primary sizes. (E) Specific surface area. (F) Hydrodynamic sizes. (g) ζ-Potentials in water.

about the potential hazards of faceted nanomaterials, especially the clarification of the exact toxicological role of the specific facets and their property−activity relationships, which usually needs a well-designed facet-dependent nanomaterial library to systematically tailor facet properties and to precisely capture their induced toxicological responses. Traditional titanium dioxide (TiO2) nanocrystals have been produced abundantly and applied widely because of their high stability and their anticorrosive and photocatalytic properties.30 Their nontoxicity further allows them to be safely applied in consumer products, such as cosmetics, sunscreen, toothpaste, skim milk, and prosthetic implants.31 However, with the emergence of faceted TiO2 nanocrystals, reactive facets (such as {001} facets) are usually designed to be more exposed on the surface of nanocrystals than inert facets (such as {101} facet) to increase the performance of various properties.32 These reactive facets are predominantly occupied by unsaturated Ti atoms, such as two-fold, four-fold, and five-fold coordinated Ti atoms, which usually exhibit high chemical reactivities, while inert facets have abundant saturated six-fold coordinated Ti atoms, showing low reactivities.33,34 In the present study, the toxic behaviors of {101} and {001} facets of TiO2 nanocrystals were

ENMs, the toxicology knowledge usually cannot keep pace with the development of sophisticated nanomaterials that are designed and endowed with novel physicochemical properties. These properties were never encountered in previous nanomaterials and will eventually challenge the current understanding of the property−activity relationship. To bridge this knowledge gap, new property−activity relationships related to these novel physicochemical properties need to be built to understand their toxicity behaviors. Faceted nanomaterials19−21 have recently been designed to expose well-defined crystallographic facets to increase their performance in catalytic,22,23 optical,24,25 magnetic,26 and electrochemical properties.27,28 The active facets usually have higher densities of unsaturated, dangling, or distorted atoms than the inert facets,29 resulting in higher surface reactivity. When nanomaterials come into contact with the biological surfaces, such as cell membranes, endosomal compartments, and organelles,5 the high surface reactivity could elicit strong interactions between nanomaterial surfaces and biological membranes, leading to severe cellular toxicological responses. Thus, the reactive facets of faceted nanomaterials potentially can affect human health; however, few reports are concerned 6063

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

Figure 2. Prediction and assessment of reactive oxygen species generation on TiO2 nanocrystals. (A) Different behaviors of water adsorption on {101} and {001} facets as calculated by DFT. The binding energy (Eb) is defined as follows: Eb = Eadsorbate/slab − (Eslab + Eadsorbate), where Eadsorbate/slab is the total energy of adsorbate and TiO2 surface, Eslab is the total energy of the TiO2 surface after H2O was adsorbed, and Eadsorbate is the total energy of adsorbed H2O. (B) DCF fluorescence emission spectra. The spectra were recorded from 500 to 600 nm (λex = 490 nm). (C) APF fluorescence emission spectra. The spectra were recorded from 480 to 600 nm (λex = 455 nm). (D) XTT absorbance spectra. The X/ XO system was used as a positive control. The absorbance spectra were recorded from 410 to 550 nm. (E) ESR spectra of DMPO−OH in aqueous solution containing 0.1 mol/L DMPO.

(TEM) images (Figure 1A) and scanning electron microscopy (SEM) images (Figure 1B) confirmed that the low ratio of HF/ H2O facilitated the formation of spherical nanocrystals (SN) with a diameter of 62 ± 10 nm. High-resolution TEM indicated that the surface of the SN was predominantly occupied by the stable {101} lattice (Supporting Information Figure S1). With the increase of HF/H2O ratios, three types of truncated octahedral bipyramid nanocrystals (FN1, FN2, and FN3) with two {001} facets and eight {101} facets were formed (Figure 1A,B and Supporting Information Figure S2), which had similar side edge lengths of 51 ± 8, 50 ± 6, and 52 ± 10 nm for FN1, FN2, and FN3, respectively, but gradually shortened lateral edge lengths of 48 ± 6, 42 ± 8, and 35 ± 10 nm. The {001} facet percentages of FN1, FN2, and FN3 were 2, 8, and 36%, respectively, while those of {101} were 98, 92, and 64%, respectively. All of these TiO2 nanocrystals show similar X-ray diffraction (XRD) patterns (Figure 1C) that could be indexed to the anatase crystalline phase according to the standard card (JCPDS No. 21-1272). The primary sizes of these nanocrystals

systematically investigated. A series of TiO2 nanocrystals with spherical and truncated octahedral bipyramid morphologies, comprising eight {101} facets on the sides and two {001} facets on the top and bottom truncation facets, were prepared to display different {101} or {001} percentages (Scheme 1); their potential toxicities, toxicity mechanisms, and property−activity relationships were systematically investigated, and the toxicological roles of well-defined crystallographic facets of TiO2 nanocrystals were identified.

RESULTS AND DISCUSSION Preparation of TiO2 Nanocrystals with Different Percentages of {101} and {001} Facets. TiO2 nanocrystals were synthesized through solvothermal methods using titanium(IV) butoxide as precursors in aqueous solution,35 and the morphology could be precisely tailored from a sphere to truncated octahedral bipyramids with different {101} or {001} facet percentages by using different ratios of hydrofluoric acid (HF)/water (H2O). Transmission electron microscopy 6064

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

Figure 3. Hemolytic activities of TiO2 nanocrystals in terms of mass and surface area dose metrics. Mouse RBCs were exposed to nanocrystals at 12.5−200 μg/mL for 2 h, and the absorbance of the released hemoglobin from cells was recorded at 541 nm. (A) Hemolytic activities according to the mass dose metrics of nanocrystals. Inset photograph shows that the hemoglobin appears red in the supernatant of RBCs incubated with 50 μg/mL nanocrystals. Mass doses shown in (A) were converted to {001}, total, and {101} surface area, as shown in (B−D), respectively; *p < 0.05, compared to the untreated cells.

potential of 2.81 eV that is capable of widely devastating the biological system.40−42 Thus, the surface HO• radicals can endow facets with active surface reactivity, and the large production of surface HO• radicals can result in high surface reactivity. In the present study, water molecule adsorption on {101} and {001} surfaces of the anatase TiO2 supercell (2 × 2 × 1) was investigated by density functional theory (DFT) using the Vienna ab initio simulation package (VASP).43,44 To better simulate the biological system, both effects of solvation and van der Waals interaction45,46 that were neglected in the previous study under the gas environment were considered in the present study.37 The calculation details are shown in the Materials and Methods section. The results indicated that the {101} surface had five-fold (Ti5c), four-fold (Ti4c), and two-fold (Ti2c) unsaturated Ti atoms, where only Ti4c and Ti2c were reachable atoms with densities of ∼2 atoms/nm2 and Ti5c was unreachable due to the space hindrance; the {001} surface was homogeneously covered by reachable Ti5c atoms with an atom density of ∼7 atoms/nm2. Water molecules were found able to be molecularly absorbed on the {101} surface with binding energies of −2.59 and −3.07 eV for Ti4c and Ti2c, while they showed nearly dissociative adsorption on the {001} surface with a much higher binding energy of −7.15 eV for Ti5c, which could dramatically distort water molecules to form HO• radicals (Figure 2A and Supporting Information Figure S4). Moreover, the higher Ti5c density (∼7/nm2) on the {001} surface compared to those of Ti2c and Ti4c (∼2/nm2) on {101} surfaces further facilitated the formation of HO• radicals. The fluorescent dye, 2′,7′-dichlorofluorescein (DCF), is a useful reagent to detect the generation of reactive oxygen species (ROS), such as HO• and superoxide radicals (O2•−), on the surface of nanoparticles in aqueous solution.47 The ROS generation on TiO2 nanocrystals was examined by fluorescence

based on the diameters (for spheres) or side edge lengths (for truncated octahedral bipyramids) are displayed in Figure 1D. The total specific surface area of these nanocrystals were determined by Brunauer−Emmett−Teller (BET) measurements ranging from 27.6 ± 2.6 to 29.4 ± 3.3 m2/g (Figure 1E). These nanocrystals could be well-dispersed in water, resulting in hydrodynamic sizes from 198.6 ± 12.9 to 250.4 ± 19.6 nm (Figure 1F). ζ-Potential measurements indicated that all of these nanocrystals had weak positive charges ranging from 2.6 ± 0.6 to 4.3 ± 0.6 mV (Figure 1G). Moreover, band gap energies of these nanocrystals determined by the diffuse reflectance UV−vis spectroscopy showed the same value of 3.15 eV (Supporting Information Figure S3), demonstrating their similar electronic properties. All of the above characterization data reveal that the achieved spherical and faceted nanocrystals had homologous crystalline structures, primary sizes, total specific surface area, hydrodynamic sizes, and surface charges, meaning that these TiO2 nanocrystals cannot induce significant differences in biological responses in terms of these physicochemical properties. However, the significantly different {101} or {001} facet percentages in these nanocrystals can potentially induce different toxicological responses due to their potentially different surface reactivities. Moreover, the distinctive shapes of these nanocrystals are potential factors to induce different toxicological responses. Hydroxyl Radical Generation on {001} Facets. Water molecules are the predominant species in biological systems. It has been proposed that water molecules can be molecularly or dissociatively absorbed on TiO2 surfaces depending on their lattice plane types,36,37 and the dissociatively absorbed water can result in the formation of surface hydroxyl radicals (HO•),38,39 which are very deleterious to cell organelles and tissues in biological systems because of their high oxidizing 6065

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

Figure 4. Cytotoxicity of TiO2 nanocrystals in terms of mass and surface area dose metrics. BEAS-2B cells were exposed to 0.4−200 μg/mL of nanocrystals for 24 h. (A) Cell viabilities assessed by MTS according to mass doses. Mass doses shown in (A) were converted to {001}, total, and {101} surface area as shown in (B−D), respectively; *p < 0.05, compared to the untreated cells.

Surface Reactivity of {101} and {001} Facets. The HO• radicals are highly reactive species able to abstract hydrogen from nearly any C−H bond and, therefore, to react with a vast range of biomacromolecules, such as lipids, proteins, and nucleic acids.42 The surface HO• on {001} facets offers TiO2 nanocrystals with high surface reactivity to interact with cell membranes when nanocrystals are in contact with cells, leading to potential lipid peroxidation and cell membrane disruption.42 The surface reactivity of nanomaterials is usually evaluated by their potential hemolytic activities on red blood cells (RBCs).51,52 Previous studies have reported that abiotic ROS on MIN-U-Sil and fumed silica particles can trigger RBC hemolysis.49,53 Mouse RBCs were exposed to 12.5−200 μg/mL of TiO2 nanocrystal suspensions in phosphate buffer saline (PBS) for 2 h, and the hemoglobin release due to membrane disruption was quantitatively determined by the absorbance at 541 nm. Figure 3A shows that FN1, FN2, and FN3 resulted in extensive dose-dependent hemoglobin releases, and the gradually increased {001} percentages in FN1, FN2, and FN3 caused the progressively increased hemoglobin release. However, SN with a homogeneous {101} lattice on the surface resulted in negligible hemoglobin release, demonstrating their weak surface reactivity. Since all four TiO2 nanocrystals showed similar and weak surface charges, their hemolytic activity could exclude the effect of surface charge. Since the whole synthesis of TiO2 nanocrystals did not include any organic molecule, it is impossible for these TiO2 nanocrystals to have active organic groups on their surface, which could exclude the mechanical interaction between the surface group of the TiO2 nanocrystal and RBC membrane. So, the hemolytic activity of faceted TiO2 nanocrystals should be ascribed to abiotic ROS. Considering the importance of {001} facets, the dose metrics of nanocrystals in Figure 3A were converted from mass to {001} surface area. The result showed that the dose−response relationship could be depicted using a well fitted curve with an R2 value of 0.98 (Figure 3B), which clearly demonstrates that the hemoglobin

enhancement of DCF at 520 nm. SN only generated a weak fluorescence enhancement on DCF, while FN1, FN2, and FN3 could induce significant and progressive fluorescence enhancements with incremental {001} percentages in these faceted nanocrystals (Figure 2B), meaning the ROS generation closely correlates to {001} facet properties. Although DCF assessments confirm the ROS generation on {001} facets, this assay is a general radical assay and cannot identify the radical type. To further consolidate the HO• generation on {001} facets as suggested by DFT calculation, another two dyes were used to distinguish between HO• and O2•− production on the nanocrystal surfaces. Abiotic HO• radicals can be specifically detected by 3′-(p-aminophenyl) fluorescein (APF),48,49 which has a maximum emission at 515 nm, while O2•− can be detected by 3-bis(2-methoxy-4-nitro-5-sulfophehyl)-2H-tetrazolium-5-carboxanilide) (XTT),49,50 which has an absorbance maximum at 465 nm. APF assays showed a trend similar to that shown in DCF assays where SN could weakly increase APF fluorescence and FN1, FN2, and FN3 significantly increased the fluorescence intensity, which was progressive with incremental {001} percentages (Figure 2C). XTT assays showed that there was no distinct enhancement in XTT absorbance for all these TiO2 nanocrystals; however, xanthine/ xanthine oxidase, a well-established O2•−-generating system, did induce XTT absorbance (Figure 2D). Use of these additional dyes clarifies that the generated ROS on faceted TiO2 nanocrystals was due to HO• rather than O2•− radicals. The electron spin resonance (ESR) spectra further confirmed that the HO• generation of typical 1:2:2:1 quartet characteristics of DMPO−HO• was observed in FN1, FN2, and FN3; their intensities were progressively increased with the increase of {001} percentages, and there was no response in SN (Figure 2E). All of these results demonstrate that {001} facets of TiO2 nanocrystals have higher potency to generate HO• radicals than {101} facets. 6066

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

Figure 5. Hierarchical oxidative stress and toxicological responses of BEAS-2B cells to spherical and faceted TiO2 nanocrystals. (A) Cellular ROS production assessed by the ROS-Glo assay. Cells were exposed to 50, 100, and 200 μg/mL TiO2 nanocrystals (SN, FN1, FN2, and FN3) for 6 h. ROS production was expressed as fold increase. (B) Nrf2 and HO-1 expression in the cells exposed to 50 μg/mL TiO2 nanocrystals for 6 h using Western blot analysis. (C) Cellular GSH levels determined by the GSH-Glo assay. Cells were exposed to 50, 100, and 200 μg/mL TiO2 nanocrystals (SN, FN1, FN2, and FN3) for 6 h. GSH levels are depicted as a percentage of untreated cells (100%); *p < 0.05, compared to untreated cells. (D) SOD activity. Cells were exposed to 50, 100, and 200 μg/mL TiO2 nanocrystals (SN, FN1, FN2, and FN3) for 6 h. The SOD activity was determined by the WST-8 method; *p < 0.05, compared to untreated cells. (E) CAT activity. Cells were exposed to 50, 100, and 200 μg/mL TiO2 nanocrystals (SN, FN1, FN2, and FN3) for 6 h. CAT activity was based on the decomposition of H2O2; *p < 0.05, compared to untreated cells. (F) Fluorescence images of cells displaying the highest toxicological responses in hierarchical oxidative stress responses. Cells were exposed to 50 μg/mL TiO2 nanocrystals for 6 h, and nuclei were stained with Hoechst 33342 (blue). Mitochondrial superoxide production, mitochondrial membrane depolarization, intracellular calcium flux, and plasma membrane rupture were detected by MitoSox Red (red), JC-1 (green), Fluo-4 (green), and PI (red). 6067

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

exposed to nanocrystals was evaluated by the ROS-Glo assay. Figure 5A shows that the increased ROS production displayed in FN1-, FN2-, and FN3-treated cells was dose-dependent and proportional to their {001} percentages. In contrast, SN did not change the ROS level. Obviously, there exists a consistent relationship between abiotic hydroxyl radicals and cellular ROS with the increase of {001} percentages. Herein, it is necessary to indicate that abiotic and biotic ROS were generated through different mechanisms. The abiotic ROS were generated through dissociation of water molecules on {001} facets of TiO2 nanocrystals, while the biotic radicals were generated through the disruption of cellular redox homeostasis because abiotic ROS can react with a series of biomolecules to destroy the cellular redox homeostasis, leading to cellular ROS generation. According to the hierarchical oxidative stress paradigm,3 the increased cellular ROS can induce multiple oxidative stress responses, with the first tier characterized by NF-E2-related factor-2 (Nrf2)-mediated phase II enzyme expression. Heme oxygenase-1 (HO-1) as a typical phase II enzyme and its induction is associated with the protection against ROS. Western blotting is used in the present study to assess Nrf2 and HO-1 expression, demonstrating increased Nrf2 and HO-1 abundance in cells exposed to 50 μg/mL nanocrystals for 6 h (Figure 5B). Importantly, the levels of Nrf2 and HO-1 expression were proportional to the {001} percentages of FN1, FN2, and FN3. The phase II enzyme expression could maintain the redox homeostasis; however, 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. Cellular GSH levels were assessed by the GSHGlo assay. Six hour exposure to faceted nanocrystals at 50, 100, and 200 μg/mL could induce a remarkable and dose-dependent decline in cellular GSH levels, proportional to their {001} percentages (Figure 5C). In contrast, exposure to SN did not reduce the GSH level. Further assessments of cellular superoxide dismutase (SOD) and catalase (CAT) activities also supported the activation of oxidative stress (Figure 5D,E), where FN3 triggered the strongest enzyme activities, followed by FN2, FN1, and SN. Cellular ROS production as well as Nrf2 and HO-1 expression increased SOD and CAT activity, and GSH depletion can activate a series of toxicological responses as results of cell death, including intracellular calcium flux, mitochondrial superoxide production, mitochondrial membrane depolarization, and plasma membrane rupture, which can be specifically detected by fluorescent dyes, Fluo-4, MitoSox Red, JC-1, and propidium iodide (PI). Fluorescence microscopy was employed to capture the fluorescent signals of toxicological responses in BEAS-2B cells treated with 50 μg/ mL spherical or faceted TiO2 nanocrystals for 24 h. Figure 5F shows that increased {001} percentages in FN1, FN2, and FN3 could trigger increased mitochondrial membrane depolarization, mitochondrial superoxide production, intracellular calcium flux, and plasma membrane leakage, while SN without a {001} facet did not exert any activation of the above toxicological responses. All of the above toxicological studies indicate that faceted TiO2 nanocrystals can cause toxicities through the oxidative stress mechanism, and {001} facets play a critical role in the escalation of oxidative stress levels. Activation of oxidative stress is very dangerous to human health, which can cause pulmonary, cardiovascular, neurodegeneration, and renal diseases.

release is closely correlated to the {001} facet property. In comparison, the hemolytic activities could not be fitted according to the doses of total specific surface area and {101} surface area (Figure 3C,D). Hemolysis assessments reveal that {001} facets of TiO2 nanocrystals have great potential to disrupt the cell membrane, exhibiting strong surface reactivity, while their {101} facets do not contribute to this activity. Toxicity Behaviors of {101} and {001} Facets. The strong surface reactivity of nanomaterials, as contributed by surface active groups,51 surface lattice defects,11 surface charges,52 and surface roughness,54 has been verified to be capable of eliciting toxicological responses at the nano-bio interface and affecting human health.6,55,56 It is necessary to investigate the potential toxicities of faceted TiO2 nanocrystals in terms of their reactive facets. Considering that inhalation is a primary route of entry for nanomaterials, normal human bronchial epithelial (BEAS-2B) cells and murine alveolar macrophages (RAW 264.7) were used as representatives of a lung target for nanocrystals to investigate the lung toxicities of faceted TiO2 nanocrystals.51,57 All of the nanocrystals could be well-dispersed in culture medium (BEGM for BEAS-2B cells and DMEM for RAW 264.7 cells), and their hydrodynamic sizes and ζ-potentials in both media are shown in Supporting Information Figure S5. After 24 h exposure to TiO 2 nanocrystals at a wide range of doses (0.4−200 μg/mL), cell viability measurement by the MTS assay showed that while SN with a homogeneous {101} lattice on the surface did not affect cell viability, faceted nanocrystals with incremental {001} facet percentages did result in increased toxicities (Figure 4A for BEAS-2B and Supporting Information Figure S6A for RAW 264.7 cells). The critical role of the {001} facet in toxicity could also be more clearly clarified in Figure 4B and Supporting Information Figure S6B, which converted the mass doses in Figure 4A and Supporting Information Figure S6A to {001} surface area doses, showing well-fitted curves with R2 values of 0.94 for BEAS-2B cells and 0.92 for RAW 264.7 cells according to the relationship of cell viability/{001} surface area. In comparison, the cell viabilities could not be fitted according to the doses of total specific surface area and {101} surface area (Figure 4C,D for BEAS-2B cells and Supporting Information Figure S6C,D for RAW 264.7 cells). These results are similar to those of the hemolytic activity, demonstrating the toxic roles of {001} facets of TiO2 nanocrystals in the activation of severe toxicities. Toxicological Mechanism of {001} Facets. Nanomaterials can cause toxicity through multiple different toxicological mechanisms depending on different physicochemical properties. In the present study, the toxicities of faceted TiO2 nanocrystals showed a close correlation with the surface reactivity of {001} facets, where the surface hydroxyl radicals exerted potent effects in the damage of cell organelles and disruption of cellular redox homeostasis, leading to direct and indirect cellular ROS production. It should be pointed out that the abiotic hydroxyl radicals could remain active even under cellular condition because the enhanced DCF fluorescence by faceted TiO2 nanocrystals was also observed in culture medium (BEGM and DMEM, Supporting Information Figure S7), which contains biological additives or serum. The oxidative stress toxicological mechanism was probably involved in the toxicity process of faceted TiO2 nanocrystals. The toxicological mechanism of TiO2 nanocrystals was further investigated in BEAS-2B cells. Cellular ROS production in BEAS-2B cells 6068

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

Figure 6. ICP-OES analysis for cellular Ti contents in BEAS-2B and RAW 264.7 cells. Cells were exposed to 6.25 μg/mL nanocrystals for 24 h, and untreated cells were used as a control. The cellular Ti contents were determined by ICP-OES.

Other Factors To Potentially Trigger Different Toxicity of TiO2 Nanocrystals. Besides {001}-facet-mediated surface reactivity, the possibilities of other physicochemical properties of faceted TiO2 nanocrystals triggering different toxicities were also considered. Since all of these TiO2 nanocrystals have homologous crystalline structures, primary sizes, total specific surface area, hydrodynamic sizes, and surface charges, the different toxicities cannot be attributed to these properties. However, the different shapes of these nanomaterials are potentially important factors affecting the interaction of nanomaterials and cells. Nanomaterials with significantly different aspect ratios have been reported showing different toxicities, which are mainly attributed to the different cellular uptake though cytoskeleton formation, and adhesion and migration are also influenced weakly.58,59 However, few studies focus on the cellular uptake profiles of faceted nanomaterials. In the present study, the cellular uptake of spherical and faceted TiO2 nanocrystals was investigated in BEAS-2B and RAW 264.7 cells, and the cellular Ti content was identified by ICPOES. After cells were exposed to 6.25 μg/mL spherical or faceted TiO2 nanocrystals for 24 h, 2.1−2.76 ng Ti/μg protein was determined in BEAS-2B cells (Figure 6A) and 0.88−1.15 ng Ti/μg protein in RAW 264.7 cells (Figure 6B). The cellular uptake is fairly uniform for all of the nanocrystals, which can rule out the influence of shapes on toxicities. To further exclude the specific effects of faceted TiO2 nanocrystals by their size, shape, or charge, another two TiO2 nanocrystals, LFN1 and LFN2, were prepared (see Supporting Information), which also had similar side edge lengths (106 ± 20.5 and 112.5 ± 22.5 nm, respectively) but significantly different lateral edge lengths (68.5 ± 13.8 and 49.6 ± 10.2 nm, respectively) (as shown in Supporting Information Figure S8A). The primary sizes of LFN1 and LFN2 were larger than FN1−FN3, and LFN2 showed a {001} surface area larger than that of LFN1. Both LFN1 and LFN2 had similar and weak surface charges (3.24 ± 0.46 and 2.83 ± 0.38 mV, respectively) in water. The cell viability assessment indicated that LFN2 exhibited stronger toxicity than LFN1 in both BEAS-2B cells and RAW 264.7 cells (as shown in Figure S8B,C). This result is consistent with the previous toxicity result achieved in FN1− FN3, demonstrating it is a {001} facet rather than size, shape, or charge that induces the severe toxicity of faceted TiO2 nanocrystals. Dissolution is also an important property for metal-based nanomaterials that cause toxicity (e.g., ZnO and CuO),57 and the different levels of metal ions released from nanomaterials

can lead to the different toxicities. The Ti dissolution of spherical or faceted TiO2 nanocrystals was investigated in cell culture medium using ICP-OES after 24 h incubation at 37 °C. Unfortunately, the Ti element in the supernatant of all of these TiO2 nanocrystals was undetectable, which is consistent with the previous report that TiO2 nanomaterials are indissolvable.12 These dissolution studies further rule out the possibility of toxicity induced by nanocrystal dissolution. Moreover, the surface defect is another potential factor that causes severe toxicity of faceted TiO2 nanocrystals. Since the high catalytic activity of faceted TiO2 nanocrystals was found in 2008 by Yang et al.,33 the facet-dependent activity due to the unique atomic arrangement of facets has been commonly accepted.60,61 However, some literature has reported that the high catalytic activity of faceted TiO2 nanocrystals was probably due to the formation of surface defects that were induced by HF treatment during the synthesis.62,63 Although the most recent study has clarified that even under the HF-free synthetic conditions the achieved faceted TiO2 nanocrystals still maintain high catalytic activity and the unique atomic arrangement of the {001} facet is still the major reason for high catalytic activity,64 there exists some possibility that faceted TiO2 nanocrystals cause the toxicity through the surface defect approach because these defects can facilitate the formation of reactive oxygen species and result in cell death. It will be necessary to perform a detailed study to clarify the role of surface defects in the toxicity of faceted TiO2 nanocrystals.

CONCLUSION Faceted TiO2 nanocrystals with the morphology of truncated octahedral bipyramids were prepared to investigate their potential toxicities, toxicity mechanisms, and property relationships in terms of the specific {101} and {001} facets. The {001} facet compared to {101} facet generates surface hydroxyl radicals easier due to the dissociative absorption of water molecules, leading to higher surface reactivity. Faceted TiO2 nanocrystals with high {001} percentages could significantly reduce cell viabilities and induce a series of toxicological responses through on the oxidative stress mechanism. MATERIALS AND METHODS Materials. All chemicals were reagent grade and purchased from Sigma-Aldrich Co. unless otherwise indicated. Water was purified from a Milli-Q water purification system (Millipore, Bedford, MA). Titanium(IV) butoxide was purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. 6069

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano

microplate reader. APF fluorescence emission spectra excited at 455 nm were recorded from 480 to 600 nm, while XTT absorbance spectra were collected from 410 to 550 nm. Hemolysis Assay. One milliliter of fresh mouse blood was mixed with 5 mL of PBS, and the resulting mixture was centrifuged at 1000 rpm for 5 min to isolate red blood cells. Achieved RBCs were resuspended in 10 mL of PBS for future use. As a negative control, 300 μL of the RBC suspension was mixed with 1200 μL of PBS, while as a positive control, 300 μL of the RBC suspension was mixed with 1200 μL of PBS containing 0.025% Triton X-100. TiO2 nanocrystal suspensions in PBS were mixed with a RBC suspension to provide a particle suspension from 12.5 to 200 μg/mL. After incubation at room temperature for 2 h, the mixture was centrifuged and the released hemoglobin in the supernatants was determined by the absorbance at 541 nm in a SpectraMax M3 microplate spectrophotometer. The percent hemolysis in each sample was calculated by dividing the difference in absorption between the sample and the negative control by the difference in absorption between the positive and negative controls, then multiplying this ratio by 100 to obtain % hemolysis. Cell Culture. Human bronchial epithelial (BEAS-2B) cells, purchased from ATCC, were cultured in bronchial epithelial basal medium, supplemented with growth factors from the SingleQuot kit to reconstitute BEGM. Mouse macrophage cell lines (RAW 264.7), purchased from ATCC, were cultured in DMEM medium containing 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. The media were changed every 2 days, and the cells were passaged by trypsinization before confluence. In either case, cells were grown to approximately 75% confluency before beginning the experiments. Cytotoxicity Assessment. MTS assays were carried out to assess the cytotoxicity of TiO2 nanocrystals. In a typical procedure, 1 × 104 BEAS-2B or RAW 264.7 cells were seeded in 96-well plates for overnight growth. Then, the medium was removed, and 100 μL of TiO2 nanocrystal suspension (0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg/mL) was added to each well of a 96-well plate. After 24 h treatment, the medium containing nanocrystals was removed, and cells were treated with 100 μL of culture medium containing 16.7% of MTS stock solution for 1 h at 37 °C in a humidified 5% CO2 incubator. The plate was centrifuged in Xiangyi L535R with a microplate rotor, and 80 μL of the supernatant was transferred from each well to a new well for measurement of the absorbance at 490 nm by a SpectraMax M3 microplate reader. Cellular ROS and GSH Assessment. Cellular ROS and GSH levels were determined by ROS-Glo and GSH-Glo assay kits, respectively, according to manufacturer’s instruction. One hundred microliters of cell suspensions (1 × 105 cells/mL) was seeded in a 96multiwell white plate and allowed to attach for overnight growth. The medium was removed, and cells were exposed to 100 μL of TiO2 nanocrystal suspension (50, 100, and 200 μg/mL) for the indicated time period. For cellular ROS assessments, after 4 h exposure, 20 μL of ROS-Glo H2O2 substrate (Promega Corporation) was added to each well, followed by 2 h incubation at 37 °C in a humidified 5% CO2 atmosphere. The reaction was stopped by adding 100 μL of ROS-Glo detection solution, and the luminescence intensity proportional to ROS amounts was determined after 20 min by a SpectraMax M3 microplate reader. For cellular GSH assessments, after 6 h exposure, the medium was removed, and 100 μL of GSH-Glo reagent was added to each well, followed by 30 min incubation at room temperature. The reaction was stopped by adding 100 μL of GSH-Glo detection solution, and the luminescence intensity proportional to GSH amounts was determined after 15 min by a SpectraMax M3 microplate reader. Western Blot Analysis for Nrf2 and HO-1 Expression. Proteins were separated by electrophoresis in a 8−16% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride Immobilion P membrane (Millipore Corp., USA). Then, the membranes were blocked for 1 h at room temperature in Trisbuffered saline containing 0.1% v/v Tween-20 (TBS/T) buffer and 5% nonfat dry milk, followed by 2 h incubation with anti-human Nrf2 or HO-1 monoclonal antibody (dilution 1:500) (ENZO Life Sciences,

Synthesis of Spherical and Faceted TiO2 Nanocrystals. The synthesis of anatase TiO2 nanocrystals was accomplished using a solvothermal method. Typically, 5 mmol of titanium(IV) butoxide was added to a mixture of HF and H2O, and the total molar amounts of HF and H2O were kept at 30 mmol. The HF/H2O ratios were set at 0.5, 1, 2, and 3 to achieve spheres and three types of truncated octahedral bipyramids with increased {001}/{101} facet ratios. The obtained mixture was stirred for 30 min before being transferred into a 100 mL Teflon-lined stainless steel autoclave. The system was then heated at 180 °C for 18 h. The produced white precipitates were washed with ethanol and water and then dried at room temperature. For all of 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 an ultrasonic bath (Health-Sonics, 110 W, 42 kHz) was employed for the stock solution before being diluted to the desired concentrations for a series of abiotic and biotic assessments. Physicochemical Characterization of TiO2 Nanocrystals. The physicochemical properties of achieved TiO2 nanocrystals were characterized by multiple standard techniques. The primary sizes and morphological features were determined by TEM (FEI Tecnai F20) and SEM (Hitachi S-4800-II). The BET method based on N2 sorption isotherms was used to determine the specific surface area by using a Micrometrics ASAP 2020 sorption instrument. X-ray diffraction patterns were recorded on a Bruker D8 Focus diffractometer with Cu Kα radiation and a Lynx Eye detector at a scanning rate of 0.02° min−1 over a range of 15−80°. A Malvern Nanosizer ZS was used to obtain ζ-potential and dynamic light scattering data of nanocrystals (50 μg/mL) dispersed in DI water. Electron spin resonance spectra were measured using a Bruker A300 EPR electron paramagnetic resonance spectrometer. The settings were the center field at 3480.00 G, with a microwave frequency at 9.83 GHz and power at 6.35 mW. The band gap energy of nanocrystals was determined by UV−vis diffuse reflectance spectroscopy using a UV− vis spectrophotometer (Cary 500, Varian Co.). Density Functional Theory Calculation. The calculations were carried out using density functional theory with the Perdew−Burke− Ernzerhof form of the generalized gradient approximation functional. The Vienna ab initio simulation package was employed. The plane wave energy cutoff was set to 400 eV. The Fermi scheme was employed for electron occupancy with an energy smearing of 0.1 eV. The first Brillouin zone was sampled by a 2 × 2 × 1 k-point mesh in the Monkhorst−Pack grid. The energy (converged to 1.0 × 10−6 eV/ atom) and force (converged to 0.01 eV/Å) were set as the convergence criterion for geometry optimization. To describe the configuration more accurately, a semiempirical DFT-D2 force-field approach including the van der Waals interaction was employed in this work. Since the magnetic atom existed, the spin polarization was considered in all calculations. The {101} and {001} surfaces were obtained by cutting the anatase TiO2 supercell (2 × 2 × 1) (the supercell structure is shown in Supporting Information Figure S4) along {101} and {001}, respectively. The {101} and {001} slabs contain 20 and 16 TiO2 units, respectively, while the structures are shown in Supporting Information Figure S4, respectively. A vacuum layer as large as 10 Å was used along c directions to avoid periodic interactions. Abiotic Assessment of Total ROS, HO•, and O2•− Generation. Total ROS, HO•, and O2•− were determined by DCF fluorescence, APF fluorescence, and XTT absorbance. The DCF stock solution containing 50 μg of H2DCFDA, 17.3 μL of ethanol, and 692 μL of a 0.01 mol/L sodium hydroxide solution was incubated at room temperature for 30 min, followed by addition of 3500 μL of phosphate buffer saline (10 mmol/L, pH 7.4) to form a final DCF working solution. The APF and XTT working solution were prepared in PBS solution at 10 and 100 μmol/L, respectively. Eighty microliters of each working solution was added to each well of a 96-multiwell black plate (Costar, Corning, NY), followed by addition of 20 μL of a 1 mg/mL nanocrystal suspension. The resulting solution was incubated at room temperature for 2 h. DCF fluorescence emission spectra excited at 490 nm were recorded from 500 to 600 nm using a SpectraMax M3 6070

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano Plymouth Meeting, PA, USA) in TBS/T buffer containing 2% nonfat dry milk at room temperature. Membranes were then washed three times with TBS/T and incubated for 2 h with horseradish peroxidase conjugated secondary antibody (dilution 1:1000) (Santa Cruz, CA, USA). After being washed, membranes were developed using an enhanced SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, USA). Superoxide Dismutase and Catalase Activity Assessments. Cells were cultured in 6-well plates at a density of 1.6 × 105 cells/well and allowed to attach for 16 h. After removal of medium, cells were exposed to 100 μL of nanocrystals (50, 100, and 200 μg/mL) for 6 h. After being washed, cells were harvested by scraping. The SOD activity of cells was determined by the WST-8-based SOD assay kit according to the manufacturer’s instructions (Beyotime, Nanjing, China).65 Briefly, 20 μL of cell supernatants was mixed with 200 μL of WST-8 working solution, and the reaction was initiated by the addition of 20 μL of enzyme working solution. Water-soluble WST-8 formazan was determined spectrophotometrically at 450 nm. One unit of SOD enzyme activity is defined as the amount of enzyme required for inhibiting the chromogen production by 50% in 1 min under assay conditions. The CAT activity of cells was estimated by their ability to split hydrogen peroxide (H2O2) using a CAT assay kit according to the manufacturer’s instructions (Beyotime, Nanjing, China).66 The reaction was then stopped by adding dichromate/acetic acid reagent, and the remaining H2O2 was determined by measuring chromic acetate at 570 nm, which is formed by reduction of dichromate/acetic acid in the presence of H2O2. One unit of CAT activity is defined as 1 μmol H2O2 decomposed/min. ICP-OES Analysis To Determine the Abundance of Cellular Uptake and Metal Dissolution of Nanocrystals. ICP-OES measurement was carried out to investigate the cellular uptake of spherical and faceted TiO2 nanocrystals by determining the Ti content in both BEAS-2B cells and RAW 264.7 cells. Cells were plated in 6well plates at a density of 1.6 × 105 cells/well and allowed to attach for 16 h. Then, 1.6 mL of 6.25 μg/mL nanocrystals was added to each well of the 6-well plates, followed by 6 h incubation. After being washed, the harvested cells through trypsinization were well digested with nitric acid for ICP-OES measurements. ICP-OES measurement was also implemented to investigate the Ti dissolution of different TiO2 nanocrystals in culture medium. Five hundred microliters of 200 μg/mL TiO2 nanocrystal suspension in culture medium was incubated at 37 °C for 24 h. The supernatant collected through centrifugation at 20 000 rpm for 30 min was further digested with nitric acid for ICP-OES measurements. Fluorescence Microscopy To Study the Hierarchical Oxidative Stress Responses. BEAS-2B cells (4 × 104) in 400 μL of culture medium were plated in each well of an 8-well chamber slide (NUNC Lab-Tek) for overnight growth at 37 °C in a humidified 5% CO2 atmosphere. After the removal of medium, cells were exposed to 50 μg/mL nanocrystals for 6 h. After being washed with PBS, cells were stained with 5 μM MitoSox Red, JC-1, Fluo-4, or propidium iodide. Cell nuclei were stained with 1 μM Hoechst 33342. After being washed, cells were fixed in 400 μL of PBS containing 4% paraformaldehyde for 2 h at room temperature. Cells were washed with PBS three times and visualized using an Olympus BX-51 optical system microscope (Tokyo, Japan) with a 40× objective. Statistical Analysis. Quantitative data were presented as the mean ± standard deviation. Statistical significance was evaluated using twotailed heteroscedastic Student’s t tests according to the TTEST function in Microsoft Excel.

TiO2 nanocrystals in culture medium, and cytotoxicity data in RAW 264.7 cell lines (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was primarily supported by National Natural Science Foundation of China (21573216, 21501170, and 21503210), Hundred Talents Program of Chinese Academy of Sciences, Science and Technology Development Project Foundation of Jilin Province (20160101304JC, 20160520134JH), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, the Start-up fund from Changchun Institute of Applied Chemistry, CAS, and Talent Development fund of Jilin, China. REFERENCES (1) Service, R. F. American Chemical Society meeting. Nanomaterials Show Signs of Toxicity. Science 2003, 300, 243. (2) Brumfiel, G. Nanotechnology: A Little Knowledge. Nature 2003, 424, 246−248. (3) Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic Potential of Materials at the Nanolevel. Science 2006, 311, 622−627. (4) Oberdorster, G.; Oberdorster, E.; Oberdorster, J. Nanotoxicology: An Emerging Discipline Evolving From Studies of Ultrafine Particles. Environ. Health Perspect. 2005, 113, 823−839. (5) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the Nano-Bio Interface. Nat. Mater. 2009, 8, 543−557. (6) Zhu, M. T.; Nie, G. J.; Meng, H.; Xia, T.; Nel, A.; Zhao, Y. L. Physicochemical Properties Determine Nanomaterial Cellular Uptake, Transport, and Fate. Acc. Chem. Res. 2013, 46, 622−631. (7) Donaldson, K.; Poland, C. A. Nanotoxicology: New Insights Into Nanotubes. Nat. Nanotechnol. 2009, 4, 708−710. (8) Bhabra, G.; Sood, A.; Fisher, B.; Cartwright, L.; Saunders, M.; Evans, W. H.; Surprenant, A.; Lopez-Castejon, G.; Mann, S.; Davis, S. A.; Hails, L. A.; Ingham, E.; Verkade, P.; Lane, J.; Heesom, K.; Newson, R.; Case, C. P. Nanoparticles Can Cause DNA Damage Across a Cellular Barrier. Nat. Nanotechnol. 2009, 4, 876−883. (9) Jiang, J.; Oberdorster, G.; Elder, A.; Gelein, R.; Mercer, P.; Biswas, P. Does Nanoparticle Activity Depend Upon Size and Crystal Phase? Nanotoxicology 2008, 2, 33−42. (10) Cohen, Y.; Rallo, R.; Liu, R.; Liu, H. H. In Silico Analysis of Nanomaterials Hazard and Risk. Acc. Chem. Res. 2013, 46, 802−812. (11) George, S.; Lin, S. J.; Ji, Z. X.; Thomas, C. R.; Li, L. J.; Mecklenburg, M.; Meng, H.; Wang, X.; Zhang, H. Y.; Xia, T.; Hohman, J. N.; Lin, S.; Zink, J. I.; Weiss, P. S.; Nel, A. E. Surface Defects on Plate-Shaped Silver Nanoparticles Contribute to Its Hazard Potential in a Fish Gill Cell Line and Zebrafish Embryos. ACS Nano 2012, 6, 3745−3759. (12) Zhang, H.; Ji, Z.; Xia, T.; Meng, H.; Low-Kam, C.; Liu, R.; Pokhrel, S.; Lin, S.; Wang, X.; Liao, Y. P.; Wang, M.; Li, L.; Rallo, R.; Damoiseaux, R.; Telesca, D.; Madler, L.; Cohen, Y.; Zink, J. I.; Nel, A. E. Use of Metal Oxide Nanoparticle Band Gap to Develop a Predictive Paradigm for Oxidative Stress and Acute Pulmonary Inflammation. ACS Nano 2012, 6, 4349−4368. (13) Puzyn, T.; Rasulev, B.; Gajewicz, A.; Hu, X. K.; Dasari, T. P.; Michalkova, A.; Hwang, H. M.; Toropov, A.; Leszczynska, D.; Leszczynski, J. Using Nano-QSAR to Predict the Cytotoxicity of Metal Oxide Nanoparticles. Nat. Nanotechnol. 2011, 6, 175−178.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b01657. Detailed information including high-resolution TEM of {101} and {001} facets, band gap energy measurements of TiO2 nanocrystals, DFT calculation of {101} and {001} facets, hydrodynamic sizes and ζ-potentials of 6071

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano (14) Lynch, I.; Weiss, C.; Valsami-Jones, E. A Strategy for Grouping f of Nanomaterials Based on Key Physico-Chemical Descriptors As a Basis for Safer-By-Design NMs. Nano Today 2014, 9, 266−270. (15) Sealy, C. ’Safe-By-Design’ Nanoparticles Show Reduced Toxicity. Nano Today 2011, 6, 113−114. (16) Campagnolo, L.; Massimiani, M.; Magrini, A.; Camaioni, A.; Pietroiusti, A. Physico-Chemical Properties Mediating Reproductive and Developmental Toxicity of Engineered Nanomaterials. Curr. Med. Chem. 2012, 19, 4488−4494. (17) Fubini, B.; Ghiazza, M.; Fenoglio, I. Physico-Chemical Features of Engineered Nanoparticles Relevant to Their Toxicity. Nanotoxicology 2010, 4, 347−363. (18) Nel, A.; Xia, T.; Meng, H.; Wang, X.; Lin, S.; Ji, Z.; Zhang, H. Nanomaterial Toxicity Testing in the 21st Century: Use of a Predictive Toxicological Approach and High-Throughput Screening. Acc. Chem. Res. 2013, 46, 607−621. (19) Zhou, K. B.; Li, Y. D. Catalysis Based on Nanocrystals with Well-Defined Facets. Angew. Chem., Int. Ed. 2012, 51, 602−613. (20) Wu, B. H.; Zheng, N. F. Surface and Interface Control of Noble Metal Nanocrystals for Catalytic and Electrocatalytic Applications. Nano Today 2013, 8, 168−197. (21) Kuang, Q.; Wang, X.; Jiang, Z. Y.; Xie, Z. X.; Zheng, L. S. HighEnergy-Surface Engineered Metal Oxide Micro- and Nanocrystallites and Their Applications. Acc. Chem. Res. 2014, 47, 308−318. (22) Han, X. G.; Jin, M. S.; Xie, S. F.; Kuang, Q.; Jiang, Z. Y.; Jiang, Y. Q.; Xie, Z. X.; Zheng, L. S. Synthesis of Tin Dioxide Octahedral Nanoparticles with Exposed High-Energy {221} Facets and Enhanced Gas-Sensing Properties. Angew. Chem., Int. Ed. 2009, 48, 9180−9183. (23) Hu, L.; Peng, Q.; Li, Y. Selective Synthesis of Co3O4 Nanocrystal with Different Shape and Crystal Plane Effect on Catalytic Property for Methane Combustion. J. Am. Chem. Soc. 2008, 130, 16136−16137. (24) Wang, L.; Li, H. B.; Xu, S. L.; Yue, Q. L.; Liu, J. F. FacetDependent Optical Properties of Nanostructured ZnO. Mater. Chem. Phys. 2014, 147, 1134−1139. (25) Huang, M. H.; Rej, S.; Chiu, C. Y. Facet-Dependent Optical Properties Revealed through Investigation of Polyhedral Au-Cu2O and Bimetallic Core-Shell Nanocrystals. Small 2015, 11, 2716−2726. (26) Luo, T.; Meng, Q. Q.; Gao, C.; Yu, X. Y.; Jia, Y.; Sun, B.; Jin, Z.; Li, Q. X.; Liu, J. H.; Huang, X. J. Sub-20 nm-Fe3O4 Square and Circular Nanoplates: Synthesis and Facet-Dependent Magnetic and Electrochemical Properties. Chem. Commun. 2014, 50, 15952−15955. (27) Tan, C. S.; Hsu, S. C.; Ke, W. H.; Chen, L. J.; Huang, M. H. Facet-Dependent Electrical Conductivity Properties of Cu2O Crystals. Nano Lett. 2015, 15, 2155−2160. (28) Yu, X. Y.; Meng, Q. Q.; Luo, T.; Jia, Y.; Sun, B.; Li, Q. X.; Liu, J. H.; Huang, X. J. Facet-Dependent Electrochemical Properties of Co3O4 Nanocrystals Toward Heavy Metal Ions. Sci. Rep. 2013, 3, 2886. (29) Pal, J.; Pal, T. Faceted Metal and Metal Oxide Nanoparticles: Design, Fabrication and Catalysis. Nanoscale 2015, 7, 14159−14190. (30) Chen, X.; Mao, S. S. Titanium Dioxide Nanomaterials: Synthesis, Properties, Modifications, and Applications. Chem. Rev. 2007, 107, 2891−2959. (31) Shi, H. B.; Magaye, R.; Castranova, V.; Zhao, J. S. Titanium Dioxide Nanoparticles: A Review of Current Toxicological Data. Part. Fibre Toxicol. 2013, 10, 15. (32) Yu, X.; Jeon, B.; Kim, Y. K. Dominant Influence of the Surface On the Photoactivity of Shape-Controlled Anatase Tio2 Nanocrystals. ACS Catal. 2015, 5, 3316−3322. (33) Yang, H. G.; Sun, C. H.; Qiao, S. Z.; Zou, J.; Liu, G.; Smith, S. C.; Cheng, H. M.; Lu, G. Q. Anatase TiO2 Single Crystals with a Large Percentage of Reactive Facets. Nature 2008, 453, 638−641. (34) Han, X.; Kuang, Q.; Jin, M.; Xie, Z.; Zheng, L. Synthesis of Titania Nanosheets with a High Percentage of Exposed (001) Facets and Related Photocatalytic Properties. J. Am. Chem. Soc. 2009, 131, 3152−3153.

(35) Liu, S. W.; Yu, J. G.; Jaroniec, M. Anatase TiO2 with Dominant High-Energy {001} Facets: Synthesis, Properties, and Applications. Chem. Mater. 2011, 23, 4085−4093. (36) Turchi, C. S.; Ollis, D. F. Photocatalytic Degradation of Organic-Water Contaminants - Mechanisms Involving Hydroxyl Radical Attack. J. Catal. 1990, 122, 178−192. (37) Vittadini, A.; Selloni, A.; Rotzinger, F. P.; Gratzel, M. Structure and Energetics of Water Adsorbed at TiO2 Anatase (101) and (001) Surfaces. Phys. Rev. Lett. 1998, 81, 2954−2957. (38) Huang, L. L.; Gubbins, K. E.; Li, L. C.; Lu, X. H. Water on Titanium Dioxide Surface: A Revisiting by Reactive Molecular Dynamics Simulations. Langmuir 2014, 30, 14832−14840. (39) Wahab, H. S.; Bredow, T.; Aliwi, S. M. MSINDO Quantum Chemical Modeling Study of Water Molecule Adsorption at NanoSized Anatase TiO2 Surfaces. Chem. Phys. 2008, 354, 50−57. (40) Du, Y. X.; Fu, Q. S.; Li, Y.; Su, Y. L. Photodecomposition of 4Chlorophenol by Reactive Oxygen Species In UV/Air System. J. Hazard. Mater. 2011, 186, 491−496. (41) Klamerth, N.; Gernjak, W.; Malato, S.; Aguera, A.; Lendl, B. Photo-Fenton Decomposition of Chlorfenvinphos: Determination of Reaction Pathway. Water Res. 2009, 43, 441−449. (42) Weidinger, A.; Kozlov, A. V. Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction. Biomolecules 2015, 5, 472−484. (43) Kresse, G.; Furthmuller, J. Efficiency of Ab-Initio Total Energy Calculations for Metals and Semiconductors Using a Plane-Wave Basis Set. Comput. Mater. Sci. 1996, 6, 15−50. (44) Kresse, G.; Furthmuller, J. Efficient Iterative Schemes for Ab Initio Total-Energy Calculations Using a Plane-Wave Basis Set. Phys. Rev. B: Condens. Matter Mater. Phys. 1996, 54, 11169−11186. (45) Grimme, S. Semiempirical GGA-Type Density Functional Constructed With a Long-Range Dispersion Correction. J. Comput. Chem. 2006, 27, 1787−1799. (46) Mathew, K.; Sundararaman, R.; Letchworth-Weaver, K.; Arias, T. A.; Hennig, R. G. Implicit Solvation Model for Density-Functional Study of Nanocrystal Surfaces and Reaction Pathways. J. Chem. Phys. 2014, 140, 084106. (47) Rushton, E. K.; Jiang, J.; Leonard, S. S.; Eberly, S.; Castranova, V.; Biswas, P.; Elder, A.; Han, X.; Gelein, R.; Finkelstein, J.; Oberdö rster, G. Concept of Assessing Nanoparticle Hazards Considering Nanoparticle Dosemetric and Chemical/Biological Response Metrics. J. Toxicol. Environ. Health, Part A 2010, 73, 445− 461. (48) Matsui, K.; Karasaki, M.; Segawa, M.; Hwang, S. Y.; Tanaka, T.; Ogino, C.; Kondo, A. Biofunctional TiO2 Nanoparticle-Mediated Photokilling of Cancer Cells Using UV Irradiation. MedChemComm 2010, 1, 209−211. (49) Zhang, H.; Pokhrel, S.; Ji, Z.; Meng, H.; Wang, X.; Lin, S.; Chang, C. H.; Li, L.; Li, R.; Sun, B.; Wang, M.; Liao, Y.-P.; Liu, R.; Xia, T.; Mädler, L.; Nel, A. E. PdO Doping Tunes Band-Gap Energy Levels as Well as Oxidative Stress Responses to a Co3O4 p-Type Semiconductor in Cells and the Lung. J. Am. Chem. Soc. 2014, 136, 6406−6420. (50) Brunet, L.; Lyon, D. Y.; Hotze, E. M.; Alvarez, P. J.; Wiesner, M. R. Comparative Photoactivity and Antibacterial Properties of C60 Fullerenes and Titanium Dioxide Nanoparticles. Environ. Sci. Technol. 2009, 43, 4355−4360. (51) Zhang, H.; Dunphy, D. R.; Jiang, X.; Meng, H.; Sun, B.; Tarn, D.; Xue, M.; Wang, X.; Lin, S.; Ji, Z.; Li, R.; Garcia, F. L.; Yang, J.; Kirk, M. L.; Xia, T.; Zink, J. I.; Nel, A.; Brinker, C. J. Processing Pathway Dependence of Amorphous Silica Nanoparticle Toxicity: Colloidal vs Pyrolytic. J. Am. Chem. Soc. 2012, 134, 15790−15804. (52) Zhang, H. Y.; Xia, T.; Meng, H.; Xue, M.; George, S.; Ji, Z. X.; Wang, X.; Liu, R.; Wang, M. Y.; France, B.; Rallo, R.; Damoiseaux, R.; Cohen, Y.; Bradley, K. A.; Zink, J. I.; Nel, A. E. Differential Expression of Syndecan-1 Mediates Cationic Nanoparticle Toxicity in Undifferentiated versus Differentiated Normal Human Bronchial Epithelial Cells. ACS Nano 2011, 5, 2756−2769. 6072

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073

Article

ACS Nano (53) Razzaboni, B. L.; Bolsaitis, P. Evidence of an Oxidative Mechanism for the Hemolytic-Activity of Silica Particles. Environ. Health Perspect. 1990, 87, 337−341. (54) Lin, Y. S.; Haynes, C. L. Impacts of Mesoporous Silica Nanoparticle Size, Pore Ordering, and Pore Integrity on Hemolytic Activity. J. Am. Chem. Soc. 2010, 132, 4834−4842. (55) Warheit, D. B.; Reed, K. L.; Sayes, C. M. A Role for Surface Reactivity In TiO2 and Quartz-Related Nanoparticle Pulmonary Toxicity. Nanotoxicology 2009, 3, 181−187. (56) Warheit, D. B.; Webb, T. R.; Colvin, V. L.; Reed, K. L.; Sayes, C. R. Pulmonary Bioassay Studies with Nanoscale and Fine-Quartz Particles in Rats: Toxicity Is Not Dependent Upon Particle Size But on Surface Characteristics. Toxicol. Sci. 2006, 95, 270−280. (57) Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H. B.; Yeh, J. I.; Zink, J. I.; Nel, A. E. Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties. ACS Nano 2008, 2, 2121−2134. (58) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Determining the Size and Shape Dependence of Gold Nanoparticle Uptake into Mammalian Cells. Nano Lett. 2006, 6, 662−668. (59) Huang, X. L.; Li, L. L.; Liu, T. L.; Hao, N. J.; Liu, H. Y.; Chen, D.; Tang, F. Q. The Shape Effect of Mesoporous Silica Nanoparticles on Biodistribution, Clearance, and Biocompatibility in Vivo. ACS Nano 2011, 5, 5390−5399. (60) Han, X. G.; Kuang, Q.; Jin, M. S.; Xie, Z. X.; Zheng, L. S. Synthesis of Titania Nanosheets with a High Percentage of Exposed (001) Facets and Related Photocatalytic Properties. J. Am. Chem. Soc. 2009, 131, 3152−3153. (61) Liu, G.; Sun, C. H.; Yang, H. G.; Smith, S. C.; Wang, L. Z.; Lu, G. Q.; Cheng, H. M. Nanosized Anatase TiO2 Single Crystals for Enhanced Photocatalytic Activity. Chem. Commun. 2010, 46, 755−757. (62) Zhang, D. Q.; Li, G. S.; Wang, H. B.; Chan, K. M.; Yu, J. C. Biocompatible Anatase Single-Crystal Photocatalysts with Tunable Percentage of Reactive Facets. Cryst. Growth Des. 2010, 10, 1130− 1137. (63) Yu, J. G.; Wang, W. G.; Cheng, B.; Su, B. L. Enhancement of Photocatalytic Activity of Mesporous TiO2 Powders by Hydrothermal Surface Fluorination Treatment. J. Phys. Chem. C 2009, 113, 6743− 6750. (64) Liu, C.; Han, X. G.; Xie, S. F.; Kuang, Q.; Wang, X.; Jin, M. S.; Xie, Z. X.; Zheng, L. S. Enhancing the Photocatalytic Activity of Anatase TiO2 by Improving the Specific Facet-Induced Spontaneous Separation of Photogenerated Electrons and Holes. Chem. - Asian J. 2013, 8, 282−289. (65) Kim, J.; Takahashi, M.; Shimizu, T.; Shirasawa, T.; Kajita, M.; Kanayama, A.; Miyamoto, Y. Effects of a Potent Antioxidant, Platinum Nanoparticle, on the Lifespan of Caenorhabditis Elegans. Mech. Ageing Dev. 2008, 129, 322−331. (66) Ahamed, M.; Akhtar, M. J.; Raja, M.; Ahmad, I.; Siddiqui, M. K. J.; AlSalhi, M. S.; Alrokayan, S. A. ZnO Nanorod-Induced Apoptosis in Human Alveolar Adenocarcinoma Cells Via P53, Survivin and Bax/ Bcl-2 Pathways: Role of Oxidative Stress. Nanomedicine 2011, 7, 904− 913.

6073

DOI: 10.1021/acsnano.6b01657 ACS Nano 2016, 10, 6062−6073