Anatase

ENMs and reagents. The CPPs obtained ZnO from Meliorum Technologies Inc. (Rochester, NY). TiO₂-P25 (81% anatase and 19% rutile) was purchased from Evonik (Parsippany, NJ); TiO₂-A was provided by P. Biswas (Washington University, St. Louis, MO); and the CPPs prepared the TiO₂-NBs as previously described (Hamilton et al. 2009 (link)). The CPPs obtained the O-MWCNT stock in powder form from Cheap Tubes Inc. (Brattleboro, VT); obtained the P-MWCNT by treating O-MWCNT with dilute acids, chelating agents, and mild conditions to minimize oxidized or damaged tubes; and created F-MWCNT through further acid treatment of P-MWCNT, which introduced carboxyl groups on 5.27% of the carbon backbone (on a per weight basis) (Chen and Mitra 2008 ; Wang et al. 2011 (link)).
The CPPs purchased low-endotoxin bovine serum albumin (BSA) from Gemini Bio-Products (West Sacramento, CA); dipalmitoylphosphatidylcholine, phorbol 12-myristate, 13-acetate (PMA), and lipopolysaccharide (LPS from Escherichia coli 0127:B8) from Sigma-Aldrich (St. Louis, MO); and 1,25-dihydroxy-vitamin D₃ from EMD Millipore (Billerica, MA). The CPPs purchased the cytotoxicity assays CellTiter 96 (MTS assay) and CytoTox 96 [LDH (lactate dehydrogenase) assay] from Promega (Madison, WI).
Preparation of ENMs in cell culture media. The CPPs prepared ENM stock solutions (5 mg/mL) from dry powder using endotoxin-free sterile water and then prepared all ENM suspensions in cell culture media using the stock solutions as needed. Briefly, the CPPs vortexed and then sonicated ENM stock solutions (with the exception of TiO₂-NB, which was stirred to prevent mechanical shear) using a water bath sonicator or cup horn sonicator (depending on laboratory availability) immediately before diluting the solutions into complete cell culture media.
Cell culture and co-incubation with EMN. The CPPs grew all cells at 37°C in a 5% CO₂ atmosphere. RLE-6TN cells, a rat alveolar type II epithelial cell line, from American Type Culture Collection (ATCC; Manassas, VA) were cultured in Ham’s F12 medium (ATCC) supplemented with l-glutamine, bovine pituitary extract (BPE), insulin, insulin growth factor (IGF)-1, transferrin, and epithelial growth factor (EGF), supplemented with 10% fetal bovine serum (FBS). THP-1 cells, a human acute monocytic leukemia cell line (ATCC) were cultured in HEPES-buffered RPMI 1640 supplemented with l-glutamine (Mediatech, Corning, NY), 0.05 mM β-mercaptoethanol, and 10% FBS (PAA Laboratories, Dartmouth, MA). BEAS-2B cells (ATCC) were cultured in bronchial epithelial growth medium (BEGM) obtained from Lonza Inc. (Walkersville, MD) supplemented with BPE, insulin, hydrocortisone, human EGF, epinephrine, triiodothyronine, transferrin, gentamicin/amphotericin-B, and retinoic acid. For the THP-1 differentiation performed in the first series of experiments (phase I), the CPPs pretreated cells with 1.62 µM (1 µg/mL) PMA for 18 hr. However, the CPPs identified excessive cell clumping and cell death during the phase I studies. Therefore, the CPPs alternatively pretreated THP-1 cells with vitamin D₃ at 150 nM overnight and then 5 nM PMA in order to obtain the differentiated macrophage-like cells used during the second series of experiments (phase II). For the IL-1β release, co-culturing THP-1 cells with 10 ng/mL LPS was necessary to initiate transcription of pro-IL-1β. The CPPs initiated aggressive phagocytic activity by adding PMA just before particle exposure.
Before ENM exposure, the CPPs cultured aliquots of 1.5 × 10⁴ cells (for THP-1 cells, 10⁵ cells were seeded into each well of a 96-well plate) in 0.2 mL of the cell culture media in 96-well plates (Costar, Corning, NY) at 37°C for 24 hr. The CPPs freshly prepared all of the ENM suspensions at final concentrations of 10, 25, 50, and 100 µg/mL in the cell culture media. After exposure of the cells to the ENMs for 24 hr at 37°C, the CPPs collected supernatants to measure LDH and IL-1β production then used the remaining cells to test cellular viability by MTS assay.
Physicochemical characterization of ENMs. The CPPs identified the primary particle size and morphology of the ENMs by using a transmission electron microscope (TEM; model 100CX) and a scanning electron microscope (SEM; model JSM-7600F) (both from JEOL Ltd., Tokyo, Japan). In addition, the CPPs characterized the particle hydrodynamic size in H₂O and cell culture media using dynamic light scattering (DLS) (Ji et al. 2010 (link)). The CPPs characterized particle crystallinity and structure using X-ray diffraction measurements and measured particle surface area by Brunauer–Emmett–Teller (BET) surface area analysis. The CPPs performed zeta-potential measurements of the ENM suspensions using a ZetaSizer Nano-ZS instrument (Malvern Instruments, Worcestershire WR, UK). Finally, the CPPs determined the elemental composition of the particles as well as ZnO dissolution rate using inductively coupled plasma mass spectrometry (ICP-MS) (model SCIEX Elan DRCII; PerkinElmer, Norwalk, CT).
Endotoxin analysis of ENMs. CPPs measured the endotoxin content of ENM stock suspensions, as well as dispersions in PBS and tissue culture media, using the colorimetric Limulus amebocyte lysate assay (Lonza Inc.). The LPS content of all ENM suspensions was < 0.3 EU/mL.
Determination of cell viability. The CPPs determined cellular viability using MTS (CellTiter 96) and LDH (CytoTox 96; both from Promega) according to the manufacturer’s protocols. To avoid the interference created by ENMs while measuring formazan absorbance at 490 nm, the CPPs introduced a centrifugation (2000 × g for 10 min) procedure in phase II experiments to collect particles in the wells after incubation with the MTS reagents. CPPs then followed this centrifugation step with a brief mixing and transfer of the supernatant to a new 96-well plate before measuring the formazan absorbance at 490 nm. The CPPs eliminated interference of any residual LDH in FBS by heat-inactivation (70°C water bath for 5 min).
ELISA for IL-1β quantification. The CPPs determined IL-1β production in the THP-1 culture supernatant using a human IL-1β ELISA kit (R&D Systems Human IL-1β DuoSet™; R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.
Statistical analysis. The CPPs used the two-way analysis of variance followed by Tukey or Bonferroni correction for multiple comparisons of means for statistical analysis of responses across ENMs and cell lines. In order to define interlaboratory comparisons across two harmonization rounds, the CPPs conducted a meta-analysis of LDH, MTS, and IL-1β assays across eight different laboratories for three cell lines (BEAS-2B, RLE-6TN, and THP-1) exposed to several ENMs (TiO₂-P25, TiO₂-A, TiO₂-NBs, ZnO, O-MWCNT, P-MWCNT, and F-MWCNT). The CPPs combined information within assays and cell lines using a robust two-stage hierarchical model of toxicity. For all quantities of interest, the CPPs obtained Monte Carlo inference by implementing a custom Gibbs sampler in the R computing environment (R Foundation for Statistical Computing, Vienna, Austria). To normalize data, the CPPs subtracted background negative control values (MTS, LDH, and IL-1β) and provided adjustments for positive control values in the case of LDH assays. Details about the statistical model used for analysis are provided in Supplemental Material, p. 8 (http://dx.doi.org/10.1289/ehp.1306561).

All measurements reported in this paper were made at a temperature of 25^°C on a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) fitted with a high-concentration zeta potential cell (ZEN1010).
Two samples were measured in this study. (i) A titanium dioxide (anatase) sample was prepared in 10 mM NaCl at a range of volume fractions (0.236×10⁻⁵ to 7.092×10⁻⁴). Dilution in an indifferent electrolyte, such as NaCl, should ensure that any changes in the zeta potential values obtained were not due to conductivity differences. (ii) A polyurethane dispersion at a volume fraction of 0.4 was kindly provided by Baxenden, a Chemtura company. Various concentrations were prepared in 5 per cent v/v triethylamine to maintain ionic strength and ensured that any differences in the measured electrophoretic mobilities were not due to changes in conductivity.
For all measurements, a field of 40 V was applied across an electrode spacing of 16 mm. Five repeat measurements on each sample were made to check the repeatability of the results obtained. All measured electrophoretic mobilities were converted into zeta potential using Smoluchowski’s formula (Smoluchowski 1921 ; Delgado et al. 2005 (link)). Sample viscosities were determined at 25^°C using an SV-10 vibroviscometer (A&D Company Ltd, Tokyo, Japan).
Size characterization of the samples was made by dynamic light-scattering (DLS) measurements using the Zetasizer Nano ZS, which uses a 4 mW He–Ne laser operating at a wavelength of 633 nm and a detection angle of 173^°. The intensity-averaged particle diameters and the polydispersity index (PDI) values (an estimate of the distribution width) were calculated from the cumulants analysis as defined in ISO13321 (International Organization for Standardization 1996 ). The intensity size distributions were obtained from analysis of the correlation functions using the general purpose algorithm in the instrument software. This algorithm is based upon a non-negative least squares fit (Lawson & Hanson 1995 ; Twomey 1997 ).

For measuring the accuracy of size and zeta potential measurement, 60 nm plain, carboxyl and amine modified particles were prepared at a concentration of 0.02 mg/ml in distilled water.
To evaluate the different sequences of preparation steps, a TiO₂ (rutile) stock solution was prepared at a concentration of 0.02 mg/ml in distilled water with or without sonication with 4.2 × 10⁵ kJ/m³ specific energy. Thirty μl of HSA (end concentration 1.5 mg/ml) or Tween 80 (end concentration 0.1%) was given to 870 μl of dispersion before or after the addition of 100 μl of a 10 × concentrated PBS solution.
The TiO₂ (rutile) dispersion was also prepared in a similar way using RPMI 1640 cell culture medium and with the addition of other dispersion stabilizers, i.e. 1.5 mg/ml mouse serum albumin, 1.5 mg/ml bovine serum albumin, 0.1% Tween 80, or 30 μl mouse serum.
The effect of different TiO₂ (rutile) and HSA concentrations was tested at TiO₂ (rutile) concentrations of 0.002, 0.02, 0.2, 2 mg/ml and HSA concentrations of 0.0015, 0.015, 0.15, 1.5, and15 mg/ml.
The stability of 0.02 mg/ml TiO₂ (rutile) dispersions made by sonication with 4.2 × 10⁵ kJ/m³ energy and addition of 1.5 mg/ml HSA followed by addition of PBS was measured for 1 week.
Dispersions were also made from TiO₂ (anatase), ZnO, SWNT, MWNT, silver, SiO_x, and SRM 2975 diesel nanoparticles by preparing a 0.02 mg/ml stock solution, sonicating with 4.2 × 10⁵ kJ/m³ specific ultrasound energy, adding of 1.5 mg/ml HSA, 0.1% Tween, or 30 μl serum prior to addition of concentrated PBS.

A known volume of hydrochloric acid (X = 10, 15, 20, 25, or 30 mL), 1 mL of concentrated sulfuric acid, and 1 mL of TBOT were added to a solution of ethanol (2 mL) and deionized water (28 mL) in a beaker and mixed evenly by stirring. Then, the NR-TiO₂ sample was placed in a 200 mL reactor and reacted with the acidified TBOT solution at 150 °C for 8 h. After the reaction, the sample was rinsed several times with deionized water and then soaked in deionized water for 3 h. Finally, the sample was annealed in a tube furnace at 400 °C for 20 min. The samples thus prepared were denoted as TiO₂(R/A-X), where R and A represent rutile and anatase phases and X represents the volume of HCl (10, 15, 20, 25, or 30 mL).

In the process of synthesis pure anatase TiO₂ NPs, 5 ml polyethylene glycol was added to a well stirred deionized (DI) water:ethanol solution with 4:1 ratio by volume (40:10 ml). Then 10 ml of C₁₂H₂₈O₄Ti was gradually dropped to this solution, while vigorously stirred at room temperature on a magnetic stirrer hot plate for further 20 min. Then, 2.5 wt.% aqueous ammonia (NH₄OH) was added dropwise to carefully controlled the pH at 7, and further stirring for 30 min. After that, increased the temperature of a solution to 60 °C and kept on stirring until a wet gel was formed, and allowed to dry at 75 °C. The final product was achieved by crushing the dried gel, ground to fine powder and pyrolized at 500 °C in a furnace for 2 h, using a heating rate of 2 °C/min. Li_xTi_1-xO₂ NPs (x = 0.05, 0.10, 0.15 and 0.20) was synthesized by a similar way, only that lithium hydroxide (LiOH) of 0.05, 0.10, 0.15 and 0.20 by wt % was added in the mixture solution before adding NH₄OH.

Titania (TiO₂) was synthesized using a low temperature sol–gel method, as shown in Fig. 1. Tetrabutyl titanate (TBOT) was slowly added to anhydrous ethanol to form a TBOT master-batch with a molar concentration of 0.59 M. A 24 mL sample of the TBOT master-batch was measured and labeled “solution A”. A 2 mL volume of diluted acetic acid solution (2% by mass) was added to a beaker containing 35 mL of anhydrous ethanol with hydrochloric acid (HCl) as a catalyst, and the pH was adjusted to give solution B. At a H⁺/Ti molar ratio of 0, 0.001, 0.2 and 0.5, the corresponding pH was 0.6, 1.0, 1.9 and 4.7, respectively. A peristaltic pump was used to deliver solution A dropwise into solution B at a rate of 3 mL/min. The mixture was then agitated in a water bath at 25 °C for 3 h to produce a sol, and following a 20 h aging period, a gel was formed. The resultant samples were dried at 120 °C for 6 h, and treated in air at 300, 400, 500, 600, and 700 °C for 2 h using a heating rate of 4 °C/min. Finally, the calcined samples were ground in agate to obtain TiO₂ powder. The samples are denoted as TN-X-Y, where X represents the pH and Y represents the calcination temperature.Figure 1

Process flow diagram for the preparation of TiO₂ nanopowder and thin films.

Titania films were also prepared. Microscope coverslips and slides were used as substrates for the TiO₂ films, which were ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and deionised water before use to remove organic and inorganic contaminants. The surfaces were then dried and made ready for use. Slides were utilized for assessing anti-bacterial adhesion whereas coverslip substrates were employed for contact angle testing. The TiO₂ powder was dissolved in anhydrous ethanol and subjected to ultrasonication for 30 min to produce a suspension with a concentration of 13 g/L. The application of the films onto the glass substrate was performed using a constant-temperature impregnation lift coater (Shanghai Sanyan Technology, SYDC-200H) with a descending speed of 3 mm/s, lifting speed of 1 mm/s, impregnation time of 30 s, retention time of 120 s, and six coatings.