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Implications of the Differential Toxicological Effects of III-V Ionic and Particulate Materials for Hazard Assessment of Semiconductor Slurries Wen Jiang, Sijie Lin, Chong Hyun Chang, Zhaoxia Ji, Bingbing Sun, Xiang Wang, Ruibin Li, Nanetta Pon, Tian Xia, and Andre E Nel ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b04847 • Publication Date (Web): 07 Nov 2015 Downloaded from http://pubs.acs.org on November 12, 2015
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Implications of the Differential Toxicological Effects of III-V Ionic and Particulate Materials for Hazard Assessment of Semiconductor Slurries
Wen Jiang†, Sijie Lin†, Chong Hyun Chang†, Zhaoxia Ji†, Bingbing Sun†, Xiang Wang†, Ruibin Li†, Nanetta Pon,† Tian Xia† ‡and André E. Nel† ‡* †
Center for Environmental Implications of Nanotechnology, California NanoSystems Institute,
University of California, 570 Westwood Plaza, Los Angeles, CA 90095, USA; ‡ Division of NanoMedicine, Department of Medicine, University of California, 10833 Le Conte Ave, Los Angeles, CA 90095, USA
*
Corresponding Author:
André E. Nel, M.D., Department of Medicine, Division of NanoMedicine, UCLA School of Medicine, 52-175 CHS, 10833 Le Conte Ave, Los Angeles, CA 90095-1680. Tel: (310) 825-6620, Fax: (310) 206-8107 E-mail:
[email protected] 1
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ABSTRACT Because of tunable band gaps, high carrier mobility and low energy consumption rates, III-V materials are attractive for the use in semiconductor wafers. However, these wafers require chemical mechanical planarization (CMP) for polishing, which leads to the generation of large quantities of hazardous waste including particulate and ionic III-V debris. Although the toxic effects of micron-sized III-V materials have been studied in vivo, no comprehensive assessment has been undertaken to elucidate the hazardous effects of sub-micron particulates and released III-V ionic components. Since III-V materials may contribute disproportionately to the hazard of CMP slurries, we obtained GaP, InP, GaAs and InAs as micron (0.2-3 µm) and nanoscale (< 100 nm) particles for comparative studies of their cytotoxic potential in macrophage (THP-1) and lung epithelial (BEAS-2B) cell lines. We found nano-sized III-V arsenides, including GaAs and InAs, could induce significantly more cytotoxicity over a 24-72 h observation period. In contrast, GaP and InP particulates of all sizes as well as ionic GaCl3 and InCl3 were substantially less hazardous. The principal mechanism of III-V arsenide nanoparticle toxicity is dissolution and shedding of toxic As(III) and, to a lesser extent, As(V) ions. GaAs dissolves in the cell culture medium as well as in acidifying intracellular compartments, while InAs only dissolves (more slowly) inside cells. Chelation of released As by 2,3-dimercapto-1-propanesulfonic acid (DMPS) interfered in GaAs toxicity. Collectively, these results demonstrate that III-V arsenides, GaAs and InAs nanoparticles, contribute in a major way to the toxicity of III-V materials that could appear in slurries. This finding is of importance for considering how to deal with the hazard potential of CMP slurries. Keywords: Size, Dissolution, Cytotoxicity, III-V materials, Nanoparticle, Semiconductor
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The high electron mobility, favorable optoelectronic properties, and low power requirement for III-V semiconductor materials, such as gallium arsenide (GaAs) and indium phosphide (InP), make them highly attractive to the semiconductor industry, where these materials are used in lasers, microcircuits, light-emitting diodes, mobile telephones, high frequency radar, optical detectors, bio-imaging agent and biosensors.1-5 However, while the market for III-V materials is expected to continue to grow in the 21st century,6-8 there is also a trend for more III-V nanomaterials being developed
3, 9, 10
by industry, including III-V wafers that require a
combination of chemical and mechanical abrasions, also known as chemical mechanical planarization (CMP) to achieve smooth material surfaces.11-13 Although CMP is not the only process generating nanoscale III-V particulates, it is the best available technique to achieve the surface topography and planarization for layering of wafers (e.g., manufacturing of electronic circuitry), with the potential to generate high volumes of hazardous waste. The CMP process involves the use of pristine slurries, including SiO2, Al2O3 or CeO2 nanoparticles, as well as oxidizers, surfactants, dispersants, corrosion inhibitors, and acid/base ingredients for grinding, lapping and polishing.13-16 A typical wafer production process may take around 6-90 seconds for each polish, involving 200-800 mL CMP slurry, 1-2 L of rinse water and >5 L of pad cleaner.10, 15 Spent slurries are generated in the process, which in addition to the presence of the abrasive nanoparticles, may include abundant III-V particulates, III-V ions, as well as other unknown substances,10, 14 which may contribute in an additive or synergistic fashion to hazard generation in humans and the environment. In order to understand the complexity of this chemical mixture, it could be helpful, as a 1st analysis, to understand the relative contribution and ranking of the major components undergoing change during the CMP process. This includes gaining an understanding of the proportional contribution of abraded III-V particulates as well as their ionic forms, following their release from the wafer surface.
To date, most of the limited studies undertaken to assess the hazard potential of III-V materials, have concentrated on in vivo toxicity testing of the micron-sized (> 1 µm) particles17-21 and their corresponding ions.19, 22-24 For instance, intratracheal instillation of 10 3
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µm GaAs particles (10-100 mg/kg) was shown to induce pulmonary inflammation,25-27 immunotoxicity,28, 29 pneumocyte hyperplasia,25 and tumors in rats.21 Similarly, intratracheal instillation of 1~2 µm InAs and InP particles was associated with severe pulmonary inflammation and systemic toxicity in hamsters, compared to lesser effects by GaAs or As2O3.30, 31 It is important to understand their hazard contribution in light of an expanding literature indicating that nanoscale metal and metal oxide particles are frequently more toxic than micronscale materials. The uncertainty of III-V toxicity triggers us to understand more about the mechanism of III-V material toxicity, even though As is recognized as a toxic pollutant by the European Union and the U.S. Environmental Protection Agency (EPA, 1982).32 Moreover, there are contradictory reports of the relative toxicity of gallium vs. indium ions.18, 24, 33, 34 Although dissolution and release of As, Ga or In ions are suspected of playing a role in the toxicity of III-V particulates in vivo,17, 30, 35-37 no direct evidence has been provided to explain the details of where the ion release takes place and how the fate and transport of different size particles may change their bioavailability and hence toxicological outcome.
In this study, we set out to elucidate the cytotoxicity of four commonly used III-V materials in semiconductor industry, GaP, GaAs, InP and InAs. Each material was acquired as micron and nanoscale particulates, in addition to the corresponding ionic forms, GaCl3, InCl3, As(III) (NaAsO2) and As(V) (Na2HAsO4). Cytotoxicity analysis was performed in macrophage and lung epithelial cell lines, for which outcome was related to particle size, rate of dissolution, cellular uptake, effects of ion chelation and intracellular uptake. We found III-V arsenides could induce cytotoxicity. Nanoscale GaAs exhibit similar toxicity potential as As(III) (NaAsO2), which was significantly more toxic than micron scale GaAs and As(V) (Na2HAsO4) at 24 h. In contrast, InAs only induced toxicity over a longer timescale of 48 and 72 h, with n-InAs being more toxic than the microscale version. The rest of the materials, including ionic Ga and In, showed no hazardous effect. GaAs toxicity could be attributed to particle dissolution and shedding of toxic As in the cell culture medium as well as in acidifying endosomal compartments. In contrast, InAs only induced cytotoxictity by 4
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intracellular dissolution. The in vitro ranking of these materials should prove useful for planning, predictive in vivo studies, as well as for the toxicological analysis of complex CMP slurries.
RESULTS Acquisition and Physicochemical Characterization of III-V Particulate Materials We purchased four types of III-V semiconductor particulates (GaP, GaAs, InP and InAs) as micron- as well as sub-micron scale materials. The transmission electron microscopy (TEM) images of these materials are shown in Figure 1, while details of their physicochemical characterization appear in Table 1. All the micron-sized particles were designated m-III-V, while the nanoparticles were referred to as n-III-V. TEM analysis demonstrated that m-GaP and m-InP particles ranged from 100 nm−3 µm and 200 nm−1.1 µm in size, respectively, while the size ranges for m-GaAs and m-InAs were 100 nm-700 nm, respectively. In contrast, the sizes of n-GaP, n-GaAs and n-InP, were all less than 10 nm, while n-InAs was larger with an average size of 80 nm. Most of the particles had irregular shapes except n-GaP and n-InP, which exhibited spherical shapes. XRD analysis confirmed the high crystallinity and purity of III-V materials as shown in Figure S1. Moreover, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis showed only trace amount of iron, calcium and magnesium in the purchased III-V materials (Table S1). Because cytotoxicity studies were conducted in aqueous cell culture media,
we also performed
physicochemical
characterization of particle size and zeta potential in the cell culture media to be used for macrophages (RPMI 1640) and epithelial cells (BEGM), as well as deionized (DI) water (Table 1). All the particles agglomerated in DI water and the cell culture media. Generally, the hydrodynamic sizes of the micron-sized particles were larger in RPMI and BEGM than DI water, while there were no size differences for the nanoparticles in cell culture media and water (Table 1). Zeta potential measurements demonstrated that most particles were negatively charged in DI water, except m-InP. While the zeta-potential did not change appreciably in RPMI (supplemented with 10% fetal bovine serum), the values were more negative in BEGM, which was supplemented with 2% bovine serum albumin (BSA). 5
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Cytotoxicity Screening of III-V Materials in THP-1 and BEAS-2B Cells Cytotoxicity assessment was performed on all III-V particulates as well as corresponding ionic solutions in differentiated THP-1 cells (macrophage-like) as well as the transformed human bronchial epithelial cell line, BEAS-2B. These cells were used to conduct comparative CellTox™ Green Cytotoxicity and ATP assays on the acquired list of materials (Figure 2). The CellTox™ Green Cytotoxicity assay assesses increased plasma membrane permeability, which pre-stages nuclear DNA staining by the dye in dead cells. The ATP assay measures the decline in cellular ATP content as a measure of decreased cell viability. THP-1 and BEAS-2B cells were treated with 12.5-100 µg/mL of each of the particle suspensions for 24 h. ZnO nanoparticles, which represent a dissolvable nanomaterial, were used as a positive control.38, 39
Cells were also exposed to ionic GaCl3, InCl3, NaAsO2 {As(III)} and Na2HAsO4 {As(V)}
solutions over the same dose range.
Cellular toxicity, as expressed according to the molar mass (Table S2), demonstrated dose-dependent toxicity for m-GaAs, n-GaAs, NaAsO2 and Na2HAsO4 in THP-1 cells (Figure 2A and 2B). ZnO had roughly similar toxicity as Na2HAsO4 at the same molar concentration. In contrast, no significant cytotoxicity was seen for GaP, InP, and InAs particles as well as representative ionic forms, GaCl3 and InCl3, at 24 h. Importantly, n-GaAs induced death of up to ~75% in THP-1 cells, while m-GaAs had a lesser effect, only achieving 35% cell death at the highest concentration (Figure 2A). Confirmation of the decrease in cell viability was sought through the use of an ATP assay, which showed the proportional inverse outcome to the CellTox™ results (Figure 2B). Use of BEAS-2B cells to perform cell death and cell viability assays, showed the same trend as in THP-1 cells (Figure 2C and 2D). However, the toxicity of n-GaAs is more similar to As(III) (NaAsO2), but significantly higher than As (V) (Na2HAsO4). When expressing the results according to mass-dose rather than a molar-dose metric, the same ranking was maintained in THP-1 and BEAS-2B cells except for ZnO because of low molecular weight compared to other materials (Figure S2, Table S2). Finally, we derived a visual display of the toxicity ranking through the use of a heat map, which shows that in both cell types the toxicity of NaAsO2 ≥ n-GaAs > 6
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Na2HAsO4 > m-GaAs, with GaP, InP, InAs, GaCl3 and InCl3 being relatively non-toxic (Figure 2E). Arsenic Shedding from GaAs Particulates Plays a Key Role in Cellular Toxicity Based on the toxicity profiles of n-GaAs, NaAsO2 and Na2HAsO4, we hypothesized that dissolution of n-GaAs or m-GaAs may contribute to toxicity of these materials. ICP-OES analysis was used to quantify the dissolution of GaAs particles in cell-free RPMI (10% FBS) and BEGM media (Figure 3A). For comparative analysis, we also assessed In and As release from InAs particles in the same media (Figure 3A). The data demonstrate that n-GaAs was highly soluble in both culture media, with >15% of the elemental Ga and As content being shed from the particles over 24 h, in contrast to the shedding 5% of their metal content by micron scale particles. The dissolution rate was slightly higher in BEGM (20%) than in RPMI (15%). For InAs, 85% of As released by nano-sized GaAs is in the trivalent (III) form, with 18Ωcm), and then reconstituted as 10 mg/mL stock solutions by bath sonication for 15 min. DLS data and zeta potential were obtained using a ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY). III-V stock suspensions (10 mg/mL) were diluted to 50 µg/mL in water or cell culture media for further analysis. The cell culture media used in vitro study, included Roswell Park Memorial Institute (RPMI) 1640 medium (supplemented with 10% FBS) and Bronchial Epithelial Growth Medium (BEGM), supplemented with 2 mg/mL BSA. The primary particle sizes and morphologies were determined by Transmission Electron Microscopy (TEM) in a JEOL 1200 EX microscope, with 80 kV accelerating voltage. Surface area was determined by Brunauer–Emmett–Teller (BET) method using QUADRASORB SI (Quantachrome, USA) instrument. Approximately 100 mg of each powder was placed in a sample cell and degassed under vacuum for 3 h at 120 °C to remove any water vapor and adsorbed gases. The N2 adsorption and desorption isotherms were recorded in the relative pressure (P/P0) range from 0.01 to 0.35 at constant temperature (78 K). The surface area was calculated using multiple point BET equation. X-ray diffraction (XRD) patterns were collected using a Panalytical X’Pert Pro diffractometer (Cu Kα radiation) with a step size of 0.02° and a counting time of 0.5 s per step, over a range of 10−80° 2θ. 15
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Assessment of Particle Dissolution and Confirmation of the Dissolved Arsenic Species ICP-OES analysis was used to detect III-V shedding from particles in various aqueous media. The stock suspensions of micron, sub-micron and pulverized GaAs, as well as InAs particulates, at 10 mg/mL, were diluted in RPMI 1640 (10% FBS) and BEGM (2 mg/mL BSA) to obtain 50 µg/mL suspensions, which were incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h. After centrifugation at 15,000 rpm for 60 minutes, the supernatants were transferred to clean tubes (SC475, Environmental Express) for acid digestion. The digestion was carried out in 10 mL trace metal grade HNO3 (65–70%) at 80 °C for 6 h in a HotBlock (SC100, Environmental Express). The temperature was increased to 95 °C to evaporate all liquids. The dried samples were cooled to room temperature and subsequently diluted by 2% (v/v) nitric acid (80 °C for 3 h) to extract the analytes. These extracts were transferred to 15 mL ICP-OES tubes and additional HNO3 was added to reach a final volume of 8 mL. A calibration curve was established using a standard III-V solution (Elements Inc., 100 mg/L in 2% HNO3). Each sample and standard was analyzed in triplicate in the presence of 2% (v/v) nitric acid. For the determination of the chemical species of arsenic in the supernatants, we used disposable commercial arsenic speciation cartridges (MetalSoft Center, USA)42 for fractionation of dissolved As in the cell culture medium into As(III) and As(V)41 with the same protocol from use instruction,42 following with the similar acid digestion procedure in the above, and then use ICP-OES to quantify the As content of each fraction to determine the dissolved content of As(III) and As(V) in the supernatant. We also dispersed micron- and nano-sized GaAs in simulated phagolysosomal fluid (PSF) and an aqueous medium, acidified by HCl (pH=4.5) for 24 h, to assess particle dissolution in an acidic environment. Similarly, for InAs, we dispersed micron- and nano-sized particles in PSF for 24, 48 and 72 h following with ICP-OES analysis for prolonged dissolution study in the lysosomal mimicking solution. III-V Material Dispersion and Cell Culture THP-1 and BEAS-2B cells were obtained from ATCC (Manassas, VA). RPMI 1640 was purchased from Invitrogen (Carlsbad, CA, USA) and supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA) to culture THP-1 cells. BEGM was obtained from 16
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Lonza (Mapleton, IL) and supplemented with growth factors (SingleQuot Kit Suppl. & Growth Factors, Lonza) for culture of BEAS-2B cells. 3×104 THP-1 cells were pretreated with 1 µg/mL phorbol 12-myristate acetate (PMA) overnight and cultured in 0.1 mL RPMI 1640 in the wells of a 96-well plate at 37 °C in a humidified 5% CO2 atmosphere. 2×104 BEAS-2B cells were cultured in 0.1 mL BEGM in 96-well plates under similar conditions. III-V particles suspensions were freshly prepared in RPMI 1640 (10% FBS) or BEGM (2 mg/mL BSA) by probe sonication (3 W) to obtain a 12.5-100 µg/mL dose range. These suspensions were added to the tissue plates. GaCl3, InCl3, NaAsO2 and Na2HAsO4•7H2O were used as representative ionic controls, with dissolvable ZnO serving as a positive nanoparticle control. CellTox™ Green Cytotoxicity and ATPlite 1step Assays Cells in 100µL culture medium were plated overnight in each well of a Costar™ 96-well black-bottom (for cell death study) or white-bottom plate (for cell viability study) (Corning, NY, USA). The medium was removed, and replenished with 100 µL suspensions of III-V particulates, ionic materials and ZnO at the indicated mass-dose concentrations for 24 h. Due to the differences in the molecular weight of the materials, we also calculated the molar concentration of the materials, as shown in Table S2. The cytotoxicity data were normalized for molar as well as mass particle concentrations as shown in Figure 2 and Figure S2, separately. For GaAs, the molar concentration range of 0.07 to 0.7 µmol/mL equals the mass-dose range of 12.5-100 µg/mL. Cell death was determined by the CellTox™ Green Cytotoxicity Assay (Promega Corporation, Madison, WI, USA). The fluorescence intensity was read on a SpectraMax M5 microplate spectrophotometer. Cell viability was assessed in Costar™ 96-well white-bottom plates, using the ATPlite 1step Assay (PerkinElmer, Boston, MA, United States) for determining cellular ATP content. After incubation with the III-V particle suspensions, ionic forms, ZnO, and pulverized GaAs particle suspensions for 24 h, the culture media were removed and cells incubated with 100 µL reconstituted ATPlite 1step reagent for 10 min. The luminescence intensity was read on a SpectraMax M5 microplate spectrophotometer. For cytotoxicity assessment of the supernatants, micron- and nano-sized GaAs and InAs particles were suspended at 50 µg/mL in culture medium for 24 h, before 17
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centrifugation at 15,000 rpm for 60 minutes. 100 µL of each supernatant was added to 96-well plates for 24 h to perform CellTox™ Green Cytotoxicity and ATPlite 1step assays, as described above. For prolonged time periods toxicity study of InAs, micron- and nano-sized particles were suspended in RPMI 1640 with mass-dose range of 12.5-100 µg/mL, and interact with differentiated THP-1 cells for 24, 48 and 72 h to perform CellTox™ Green Cytotoxicity and ATPlite 1step assays. n-GaAs was chosen as positive control, while n-GaP served as negative control in the same concentration. Using a Chelating Agent to Inhibit Cytotoxicity 12 µL of a freshly made 2,3-dimercapto-1-propanesulfonic acid (DMPS) solution (5 mg/mL) was added to 888 µL of each of the supernatants collected from GaAs, InAs or pulverized GaAs (m-GaAs0-4) particles. After vortexing for 15s, the DMPS supplemented supernatants were incubated with the cells for 24 h in 96-well plate. Cell death and viability were assessed as described above. ICP-OES and TEM Analysis to Study Cellular Uptake of the III-V Materials Fresh prepared 50 µg/mL III-V suspensions were incubated with THP-1 and BEAS-2B cells for 24 h in 6-well plates (Costar, Corning, NY), using 1×106 cells per well. Cells were washed 3 times with PBS, scraped off by a Corning® cell lifter, and then centrifuged at 1500 rpm for 5 min. Half parts of the cell pellets were digested in 10 mL HNO3 (65–70%) at 80 °C for 6 h for ICP-OES analysis of metal content, while the rest of the pellets were used for TEM analysis, after prefixing for 1 h in 4% paraformaldehyde and 1% OsO4. The latter samples were dehydrated in a graded ethanol series before propylene oxide treatment, Epon embedding, sectioning and placement on formvar-coated copper grids. The sections were stained with uranyl acetate and reynolds lead citrate, and examined on a JEOL 100 CX TEM at 80 kV in the UCLA BRI Electron Microscopy Core. Statistical Analysis All the experiments were performed in triplicate and results expressed as mean ± standard deviation (SD). Statistical significance was evaluated using two-tailed heteroscedastic Student’s t-tests, using the TTEST function in Microsoft Excel. The results were considered 18
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statistically significant at p
