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Historically, silver (Ag) compounds have been used in numerous \ue001elds to prevent microbial growth. Like many nonessential heavy met\u00ad als, Ag is a natural biocide, but compared with titanium, zinc, and copper, Ag nano\u00ad particles (Ag\u00adnps) show the highest anti\u00ad microbial e\ue003\ue003icacy against bacteria, viruses, and other eukaryotic micro organisms (Gong et al. 2007). \ue002he Phoenicians coated milk bottles with Ag to inhibit bacterial growth; doctors have adminis tered drops o\ue003 Ag nitrate solutions to newborn babies to prevent neo\u00ad natal conjunctivitis (Crede 1881); and Ag sul\ue003adiazine creams have long been consid\u00ad ered the standard o\ue003 care \ue003or the preven\u00ad tion o\ue003 widespread bacterial growth on burn patients\u2019 denuded skin (Moyer et al. 1965). Both dietary supplements and homemade varieties o\ue003 Ag colloids have been sold \ue003or decades as a \u201ccure\u00adall\u201d \ue003or such diseases as tuberculosis, syphilis, scarlet \ue003ever, shingles, herpes, pneumonia, and arthritis (National Center \ue003or Complementary and Alternative Medicine 2009). Furthermore, advances in nanotechnology have \ue003acilitated the increase o\ue003 Ag\u00adcontaining merchandise available to the public, making Ag the most used nano\u00ad material o\ue003 all manu\ue003acturer\u00adidenti\ue003ied
products in the world (Project on Emerging Nanotechnologies 2009). Products such as room deodorizing sprays, acne creams, cloth\u00ad ing that prevents body odor, baby wipes, and paci\ue003iers all exploit the natural anti\u00ad microbial activity o\ue003 Ag (Project on Emerging Nanotechnologies 2009). In a study inves\u00ad tigating the release o\ue003 Ag\u00adnps \ue003rom com\u00ad mercially available sock \ue003abric, Benn and Westerho\ue000 (2008) showed that socks could contain up to 1,360 \u00b5g Ag/g per sock and could release as much as 1.3 \u00b5g/mL o\ue003 Ag into distilled water.
Ingestion o\ue003 Ag can cause argyria, the benign condition characterized by the bluish\u00ad graying o\ue003 the skin that occurs through the pre\ue003erential deposition o\ue003 Ag in the basal lam\u00ad ina o\ue003 so\ue003t tissues such as the skin, liver, and spleen (Fung and Bowen 1996) and blood vessels, gastro intestinal tract, liver, and kidney (Danscher 1980). Although argyria is most commonly reported clinically a\ue003ter exces\u00ad sive Ag ingestion, Ag deposition has been seen a\ue003ter treatment o\ue003 burned skin with Ag sul\ue003a diazine (Lee and Lee 1994; Marshall and Schneider 1977; \ue002emple and Farooqi 1985). In response to argyria, but not to Ag toxic\u00ad ity, the National Institute \ue003or Occupational
Sa\ue003ety and Health (2003) set a daily exposure limit \ue003or all \ue003orms o\ue003 Ag at 0.01 mg/m3, and the U.S. Environmental Protection Agency (2003) established the oral re\ue003erence dose at 0.005 mg/kg/day.
Studies indicating Ag toxicity exist \ue003rom as early as 1983, when Rungby and Danscher (1983) showed that Ag salts intra peritoneally adminis tered to rats can accumulate in neurons and in protoplasmic glial cells o\ue003 the brain and spinal cord. In vitro cell line studies have shown decreased mitochondrial \ue003unction a\ue003ter expo\u00ad sure to Ag\u00adnps in murine neuro blastoma cells (Schrand et al. 2008), hepatic cells (Hussain et al. 2005), germline stem cells (Braydich\u00ad Stolle et al. 2005), human skin carcinoma cells (Arora et al. 2008), and human epidermal kera\u00ad tinocytes (HEKs) and \ue001broblasts (Burd et al. 2007). Although in vivo studies have not been per\ue003ormed with Ag\u00adnps, poly vinyl pyrrolidone\u00ad stabilized Ag\u00adnps with a mean size o\ue003 25 nm were shown to penetrate into the upper lay\u00ad ers o\ue003 the epidermis in excised human skin in static di\ue000usion cells (Larese et al. 2009). Other nanomaterials, such as quantum dots (QDs) and \ue003ullerenes (Rouse et al. 2006; Ryman\u00ad Rasmussen et al. 2006; Zhang and Monteiro\u00ad Riviere 2008; Zhang et al. 2008), as well as zinc oxide (Cross et al. 2007; Gamer et al. 2006), are able to penetrate into the stratum corneum; thus, examination o\ue003 the ability o\ue003 Ag\u00adnps to penetrate the skin is warranted.
Our objectives in this study were to determine the optimal viability assay \ue003or use with Ag\u00adnps in order to assess their toxicity
Address correspondence to N.A. Monteiro\u00adRiviere, North Carolina State University, Center \ue003or Chemical \ue002oxicology Research and Pharmacokinetics, 4700 Hillsborough St., Raleigh, NC 27606 USA. \ue002elephone: (919) 513\u00ad6426. Fax: (919) 513\u00ad6358. E\u00admail: email@example.com
We thank A. Siekkinen (nanoComposix) \ue003or \ue003abri\u00ad cation o\ue003 the Ag\u00adnps and A. Neigh (nanoComposix) \ue003or characterization o\ue003 the Ag\u00adnps.
\ue002his research was supported by U.S. Air Force O\ue000ce o\ue003 Scienti\ue001c Research (USAFOSR) grants FA 9550\u00ad08\u00ad1\u00ad0182, FA8651\u00ad06\u00adC\u00ad0136, and FA8650\u00ad 08\u00adC\u00ad6853.
S.J.O. is employed by nanoComposix, which under a USAFOSR grant, \ue003abricated and charac\u00ad terized Ag\u00adnps and provided all Ag\u00adnps used in this study. Te remaining authors declare they have no competing \ue001nancial interests.
50, and 80 nm particle diameter), washed/uncoated (20, 50, and 80 nm), and carbon-coated (25 and 35 nm)]. Skin was dosed topically \ue003or 14 consecutive days. HEK viability was assessed by M\ue002\ue002, alamarBlue (aB), and Cell\ue002iter 96 AQueous One (96AQ). Release o\ue003 the pro in\ue001ammatory mediators interleukin (IL)-1\u03b2, IL-6, IL-8, IL-10, and tumor necrosis \ue003actor-\u03b1 (\ue002NF-\u03b1) were meas ured.
signifcant dose-dependent decrease (p < 0.05) at 0.34 \u00b5g/mL with aB and 96AQ and at 1.7 \u00b5g/mL with M\ue002\ue002. However, both the washed Ag-nps and carbon-coated Ag-nps showed no signi\ue003i- cant decrease in viability at any concentration assessed by any o\ue003 the three assays. For each o\ue003 the unwashed Ag-nps, we noted a signifcant increase (p < 0.05) in IL-1\u03b2, IL-6, IL-8, and \ue002NF-\u03b1 con- centrations. We observed localization o\ue003 all Ag-nps in cyto plasmic vacuoles o\ue003 HEKs. Macroscopic observations showed no gross irritation in porcine skin, whereas microscopic and ultrastructural observations showed areas o\ue003 \ue003ocal in\ue001ammation and localization o\ue003 Ag-nps on the sur\ue003ace and in the upper stratum corneum layers o\ue003 the skin.
tured Ag\u00adnps on the market, we used eight di\ue003\u00ad \ue003erent \ue003orms o\ue003 Ag\u00adnps with di\ue000erent sizes and sur\ue003ace conditions (\ue002able 1). All Ag\u00adnps used in this study were supplied by nanoComposix (San Diego, CA): commercially used unwashed and uncoated Ag\u00adnps suspended in deionized water with diameters o\ue003 20, 50, and 80 nm (\u201cunwashed\u201d); washed and uncoated Ag\u00adnps suspended in deionized water with diameters o\ue003 20, 50, and 80 nm (\u201cwashed\u201d); and commer\u00ad cially used dried carbon\u00adcoated Ag\u00adnps with diameters o\ue003 25 and 35 nm (\u201ccarbon\u00adcoated\u201d). Te sizes o\ue003 each type o\ue003 Ag\u00adnps were deter\u00ad mined by the manu\ue003acturer and con\ue001rmed in this study by dynamic light scattering (DLS) and transmission electron microscopy (\ue002EM).
Both the unwashed and washed Ag\u00adnps were synthesized by ammonium hydroxide\u2013 catalyzed growth o\ue003 Ag onto 5\u00adnm gold seed particles. Concentration o\ue003 the particles was achieved via tangential \ue003low \ue003iltration. \ue002he unwashed Ag\u00adnps solution contained approxi\u00ad mately 5.55 mg/mL \ue003ormaldehyde solvent and methanol by\u00adproduct \ue003rom their \ue003orma\u00ad tion. Tese unwashed Ag\u00adnps were then ultra\u00ad centri\ue003uged to obtain the solution super natant
\ue003or toxicity testing (\u201cas synthesized\u201d super\u00ad natant). Te Ag\u00adnps were then serially washed with 20 vol equivalents o\ue003 2 mM phosphate bu\ue003\ue003er (pH 7.5) and the 5, 10, 15, and 20 washing permeates were collected. Te wash\u00ad ing permeates contained 20\u201350 ppb dissolved Ag. Te colloidal Ag\u00adnps were stored at 4\u00b0C in the dark. Te carbon\u00adcoated Ag\u00adnps, synthe\u00ad sized by pulsed plasma reactor and coated with poly aromatic graphitic carbon, were supplied as a powder and stored at room temperature.
natal HEKs (Lonza, Walkersville, MD) were grown in Keratinocyte Growth Medium\u00ad2 (KGM\u00ad2; Lonza) in cell culture fasks (75 cm2; 1,000,000 cells) to approximately 80% con\ue003luency in a 37\u00b0C humidi\ue003ied 5% CO2 incubator. Te cells were passed into clear or black 96\u00adwell microplates (12,500 cells/well; 200 \u00b5L) in which the peripheral wells con\u00ad tained only KGM\u00ad2 to prevent the evapora\u00ad tion o\ue003 treatment medium. Between 18 and 24 hr later, a\ue003ter reaching approximately 80% confuency, the HEKs were exposed to either KGM\u00ad2 (control) or serial dilutions o\ue003 each Ag\u00adnps type \ue003or the \ue003ollowing experiments.
particle (no\u00adcell) control (nanoparticles in collagen\u00adcoated wells with no HEKs) and a nanoparticle/cell control (nanoparticles exposed to HEK\u00admetabolized assay dye) were run in
parallel with each viability assay as described by Monteiro\u00adRiviere et al. (2009) to assess the interactions between the viability assays and the Ag\u00adnps [see Supplemental Material avail\u00ad able online (doi:10.1289/ehp.0901398.S1 via http://dx.doi.org/)].
an initial dose\u2013response study to assess the concentrations o\ue003 Ag\u00adnps that could a\ue003\ue003ect HEK a\ue003ter 24\u00adhr exposure. Most o\ue003 the col\u00ad loidal Ag\u00adnps tested were supplied in both low volume and concentration, which limited the highest HEK dosing concentration to 1.7 \u00b5g/mL. Combined with KGM\u00ad2 medium, a 1.7 \u00b5g/mL solution o\ue003 the Ag\u00adnps was serially diluted (1:5) to provide concentrations rang\u00ad ing \ue003rom 1.7 to 0.000544 \u00b5g/mL. Te e\ue000ect o\ue003 the Ag\u00adnps on HEK viability was assessed by three di\ue000erent toxicity assays: 3\u00ad(4,5\u00addimethyl\u00ad thiazol\u00ad2\u00adyl)\u00ad2,5\u00addiphenyltetrazolium bro\u00ad mide (M\ue002\ue002; Sigma\u00adAldrich, St. Louis, MO), alamarBlue (aB; Molecular Probes, Invitrogen, Eugene, OR), and Cell\ue002iter 96 AQueous One (96AQ; Promega, Madison, WI) [\ue003or details, see Supplemental Material (doi:10.1289/ ehp.0901398.S1 via http://dx.doi.org/)].
We tested dosing concentrations o\ue003 the washed and carbon\u00adcoated Ag\u00adnps up to 42.5 \u00b5g/mL but saw no response. Additionally, to di\ue003\ue003erentiate the potential cytotoxicity between the particles and the contaminants present in the colloidal solution, HEKs were treated with the \u201cas synthesized\u201d supernatant and the 5, 10, 15, and 20 washing perme\u00ad ate \ue003or 24 hr at concentrations ranging \ue003rom 1.7 \u00b5g/mL to 0.068 \u00b5g/mL.
o\ue003 Ag\u00adnps that showed toxicity, we conducted cytokine analysis to determine their pro\u00ad infammatory potential by assessing the release o\ue003 interleukin (IL)\u00ad8, IL\u00ad6, tumor necrosis \ue003actor\u00ad\u03b1 (\ue002NF\u00ad\u03b1), IL\u00ad10, and IL\u00ad1\u03b2; the procedure is described in the Supplemental Material (doi:10.1289/ehp.0901398.S1).
pared the e\ue003\ue003ects o\ue003 unwashed Ag\u00adnps with those o\ue003 washed Ag\u00adnps in vivo. Assuming the two smallest Ag\u00adnps could penetrate the skin, the comparison was limited to the 20\u00ad and 50\u00adnm washed and unwashed samples. Pigs were dosed on the back skin with Ag\u00adnps solutions ranging \ue003rom 34.0 to 0.34 \u00b5g/mL [\ue003or details, see Supplemental Material (doi:10.1289/ehp.0901398.S1)]. Skin was evaluated \ue003or erythema and edema according to the Draize system (Draize et al. 1944): \ue003or erythema: 0, no change; 1, very slight change; 2, pale red in de\ue001ned area; 3, de\ue001nite red in well\u00adde\ue003ined area; 4, crimson red; and \ue003or edema: 0, no change; 1, very slight change; 2, slight change with edges barely de\ue003ined; 3, moderate change, with area raised 1 mm; 4, severe change, with area raised > 1 mm and extending beyond the exposure area. All
phologic alterations during the in vivo study, we harvested tissue samples a\ue003ter the pigs were euthanized and processed them rou\u00ad tinely \ue003or light microscopy [see Supplemental Material (doi:10.1289/ehp.0901398.S1)]. Approximately 1\u00adcm sections were evaluated \ue003or intercellular and intracellular epidermal edema, dermal edema, and in\ue003lammation using the \ue003ollowing scoring system: 0, no change; slight, infammation on less than hal\ue003 the sample; moderate, infammation on hal\ue003 the sample; severe, in\ue003lammation on more than hal\ue003 the sample.
size analysis was conducted with both DLS and \ue002EM to con\ue003irm the manu\ue003acturer\u00ad identi\ue001ed diameters and sur\ue003ace characteriza\u00ad tion [\ue003or details, see Supplemental Material (doi:10.1289/ehp.0901398.S1)]. \ue002o localize Ag\u00adnps uptake in vitro, HEKs were grown to approximately 70% con\ue003luency in cell cul\u00ad ture fasks (25 cm2) and treated \ue003or 24 hr with each Ag particle at 1.7 \u00b5g/mL in KGM\u00ad2. Te cells and skin samples were processed rou\u00ad tinely \ue003or \ue002EM (\ue003or details, see Supplemental Material). We viewed all \ue002EM samples on an FEI/Philips EM 208S \ue002EM (FEI, Hillsboro, OR), operating at an accelerating voltage o\ue003 80 kV. Additionally, unstained samples were analyzed by X\u00adray microanalysis [energy dispersive X\u00adray spectroscopy (EDS)] with a Hitachi HF2000 FE \ue002EM (Hitachi High \ue002echnologies America Inc., Lexington, KY)
mean values \ue003or HEK percent viability (nor\u00ad malized by viability) and cytokine concen\u00ad tration \ue003or each treatment and determined the signi\ue001cant di\ue000erences (p < 0.05) using the PROC GLM procedure (SAS, version 9.1 \ue003or Windows; SAS Institute Inc., Cary, NC). When signi\ue001cant di\ue000erences were \ue003ound, we per\ue003ormed multiple comparisons using the \ue002ukey\u2019s studentized range highest signi\ue001cant di\ue003\ue003erence test, withp < 0.05 as the level o\ue003 signi\ue001cance. Dunnett\u2019st\u00adtest was per\ue003ormed to determine the signi\ue001cance atp < 0.5 o\ue003 di\ue000er\u00ad ences between control and treatment groups. Data are expressed as the mean \u00b1 SEM o\ue003 two plates (n = 6/plate).
rizes the control studies \ue003or the three viability assays, listing the absorbance or fuorescence value \ue003or each Ag\u00adnps type that shows a signi\ue001\u00ad cant inter action. For M\ue002\ue002, at 1.7 \u00b5g/mL both the 25\u00adnm (Figure 1A) and 35\u00adnm (Figure 1B) carbon\u00adcoated Ag\u00adnps nanoparticle/cell con\u00ad trols show a statistically signi\ue003icant increase in absorbance a\ue003ter exposure o\ue003 Ag\u00adnps to cell\u00adreacted assay dye. \ue002he nanoparticle (no cell) controls showed a statistically signi\ue001cant increase in absorbance at 1.7 \u00b5g/mL with the 20\u00adnm unwashed Ag\u00adnps \ue003or 96AQ and M\ue002\ue002 but not \ue003or aB (Figure 1C).
(doi:10.1289/ehp.0901398.S1). We saw no signi\ue003icant changes in the \ue003luorescence values \ue003or aB \ue003or the 20\u00ad, 50\u00ad, or 80\u00adnm unwashed Ag\u00adnps (see Supplemental Material, Figure 1a). \ue002he unwashed 50\u00ad and 80\u00adnm Ag\u00adnps caused a signi\ue001cant increase in absor\u00ad bance values \ue003or M\ue002\ue002 and 96AQ but not \ue003or aB (see Supplemental Material, Figure 1b,c). Te fuorescence value \ue003or aB increased sig\u00ad ni\ue001cantly at 0.034 \u00b5g/mL \ue003or both 20\u00ad and 50\u00adnm washed Ag\u00adnps and at 1.7 \ue003or 80\u00adnm washed Ag\u00adnps (see Supplemental Material, Figure 2a). Te absorbance value \ue003or 96AQ changed signi\ue001cantly at 1.7 \u00b5g/mL \ue003or each o\ue003 the 20\u00ad, 50\u00ad, and 80\u00adnm washed Ag\u00adnps (see Supplemental Material, Figure 2b). Te 80\u00adnm washed Ag\u00adnps caused a signi\ue001\u00ad cant increase in absorbance value \ue003or M\ue002\ue002 at 1.7 \u00b5g/mL (see Supplemental Material, Figure 2c). \ue002he \ue003luorescence value \ue003or aB increased signi\ue001cantly at 1.7 \u00b5g/mL \ue003or 35\u00adnm carbon\u00adcoated Ag\u00adnps (see Supplemental Material, Figure 3a). \ue002he absorbance value increased signi\ue001cantly \ue003or 96AQ at 1.7 \u00b5g/mL \ue003or 25\u00adnm carbon\u00adcoated Ag\u00adnps and at 0.034 \u00b5g/mL \ue003or 35\u00adnm carbon\u00adcoated Ag\u00adnps (see Supplemental Material, Figure 3b). We observed a signi\ue003icant increase in the absorbance value \ue003or M\ue002\ue002 at 1.7 \u00b5g/mL \ue003or 25\u00ad and 35\u00adnm carbon\u00adcoated Ag\u00adnps (see Supplemental Material, Figure 3c).
concentrations ranging \ue003rom 0.000544 to 1.7 \u00b5g/mL and exposures o\ue003 24 hr, treatment with unwashed Ag\u00adnps resulted in a dose\u00ad dependent decrease in viability with all three
*p < 0.05; nanoparticle/cell controls were compared \ue001or each concentration be\ue001ore and a\ue001ter Ag-nps, nanoparticle controls were run with multiple comparisons between concentrations, and each Ag-nps type was assessed independently. InD, di\ue001\ue001erent letters denote signi\ue000cant di\ue001\ue001erences \ue001or each nanoparticle at each concentration (p < 0.05); each Ag-nps type was assessed independently.
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