Ag-nps have been integrated into hundreds of products that affect the daily lives of millions of people in many countries. Their main use is for disinfection in wound care and in products such as odor-reducing clothing, acne creams, and face masks. Most of these products come into direct contact with skin, the largest organ of the human body, and could serve as a potential route for nanoparticle penetration. Therefore, the relationship of Ag-nps in skin needs to be investigated with particular focus on their irritation potential, toxicity, and penetration into skin and skin cells. In this study we evaluated the cytotoxic potential of Ag-nps of varying sizes and surface conditions in HEK cells, their penetrating capacity into porcine skin after topical repetitive daily dosing for 2 weeks, and the localization of the Ag-nps within HEKs and porcine skin.
The use of several viability assays is important to determine the optimal assay to assess Ag-nps toxicity; therefore, mortality of HEKs after Ag-nps exposure was evaluated with three different assays that use colorimetric or fluorescent dyes as markers to determine cell viability by assessing cell metabolism.
Nanomaterials, such as single-walled carbon nanotubes (Zhang et al. 2007
), carbon black (Monteiro-Riviere and Inman 2006
), fullerenes, and QDs (Monteiro-Riviere et al. 2009
), are capable of interfering with dye and dye products in viability assays through the adsorption of cell medium constituents and cytokines. We assessed the potential interactions between assays and Ag-nps using nanoparticle and nanoparticle/cell controls, which showed an increase in absorbance and fluorescence values at the highest concentration. The increase in absorbance and fluorescence values could cause the toxicity of the Ag-nps in HEKs to be underestimated. Additionally, the nanoparticle/cell control showed that both the 25- and 35-nm carbon-coated Ag-nps interfered with the MTT assay at the 1.7 μg/mL concentration because of the increase in absorbance values after incubation of the reduced formazan product with Ag-nps (). Overall, all assays were affected by Ag-nps; based on its fluorescence values, aB may be the best viability assay to use when conducting experiments with Ag-nps.
MTT, aB, and 96AQ viability assays did not show toxicity for the 25- and 35-nm carbon-coated Ag-nps or for the 20-, 50-, or 80-nm washed Ag-nps. All three assays also showed that the 20-, 50-, and 80-nm unwashed Ag-nps contributed to a decrease in HEK viability 24 hr after exposure to the 0.34–1.7 μg/mL concentrations, but there was no size-dependent decrease in viability. However, the difference in toxicity between the unwashed and washed Ag-nps is inferred to be due to the presence of contaminants in the unwashed solution such as formaldehyde, which has shown to have cytotoxic effects on cell culture (Ku and Billings 1984
). In the present study, these residual contaminants were removed by the fifth washing step, as indicated by the lack of cell death after exposure to the 5, 10, 15, and 20 washing supernatants. We also found that Ag-nps of different sizes, surface conditions, and synthesis methods are all internalized into membrane-bound vacuoles in HEKs, without a decrease in viability, after 24 hr.
cell line studies have shown that 25 μg/mL of 25-nm Ag-nps in murine neuroblastoma cells decreases mitochondrial function and causes the production of reactive oxygen species that could potentially contribute to neurodegenerative diseases (Schrand et al. 2008
). A significant decrease in mitochondrial function has been shown in hepatic cells after single exposures to 15- and 100-nm Ag-nps at concentrations ranging from 5 to 50 μg/mL (Hussain et al. 2005
), in germline stem cells exposed to 10 μg/mL of 15-nm Ag-nps (Braydich-Stolle et al. 2005
), and in HEKs and fibroblasts after exposure to approximately 15 μg/mL Ag-nps extracted from commercially available Ag-based wound dressings with Ag content ranging from 13 to 934 μg/cm2
(Burd et al. 2007
). Interactions between Ag-nps ranging in size from 7 to 20 nm and human skin carcinoma cells showed a decrease in mitochondrial function and the onset of apoptosis at concentrations of 0.78 μg/mL and 1.56 μg/mL, respectively (Arora et al. 2008
). The toxic concentrations of the 20-, 50-, and 80-nm unwashed Ag-nps (0.34–1.7 μg/mL) are slightly more sensitive in HEKs compared with in vitro
toxicity studies conducted by others in different cell lines, although it is important to consider such factors as agglomeration, surface conditions, size, cell lines, and interactions with the assay dye products when comparing across studies.
Keratinocytes produce proinflammatory cytokines, such as IL-8, IL-6, TNF-α, and IL-1β, that serve as mediators for inflammatory and immunologic reactions in skin exposed to irritants (Allen et al. 2000
; Barker et al. 1991
; Corsini and Galli 2000
; Grone 2002
; Monteiro-Riviere et al. 2003
; Nickoloff et al. 1991
). Although different toxicants may elicit different responses in HEKs, studies in our laboratory have shown cytokine release by HEKs in response to jet fuel exposure (Allen et al. 2000
; Chou et al. 2002
; Monteiro-Riviere et al. 2003
), multiwalled carbon nanotubes (Monteiro-Riviere et al. 2005
), 6-aminohexanoic acid-functionalized single-walled carbon nanotubes (Zhang et al. 2007
), fullerenes (Rouse et al. 2007
), and QDs (Ryman-Rasmussen et al. 2006
; Zhang et al. 2008
). The inflammatory potential of Ag-nps was confirmed by the increases in IL-1β, IL-6, IL-8, and TNF-α detected in the media from HEK cell cultures exposed to 0.34 μg/mL of each of the unwashed Ag-nps.
Nanomaterials are also capable of being internalized into cells and penetrating through skin; QD-621 have the ability to penetrate into the intercellular lipid layers of the stratum corneum of porcine skin (Zhang et al. 2008
); QD-565 and QD-655, with diverse physiochemical properties, have been shown to penetrate into the dermis of abraded skin (Zhang and Monteiro-Riviere 2008
); and derivatized fullerenes are localized within the intercellular space of the stratum granulosum layer of flexed excised porcine skin (Rouse et al. 2006
). Topical application of 26–30 nm zinc oxide in a sunscreen formulation on in vitro
human skin localized nanoparticles in the upper stratum corneum with minimal penetration (Cross et al. 2007
), and microfine zinc oxide, with a mean size of 80 nm, and agglomerates of titanium dioxide < 160 nm did not penetrate the porcine stratum corneum layer of in vitro
static diffusion cells (Gamer et al. 2006
Porcine skin is an excellent model for studying penetration of human skin because its thickness and absorption rates are comparable to those of human skin (Bronaugh et al. 1982
; Monteiro-Riviere and Riviere 1996
; Reifenrath et al. 1984
). In the present study, we were surprised that after 14 consecutive days of topical dosing, the Ag-nps did not cause any macroscopic irritation, although the gray appearance of the skin due to the deposition of Ag-nps may have masked any subtle signs of erythema. When viewed microscopically, focal inflammation and edema increased with an increase in Ag-nps concentration. The highest concentration consistently caused epidermal hyperplasia with elongated extension of rete pegs down into the dermis, which is typical of a chronic irritation reaction as reported with exposure to jet fuels (Monteiro-Riviere et al. 2001
; Muhammad et al. 2005
). TEM depicted the localization of Ag-nps only in the superficial layers of the stratum corneum, which was similar to results found in a static cell diffusion study (Larese et al. 2009
), and suggests that ionic flux into the epidermis could attribute to focal inflammation. Many Ag-nps that were not bound to the skin were washed away during both the light and electron microscopy processing techniques, yet their location is confirmed with other nanoparticles that were not shown to penetrate into the deeper epidermis (Cross et al. 2007
; Zhang and Monteiro-Riviere 2008