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Gold Nanoparticles

Gold Nanomaterials in Consumer Cosmetics Nanoproducts: Analyses, Characterization, and Dermal Safety Assessment Mingjing Cao, Jiayang Li, Jinglong Tang, Chunying Chen,* and Yuliang Zhao*

Establishment of analytical methods of engineered nanomaterials in consumer products for their human and environmental risk assessment becomes urgent for both academic and industrial needs. Owing to the difficulties and challenges around nanomaterials in complex media, proper chemical separation and biological assays of nanomaterials from nanoproducts needs to be firstly developed. Herein, a facile and rapid method to separate and analyze gold nanomaterials in cosmetics is reported. Gold nanomaterials are successfully separated from different facial or eye creams and their physiochemical properties are analyzed by quantitative and qualitative state-of-the art techniques with high sensitivity or high spatial resolution. In turn, a protocol including quantification of gold by inductively coupled plasma mass spectrometry and thorough characterization of morphology, size distribution, and surface property by electron microscopes, atomic force microscope, and X-ray photoelectron spectroscope is developed. Subsequently, the preliminary toxicity assessment indicates that gold nanomaterials in cosmetic creams have no observable toxicity to human keratinocytes even after 24 h exposure up to a concentration of 200 µg mL−1. The environmental scanning electron microscope reveals that gold nanomaterials are mostly attached on the cell membrane. Thus, the present study provides a full analysis protocol for toxicity assessment of gold nanomaterials in consumer products (cosmetic creams).

1. Introduction Due to the good performance of engineered nanomaterials (ENMs) as well as their commercial interests, more and more consumer products containing ENMs which are called nano-

M. Cao, Dr. J. Li, J. Tang, Prof. C. Chen, Prof. Y. Zhao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience National Center for Nanoscience and Technology of China University of Chinese Academy of Sciences No. 11, Beiyitiao, Zhongguancun, Beijing 100190, P. R. China E-mail: [email protected]; [email protected] DOI: 10.1002/smll.201601574

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products have been emerging in the marketplace. According to a survey launched by the Project on Emerging Nanotechnologies, the updated Nanotechnology Consumer Products Inventory now contains 1814 consumer products from 622 companies that have been introduced to the marketplace since 2005.[1] The nanoproducts being sold in the market include cosmetics, clothing, food, electronic equipment, and so forth. Among the ENMs contained in consumer products, there are nano-sized metals, metal oxides, quantum dots, polymers, carbon nanomaterials. In addition, cosmetic industry is a popular field in which ENMs are applied.[2,3] Cosmetics including sunscreens and personal care products make up 34% of the updated Nanotechnology Consumer Products Inventory. In addition to the two common types of ENMs (TiO2 and ZnO) used in sunscreens, other materials in nanosize have also begun to be applied to cosmetics, which include

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metals (gold and silver), metal oxides, liposomes, nanocapsule, solid lipid nanoparticles, nanocrystals, dendrimers, cubosomes, niosomes, and fullerenes.[2] Since the ENMs with novel properties can enhance cosmetics properties, such as better skin penetration, transparency, unique texture, color, solubility, and enhanced UV protection, their use in cosmetic field have attracted great attentions over the past few years. For instance, one famous cosmetic brand whose cost as much as $1398 for a face mask contains gold nanomaterials in its formula and claims that these nanoparticles have antiinflammatory and antioxidant properties, drive tissue regeneration, restore skin elasticity, and reduce the signs of stress and aging. For another example, L’Oreal which is the world’s largest cosmetics company devotes around $600 million dollars accounting for 3.5% of its total revenues to patents about nanocosmetics.[4] However, nanoproducts often raise increasing safety concerns than others because of smaller size of ENMs and bigger possibility of entering human body.[5,6] Inadequate information about health impact and safety of ENMs in cosmetics industry has been arising considerable safety concerns.[4,7–10] According to an estimation made by Keller group in 2014 in the US,[11] ZnO nanoparticles, with the rate of 1800–2100 metric tons year−1, and TiO2 nanoparticles, with the rate of 870–1000 metric tons year−1, were released into the environment from the use of personal care products. Friends of the Earth Australia claimed that nanomaterials pose new threats to human health and called for a halt to the use and sales of nano-based cosmetics until the risks associated with the ENMs in cosmetics are properly assessed and their safety are established.[12] Moreover, US FDA and EU all made regulations for the safety of nanomaterials in cosmetic products.[13,14] Quite a few of researches have been carried out on how to characterize TiO2 and ZnO nanoparticles in sunscreens and to assess the toxicity and health impact of nano-sunscreens.[15–24] Many in vitro studies demonstrated that more reactive oxygen species which can induce cytotoxicity and DNA damage could be generated by TiO2 and ZnO nanoparticles in sunscreens especially when they are exposed in the UV light.[18,20] Similar studies aiming at gold nanomaterials-contained cosmetics have yet to be located in primary literatures. However, Paller and co-workers from Northwestern University have found that after applying gold nanoparticles to mouse and human skin models, the vast majority of the nanoparticles penetrate through several layers of skin within a few hours.[25] Besides, given the fact that there are 12 cosmetic products containing gold nanomaterials in the updated Nanotechnology Consumer Products Inventory[1,26] including nano gold whitening mask, daily facial, and eye creams and no studies focusing on gold nanomaterials contained in cosmetics, it is badly necessary to fill the gap of assessing the risk of gold nanomaterials in cosmetics. A large number of studies about the toxicity assessment of nanomaterials have been reported, from which we can conclude that the safety of nanoparticles is largely affected by their physicochemical properties, such as morphology, shape, size, chemical composition, and surface chemistry.[27–32] Besides, in the report entitled “Scientific Committee on Consumer Products (SCCP) Opinion on Safety of Nanomaterials small 2016, 12, No. 39, 5488–5496

in Cosmetic Products,” SCCP regulated several physical and chemical characteristics for risk assessment and emphasized the role of physicochemical parameters of nanomaterials in the safety evaluation.[14] For instance, the surface charge plays a significant role in the systemic response, while the shape and surface modification determine the uptake, transport, and accumulation in systemic circulation. Therefore, in our study the physicochemical characteristics of gold nanomaterials contained in cosmetics are of great importance to assess their safety. Nonetheless, the matrices in which gold nanomaterials are embedded are so complicated and indistinguishable that in situ characterization seems impossible. For example, Lorenz et al. characterized the TiO2 in commercial sunscreens after dissolved the sunscreens with water and methanol by using scanning electron microscope,[19] but it was difficult to distinguish the particles one by one and make a true statistic of size distribution. In order to characterize and conduct the further safety assessment of ENMs, separation of ENMs from nanocosmetics is taken for granted. Several methods to separate ENMs from complex matrices have been provided over the last years, such as cloud point extraction, field-flow fractionation (FFF), ultrafiltration, chromatographic techniques, and capillary electrophoresis.[33,34] FFF and hydrodynamic chromatography were used to separate TiO2 and ZnO nanoparticles from sunscreens.[16,35–37] However, the aggregation of nanoparticles may occur in the column so that the morphology and size distribution may not be obtained precisely. Additionally, although FFF can be coupled with other analytical tools such as mass spectroscopy and UV–vis spectrophotometer to provide more information, the sample treatment is largely dependent on the type of original samples and should be optimized through many attempts. Consequently, a facile yet feasible separation method is crucial to accurately characterize physicochemical properties of gold nanomaterials in cosmetics. Due to the difficulties and challenges around analysis of ENMs in complex media and the limitations of each available technique, a suite of protocols need to be developed for a sample in order to produce reliable data. The strategy we proposed in present study is to establish a simple, effective, and reliable method to separate and characterize the gold nanomaterials in commercial cosmetics (see Scheme 1). Firstly, total gold amount in creams was quantified by inductively coupled plasma mass spectrometry (ICP-MS) after wet digestion. And then the gold nanomaterials were separated from cosmetic complex ingredients by solvent extraction procedure. Subsequently, analyses of physicochemical parameters and preliminary cytotoxicity test of extracted gold nanomaterials were carried out, which is valuable and essential for the safety assessment of ENMs in cosmetics.

2. Results and Discussion 2.1. Selected Cosmetic Creams Containing Gold Nanomaterials Four typical commercial cosmetic creams of different leading international brands were chosen to be studied and labeled


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Scheme 1. Thorough characterization, quantification, and identification of gold nanomaterials in consumer cosmetic products and their cytotoxicity evaluation.

as Cream-1, Cream-2, Cream-3, and Cream-4. The four cosmetics are facial creams for moisturizing, brightening, and firming purposes (Cream-1, Cream-3) and eye creams possessing the functions of anti-wrinkle and removing darkness and puffiness for eye perfection (Cream-2, Cream-4), whose ingredients all include gold which are labeled as “nanoscale.” It is claimed that these creams containing gold nanomaterials have anti-inflammatory and antioxidant properties, drive tissue regeneration, restore skin elasticity, and reduce the signs of stress and aging.

2.2. Quantiﬁcation of Total Amounts of Gold and Other Elements The content of five elements (i.e., Au, Ti, As, Pb, Hg) determined by ICP-MS after wet digestion is reported in Table 1. Data is expressed in mg element (kg cream)−1. Three parallel samples were prepared for each cream, whereas the repeatability is quite satisfied. The limit of quantification (LOQ) of Au was 140.03 pg mL−1 when the signal/noise ratio was 10. The limit of detection (LOD) of Au which reflects the ability of the instrument was calculated as three times standard deviation of eight repetitive measurements of sample blanks and the result was 13.86 pg mL−1. Similarly, LOQ of Ti, As, Pb, Hg were figured out, which were 27.54, 3.81, 2.43, and 2.79 ng mL−1, and LOD of Ti, As, Pb, Hg were 2.61, 0.61, 1.23, and 0.62 ng mL−1, respectively. In addition, the well-dispersed cream samples were spiked by standard gold solution and then subjected for the wet digestion. The purpose of the spiked samples is to cal-

culate analyte recovery. The recovery of Au from cream was quite high as (96.53 ± 6.35)%. To our surprise, the content of Ti in Cream-3 and Cream-4 was several orders of magnitudes higher than that in Cream-1 and Cream-2, while Cream-1 and Cream-2 contained higher amount of gold. Consequently, we chose Cream-1 and Cream-2 to carry out the further separation and characterization of gold nanomaterials. The EU, US, and China regulations on the standard of hazardous substances in cosmetics ingredients (i.e., As, Hg, and Pb) specify the limit of As, Hg, and Pb as 10, 1, and 40 mg kg−1, respectively (for the eye products, the limit of Hg is 65 mg kg−1).[38–40] Thus, the content of Hg in Cream-1 was slightly excessive, while all the others met the specification.

2.3. The Yield to Obtain Gold Nanomaterials from Cosmetic Creams Due to the complex ingredients of cosmetic creams, it is difficult to characterize the primary gold nanomaterials in situ. In order to analyze the gold nanomaterials, it is necessary to separate gold nanomaterials from intricate system. However, separation of the gold nanomaterials is not an easy task. Firstly, some natural extracts are included in the ingredients, such as aloe barbadensis and root extractives. In addition, formulas of cosmetic creams also contain non-water-soluble organics, such as polydimethylsiloxane and esters. Secondly, the physical and chemical properties of gold nanomaterials should be kept during the process of separation. These factors all result in the difficulty to separate the gold nanomaterials from cosmetic creams.

Table 1. Determination of gold and other elements in the four brands of cosmetic creams. (Data is reported as mean ± SD, n = 3). Sample

Sample mass [mg]

Au [mg kg−1]

Ti [mg kg−1]

As [mg kg−1]

Pb [mg kg−1]

Hg [mg kg−1]

Cream-1

100

82.05 ± 5.19

6.99 ± 3.63

0.49 ± 0.12

0.75 ± 0.17

3.38 ± 0.60

Cream-2

100

78.86 ± 9.14

24.57 ± 0.97

0.37 ± 0.22

7.64 ± 0.88

1.45 ± 0.26

Cream-3

100

0.75 ± 0.05

2547.76 ± 238.85

0.14 ± 0.02

5.24 ± 1.16

0.18 ± 0.03

Cream-4

100

0.051 ± 0.006

911.50 ± 81.35

0.036 ± 0.009

0.064 ± 0.044

0.16 ± 0.038



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It has been demonstrated that solvent extraction method could keep the original state of nanomaterials in complex system in the process of extraction.[41] The method used to separate gold nanomaterials from cosmetic creams is based on the solubility difference of ingredients in hexane, methanol, and deionized water. Detailed steps are described in the Experimental Section and Supporting Information. Eventually, the precipitates were the desired products and redispersed in deionized water for future use. Supernatants in each step and the final sediment were collected for the determination of the extraction efficiency by measuring the content of gold with ICP-MS. The results indicate that the supernatants of each step contained very few gold nanomaterials and the vast majority of gold exist in the sediment of final step. The yield of the separation method was figured out, which were 99.8% for Cream-1 and 92.4% for Cream-2. The high yield of separation demonstrated that separation method of gold nanomaterials from cosmetic creams was efficient and satisfied. Besides gold nanomaterials, other metal nanomaterials, such as silver and platinum nanoparticles, and metal oxides nanomaterials have been used in cosmetic products for their unique activities.[2] The facile and effective method could also be applied to separate these nanomaterials from cosmetics system.

2.4. Characterization of the Separated Gold Nanomaterials from Cosmetic Creams It has been demonstrated that particle size, shape, morphology, and surface property are the main factors influencing the toxicity of nanoparticles. Electron microscope and scanning probe microscope techniques are famous for high spatial resolution of atomic scale and powerful tools to determine the size, shape, and morphology.[42] Consequently, we made further efforts to characterize particle size

distribution, physical form and morphology by transmission electron microscope (TEM), scanning electron microscope (SEM), and atomic force microscope (AFM). Given the fact that gold nanomaterials are present in the form of tiny foils and disappear after rubbed on skin, the separated gold nanomaterials were ultrasonicated in deionized water until the tiny gold foils disappeared to simulate the process of rubbing cream on skin before the physicochemical properties were characterized. Figure 1A,E and Figure S2 in the Supporting Information show TEM images of the gold nanomaterials separated from Cream-1 and Cream-2. The elemental analyses of the TEM images were determined by energy dispersion X-ray spectroscopy (EDS) which are shown in Figure 1B,F. TEM images and EDS spectra suggested that the gold nanomaterials in Cream-1 and Cream-2 were of slightly regular sheet shape. In order to quantify the size distribution, more than 100 fragments in TEM images from each sample were measured by ImageJ. Figure 1C,D,G,H shows the length and width distribution histograms of the gold nanomaterials separated from Cream-1 and Cream-2, respectively. Statistical results indicate that gold nanomaterials in Cream-1 have the length of (1.23 ± 0.59) µm and the width of (0.70 ± 0.28) µm, while most of the gold nanomaterials in Cream-2 have the length of (4.59 ± 1.69) µm and the width of (3.13 ± 1.33) µm. The SEM images and EDS spectra shown in Figure 2 suggest that the physical forms of gold nanomaterials in Cream-1 (Figure 2A–C) and Cream-2 (Figure 2D–F) were ultrathin flakes in shape, which has an agreement with the results of TEM. As we can find from the above results, the length and width of the flakes are all in the range of several micrometers. Why do the manufacturers label the gold in nanoscale? Subsequently, we made further efforts to measure the thickness of the flakes by using AFM. Figure 3 shows the AFM images and demonstrate the thickness distribution of gold

Figure 1. The TEM images, EDS spectra, and size distribution of gold nanomaterials separated from cosmetic creams. A) Representative TEM image, B) EDS spectrum and C,D) size distribution histogram obtained from 133 fragments of gold nanomaterials separated from Cream-1. E) Representative TEM image, F) EDS spectrum and G,H) size distribution histogram obtained from 162 fragments of gold nanomaterials separated from Cream-2.

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Figure 2. SEM image and EDS spectra of gold nanomaterials separated from A–C) Cream-1 and D–F) Cream-2. B and E are the magnified images.

nanomaterials in Cream-1 (Figure 3A,C) and Cream-2 (Figure 3B,D). Among them, 39.3% of the gold nanomaterials in Cream-1 were less than 10 nm and 49.1% were between 10 and 60 nm, whereas around 74.3% of the gold nanomaterials in Cream-2 were less than 100 nm in the thicknesses. According to the definition of nanoparticles made by the International Organization for Standardization, it is more appropriate to use the term “nanosheets” instead of “nanomaterials.” Surface property is another important factor dominating the toxicity of nanoparticles. It was determined by

measuring the surface charge using a Malvern ZetaSizer and the valence state of gold with X-ray photoelectron spectroscope (XPS). The zeta potentials of gold nanosheets separated from Cream-1 and Cream-2 were (−1.28 ± 4.07) and (−2.91 ± 6.98) mV, respectively, which showed the nearly neutral surface charge of gold nanosheet present in Cream-1 and Cream-2. Figure 4 reveals the surface component and valence state of gold nanosheets separated from Cream-1 (black line) and Cream-2 (red line) obtained by XPS. The XPS spectra in Figure 4A,C indicates that there were no other elements besides Au on the surface of the gold nanosheets.

Figure 3. AFM images and the thickness distribution of gold nanosheets separated from cosmetic creams. A) Representative AFM image and C) thickness distribution histogram obtained from 113 fragments of gold nanosheets separated from Cream-1. B) Representative AFM image and D) thickness distribution histogram obtained from 113 fragments of gold nanosheets separated from Cream-2.



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Figure 4. XPS analyses of the gold nanosheet. A,C) XPS spectra and B,D) the Au4f peaks at high resolution of gold nanosheets separated from Cream-1 (black line) and Cream-2 (red line).

Furthermore, from the spectra of Au4f in Figure 4B,D, we found that the difference between Au 4f5/2 and 4f7/2 peaks was 3.65 eV which was as same as that of the Au in zero valence state.[43] In brief, the results of XPS indicated that there may not be any modifiers on the surface of gold nanosheets, which is in accord with the results of zeta potential.

2.5. Cytotoxicity Assessment of the Separated Gold Nanosheets in Cosmetic Creams Generally, cosmetic products enter human body via transcutaneous administration. For this reason, human keratinocytes (HaCaT cells) were chosen to estimate the potential dermal toxicity of gold nanosheets. We designed the experimental concentrations based on the assumptions that average daily usage of face creams and eye creams are 2.0 and 0.5 g, respectively.[44] More details about the development of the calculation equations can be found in Supporting Information. The final calculation results are shown in Table S2 in the Supporting Information. The experimental concentrations of gold nanosheets separated from Cream-1 and Cream-2 were set as 0, 10, 20, 40, 80, 100, 150, 200 µg mL−1 and the maximum one can be equal to about eight months’ consumption of Cream-1 and six months’ consumption of Cream-2. The toxicity was assessed quantitatively by measuring the cell viability and qualitatively by staining the dead cells with propidium iodide (PI) small 2016, 12, No. 39, 5488–5496

fluorescent dyes. Figure 5A,B shows that gold nanosheets separated from Cream-1 and Cream-2 did not induce any evident loss of cell viability even after 24 h of exposure up to a concentration of 200 µg mL−1. The fluorescent images of the stained dead cells incubated for 24 h at different concentrations in Figures S1A and S1B in the Supporting Information were another evidence of no observable toxicity of the gold nanosheets in cosmetic creams to HaCaT cells. Our results are in line with previous findings related to the toxicity of gold nanomaterials. Our group has developed a theranostic nanoplatform based on gold nanorods and demonstrated the safety of gold nanorods.[45,46] Besides, Bartczark et al. investigated the cytotoxicity of gold nanospheres, nanorods, hollow gold spheres, and core/shell silica/gold nanocrystals to endothelial cells and found noncytotoxic effects.[47]

2.6. Characterization of the Interaction between Gold Nanosheets and Human Keratinocytes by Using Environmental Scanning Electron Microscope (ESEM) ESEM is an imaging technique affording higher resolution than conventional optical microscope, which was used for further investigation of direct interaction or possible penetration of gold nanomaterials with human keratinocytes at high spatial resolution of tens-of-nanometer scale.[48–50] There is weak interaction between gold nanosheets and human keratinocytes as shown in ESEM images of Figure 6. Cellular



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of (4.59 ± 1.69) µm and the width of (3.13 ± 1.33) µm with the thicknesses less than 100 nm (74.3%). Moreover, zeta potential and XPS indicated that there may not be any modifiers or coating on the surface of gold nanosheets. Furthermore, assessment of cell viability demonstrated gold nanosheets in cosmetic creams were relatively safe for HaCaT cells under our experimental conditions. Additionally, ESEM revealed that gold nanosheets in cosmetic creams were only attached onto the surface of human keratinocytes. However, our human body is a systemic organism and the possibility of gold nanosheets passing through human skin barrier to enter into other organs need further in vivo evaluation in the future. Thus, the present studies proposed a simple but effective and reliable protocol for separating and characterizing gold nanomaterials from cosmetic creams and also provide new insights into the comprehensive understanding of physicochemical properties and toxicity assessment of cosmetic creams containing nanomaterials.

4. Experimental Section

Figure 5. Cell viability of gold nanosheets in A) Cream-1 and B) Cream-2 to HaCaT cells after incubation of 24 h at different concentrations (0, 10, 20, 40, 80, 100, 150, 200 µg mL−1).

morphology of the cells treated with gold nanosheets in Cream-1 and Cream-2 were different from those in control group. Gold nanosheets were attached to the surface of HaCaT cells, while the cellular surface was stretched by the gold nanosheets. We deduce the morphological variation may result from the interactions between gold nanosheets and sulfur in cell membrane. These results suggest that the relatively big gold nanosheets cannot enter into the cells, which may be the reason of low cytotoxicity.

3. Conclusion In summary, we developed a practical protocol including separation, quantification, and characterization of gold nanomaterials in commercially available cosmetic creams. The total content of Au in Cream-1 and Cream-2 was 82.05 and 78.86 mg kg−1, respectively. A sequential solvent extraction gives >92% extraction efficiency of gold nanomaterials from the cream products. Thorough characterization of morphology, size distribution, and surface property by electron microscopes and atomic force microscope indicated that most of gold nanomaterials in Cream-1 have the length of (1.23 ± 0.59) µm and the width of (0.70 ± 0.28) µm with thickness of less than 10 nm (39.3%) and 10–60 nm (49.1%), while most of the gold nanomaterials in Cream-2 roughly have the length


Materials and Reagents: The four cosmetic creams of different brands were purchased from the supermarkets. Chemical reagents used in this work were concentrated nitric acid (70%, MOS grade), hydrogen peroxide (30%, MOS grade), hydrofluoric acid (40%, MOS grade), hydrochloric acid (37%, MOS grade), hexane (95%, AR), methanol (99.5%, AR), and deionized water. Extraction solvents, i.e., methanol and hexane, have negligible contaminant metals. All the chemicals were commercially available and used without further purification. Deionized water with resistivity of 18.2 MΩ was obtained from a Millipore Milli-Q plus system. HaCaT cells were purchased from American type culture collection. Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories in Japan. Dulbecco’s modified eagle’s medium, trypsin, and fetal bovine serum were purchased from Wisent, China. PI were purchased from Donjindo (Beijing, China). Separation and Measurement of Gold Nanomaterials in Cosmetic Creams: Measurement of gold and other elements in cosmetic creams was accomplished by ICP-MS. Method of wet digestion and operating conditions are described in Table S1 in the Supporting Information. In consideration of the species of ingredients, nonpolar hexane and polar methanol and water were chosen to dissolve the components except for gold, based on which a facial and effective solvent extraction method was developed. The procedure of preparation is shown in Scheme S1 in the Supporting Information, and details are described in the Supporting Information. Characterization of Gold Nanomaterials Separated from Cosmetic Creams: After gold nanomaterials were separated from cosmetic creams, morphology, size distribution, surface charge, and chemical valence states were characterized. Morphology and size were characterized by a FEI Tecnai G2 20 S-TWIN TEM (FEI Corporation, US) operating at 200 kV. The presence of Au element and successful separation of gold nanomaterials were confirmed by EDS. The shape and morphology were also determined by a FEI NOVA Nano SEM 430+ EDS (FEI Corporation, US). The morphology and thickness characterization were performed by a Veeco Dimension 3100 AFM (Veeco Corporation, US). Zeta potentials were measured


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Figure 6. ESEM images and corresponding magnified images of HaCaT cells of A,B) control group, of cells treated with gold nanosheets in C,D) Cream-1 and E,F) Cream-2. Scale bars are shown in the images.

by using a Malvern ZetaSizer Nano ZS instrument (Malvern Instruments, UK) in water solution at room temperature. Surface properties, like the component on the surface of gold nanomaterials and the valence state of gold element, were determined by a Thermo Fisher ESCALAB250Xi XPS (Thermo Fisher Scientific, China). The detailed sample preparations are described in Supporting Information. Cytotoxicity Assessment of Gold Nanomaterials Separated from Cosmetic Creams: HaCaT cells were chosen to estimate the potential skin toxicity of gold nanomaterials in cosmetic creams. The cytotoxicity of gold nanomaterials in cosmetic creams was evaluated quantitatively by assessing cell viability with a CCK-8 kit and qualitatively by staining the dead cells with PI fluorescent dye

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detected by a laser confocal microscope (Ultra VIEW VoX, PerkinElmer, United States). The interaction between gold nanomaterials and human keratinocytes was characterized by using ESEM (FEI Quanta 200 FEG, US). The details are described in Supporting Information.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.



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Acknowledgments This work was supported by the Ministry of Science and Technology of China (2016YFA0201600), the National Natural Science Foundation of China (21320102003, 21403043, 91543206), Major Project of the National Social Science Fund (Grant No. 12&ZD117) “Ethical issues of high-tech” and the National Science Fund for Distinguished Young Scholars (11425520).

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Received: May 10, 2016 Revised: July 6, 2016 Published online: August 26, 2016

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