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Safety of the application of nanosilver and nanogold in topical cosmetic preparations
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Safety of the application of nanosilver and nanogold in topical cosmetic preparations
T
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Jolanta Pulit-Prociaka, , Aleksandra Grabowskaa, Jarosław Chwastowskia, Tomasz M. Majkab, Marcin Banacha a
Faculty of Chemical Engineering and Technology, Institute of Chemistry and Inorganic Technology, Cracow University of Technology, Warszawska 24, Cracow 31-155, Poland b Faculty of Chemical Engineering and Technology, Department of Chemistry and Technology of Polymers, Cracow University of Technology, Warszawska 24, Cracow 31155, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanosilver Nanogold Oil in water emulsions Biocidal properties Model dermal membrane Permeation studies
The safety of the use of cosmetic preparations with silver or gold nanoparticles was assessed. This study describes the methodology and results of research involving the generation of suspensions of silver and gold nanoparticles and creams with added silver or gold at concentrations of 20, 65, 110, 155, and 200 mg/kg. The silver nanoparticles ranged from 8 to 140 nm, and the gold nanoparticles, measured 13–99 nm. The sizes were determined using dynamic light scattering. The presence of metallic nanoparticles in the obtained oil-in-water emulsions was confirmed with UV–vis spectroscopy and transmission electron microscopy with an X-ray scattering spectrometer (TEM-EDX). Additionally, based on the TEM-EDX results, it was possible to analyse the distributions of the silver nanoparticles in the tested cosmetic emulsions. Microbiological tests showed that both the silver and gold nanoparticle emulsion possessed satisfactory fungicidal properties. Based on viscosity curves, the lowest estimated yield limits were achieved by the reference cream and the creams with the gold and silver nanoparticles at concentrations of 20 and 65 mg/kg, respectively, which improved their consistencies and distributions on the skin. The best appraisals from the respondents in terms of consistency, absorption, oiling, colour, and smell were received for the emulsion containing 200 mg/kg gold nanoparticles. The worst assessment in terms of uniformity, colour, and smell were obtained for the emulsion with 200 mg/kg silver nanoparticles. However, the most important aspect of this study was the assessment of the permeabilities of the metallic nanoparticles through imitation skin in the form of dermal membranes. The highest permeabilities were confirmed for the creams with metallic nanoparticles present at 110-–200 mg/kg. This permeability is an issue of concern given the toxic properties of metallic nanoparticles for living organisms.
1. Introduction Throughout history, silver has been used as a raw material for the production of coins, silverware, and other decorative items. This noble metal has also been used in medicine and as an antibacterial agent. To prevent diseases and other illnesses, the ancient Greeks drank water from silver cups [1]. Due to dynamically developing nanotechnology, silver nanoparticles are currently used in many applications. These nanoparticles play a particularly important role as a component of disinfecting liquids and fungicides [2]. Nanosilver has also been added to dressings, bandages, anti-infectious agents, catheters, and implants (e.g., artificial heart valves). Soaps with nanosilver are widely used in
the treatment of acne as well as dermatitis because they regulate sebaceous glands. In the construction industry, nanosilver particles are added to insulation, roofing, and anti-bacterial paints [3]. The enrichment of textile products with silver nanoparticles also inhibits the development of pathogenic microorganisms [4]. In ancient times, people used gold due to faith in its miraculous healing power. Currently, nanogold is being used in various fields of science and technology, such as electrical engineering, catalysis, cosmetology, medicine, and pharmacology [5]. Gold nanoparticles are used as a carrier for anti-cancer drugs and in thermotherapy targeting cancer cells. Gold nanopigments have also been used as contrast agents in computed tomography and in infrared thermal imaging.
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Corresponding author. E-mail addresses: [email protected] (J. Pulit-Prociak), [email protected] (A. Grabowska), [email protected] (J. Chwastowski), [email protected] (T.M. Majka), [email protected] (M. Banach). https://doi.org/10.1016/j.colsurfb.2019.110416 Received 1 March 2019; Received in revised form 26 July 2019; Accepted 31 July 2019 Available online 01 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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type of research were verified. This study also analysed the release of nanoparticles and their potential for penetration through a model dermal membrane.
Additionally, gold in the form of spheroidal nanoparticles is used as a carrier of RNA and in advanced medical diagnosis. Gold nanopigments exhibit high photoluminescence that is used to create three-dimensional images in vivo. In the cosmetics industry, manufacturers are adding the “nano” prefix to cosmetic names with increasing frequency, even when the presence of nanoparticles has not been confirmed and the term “nano” is intended to attract more customers. This widespread influence of nanotechnology in the cosmetics industry results from the fact that formulations containing nanometric particles have more desirable properties, i.e., deeper colours, better transparencies, and increased solubility. Nanoparticles in cosmetic products can act as active substances, carriers, consistency-improving substances, effectiveness-enhancing substances, and antimicrobial agents. Nanoparticles are added to permanent make-up formulations as well as anti-aging creams and toothpastes [6]. Due to their antimicrobial properties, nanosilver and nanogold have quickly entered into everyday use. Silver and gold nanoparticles in particular have found uses as active ingredients in creams, soaps, shower gels, masks, and other cosmetics with biocidal activities. Nanosilver shampoos are recommended for the treatment of dandruff, itchy scalp, and excessive sebum production. Antiperspirants with silver nanoparticles are designed to eliminate the unpleasant odour of sweat. Manufacturers of intimate hygiene fluids with silver and copper nanoparticles indicate that the products accelerate the regeneration of minor wounds and inhibit infections. The producers of mask with gold nanoparticles claim that nanogold contributes to scattering its active ingredients [7]. There are also available creams with nanogold particles. Thanks to nanotechnology, the precious elements reach the cellular level where they play the role of the ultimate healing and preserving force [8]. A toothpaste that contains nanoparticulate gold is also available. Thanks to the presence of nanogold, it provides an antibacterial effect, which is particularly desired in the treatment of the mouth [9].Studies have demonstrated that nearly all major cosmetic manufacturers draw on nanotechnology in the design of their products. One of the cosmetics giants that produces mainly colour cosmetics entered the NanoMarket in 2006 and offer a range of products containing nanoparticles. The world's largest cosmetics company, utilises approximately $600 million of its $17 billion revenue to patent the use of nanoparticles in cosmetic products [10]. Commercial products containing nanomaterials have become a substantial source of nanoparticles that are subsequently released into ecosystems and lead to the accumulation of nanoparticles in living organisms. A number of studies has assessed the toxicities of metallic nanoparticles, particularly metallic silver, to living organisms. Metallic silver nanoparticles have been found to cause discolorations of the bodies of Daphnia, and 100% mortality results from 48 h of contact with a suspension with a concentration of 200,000 mg/L [11]. Following contact with metallic nanosilver suspension, Eurasian perch exhibit reduced tolerance to hypoxia [12]. A zebrafish population treated with a suspension of metallic silver nanoparticles at a concentration of 1.18 mg/L showed 50% mortality, decreased heart rate, pericardial oedema, and general degeneration of the organisms [13]. Providing chickens with water containing metallic silver nanoparticles at a concentration of 12 mg/L has been noted to result in the accumulation of nanoparticles in the hepatocytes and central vein syndrome [14]. Considering the data showing the negative effect of nanoparticles on living organisms, it is important to account for the safety of their application in the production of various cosmetic preparations. The aim of this study was to develop recipes for cosmetic preparations containing silver or gold nanoparticles at different concentrations, and to assess their physicochemical and application properties. Experiments were performed to verify the antifungal properties of the obtained products. Due to the presence of various types of cutaneous mycoses in humans, the effects of the addition of nanoparticles to creams on their antimicrobial activities against model fungi that are routinely used in this
2. Materials and methods 2.1. Materials The following compounds were used in this study: cetearyl alcohol (p.p.a.), glycerol (p.p.a.), triethanolamine (≥ 99.0%), stearic acid (≥ 95.0%), potassium sorbate (p.p.a.), disodium EDTA (p.p.a.), soy lecithin (p.p.a.), silicone oil (p.p.a., 1000 cSt), castol oil (p.p.a.), tocopherol (p.p.a.), sodium carboxymethylcellulose (ultra-high viscosity, highly purified), chloroauric acid (≥ 99.999%), silver nitrate (≥ 99.9%), tannic acid (p.p.a.), sodium chloride (≥ 99.5%), potassium chloride (≥ 99.5%), calcium chloride (99.0%), and sodium hydroxide (≥ 98.0%). All compounds were obtained from Sigma-Aldrich (Germany). The following compounds were used in the microbiological tests: peptone, yeast extract, sucrose (≥ 98.0%), and agarose. All compounds were obtained from Sigma-Aldrich. The Aspergillus niger and Saccharomyces cerevisiae strains used in the study were provided by the National Collection of Yeast Cultures (England). All solutions were prepared using deionised water (Polwater, 0.18 μS). 2.2. Methods 2.2.1. Preparation of the silver and gold nanoparticle suspensions The suspensions of both silver and gold nanoparticles were obtained in a one-step chemical reduction process using tannic acid as a reducing and stabilising substance. First, aqueous solutions of silver nitrate (5.15 × 10−3 mol/L), chloroauric acid (2.82 × 10−3 mol/L), tannic acid (9.27 × 10−3 mol/L to generate nanosilver and 5.08 × 10−3 mol/ L to generate nanogold), and sodium hydroxide (1 mol/L) were prepared. To obtain suspensions of silver and gold nanoparticles at a concentration of 500 mg/L, 10 mL of tannic acid solution was added to a beaker containing 90 mL of silver nitrate or chloroauric acid solution. The mixture was stirred at ambient temperature (550 rpm). The molar ratio of the tannic acid to both the silver and gold ions was 0.2:1.0. Thereafter, sodium hydroxide was added to obtain a pH of 7, and the solution was stirred for an additional 10 min. The obtained suspensions were subjected to UV–vis spectrophotometry analysis. A single-beam spectrophotometer (Rayleigh UV-1800) connected to a computer loaded with spectra UVSoftware with 1.0-cm quartz cells was used. The spectra were obtained with the following instrumental parameters: wavelength range: 300–800 nm; scan speed: slow; sampling interval: 1.0 nm; and spectral slit width: 2 nm. Dynamic light scattering (DLS) was applied to determine the particle size. Zetasizer Nano ZS (Malvern Instruments Ltd.). The machine was operating with a He–Ne laser and connected to a computer loaded with the software Malvern Panalytical. The results were the means of triplicate runs, and at least 10 measurements were made in each run. The refractive indexes (RI) for silver nanoparticles of 0.240 and for gold nanoparticles of 0.180 were used. The viscosity of the samples were equal to 0.8872 cP, which is appropriate for water, i.e., the sample dispersant. The suspensions were vortexed and transferred to 2.0-cm disposable polystyrene sizing cuvettes. The actual concentrations of the silver and gold nanoparticles in the aqueous suspensions were analysed by means of inductively-coupled atomic emission spectroscopy (ICP-OES) that was conducted on a Plasm 40 apparatus (Perkin Elmer Co.) with a method detection limit of 0.1 μg/L. 2.2.2. Preparation of the oil-in-water (O/W) creams with metal nanoparticles The components of both the aqueous and oil phases were weighed (Table 1). The aqueous phase contained the required amount of 2
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Table 1 Numerical data for creams with metallic nanoparticles. EMULSION RECIPES FOR 50 G OF PRODUCT Compound (INCI name) Aqueous phase Aqua or metallic nanoparticle suspension* Glycerol Carboxymethylcellulose Triethanoloamine Potassium sorbate Disodium EDTA Oil phase Dimethicone Castol oil
Function
Content [%]
Mass [g]
solvent solvent, humectant, moisturising agent rheology modifier pH modifier preservative sequestrant
60 6
30.0 3.0
0.2 1.5 0.2 0.1
0.1 0.75 0.1 0.05
3 3
1.5 1.5
5 6 10
2.5 3.0 5.0
5 q.s.
2.5 q.s.
protecting and moisturising agent moisturising agent, transepidermal water loss (TEWL) reducer Lecithin emulsifier Stearic acid stabilising agent Cetearyl alcohol consistency regulator, fatty substance Tocopheryl acetate antioxidant Fragrance oil (hibiscus flower) fragrance *METALLIC NANOPARTICLES SUSPENSION USED TO PREPARE EMULSION WITH SILVER OR GOLD NANOPARTICLES Metal concentration in the final product [mg/kg] Volume of nanosilver or nanogold suspension at a concentration of 500 mg/L [mL] 20 2.0 65 6.5 110 11.0 155 15.5 200 20.0
Volume of water [mL] 28.0 23.5 19.0 14.5 10.0
RESULTS OF ANTIMICROBIAL PROPERTIES Aspergillus niger Time
Nano Ag (mg/kg) 20 65 110 155 Growth inhibition comparing to Reference Cream [%] 48 h 4 35 29 38 72 h 2 10 11 12 Saccharomyces cerevisiae Time Nano Ag (mg/kg) 20 65 110 155 Growth inhibition comparing to Reference Cream [%] 48 h 6 7 5 10 72 h 0 4 4 4
200
Nano Au (mg/kg) 20 65
110
155
200
36 11
15 2
21 7
22 10
20 10
200
Nano Au (mg/kg) 20 65
110
155
200
11 5
0.5 0
1 0
4 3.5
5 5
20 8
0 1
aqueous phase of pure water instead of a metallic suspension. The obtained emulsions were subjected to pH measurements and UV–vis and viscosity analyses (rotary rheometer, Haake Mars III, Thermo Scientific, Waltham, MA, USA). The studies were performed in a plate-plate arrangement with a 1-mm gap between the plates. The diameter of the plate was 60 mm. The tests were performed in a controlled-rotation system (control rate: CR) at 25 °C using a titanium rotor with a rotational speed of 10 rpm. The viscosities at the points of the two shear rates were measured; i.e., γ1 = 10 rpm and γ2 = 100 rpm. Flow curves expressing the dependencies of the shear stress (τ) and viscosity (η) on the shear rate (γ) were also obtained. The study was conducted in a range of shear speeds of 10–500 rpm. Additionally, microphotographs of the obtained samples were taken using high-resolution transmission electron microscopy with EDX and the elemental mapping mode. The study was performed using a Tecnai transmission electron microscope (TEM G2 F20X-Twin 200 kV, FEI). To identify the chemical elements, energy-dispersive X-ray spectroscopy (EDX, RTEM model SN9577, 134 eV, EDAX) was used. The spectra were recorded in the designated areas and along the lines. The samples were prepared as follows: a few milligrams of each cream were dispersed in ethanol (99.8%, anhydrous), then a drop of dispersion was applied on carboncoated copper mesh on which there were holes (Lacey type Cu 400 mesh, Plano). The dispersing agent was evaporated at ambient temperature.
previously prepared silver or gold nanoparticle suspension. The silver and gold nanoparticle suspensions were used at a suitable concentrations, i.e., 20, 65, 110, 155, or 200 mg/kg. The minimum effective concentrations of the metallic nanoparticles (both silver and gold) contained in the cosmetic products were equal to 10 mg/kg. Based on the state of art, cosmetic products contain silver or gold nanoparticles with concentrations typically vary from 10 ppm to 100 ppm [15–21] and even reach 35,000 ppm (3.5%) in some cases [22]. However, according to the Opinion on Colloidal Silver (nano) issued by the Scientific Committee on Consumer Safety (SCCS), the upper limit of silver concentration should not exceed 10,000 ppm (1%). This value was based on the reasonably foreseeable exposure conditions [23]. The authors intended to assess the properties and safety of the use of creams containing a wide range of concentrations of nanoparticles. Thus, the range from 20 to 200 ppm that corresponds with the typical effective contents of metallic nanoparticles was chosen. Both phases were heated in water baths (aqueous phase up to 70 °C and oil phase up to 72 °C) and mechanically stirred (600 rpm) for 20 min after the appropriate temperature was reached. The oil phase was introduced into the aqueous phase to form an emulsion. When the temperature dropped to 42 °C, tocopherol and a perfume oil (hibiscus flower) were added. After reaching room temperature, the emulsion was poured into a plastic container, and the product was maintained at room temperature. The reference sample was obtained using an 3
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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2.2.3. Assessments of the release of nanoparticles from topical formulations and analyses of their accumulations in model bodily fluids An aqueous receptor fluid, i.e., Ringer’s solution, was prepared as previously described [24]. The Strat-M® Membrane for Transdermal Diffusion Testing was used. The kit consisted of a magnetic stirrer with a heating plate, a water bath, a container with 10 mL of receptor fluid, and a glass column to which the model dermal membrane was attached. Three grams of the prepared creams and 5 mL of deionised water were placed in the column and allowed to adhere to the model membrane. The temperature of the water bath was set at 37 °C. After reaching the appropriate water temperature, the previously prepared set with the membrane was embedded in such a manner that the smooth surface of the membrane was in contact with the receptor fluid. The mixture was stirred at 200 rpm. The identification of the nanometals in the receptor fluid was performed with a UV–vis spectrophotometer after 48 h. The actual concentrations of silver and gold nanoparticles in the receptor fluids were analysed by means of inductively-coupled atomic emission spectroscopy (ICP-OES).
of 8 nm, and 23.2% of the nanoparticles had an average size of 140 nm. The average diameter of 98% of the gold nanoparticles was 99 nm, and 2% of the nanoparticles had an average size equal to 15 nm. The measured electrokinetic potential of both silver and gold nanoparticles was equal to -19,8 mV and -34,6 mV, respectively. The native shapes and dispersion levels of both the silver and gold nanoparticles are presented in Fig. 1C. The results are in line with the DLS analysis. The majority of the spherical silver nanoparticles with diameters below 10 nm can be seen. The larger clusters are also visible and confirm the presence of silver agglomerates with sizes greater than 120 nm. Spherical gold nanoparticles, which are characterised by sizes equal to approximately 100 nm are mainly revealed in the TEM image. However, smaller particles with diameters of 15 nm are also included in the analysed material. 3.2. Creams with metal nanoparticles Fig. 2 shows the photograph of obtained creams. On the left is a reference cream with no added metallic nanoparticles. The upper row contains creams with silver nanoparticles at concentrations ranging from 20 to 200 mg/kg, and the lower row contains creams with gold nanoparticles at concentrations ranging from 20 to 200 mg/kg. The colour intensities of the products reflect the metallic nanoparticle contents. The aqueous phase comprised 34% and the oil phase comprised 16%. A mixture of stearic acid and triethanolamine was used as an anionic emulsifier, thus the obtained emulsions can be to be the O/W type. Additionally, after spreading the cream on the skin surface, slight foaming of the emulsions was observed, which is indicative of O/W moisturising emulsions according to the literature [25]. The pH values of the cosmetic emulsions were measured in three replicates from which the arithmetic means were calculated. The average pH values for the emulsions with the various concentrations of silver and gold nanoparticles ranged from 6.85 to 7.28. According to the literature, cosmetic preparations dedicated to skin care should have pH values similar to that of the skin. Skin pH values range from 5.0 to 6.5 and depend on the composition of the lipid coat [26]. However, Lambers et al. studied the pH values of skin before and after refraining from showering and using cosmetics for 24 h. The mean pH value dropped from 5.12 ± 0.56 to 4.93 ± 0.45. This means that the natural skin pH is approximately 5.0. Both the application of cosmetic products and the application of tap water causes an increase in the skin pH (in Europe, the pH of tap water is approximately 8.0). It has been confirmed that skin with a pH below 5.0 is characterised by a better general condition than skin with a pH above 5.0. These conclusions concern the basic biophysical parameters of skin, such as its moisturising and flaking. It has also been demonstrated that a higher pH contributes to the dispersal of the natural microbiological film from the skin [27]. Other studies have demonstrated that the amount of propionibacteria, which are known to cause acne, which increases in alkaline conditions [28,29]. Therefore, the pH values of the tested cosmetic emulsion were slightly higher than the range given for the skin in the literature. The presence of sodium hydroxide in the silver and gold nanoparticle suspensions may have contributed to the increased pH values of the obtained emulsions. These results are presented in Fig. 3A. To perform the spectrophotometric analysis, the cream suspensions had to be prepared by 15-fold dilution with deionised water. The spectrophotometric spectra of the cream suspensions were obtained by subtracting the background, which consisted of the curve of the reference sample suspension. The ragged curves resulted from the high organic matter content of the analysed samples. The spectrophotometric analysis indicated the successful incorporation of metallic nanoparticles into the creams. As the concentrations of metal increased, the absorbances of the characteristic peaks increased, which was consistent with the expectations. These results are shown in Fig. 3B. The
2.2.4. Microbiological studies Sterilised YPD growth medium (yeast extract, casein peptone, sucrose, and agar;10 mL) was placed in a Petri dish. Following solidification, 50 μl of the test sample and 50 μl of an Aspergillus niger or Saccharomyces cerevisiae cell suspension were added. The preparation was evenly distributed over the entire surface of the medium to uniformly distribute the test sample. The cultures prepared in this manner were placed in an incubator at 30 °C. After 24, 48, and 72 h, the growths of the microorganisms treated with the preparations were evaluated. 2.2.5. Study of human participants Tests of the organoleptic properties of the emulsion in the reference form and with the silver and gold nanoparticles at 20 and 200 mg/kg were performed with seven men and seven women. The task of the participants was to describe the smell, appearance, and feel of the cosmetic emulsions. Then, to determine the density and consistency of the emulsion, the participants were asked to dip a finger in the cream at an angle of 45–60 degrees and pull it out. The adhesion rating was determined based on the ease of picking up the emulsion on the fingertip. Uniformity was assessed based on homogeneity and the absence of air bubbles in the emulsion upon spreading on the surface of the skin using circular movements. The spreading of the emulsion on the surface of the skin was used to assess the degree of spreadability. The rate of the absorption of the emulsion by the skin was also evaluated. To evaluate the greasiness, following the application of the emulsion, the participants noted whether a greasy film remained on the skin. Finally, the degree of skin smoothing following the application of the emulsion was evaluated and compared with that of a non-lubricated area. The evaluations of the sensory analysis were performed on a scale that ranged from 1 to 5 where 1 indicated the least favourable properties of the product, and 5 indicated the most favourable properties. 3. Results 3.1. Silver and gold nanoparticle suspensions The resulting suspensions were homogeneous. The suspension of silver nanoparticles was characterised by a dark yellow colour and the suspension containing gold nanoparticles exhibited a dark violet colour. The results of the spectrophotometric analysis are presented in Fig. 1A. The characteristic peaks are clearly visible. For the nanosilver and nanogold suspensions, the peak maxima were located at wavelengths of 421 and 533 nm, respectively. Based on ICP-OES analysis, the actual concentration of silver and gold nanoparticles were equal to 498 and 501 mg/L respectively. Fig. 1B presents the results of the nanoparticles sizes analysis and reveals that 76.8% of the silver nanoparticles had an average diameter 4
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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Fig. 1. A) UV–vis spectroscopy results for nanosilver and nanogold suspensions diluted to 20 mg/kg, B) Histograms of the distribution of the nanoparticle sizes, C) TEM images of silver and gold nanoparticles.
Fig. 2. Photographs of the obtained cosmetic emulsions.
emulsion contacts the skin can be determined. The upper value of the viscosity limit determines the final impression following the application of the emulsion [33,34]. This value can also be an indicator of the tendency of the cosmetic preparation to delaminate during storage. Fig. 4B presents the viscosity dependence (η) on the shear rate (γ). Based on these results, it can be concluded that the viscosity decreased with increasing shear rate. According to the literature, this pattern is characteristic of, among others things, non-Newtonian liquids that are diluted and subjected to shear, including, for example, emulsions [35]. Fig. 4C shows the results of the viscosity analysis for two specific shear rates. No viscosity dependence on the nanometal content was observed for the creams tested. The viscosities of the cosmetic emulsions were greater when the low shear rate was used. The size and distributions of the metal nanoparticles embedded in the creams at the concentration of 200 mg/kg were examined using transmission electron microscopy with an X-ray scattering spectrometer (TEM-EDX). Fig. 5 presents microphotographs of the studied samples. The arrangement of gold particles was homogeneous. The observed size of the nanoparticles were consistent with the results of the DLS analysis. The presence of both silver and gold nanoparticles was confirmed using EDX analysis. The reference cream consisted of carbon, oxygen, silica, chlorine, and potassium. The spectra obtained for the creams with the metallic nanoparticles also contained peaks indicating silver and gold.
red-shift effect seen for creams with nanosilver proves that when embedded into the structure of the cream, the silver nanoparticles agglomerate. This phenomenon may occur in the phase of introducing the silver suspension into the cream mixture. The increased size of the nanoparticles embedded in the cream (200 mg/kg) is also seen in Fig. 5B. The main size of the silver nanoparticles contained in the water suspension was equal to 8 nm (which was confirmed by both DLS and TEM analyses). However, after introducing them into the cream, their sizes were larger and equal to 30 nm. These observations are in line with the results obtained by Lizoń et al. [30] and Bu et al. [31]. Compared with nanogold, the agglomeration of silver nanoparticles may be due to the fact that the native silver nanoparticles were characterised by lower absolute electrokinetic potential values, which express the stability of nanoparticles. Examination of the rheological properties of a cosmetic preparation allows for the characterisation of its consistency and ease of spreading on the skin [32]. Fig. 4A presents graphs of the dependence of the shear stress (τ) on the shear rate (γ). There are visible differences between the flow curves. For the emulsions with silver or gold nanoparticles, the shear stress decreased with increasing shear rate from 10 to 500 rpm. This finding contracts with that for the reference emulsion for which a slight increase in the shear stress is visible. The lowest yield points were found for the creams with gold and silver nanoparticles at the concentration of 20 and 65 mg/kg, respectively, and for the reference emulsion. According to the literature, emulsions with low viscosity limits are characterised by better consistency and spreading on the skin surface. Based on the viscosity limit, the first impression when the 5
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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Fig. 3. A) pH values of the obtained creams (n = 3), B) Plots of the dependencies of the absorbances on the wavelengths for the creams with silver and gold nanoparticles.
Their regular use leads to their release into the environment in significant amounts. Most of these products are biologically active and characterised by high stability and the ability to accumulate, which poses threats to natural ecosystems and human health [36]. Cosmetics containing metallic nanoparticles comprise a significant contribution to the environmental levels of these nanoparticles. According to Giese et al., most released nanosilver particles originate from textiles. However, cleaning products and cosmetics are also important sources of nanosilver particles that are present in wastewater [37]. Producers of cosmetics with metallic nanoparticles should be aware of the potential risks that are related to their applications in consumer products. Filon et al. [38] studied the literature regarding the influence of physicochemical properties of nanosilver particles on their penetration through the damaged or unimpaired skin membrane. Theses authors figured out that the rate of penetration depends mainly on the form of the nanoparticles (i.e., metallic or non-metallic). The sizes of the nanoparticles were the second-most-important factor. Nanosilver particles with diameters below 20 nm are able to penetrate both intact and damaged skin, whereas particles with sizes between 21 and 45 nm can only go into damaged skin. Larger particles are not able to penetrate either damaged or intact skin. Also, Filon et al. [39] studied the influence of nanosilver on human health. Researchers conducted analyses of the in-
3.3. Analysis of nanoparticle release from topical formulations and nanoparticle accumulation in model bodily fluids The results of spectrophotometric analysis of a receptor fluid after 48 h are presented in Fig. 6. Peaks characteristic of silver nanoparticles appeared between 400 and 500 nm, and peaks characteristic of gold nanoparticles appeared in the 500–600 nm range. Characteristic peaks were recorded for the cream samples with nanosilver at concentrations of 110, 155, and 200 mg/kg. The highest peak was recorded at the wavelength of 477 nm with an absorbance of 0.427. For gold, the highest peak was recorded at the wavelength of 533 nm with an absorbance of 0.352. Gold nanoparticles were identified in all creams samples with nanogold. Higher concentrations of metallic nanoparticles in the creams resulted in higher absorbance values. The actual concentration of silver in receptor fluids are: 2.22 mg/kg (for cream with nanoAg 110 mg/kg), 2.71 (for cream with nanoAg 155 mg/kg) and 4.15 mg/kg (for cream with nanoAg 200 mg/kg). The concentration of gold in receptor fluids for creams with nanoAu 20–200 mg/kg are: 1.65, 2.08, 2.83, 4.46 and 5.20 mg/kg. These results indicated the presence of metallic nanoparticles in the receptor fluid, which resulted from their ability to penetrate the model membrane. Cosmetic products are applied extensively throughout the world. 6
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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Fig. 4. A) Flow curves of the dependence of the shear stress (τ) on the shear rate (γ), B) Viscosity dependence (η) on the shear rate (γ), C) Viscosity analyses for two specific shear rates (n = 100).
3.4. Microbiological studies
vivo penetration of nanosilver through human skin. Similar to our studies, a physiological solution was used as a receptor fluid. PVP-coated silver nanoparticles were dispersed in artificial sweat, and this donor suspension was applied in the study which lasted for 24 h. It was found that in case of damaged skin, the rate of penetration of silver was equal to 3.31%, and the resulting value did not exceed 1% when the intact skin was applied in the study. Tak et al. [40] performed research on the influence of the shape of silver nanoparticles and their penetration through model skin. These authors compared triangular spherical and rod-like nanoparticles that were subjected to in vitro and in vivo studies. They found that the rod-like nanoparticles are characterised by the highest rate of penetration in both types of studies. These authors suggest that it is highly possible that particles in this shape may accumulate in the dermal layers, which may lead to occurrence of many side-effects, such as argyria. Due to the basal plane in triangular nanoparticles, their penetration is the slowest. The authors conclude that these nanoparticles of this shape have the ideal form of a nanoparticulate biocidal agent because they exhibit satisfactory antibacterial properties. Gupta et al. [41] conducted simulations on the comparisons of the penetration rates of gold nanoparticles coated with neutral, cationic or anionic agents. The sizes of the particles also differed. They also figured out that all neutral particles penetrated through the model dermal membrane and reached the second layer. Both anionic and cationic gold nanoparticles were able to go through the membrane in which they were accumulated. The penetration rate was increased with the decreasing the size of the nanogold particles.
Results of the microbiological tests are presented in Table 1 There were no differences on the first day of incubation. After 48 and 72 h of incubation, inhibition of the growth of Aspergillus niger was observed for the creams with silver and gold nanoparticles at concentrations of 65, 110, 155, and 200 mg/kg. In the case of Saccharomyces cerevisiae, antifungal activity after 48 h of incubation was observed for the creams with silver nanoparticles at all concentrations and for the creams with gold nanoparticles at the concentrations of 155 and 200 mg/kg. After 72 h of incubation, the same nanogold creams samples exhibited good antifungal properties; however, the 20-mg/kg nanosilver cream no longer exhibited antifungal activity Minimal inhibition concentrations for creams with nanoparticles were as follows: MIC35% = 65 mg/kg for creams with nanosilver against A. niger after 48 h; MIC6% = 20 mg/kg for creams with nanosilver against S. cerevisiae after 48 h; MIC20% = 65 mg/kg for creams with nanogold against A. niger after 48 h; MIC4% = 155 mg/kg for creams with nanogold against S. cerevisiae after 48 h. Marslin et al. obtained silver nanoparticles using Withania somnifera leaves as a source of reducing and stabilising factors and used these nanoparticles to generate cosmetic creams. Compared with creams containing silver nitrate, these creams with nanosilver particles exhibited greater activities against human pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Candida albicans. According to these authors, this type of product may be applied in the cosmetic industry [42].
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Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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Fig. 5. Microphotographs of the examined creams with the EDX collective spectra of the identified elements: A) Reference cream, B) cream with silver nanoparticles, C) cream with gold nanoparticles.
creamy consistency. The emulsions spread well on the surface of the skin and were absorbed quickly without leaving a greasy film on the skin. Compared with the reference sample, the best assessments in terms of smoothing the skin, consistency, adhesion, lubrication, application, and absorption were obtained for the 200-mg/kg gold nanoparticle cream. However, the best assessment in terms of homogeneity was obtained for the 20-mg/kg silver nanoparticles cream. The200-mg/ kg nanosilver cream performed worse than other emulsions. The
3.5. Studies with human participants Fig. 7A presents the collective results of the averaged ratings from the participants for the creams with silver and gold nanoparticles at concentrations of 20 and 200 mg/kg and for the reference cream. In the general assessments from the participants, all of the creams were characterised by satisfactory properties. The emulsions did not spill after a finger was dipped into the container, and they exhibited a
Fig. 6. UV–vis analysis of the receptor fluid. 8
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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Fig. 7. A) Sensory profile of the tested creams, B) Smell and colour assessments (n = 14).
Declaration of Competing Interest
participants gave this cream the lowest homogeneity score due to the presence of numerous visible air bubbles. This emulsion also left a greasy film after it was spread on the skin. The result of the average smell and colour sensory evaluations are presented in Fig. 7B. The most mild and pleasant scent and most interesting colour were attributed to the 200-mh/kg gold nanoparticle cream. The lowest grades were obtained for the 200-mg/kg silver nanoparticle cream.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] U.S. EPA, Nanomaterial Case Study: Nanoscale Silver in Disinfectant Spray (External Review Draft), U.S. Environmental Protection Agency, Washington, DC, 2010 EPA/600/R-10/081. [2] X.F. Zhang, Z.G. Liu, W. Shen, S. Gurunathan, Int. J. Mol. Sci. 17 (2016) 1534. [3] C. Horner, A. Kumar and K. Nieradka, Nanosilver as a biocide in building materials, US patent US20060272542A1. [4] M. Rai, A. Yadav, A. Gade, Biotechnol. Adv. 27 (2009) 76. [5] M. Bachelet, Mater. Sci. Technol. 32 (2016) 794. [6] C. Contado, Front. Chem. 3 (2015) 48. [7] Quan Zhou Hu Zheng Nano Technology Co., Ltd, Quan Zhou Hu Zheng Nano Technology Co., Ltd.® Nano-gold Mask, available on https://www.nanotechproject. org/cpi/products/quan-zhou-hu-zheng-nano-technology-co-ltd-r-nano-gold-mask/. [8] Chantecaille offer, Nano Gold Energizing Cream, available on https://www.saksfifthavenue.com/chantecaille-nano-gold-energizing-cream/product/ 0435297663735?sid=120A0B312FA8&R=656509702909&FOLDER < > folder_ id=282574492711986&ASSORTMENT < > ast_id=1408474399545537& bmUID=1239646676269&P_name=Chantecaille. [9] Lexon Nanotech, Inc. offer, Nanorama - Gold Toothpaste, available on https:// www.nanotechproject.org/cpi/products/nanorama-gold-toothpaste/. [10] S. Raj, S. Jose, U.S. Sumod, M. Sabitha, J. Pharm. Bioallied Sci. 4 (2012) 186. [11] S. Asghari, S.A. Johari, J.H. Lee, Y.S. Kim, Y.B. Jeon, H.J. YB, M.C. Moon, I.J. Yu, Int. J. Nanobiotechnology Pharm. 10 (2012) 14. [12] K. Bilberg, H. Malte, T. Wang, E. Baatrup, Aquat. Toxicol. 96 (2010) 159. [13] A. Massarsky, L. Dupuis, J. Taylor, S. Eisa-Beygi, L. Strek, V.L. Trudeau, T.W. Moon, Chemosphere 92 (2013) 59. [14] A. Loghman, S.H. Iraj, D.A. Naghi, M. Pejman, Afr. J. Biotechnol. 11 (2012) 6207. [15] Nano Siver offer, The Best Nano Colloidal Silver - 16 Oz -20 PPM Colloidal Silver, available on amazon.com. [16] Atlantean Alchemy offer, Colloidal Silver Half Gallon 60 PPM, available on amazon. com. [17] Silver Mountain Minerals offer, Liquid Silver 16 oz.240 PPM, Silver Mountain Minerals, available on amazon.com. [18] Nano Siver offer, Nano Silver colloidal silver 10 PPM, available on amazon.com.
4. Conclusions Stable cosmetic formulations (i.e., creams) with silver or gold nanoparticles at different concentrations were obtained. The presences of the nanoparticles in the creams were confirmed with analytical and microscopic techniques. There is a difference in embedding silver and gold nanoaparticles into the structure of a cream. Silver introduced to the cream mixture agglomerates which is confirmed by a red shift that is visible in the spectra of creams with nanosilver. This is also revealed by TEM images of creams with nanoAg. In contrast, gold nanoparticles do not agglomerate after introduction to cream mixtures. This phenomenon is related to the values of the electrokinetic potentials of native metallic nanoparticles. The high absolute value that is characteristic of native nanogold particles confirms their high stability. Thanks to the greater value of the electrokinetic potential located on the surface of gold nanoparticles, they stay stable even after introducing them into cream mixture. This contrasts with silver nanoparticles, which agglomerate easier. The microbiological tests revealed that the investigated creams with both silver and gold nanoparticles were characterised by different fungicidal properties against Aspergillus niger and Saccharomyces cerevisiae. The permeabilities of the metallic nanoparticles through the dermal membrane were confirmed for the samples with concentrations of 110–200 mg/kg, which represents an issue of concern given the harmful effects of metallic nanoparticles on living organisms. 9
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[31] Y. Bu, S.W. Lee, Int. J. Nanomedicine 10 (2015) 47. [32] S. Dworakowska, D. Bogdał, T.M. Majka, K. Pielichowski, Czas. Tech. 26 (2012) 9/M. [33] E.K. Park, K.W. Song, Korea. Aust. Rheol. J. 22 (2010) 279. [34] A. Garg, D. Aggarwal, S. Garg, A.K. Singla, Pharm. Technol. North Am. 26 (2002) 84. [35] S. Derkach, Adv. Colloid Interface Sci. 151 (2009) 1. [36] C. Juliano, G.A. Magrini, Cosmetics 4 (2017) 11. [37] B. Giese, F. Klaessig, B. Park, R. Kaegi, M. Steinfeldt, H. Wigger, A. von Gleich, F. Gottschalk, Sci. Rep. 8 (2018) 1565. [38] F.L. Filon, M. Mauro, G. Adami, M. Bovenzi, M. Crosera, Regul. Toxicol. Pharmacol. 72 (2015) 310. [39] F.L. Filon, F. D’Agostin, M. Crosera, G. Adami, N. Renzi, M. Bovenzi, G. Maina, Toxicology 255 (2009) 33. [40] Y.K. Tak, S. Pal, P.K. Naoghare, S. Rangasamy, J.M. Song, Sci. Rep. 5 (2015) 1690. [41] R. Gupta, B. Rai, Sci. Rep. 7 (2017) 45292. [42] G. Marslin, R.K. Selvakesavan, G. Franklin, B. Sarmento, A.C.P. Dias, Int. J. Nanomed. Nanosurg. 10 (2015) 5955.
[19] Silver Miracles offer, Colloidal Silver Gel 75 ppm, available on amazon.com. [20] My Doctor Suggests offer, Organic Exfoliating Silver Soap 30 ppm, available on amazon.com. [21] Generic offer, Colloidal Silver Soap Silver Biotics Gel 20 ppm, available on amazon. com. [22] Silver pure offer, 3-1/2% Nanosilver (35,000 ppm) Infused Organic Skin Creams, 35,000 ppm, available on silverpure.com. [23] Opinion on Colloidal Silver (nano), (2018) SCCS/1596/18, 24-25 October. [24] Ringer’s solution, Cold Spring Harb. Protoc. (2008). [25] B. Tal- Figiel, W. Figiel, A. Michno, Proc. Univ. Appl. Chem. Biotechnol. 54 (2015) 051. [26] J. Blaak, R. Wohlfart, N.Y. Schürer, JCDSA 1 (2011) 50. [27] H. Lambers, S. Piessens, A. Bloem, H. Pronk, P. Finkel, Int. J. Cosmet. Sci. 22 (2006) 359. [28] J. Tarun, J. Susan, J. Suria, V.J. Susan, S. Criton, Indian J. Dermatol. 59 (2014) 442. [29] J. Blaak, P. Staib, Curr. Probl. Dermatol. 54 (2018) 132. [30] A. Lizoń, R. Drożdż, Przeglä…d Med. Uniw. Rzesz. I Nar. Inst. Lekã³w W Warszawie 75 (2018) 457.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Safety of the application of nanosilver and nanogold in topical cosmetic preparations
T
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Jolanta Pulit-Prociaka, , Aleksandra Grabowskaa, Jarosław Chwastowskia, Tomasz M. Majkab, Marcin Banacha a
Faculty of Chemical Engineering and Technology, Institute of Chemistry and Inorganic Technology, Cracow University of Technology, Warszawska 24, Cracow 31-155, Poland b Faculty of Chemical Engineering and Technology, Department of Chemistry and Technology of Polymers, Cracow University of Technology, Warszawska 24, Cracow 31155, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanosilver Nanogold Oil in water emulsions Biocidal properties Model dermal membrane Permeation studies
The safety of the use of cosmetic preparations with silver or gold nanoparticles was assessed. This study describes the methodology and results of research involving the generation of suspensions of silver and gold nanoparticles and creams with added silver or gold at concentrations of 20, 65, 110, 155, and 200 mg/kg. The silver nanoparticles ranged from 8 to 140 nm, and the gold nanoparticles, measured 13–99 nm. The sizes were determined using dynamic light scattering. The presence of metallic nanoparticles in the obtained oil-in-water emulsions was confirmed with UV–vis spectroscopy and transmission electron microscopy with an X-ray scattering spectrometer (TEM-EDX). Additionally, based on the TEM-EDX results, it was possible to analyse the distributions of the silver nanoparticles in the tested cosmetic emulsions. Microbiological tests showed that both the silver and gold nanoparticle emulsion possessed satisfactory fungicidal properties. Based on viscosity curves, the lowest estimated yield limits were achieved by the reference cream and the creams with the gold and silver nanoparticles at concentrations of 20 and 65 mg/kg, respectively, which improved their consistencies and distributions on the skin. The best appraisals from the respondents in terms of consistency, absorption, oiling, colour, and smell were received for the emulsion containing 200 mg/kg gold nanoparticles. The worst assessment in terms of uniformity, colour, and smell were obtained for the emulsion with 200 mg/kg silver nanoparticles. However, the most important aspect of this study was the assessment of the permeabilities of the metallic nanoparticles through imitation skin in the form of dermal membranes. The highest permeabilities were confirmed for the creams with metallic nanoparticles present at 110-–200 mg/kg. This permeability is an issue of concern given the toxic properties of metallic nanoparticles for living organisms.
1. Introduction Throughout history, silver has been used as a raw material for the production of coins, silverware, and other decorative items. This noble metal has also been used in medicine and as an antibacterial agent. To prevent diseases and other illnesses, the ancient Greeks drank water from silver cups [1]. Due to dynamically developing nanotechnology, silver nanoparticles are currently used in many applications. These nanoparticles play a particularly important role as a component of disinfecting liquids and fungicides [2]. Nanosilver has also been added to dressings, bandages, anti-infectious agents, catheters, and implants (e.g., artificial heart valves). Soaps with nanosilver are widely used in
the treatment of acne as well as dermatitis because they regulate sebaceous glands. In the construction industry, nanosilver particles are added to insulation, roofing, and anti-bacterial paints [3]. The enrichment of textile products with silver nanoparticles also inhibits the development of pathogenic microorganisms [4]. In ancient times, people used gold due to faith in its miraculous healing power. Currently, nanogold is being used in various fields of science and technology, such as electrical engineering, catalysis, cosmetology, medicine, and pharmacology [5]. Gold nanoparticles are used as a carrier for anti-cancer drugs and in thermotherapy targeting cancer cells. Gold nanopigments have also been used as contrast agents in computed tomography and in infrared thermal imaging.
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Corresponding author. E-mail addresses: [email protected] (J. Pulit-Prociak), [email protected] (A. Grabowska), [email protected] (J. Chwastowski), [email protected] (T.M. Majka), [email protected] (M. Banach). https://doi.org/10.1016/j.colsurfb.2019.110416 Received 1 March 2019; Received in revised form 26 July 2019; Accepted 31 July 2019 Available online 01 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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type of research were verified. This study also analysed the release of nanoparticles and their potential for penetration through a model dermal membrane.
Additionally, gold in the form of spheroidal nanoparticles is used as a carrier of RNA and in advanced medical diagnosis. Gold nanopigments exhibit high photoluminescence that is used to create three-dimensional images in vivo. In the cosmetics industry, manufacturers are adding the “nano” prefix to cosmetic names with increasing frequency, even when the presence of nanoparticles has not been confirmed and the term “nano” is intended to attract more customers. This widespread influence of nanotechnology in the cosmetics industry results from the fact that formulations containing nanometric particles have more desirable properties, i.e., deeper colours, better transparencies, and increased solubility. Nanoparticles in cosmetic products can act as active substances, carriers, consistency-improving substances, effectiveness-enhancing substances, and antimicrobial agents. Nanoparticles are added to permanent make-up formulations as well as anti-aging creams and toothpastes [6]. Due to their antimicrobial properties, nanosilver and nanogold have quickly entered into everyday use. Silver and gold nanoparticles in particular have found uses as active ingredients in creams, soaps, shower gels, masks, and other cosmetics with biocidal activities. Nanosilver shampoos are recommended for the treatment of dandruff, itchy scalp, and excessive sebum production. Antiperspirants with silver nanoparticles are designed to eliminate the unpleasant odour of sweat. Manufacturers of intimate hygiene fluids with silver and copper nanoparticles indicate that the products accelerate the regeneration of minor wounds and inhibit infections. The producers of mask with gold nanoparticles claim that nanogold contributes to scattering its active ingredients [7]. There are also available creams with nanogold particles. Thanks to nanotechnology, the precious elements reach the cellular level where they play the role of the ultimate healing and preserving force [8]. A toothpaste that contains nanoparticulate gold is also available. Thanks to the presence of nanogold, it provides an antibacterial effect, which is particularly desired in the treatment of the mouth [9].Studies have demonstrated that nearly all major cosmetic manufacturers draw on nanotechnology in the design of their products. One of the cosmetics giants that produces mainly colour cosmetics entered the NanoMarket in 2006 and offer a range of products containing nanoparticles. The world's largest cosmetics company, utilises approximately $600 million of its $17 billion revenue to patent the use of nanoparticles in cosmetic products [10]. Commercial products containing nanomaterials have become a substantial source of nanoparticles that are subsequently released into ecosystems and lead to the accumulation of nanoparticles in living organisms. A number of studies has assessed the toxicities of metallic nanoparticles, particularly metallic silver, to living organisms. Metallic silver nanoparticles have been found to cause discolorations of the bodies of Daphnia, and 100% mortality results from 48 h of contact with a suspension with a concentration of 200,000 mg/L [11]. Following contact with metallic nanosilver suspension, Eurasian perch exhibit reduced tolerance to hypoxia [12]. A zebrafish population treated with a suspension of metallic silver nanoparticles at a concentration of 1.18 mg/L showed 50% mortality, decreased heart rate, pericardial oedema, and general degeneration of the organisms [13]. Providing chickens with water containing metallic silver nanoparticles at a concentration of 12 mg/L has been noted to result in the accumulation of nanoparticles in the hepatocytes and central vein syndrome [14]. Considering the data showing the negative effect of nanoparticles on living organisms, it is important to account for the safety of their application in the production of various cosmetic preparations. The aim of this study was to develop recipes for cosmetic preparations containing silver or gold nanoparticles at different concentrations, and to assess their physicochemical and application properties. Experiments were performed to verify the antifungal properties of the obtained products. Due to the presence of various types of cutaneous mycoses in humans, the effects of the addition of nanoparticles to creams on their antimicrobial activities against model fungi that are routinely used in this
2. Materials and methods 2.1. Materials The following compounds were used in this study: cetearyl alcohol (p.p.a.), glycerol (p.p.a.), triethanolamine (≥ 99.0%), stearic acid (≥ 95.0%), potassium sorbate (p.p.a.), disodium EDTA (p.p.a.), soy lecithin (p.p.a.), silicone oil (p.p.a., 1000 cSt), castol oil (p.p.a.), tocopherol (p.p.a.), sodium carboxymethylcellulose (ultra-high viscosity, highly purified), chloroauric acid (≥ 99.999%), silver nitrate (≥ 99.9%), tannic acid (p.p.a.), sodium chloride (≥ 99.5%), potassium chloride (≥ 99.5%), calcium chloride (99.0%), and sodium hydroxide (≥ 98.0%). All compounds were obtained from Sigma-Aldrich (Germany). The following compounds were used in the microbiological tests: peptone, yeast extract, sucrose (≥ 98.0%), and agarose. All compounds were obtained from Sigma-Aldrich. The Aspergillus niger and Saccharomyces cerevisiae strains used in the study were provided by the National Collection of Yeast Cultures (England). All solutions were prepared using deionised water (Polwater, 0.18 μS). 2.2. Methods 2.2.1. Preparation of the silver and gold nanoparticle suspensions The suspensions of both silver and gold nanoparticles were obtained in a one-step chemical reduction process using tannic acid as a reducing and stabilising substance. First, aqueous solutions of silver nitrate (5.15 × 10−3 mol/L), chloroauric acid (2.82 × 10−3 mol/L), tannic acid (9.27 × 10−3 mol/L to generate nanosilver and 5.08 × 10−3 mol/ L to generate nanogold), and sodium hydroxide (1 mol/L) were prepared. To obtain suspensions of silver and gold nanoparticles at a concentration of 500 mg/L, 10 mL of tannic acid solution was added to a beaker containing 90 mL of silver nitrate or chloroauric acid solution. The mixture was stirred at ambient temperature (550 rpm). The molar ratio of the tannic acid to both the silver and gold ions was 0.2:1.0. Thereafter, sodium hydroxide was added to obtain a pH of 7, and the solution was stirred for an additional 10 min. The obtained suspensions were subjected to UV–vis spectrophotometry analysis. A single-beam spectrophotometer (Rayleigh UV-1800) connected to a computer loaded with spectra UVSoftware with 1.0-cm quartz cells was used. The spectra were obtained with the following instrumental parameters: wavelength range: 300–800 nm; scan speed: slow; sampling interval: 1.0 nm; and spectral slit width: 2 nm. Dynamic light scattering (DLS) was applied to determine the particle size. Zetasizer Nano ZS (Malvern Instruments Ltd.). The machine was operating with a He–Ne laser and connected to a computer loaded with the software Malvern Panalytical. The results were the means of triplicate runs, and at least 10 measurements were made in each run. The refractive indexes (RI) for silver nanoparticles of 0.240 and for gold nanoparticles of 0.180 were used. The viscosity of the samples were equal to 0.8872 cP, which is appropriate for water, i.e., the sample dispersant. The suspensions were vortexed and transferred to 2.0-cm disposable polystyrene sizing cuvettes. The actual concentrations of the silver and gold nanoparticles in the aqueous suspensions were analysed by means of inductively-coupled atomic emission spectroscopy (ICP-OES) that was conducted on a Plasm 40 apparatus (Perkin Elmer Co.) with a method detection limit of 0.1 μg/L. 2.2.2. Preparation of the oil-in-water (O/W) creams with metal nanoparticles The components of both the aqueous and oil phases were weighed (Table 1). The aqueous phase contained the required amount of 2
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Table 1 Numerical data for creams with metallic nanoparticles. EMULSION RECIPES FOR 50 G OF PRODUCT Compound (INCI name) Aqueous phase Aqua or metallic nanoparticle suspension* Glycerol Carboxymethylcellulose Triethanoloamine Potassium sorbate Disodium EDTA Oil phase Dimethicone Castol oil
Function
Content [%]
Mass [g]
solvent solvent, humectant, moisturising agent rheology modifier pH modifier preservative sequestrant
60 6
30.0 3.0
0.2 1.5 0.2 0.1
0.1 0.75 0.1 0.05
3 3
1.5 1.5
5 6 10
2.5 3.0 5.0
5 q.s.
2.5 q.s.
protecting and moisturising agent moisturising agent, transepidermal water loss (TEWL) reducer Lecithin emulsifier Stearic acid stabilising agent Cetearyl alcohol consistency regulator, fatty substance Tocopheryl acetate antioxidant Fragrance oil (hibiscus flower) fragrance *METALLIC NANOPARTICLES SUSPENSION USED TO PREPARE EMULSION WITH SILVER OR GOLD NANOPARTICLES Metal concentration in the final product [mg/kg] Volume of nanosilver or nanogold suspension at a concentration of 500 mg/L [mL] 20 2.0 65 6.5 110 11.0 155 15.5 200 20.0
Volume of water [mL] 28.0 23.5 19.0 14.5 10.0
RESULTS OF ANTIMICROBIAL PROPERTIES Aspergillus niger Time
Nano Ag (mg/kg) 20 65 110 155 Growth inhibition comparing to Reference Cream [%] 48 h 4 35 29 38 72 h 2 10 11 12 Saccharomyces cerevisiae Time Nano Ag (mg/kg) 20 65 110 155 Growth inhibition comparing to Reference Cream [%] 48 h 6 7 5 10 72 h 0 4 4 4
200
Nano Au (mg/kg) 20 65
110
155
200
36 11
15 2
21 7
22 10
20 10
200
Nano Au (mg/kg) 20 65
110
155
200
11 5
0.5 0
1 0
4 3.5
5 5
20 8
0 1
aqueous phase of pure water instead of a metallic suspension. The obtained emulsions were subjected to pH measurements and UV–vis and viscosity analyses (rotary rheometer, Haake Mars III, Thermo Scientific, Waltham, MA, USA). The studies were performed in a plate-plate arrangement with a 1-mm gap between the plates. The diameter of the plate was 60 mm. The tests were performed in a controlled-rotation system (control rate: CR) at 25 °C using a titanium rotor with a rotational speed of 10 rpm. The viscosities at the points of the two shear rates were measured; i.e., γ1 = 10 rpm and γ2 = 100 rpm. Flow curves expressing the dependencies of the shear stress (τ) and viscosity (η) on the shear rate (γ) were also obtained. The study was conducted in a range of shear speeds of 10–500 rpm. Additionally, microphotographs of the obtained samples were taken using high-resolution transmission electron microscopy with EDX and the elemental mapping mode. The study was performed using a Tecnai transmission electron microscope (TEM G2 F20X-Twin 200 kV, FEI). To identify the chemical elements, energy-dispersive X-ray spectroscopy (EDX, RTEM model SN9577, 134 eV, EDAX) was used. The spectra were recorded in the designated areas and along the lines. The samples were prepared as follows: a few milligrams of each cream were dispersed in ethanol (99.8%, anhydrous), then a drop of dispersion was applied on carboncoated copper mesh on which there were holes (Lacey type Cu 400 mesh, Plano). The dispersing agent was evaporated at ambient temperature.
previously prepared silver or gold nanoparticle suspension. The silver and gold nanoparticle suspensions were used at a suitable concentrations, i.e., 20, 65, 110, 155, or 200 mg/kg. The minimum effective concentrations of the metallic nanoparticles (both silver and gold) contained in the cosmetic products were equal to 10 mg/kg. Based on the state of art, cosmetic products contain silver or gold nanoparticles with concentrations typically vary from 10 ppm to 100 ppm [15–21] and even reach 35,000 ppm (3.5%) in some cases [22]. However, according to the Opinion on Colloidal Silver (nano) issued by the Scientific Committee on Consumer Safety (SCCS), the upper limit of silver concentration should not exceed 10,000 ppm (1%). This value was based on the reasonably foreseeable exposure conditions [23]. The authors intended to assess the properties and safety of the use of creams containing a wide range of concentrations of nanoparticles. Thus, the range from 20 to 200 ppm that corresponds with the typical effective contents of metallic nanoparticles was chosen. Both phases were heated in water baths (aqueous phase up to 70 °C and oil phase up to 72 °C) and mechanically stirred (600 rpm) for 20 min after the appropriate temperature was reached. The oil phase was introduced into the aqueous phase to form an emulsion. When the temperature dropped to 42 °C, tocopherol and a perfume oil (hibiscus flower) were added. After reaching room temperature, the emulsion was poured into a plastic container, and the product was maintained at room temperature. The reference sample was obtained using an 3
Colloids and Surfaces B: Biointerfaces 183 (2019) 110416
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2.2.3. Assessments of the release of nanoparticles from topical formulations and analyses of their accumulations in model bodily fluids An aqueous receptor fluid, i.e., Ringer’s solution, was prepared as previously described [24]. The Strat-M® Membrane for Transdermal Diffusion Testing was used. The kit consisted of a magnetic stirrer with a heating plate, a water bath, a container with 10 mL of receptor fluid, and a glass column to which the model dermal membrane was attached. Three grams of the prepared creams and 5 mL of deionised water were placed in the column and allowed to adhere to the model membrane. The temperature of the water bath was set at 37 °C. After reaching the appropriate water temperature, the previously prepared set with the membrane was embedded in such a manner that the smooth surface of the membrane was in contact with the receptor fluid. The mixture was stirred at 200 rpm. The identification of the nanometals in the receptor fluid was performed with a UV–vis spectrophotometer after 48 h. The actual concentrations of silver and gold nanoparticles in the receptor fluids were analysed by means of inductively-coupled atomic emission spectroscopy (ICP-OES).
of 8 nm, and 23.2% of the nanoparticles had an average size of 140 nm. The average diameter of 98% of the gold nanoparticles was 99 nm, and 2% of the nanoparticles had an average size equal to 15 nm. The measured electrokinetic potential of both silver and gold nanoparticles was equal to -19,8 mV and -34,6 mV, respectively. The native shapes and dispersion levels of both the silver and gold nanoparticles are presented in Fig. 1C. The results are in line with the DLS analysis. The majority of the spherical silver nanoparticles with diameters below 10 nm can be seen. The larger clusters are also visible and confirm the presence of silver agglomerates with sizes greater than 120 nm. Spherical gold nanoparticles, which are characterised by sizes equal to approximately 100 nm are mainly revealed in the TEM image. However, smaller particles with diameters of 15 nm are also included in the analysed material. 3.2. Creams with metal nanoparticles Fig. 2 shows the photograph of obtained creams. On the left is a reference cream with no added metallic nanoparticles. The upper row contains creams with silver nanoparticles at concentrations ranging from 20 to 200 mg/kg, and the lower row contains creams with gold nanoparticles at concentrations ranging from 20 to 200 mg/kg. The colour intensities of the products reflect the metallic nanoparticle contents. The aqueous phase comprised 34% and the oil phase comprised 16%. A mixture of stearic acid and triethanolamine was used as an anionic emulsifier, thus the obtained emulsions can be to be the O/W type. Additionally, after spreading the cream on the skin surface, slight foaming of the emulsions was observed, which is indicative of O/W moisturising emulsions according to the literature [25]. The pH values of the cosmetic emulsions were measured in three replicates from which the arithmetic means were calculated. The average pH values for the emulsions with the various concentrations of silver and gold nanoparticles ranged from 6.85 to 7.28. According to the literature, cosmetic preparations dedicated to skin care should have pH values similar to that of the skin. Skin pH values range from 5.0 to 6.5 and depend on the composition of the lipid coat [26]. However, Lambers et al. studied the pH values of skin before and after refraining from showering and using cosmetics for 24 h. The mean pH value dropped from 5.12 ± 0.56 to 4.93 ± 0.45. This means that the natural skin pH is approximately 5.0. Both the application of cosmetic products and the application of tap water causes an increase in the skin pH (in Europe, the pH of tap water is approximately 8.0). It has been confirmed that skin with a pH below 5.0 is characterised by a better general condition than skin with a pH above 5.0. These conclusions concern the basic biophysical parameters of skin, such as its moisturising and flaking. It has also been demonstrated that a higher pH contributes to the dispersal of the natural microbiological film from the skin [27]. Other studies have demonstrated that the amount of propionibacteria, which are known to cause acne, which increases in alkaline conditions [28,29]. Therefore, the pH values of the tested cosmetic emulsion were slightly higher than the range given for the skin in the literature. The presence of sodium hydroxide in the silver and gold nanoparticle suspensions may have contributed to the increased pH values of the obtained emulsions. These results are presented in Fig. 3A. To perform the spectrophotometric analysis, the cream suspensions had to be prepared by 15-fold dilution with deionised water. The spectrophotometric spectra of the cream suspensions were obtained by subtracting the background, which consisted of the curve of the reference sample suspension. The ragged curves resulted from the high organic matter content of the analysed samples. The spectrophotometric analysis indicated the successful incorporation of metallic nanoparticles into the creams. As the concentrations of metal increased, the absorbances of the characteristic peaks increased, which was consistent with the expectations. These results are shown in Fig. 3B. The
2.2.4. Microbiological studies Sterilised YPD growth medium (yeast extract, casein peptone, sucrose, and agar;10 mL) was placed in a Petri dish. Following solidification, 50 μl of the test sample and 50 μl of an Aspergillus niger or Saccharomyces cerevisiae cell suspension were added. The preparation was evenly distributed over the entire surface of the medium to uniformly distribute the test sample. The cultures prepared in this manner were placed in an incubator at 30 °C. After 24, 48, and 72 h, the growths of the microorganisms treated with the preparations were evaluated. 2.2.5. Study of human participants Tests of the organoleptic properties of the emulsion in the reference form and with the silver and gold nanoparticles at 20 and 200 mg/kg were performed with seven men and seven women. The task of the participants was to describe the smell, appearance, and feel of the cosmetic emulsions. Then, to determine the density and consistency of the emulsion, the participants were asked to dip a finger in the cream at an angle of 45–60 degrees and pull it out. The adhesion rating was determined based on the ease of picking up the emulsion on the fingertip. Uniformity was assessed based on homogeneity and the absence of air bubbles in the emulsion upon spreading on the surface of the skin using circular movements. The spreading of the emulsion on the surface of the skin was used to assess the degree of spreadability. The rate of the absorption of the emulsion by the skin was also evaluated. To evaluate the greasiness, following the application of the emulsion, the participants noted whether a greasy film remained on the skin. Finally, the degree of skin smoothing following the application of the emulsion was evaluated and compared with that of a non-lubricated area. The evaluations of the sensory analysis were performed on a scale that ranged from 1 to 5 where 1 indicated the least favourable properties of the product, and 5 indicated the most favourable properties. 3. Results 3.1. Silver and gold nanoparticle suspensions The resulting suspensions were homogeneous. The suspension of silver nanoparticles was characterised by a dark yellow colour and the suspension containing gold nanoparticles exhibited a dark violet colour. The results of the spectrophotometric analysis are presented in Fig. 1A. The characteristic peaks are clearly visible. For the nanosilver and nanogold suspensions, the peak maxima were located at wavelengths of 421 and 533 nm, respectively. Based on ICP-OES analysis, the actual concentration of silver and gold nanoparticles were equal to 498 and 501 mg/L respectively. Fig. 1B presents the results of the nanoparticles sizes analysis and reveals that 76.8% of the silver nanoparticles had an average diameter 4
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Fig. 1. A) UV–vis spectroscopy results for nanosilver and nanogold suspensions diluted to 20 mg/kg, B) Histograms of the distribution of the nanoparticle sizes, C) TEM images of silver and gold nanoparticles.
Fig. 2. Photographs of the obtained cosmetic emulsions.
emulsion contacts the skin can be determined. The upper value of the viscosity limit determines the final impression following the application of the emulsion [33,34]. This value can also be an indicator of the tendency of the cosmetic preparation to delaminate during storage. Fig. 4B presents the viscosity dependence (η) on the shear rate (γ). Based on these results, it can be concluded that the viscosity decreased with increasing shear rate. According to the literature, this pattern is characteristic of, among others things, non-Newtonian liquids that are diluted and subjected to shear, including, for example, emulsions [35]. Fig. 4C shows the results of the viscosity analysis for two specific shear rates. No viscosity dependence on the nanometal content was observed for the creams tested. The viscosities of the cosmetic emulsions were greater when the low shear rate was used. The size and distributions of the metal nanoparticles embedded in the creams at the concentration of 200 mg/kg were examined using transmission electron microscopy with an X-ray scattering spectrometer (TEM-EDX). Fig. 5 presents microphotographs of the studied samples. The arrangement of gold particles was homogeneous. The observed size of the nanoparticles were consistent with the results of the DLS analysis. The presence of both silver and gold nanoparticles was confirmed using EDX analysis. The reference cream consisted of carbon, oxygen, silica, chlorine, and potassium. The spectra obtained for the creams with the metallic nanoparticles also contained peaks indicating silver and gold.
red-shift effect seen for creams with nanosilver proves that when embedded into the structure of the cream, the silver nanoparticles agglomerate. This phenomenon may occur in the phase of introducing the silver suspension into the cream mixture. The increased size of the nanoparticles embedded in the cream (200 mg/kg) is also seen in Fig. 5B. The main size of the silver nanoparticles contained in the water suspension was equal to 8 nm (which was confirmed by both DLS and TEM analyses). However, after introducing them into the cream, their sizes were larger and equal to 30 nm. These observations are in line with the results obtained by Lizoń et al. [30] and Bu et al. [31]. Compared with nanogold, the agglomeration of silver nanoparticles may be due to the fact that the native silver nanoparticles were characterised by lower absolute electrokinetic potential values, which express the stability of nanoparticles. Examination of the rheological properties of a cosmetic preparation allows for the characterisation of its consistency and ease of spreading on the skin [32]. Fig. 4A presents graphs of the dependence of the shear stress (τ) on the shear rate (γ). There are visible differences between the flow curves. For the emulsions with silver or gold nanoparticles, the shear stress decreased with increasing shear rate from 10 to 500 rpm. This finding contracts with that for the reference emulsion for which a slight increase in the shear stress is visible. The lowest yield points were found for the creams with gold and silver nanoparticles at the concentration of 20 and 65 mg/kg, respectively, and for the reference emulsion. According to the literature, emulsions with low viscosity limits are characterised by better consistency and spreading on the skin surface. Based on the viscosity limit, the first impression when the 5
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Fig. 3. A) pH values of the obtained creams (n = 3), B) Plots of the dependencies of the absorbances on the wavelengths for the creams with silver and gold nanoparticles.
Their regular use leads to their release into the environment in significant amounts. Most of these products are biologically active and characterised by high stability and the ability to accumulate, which poses threats to natural ecosystems and human health [36]. Cosmetics containing metallic nanoparticles comprise a significant contribution to the environmental levels of these nanoparticles. According to Giese et al., most released nanosilver particles originate from textiles. However, cleaning products and cosmetics are also important sources of nanosilver particles that are present in wastewater [37]. Producers of cosmetics with metallic nanoparticles should be aware of the potential risks that are related to their applications in consumer products. Filon et al. [38] studied the literature regarding the influence of physicochemical properties of nanosilver particles on their penetration through the damaged or unimpaired skin membrane. Theses authors figured out that the rate of penetration depends mainly on the form of the nanoparticles (i.e., metallic or non-metallic). The sizes of the nanoparticles were the second-most-important factor. Nanosilver particles with diameters below 20 nm are able to penetrate both intact and damaged skin, whereas particles with sizes between 21 and 45 nm can only go into damaged skin. Larger particles are not able to penetrate either damaged or intact skin. Also, Filon et al. [39] studied the influence of nanosilver on human health. Researchers conducted analyses of the in-
3.3. Analysis of nanoparticle release from topical formulations and nanoparticle accumulation in model bodily fluids The results of spectrophotometric analysis of a receptor fluid after 48 h are presented in Fig. 6. Peaks characteristic of silver nanoparticles appeared between 400 and 500 nm, and peaks characteristic of gold nanoparticles appeared in the 500–600 nm range. Characteristic peaks were recorded for the cream samples with nanosilver at concentrations of 110, 155, and 200 mg/kg. The highest peak was recorded at the wavelength of 477 nm with an absorbance of 0.427. For gold, the highest peak was recorded at the wavelength of 533 nm with an absorbance of 0.352. Gold nanoparticles were identified in all creams samples with nanogold. Higher concentrations of metallic nanoparticles in the creams resulted in higher absorbance values. The actual concentration of silver in receptor fluids are: 2.22 mg/kg (for cream with nanoAg 110 mg/kg), 2.71 (for cream with nanoAg 155 mg/kg) and 4.15 mg/kg (for cream with nanoAg 200 mg/kg). The concentration of gold in receptor fluids for creams with nanoAu 20–200 mg/kg are: 1.65, 2.08, 2.83, 4.46 and 5.20 mg/kg. These results indicated the presence of metallic nanoparticles in the receptor fluid, which resulted from their ability to penetrate the model membrane. Cosmetic products are applied extensively throughout the world. 6
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Fig. 4. A) Flow curves of the dependence of the shear stress (τ) on the shear rate (γ), B) Viscosity dependence (η) on the shear rate (γ), C) Viscosity analyses for two specific shear rates (n = 100).
3.4. Microbiological studies
vivo penetration of nanosilver through human skin. Similar to our studies, a physiological solution was used as a receptor fluid. PVP-coated silver nanoparticles were dispersed in artificial sweat, and this donor suspension was applied in the study which lasted for 24 h. It was found that in case of damaged skin, the rate of penetration of silver was equal to 3.31%, and the resulting value did not exceed 1% when the intact skin was applied in the study. Tak et al. [40] performed research on the influence of the shape of silver nanoparticles and their penetration through model skin. These authors compared triangular spherical and rod-like nanoparticles that were subjected to in vitro and in vivo studies. They found that the rod-like nanoparticles are characterised by the highest rate of penetration in both types of studies. These authors suggest that it is highly possible that particles in this shape may accumulate in the dermal layers, which may lead to occurrence of many side-effects, such as argyria. Due to the basal plane in triangular nanoparticles, their penetration is the slowest. The authors conclude that these nanoparticles of this shape have the ideal form of a nanoparticulate biocidal agent because they exhibit satisfactory antibacterial properties. Gupta et al. [41] conducted simulations on the comparisons of the penetration rates of gold nanoparticles coated with neutral, cationic or anionic agents. The sizes of the particles also differed. They also figured out that all neutral particles penetrated through the model dermal membrane and reached the second layer. Both anionic and cationic gold nanoparticles were able to go through the membrane in which they were accumulated. The penetration rate was increased with the decreasing the size of the nanogold particles.
Results of the microbiological tests are presented in Table 1 There were no differences on the first day of incubation. After 48 and 72 h of incubation, inhibition of the growth of Aspergillus niger was observed for the creams with silver and gold nanoparticles at concentrations of 65, 110, 155, and 200 mg/kg. In the case of Saccharomyces cerevisiae, antifungal activity after 48 h of incubation was observed for the creams with silver nanoparticles at all concentrations and for the creams with gold nanoparticles at the concentrations of 155 and 200 mg/kg. After 72 h of incubation, the same nanogold creams samples exhibited good antifungal properties; however, the 20-mg/kg nanosilver cream no longer exhibited antifungal activity Minimal inhibition concentrations for creams with nanoparticles were as follows: MIC35% = 65 mg/kg for creams with nanosilver against A. niger after 48 h; MIC6% = 20 mg/kg for creams with nanosilver against S. cerevisiae after 48 h; MIC20% = 65 mg/kg for creams with nanogold against A. niger after 48 h; MIC4% = 155 mg/kg for creams with nanogold against S. cerevisiae after 48 h. Marslin et al. obtained silver nanoparticles using Withania somnifera leaves as a source of reducing and stabilising factors and used these nanoparticles to generate cosmetic creams. Compared with creams containing silver nitrate, these creams with nanosilver particles exhibited greater activities against human pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Candida albicans. According to these authors, this type of product may be applied in the cosmetic industry [42].
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Fig. 5. Microphotographs of the examined creams with the EDX collective spectra of the identified elements: A) Reference cream, B) cream with silver nanoparticles, C) cream with gold nanoparticles.
creamy consistency. The emulsions spread well on the surface of the skin and were absorbed quickly without leaving a greasy film on the skin. Compared with the reference sample, the best assessments in terms of smoothing the skin, consistency, adhesion, lubrication, application, and absorption were obtained for the 200-mg/kg gold nanoparticle cream. However, the best assessment in terms of homogeneity was obtained for the 20-mg/kg silver nanoparticles cream. The200-mg/ kg nanosilver cream performed worse than other emulsions. The
3.5. Studies with human participants Fig. 7A presents the collective results of the averaged ratings from the participants for the creams with silver and gold nanoparticles at concentrations of 20 and 200 mg/kg and for the reference cream. In the general assessments from the participants, all of the creams were characterised by satisfactory properties. The emulsions did not spill after a finger was dipped into the container, and they exhibited a
Fig. 6. UV–vis analysis of the receptor fluid. 8
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Fig. 7. A) Sensory profile of the tested creams, B) Smell and colour assessments (n = 14).
Declaration of Competing Interest
participants gave this cream the lowest homogeneity score due to the presence of numerous visible air bubbles. This emulsion also left a greasy film after it was spread on the skin. The result of the average smell and colour sensory evaluations are presented in Fig. 7B. The most mild and pleasant scent and most interesting colour were attributed to the 200-mh/kg gold nanoparticle cream. The lowest grades were obtained for the 200-mg/kg silver nanoparticle cream.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] U.S. EPA, Nanomaterial Case Study: Nanoscale Silver in Disinfectant Spray (External Review Draft), U.S. Environmental Protection Agency, Washington, DC, 2010 EPA/600/R-10/081. [2] X.F. Zhang, Z.G. Liu, W. Shen, S. Gurunathan, Int. J. Mol. Sci. 17 (2016) 1534. [3] C. Horner, A. Kumar and K. Nieradka, Nanosilver as a biocide in building materials, US patent US20060272542A1. [4] M. Rai, A. Yadav, A. Gade, Biotechnol. Adv. 27 (2009) 76. [5] M. Bachelet, Mater. Sci. Technol. 32 (2016) 794. [6] C. Contado, Front. Chem. 3 (2015) 48. [7] Quan Zhou Hu Zheng Nano Technology Co., Ltd, Quan Zhou Hu Zheng Nano Technology Co., Ltd.® Nano-gold Mask, available on https://www.nanotechproject. org/cpi/products/quan-zhou-hu-zheng-nano-technology-co-ltd-r-nano-gold-mask/. [8] Chantecaille offer, Nano Gold Energizing Cream, available on https://www.saksfifthavenue.com/chantecaille-nano-gold-energizing-cream/product/ 0435297663735?sid=120A0B312FA8&R=656509702909&FOLDER < > folder_ id=282574492711986&ASSORTMENT < > ast_id=1408474399545537& bmUID=1239646676269&P_name=Chantecaille. [9] Lexon Nanotech, Inc. offer, Nanorama - Gold Toothpaste, available on https:// www.nanotechproject.org/cpi/products/nanorama-gold-toothpaste/. [10] S. Raj, S. Jose, U.S. Sumod, M. Sabitha, J. Pharm. Bioallied Sci. 4 (2012) 186. [11] S. Asghari, S.A. Johari, J.H. Lee, Y.S. Kim, Y.B. Jeon, H.J. YB, M.C. Moon, I.J. Yu, Int. J. Nanobiotechnology Pharm. 10 (2012) 14. [12] K. Bilberg, H. Malte, T. Wang, E. Baatrup, Aquat. Toxicol. 96 (2010) 159. [13] A. Massarsky, L. Dupuis, J. Taylor, S. Eisa-Beygi, L. Strek, V.L. Trudeau, T.W. Moon, Chemosphere 92 (2013) 59. [14] A. Loghman, S.H. Iraj, D.A. Naghi, M. Pejman, Afr. J. Biotechnol. 11 (2012) 6207. [15] Nano Siver offer, The Best Nano Colloidal Silver - 16 Oz -20 PPM Colloidal Silver, available on amazon.com. [16] Atlantean Alchemy offer, Colloidal Silver Half Gallon 60 PPM, available on amazon. com. [17] Silver Mountain Minerals offer, Liquid Silver 16 oz.240 PPM, Silver Mountain Minerals, available on amazon.com. [18] Nano Siver offer, Nano Silver colloidal silver 10 PPM, available on amazon.com.
4. Conclusions Stable cosmetic formulations (i.e., creams) with silver or gold nanoparticles at different concentrations were obtained. The presences of the nanoparticles in the creams were confirmed with analytical and microscopic techniques. There is a difference in embedding silver and gold nanoaparticles into the structure of a cream. Silver introduced to the cream mixture agglomerates which is confirmed by a red shift that is visible in the spectra of creams with nanosilver. This is also revealed by TEM images of creams with nanoAg. In contrast, gold nanoparticles do not agglomerate after introduction to cream mixtures. This phenomenon is related to the values of the electrokinetic potentials of native metallic nanoparticles. The high absolute value that is characteristic of native nanogold particles confirms their high stability. Thanks to the greater value of the electrokinetic potential located on the surface of gold nanoparticles, they stay stable even after introducing them into cream mixture. This contrasts with silver nanoparticles, which agglomerate easier. The microbiological tests revealed that the investigated creams with both silver and gold nanoparticles were characterised by different fungicidal properties against Aspergillus niger and Saccharomyces cerevisiae. The permeabilities of the metallic nanoparticles through the dermal membrane were confirmed for the samples with concentrations of 110–200 mg/kg, which represents an issue of concern given the harmful effects of metallic nanoparticles on living organisms. 9
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[19] Silver Miracles offer, Colloidal Silver Gel 75 ppm, available on amazon.com. [20] My Doctor Suggests offer, Organic Exfoliating Silver Soap 30 ppm, available on amazon.com. [21] Generic offer, Colloidal Silver Soap Silver Biotics Gel 20 ppm, available on amazon. com. [22] Silver pure offer, 3-1/2% Nanosilver (35,000 ppm) Infused Organic Skin Creams, 35,000 ppm, available on silverpure.com. [23] Opinion on Colloidal Silver (nano), (2018) SCCS/1596/18, 24-25 October. [24] Ringer’s solution, Cold Spring Harb. Protoc. (2008). [25] B. Tal- Figiel, W. Figiel, A. Michno, Proc. Univ. Appl. Chem. Biotechnol. 54 (2015) 051. [26] J. Blaak, R. Wohlfart, N.Y. Schürer, JCDSA 1 (2011) 50. [27] H. Lambers, S. Piessens, A. Bloem, H. Pronk, P. Finkel, Int. J. Cosmet. Sci. 22 (2006) 359. [28] J. Tarun, J. Susan, J. Suria, V.J. Susan, S. Criton, Indian J. Dermatol. 59 (2014) 442. [29] J. Blaak, P. Staib, Curr. Probl. Dermatol. 54 (2018) 132. [30] A. Lizoń, R. Drożdż, Przeglä…d Med. Uniw. Rzesz. I Nar. Inst. Lekã³w W Warszawie 75 (2018) 457.
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