Concerns have been raised about the safety of preservatives, which are very important in most cosmetic preparations. The antibacterial effect of silver (Ag) has been recognized; however, Silver has some limitations as a preservative, such as its effect on salts. In this study, we investigated the effect of recently synthesized nanosilver on microorganisms, the permeability of the human skin and the cytotoxicity of AgNPs in human keratinocytes on irradiation. ultraviolet UVB. AgNPs were found to be very stable and they did not settle for more than 1 year. Nanosilver showed sufficient preservative efficacy against mixed bacteria and mixed fungi, and did not penetrate normal human skin. At a concentration of 0.002–0.02 ppm, nano silver has no effect on HaCaT keratinocytes and does not increase cell death caused by UVB ultraviolet rays. These results suggest that nanosilver have the potential to be used as preservatives in cosmetics.
Preservatives are necessary to prevent primary microbiological contamination during the preparation and manufacture of cosmetics, and to prevent secondary microbiological contamination after production when the consumer opens and cans. As cosmetic preservatives, compounds such as phenoxyethanol and parabens have been used separately or in different combinations.
However, these antibacterial compounds not only temporarily irritate the skin, but also increase sensitivity to ultraviolet (UV) rays. In fact, we have shown that methylparaben (MP) within the recommended concentration range can be toxic when exposed to UVB rays. On the other hand, the antibacterial activity of small amounts of metals, first recognized in the 19th century, is the basis for many antibacterial processes and products.
Of these, silver (Silver) and Silver based compounds have been widely used and used to control bacterial growth in a variety of applications. These phenomena significantly hinder the use of Silver as a cosmetic preservative, as its antibacterial activities are compromised when Silver is separated from cosmetic ingredients. Many of these problems can be overcome using nanosilver (AgNPs). Reducing the particle size also improves biocompatibility, and this has led to an increasing focus on the medical applications of nanotechnology. Because we are interested in the potential use of nanosilver as preservatives in pharmaceuticals or cosmetics, the aim of our research is to find out if AgNPs have antimicrobial activity against many microorganisms. or whether AgNPs are safe for the skin.
Test method for nanosilver
Nanosilver sample
AgNPs seeds were obtained from Johzen Ltd. (Kyoto, Japan). The concentration of Silver in the initial solution of AgNPs is 50 parts per million (ppm; pure water 96,995%, sodium bicarbonate 1%, cellulose gum 2%, Silver 0.005%).
Surface zeta potential, size and size distribution of AgNPs were analyzed by laser scattering method (ELS-Z; Otsuka Electronics Co., Ltd., Osaka, Japan).
Check the antibacterial activity of nano silver
The antibacterial activity of AgNPs was evaluated based on bacterial suspension (Escherichia coli, American Type Culture Collection [ATCC] 8739; Pseudomonas aeruginosa, ATCC 9027; Staphylococcus aureus, ATCC 6538), fungal suspension (yeasts and molds ) (Candida albicans, ATCC 10231; Aspergillus niger, ATCC 16404; Penicillium citrium, ATCC 9849; Aureobasidium pullulans, IFO 6353), and waste suspension (filtered kitchen drainage). Each strain was inoculated on the surface of slant agar. Soy casein digestion agar was used for bacterial growth and potato dextrose agar medium for yeast and mold growth. Bacterial cultures were incubated at 30 ° C for 20 hours, yeast culture at 25 ° C for 48 hours, and mold culture at 25 ° C for 1 week. The microorganisms were harvested aseptically using a platinum ring followed by a suspension in sterile physiological saline, and the number of viable microorganisms was adjusted to approximately 108 microorganisms per ml. . These suspensions are used as inoculants, while the filtered stove drainage is used directly as the inoculum.
For the test, 20 g of AgNPs (1.0 ppm, made up of nano 50 ppm Silver by dilution with pure water) were transferred sterile to each of the three sterilized vials, and each vial was Inoculate 0.2 mL of microbiological suspension. Then, the inoculated vials of the bacterial suspension were incubated at 30 ° C, and the inoculated vials of the fungal suspension were incubated at 25 ° C. The number of viable microorganisms was determined at 7, 14 and 21 days. by the pouring plate method.
The penetration through the skin of nanosilver
Nanosilver is evaluated in a dedicated skin pass test using human skin. Sample preparation was carried out by BIOalternatives (Gençay, France). Pieces of human skin (4 cm × 4 cm) were prepared from a healthy subject during plastic surgery (female donor, 49 years old).
The quality of the skin is observed with the naked eye and the altered areas are removed. Each piece of skin is placed as a barrier between the two halves of the diffuse cell, with the horny layer facing the donor chamber. The receptor compartment is filled with 8 mL of receptor liquid (phosphate buffered brine, PBS), covered and allowed to reach the correct temperature.
The skin sample is kept at 32 ° C by circulating the temperature controlled liquid in the jacket around the receptor chamber. Nanosilver with a concentration of 0.5 ppm or 50 ppm are applied to the skin surface (55 μL / cm2). The test was carried out for 24 hours for three times. At 1, 6, 12 and 24 hours, the medium under the skin is harvested and immediately frozen at -80 ° C until the Silver concentration is measured.
Silver concentration was determined by inductively combined plasma mass spectrometry (ICP-MS) after microwave digestion. Equipment used in ICP-MS is SPI-9000 (SII NanoTechnology Inc., Tokyo, Japan)
After the skin penetration test, the skin was frozen at -80 ° C until the Silver concentration in the skin tissue was measured. The silver concentration of the skin was determined by ICP Atomic Emission Spectroscopy (ICP-AES) after microwave digestion. The device used in ICP-AES is the ICPS8000 (Shimadzu Corp., Kyoto, Japan).
Horn cells in normal humans
HaCaT cells are a naturally occurring human immortal cell line and provided by Dr. Kato of the Department of Dermatology of Kyoto Prefectural Medical University (Kyoto, Japan). HaCaT horn cells were cultured in an 80 cm2 cell culture flask and were maintained in a modified Dulbecco essential medium (Gibco-BRL, Gaithersburg, Maryland) supplemented with 5% pregnant bovine serum ( Equitech-Bio Inc., Kerrvill, Texas), 2 mM glutamine, and 100 U / mL penicillin / streptomycin (Gibco) at 37 ° C in a moist environment containing 5% CO2. Cells were divided every 7 days at a ratio of 1:10.
UVB irradiation after treatment with nanosilver or methylparaben
The UVB source consists of six fluorescent luminaires (FL-20SE-30; Toshiba Medical Supply, Tokyo, Japan) with an emission spectrum of 275–375 nm, mainly in the UVB range, peaks at 305 nm and includes a small amount of UVA and UVC (energy: UVA, 30%; UVB, 54%; UVC, 0.2%). UVB radiation is measured with a UV radiation meter (UVR3036 / S2; Topcon Corp., Tokyo, Japan).
HaCaT keratinocytes were cultured in 35 mm cell culture plates until confluence and incubated for 24 h in the absence or presence of AgNPs or corresponding MPs. After treatment with AgNPs or MP, the cells were exposed to UVB (30 mJ / cm2).
Before UVB irradiation, the medium was purified and replaced with PBS. After irradiation, cells were incubated in cell cultures without AgNPs or MP seeds for 24 hours.
Microscopic analysis of dead cells
Apoptosis caused by AgNPs or MP with and without UVB was determined by fluorescence microscopy after staining with Hoechst 33342 (HO342) and propidium iodide (PI). After 24 hours of UVB irradiation, cells were washed twice with PBS and incubated with 10 μg / mL HO342 dye for 15 minutes at 37 ° C and with 10 μg / mL PI for 10 minutes at 37 ° C. Staining cells Dual color was examined with a reverse fluorescence microscope IX70-23FL / DIC-SP (Olympus, Tokyo, Japan).
The snapshot (MPEG format) is taken from four random fields. Living cells, viable cells and early apoptotic cells (which retain membrane function) receive the blue dye (HO342). The apoptosis is morphologically characterized by a concentrated chromatin. Red stained cells (PI) are considered end-stage apoptotic cells (solid chromatin) or necrotic cells.
The results of research on nanosilver in cosmetics
Features of AgNPs synthesized
AgNPs solution has light yellow color, no change of color, no sedimentation after being stored for more than 1 year. The surface zeta potential of AgNPs was measured with ELS-Z (Otsuka Electronics Co., Ltd.). The maximum potential peaks of AgNPs were measured at -32.76 mV (Figure 1). The shape and size distribution of AgNPs were analyzed by laser scattering. highly monochromatic dispersion with mean diameter of 730.5 nm and standard deviation of 14.70 nm (n = 5, Figure 2). coated with cellulose gum and water molecules.
The zeta potential and the particle size distribution of nanosilver
Antimicrobial activity of nanosilver
Antimicrobial tests are performed using a mixture of bacterial solution, mixture of fungal solution and waste suspension (filtered kitchen drain) treated with AgNPs at concentration 1, 0 ppm. Table 1 shows the number of microorganisms in the inoculant suspension. AgNPs at 1.0 ppm showed adequate storage efficiency (CFU / g b101) for mixed bacteria and mixed fungi (Table 2).
AgNPs penetration through the skin
We evaluated whether nanosilver could penetrate human skin in a specialized transdermal test using human skin. Silver nanoparticles are rated at 0.5 ppm or 50 ppm. AgNPs were applied on the human skin surface (55 μL / cm2), and the concentration of Silver was measured in the environment below the skin at 1, 6, 12 and 24 hours after exposure. Silver was not detected at any point in time (Table 3). Furthermore, Silver in skin tissue was not detected at a dosage of 0.5 ppm or 50 ppm at any one time.
Effects of AgNPs on cell death caused by UVB radiation
We confirm that nanosilver do not penetrate human skin. However, when the protective function of human skin is interrupted by a wound or sunburn, AgNPs can penetrate the skin. Therefore, we studied the cytotoxic effects of AgNPs with and without UVB irradiation in keratinocytes. To evaluate the type of dead cells in HaCaT keratinocytes, we used a double staining method using HO342 and PI. The obtained images indicated that small amounts of apoptosis and necrosis were present, and that AgNPs at concentrations 0.002–0.02 ppm had no effect on cell death. Irradiation of UVB rays at 30 mJ / cm2 causes death (mainly terminal death). However, nanosilver at a concentration of 0.002–0.02 ppm did not increase the chances of cell death due to UVB rays of HaCaT keratinocytes (Figures 3, A). MP at a concentration of 0.003% to 0.03% has no effect on cell death; however, 0.003% MP increased UVB-induced cell death in HaCaT keratinocytes (Figures 3, B).
Figure 3. HaCaT keratinocytes stained with Hoechst 33342 (blue) and propidium iodide (red). Blue-stained cells have the function of cell membranes intact. Nuclear cells are early apoptotic cells (green) and late apoptotic cells (red). Image represents four independent experiments. (A) UVB-induced HaCaT death in keratinocytes treated with AgNPs (Silver) particles. (B) apoptosis induced by UVB rays in HaCaT keratinocytes treated with methylparaben (MP).
Discuss
The results of this study showed that nanosilver is a safe and stable preservative, and they are effective against a wide variety of microorganisms. It is known that Silver ions and Silver-based compounds have a strong antimicrobial effect. 3.7 However, these Silver-based compounds gradually precipitate in the solutions. When Silver is separated from cosmetic ingredients, its antibacterial effect is affected. The AgNPs particle solution used in this study is very stable and does not settle after being stored for more than 1 year.
Hence, many limitations related to Silver can be overcome through the use of AgNPs. The inhibitory effect of nano-silver on microorganisms has been studied. It was reported that the antibacterial activity of AgNPs against E. coli was concentration dependent and that AgNPs disrupted the membrane structure of E. coli.
Recently, Danilczuk et al. Reported that Tiny Silver produces free radicals, detected by electron spin resonance. In addition, Kim et al. Also reported that the antibacterial mechanism of AgNPs is involved in the formation of free radicals.
It is not clear whether the nanoparticles are absorbed or penetrated into human skin. If nanosilver cannot penetrate human skin, they can be considered very safe. To confirm this point, we performed a transdermal test using human skin. The results showed that AgNPs were not capable of penetrating human skin, which in turn meant that they functioned only as a preservative and did not stimulate keratinocytes.
However, when the barrier function of human skin is disrupted, the AgNPs on the skin’s surface can penetrate the skin. Therefore, we studied the effects of AgNPs on keratinocytes more in depth. In this study, AgNPs 1.0 ppm showed antibacterial activities, and AgNPs 50 ppm could not penetrate the skin’s surface. However, if the skin is somehow damaged (eg an allergy), it is possible that 0.2% to 2% of AgNPs can penetrate the skin. nanosilver concentrations of 0.002–0.02 ppm did not show any cytotoxicity to keratinocytes and did not affect cell death caused by UVB rays.
Conversely, although MP is considered a safe preservative, it increases cell death caused by UVB rays. This result supports a previous report in which we demonstrated the harmful potential of MP in normal human skin keratinocytes exposed to UVB rays in vitro.
The use of MP in cosmetic products is allowed at a maximum concentration of 0.8% (wt / wt) According to the regulations of the Danish and European Economic Communities, and the typical concentration of MP in the US product is below 0.32%. Previous studies have shown that about 1% of the applied MP reaches the bottom layer of the cuticle in the Yucatan mini pig. Consequently, an actual concentration of MP (0.003%) increases cell viability caused by UVB rays. Combined with previous reports, this study confirms the great potential of AgNPs for use as a preservative in cosmetics compared to MP. Nanosilver formulated by Johzen Co., Ltd. (Kyoto, Japan) is very effective at low concentrations, does not easily penetrate the skin barrier and has no adverse effects on ketone cells.
