Nanoparticles (NPs) are promising products in industry and medicine due to their unique physicochemical properties. In particular, zinc oxide (ZnO) NPs are extensively incorporated into sunscreens to protect the skin from exposure to ultraviolet radiation. However, there are several health concerns about skin penetration and the resultant toxicity. As methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the toxicity of ZnO NPs toward humans. In this study, we developed a platform to simultaneously detect skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs. First, we generated a stable reporter cell line expressing green fluorescent protein (GFP) under the control of interleukin-8 (IL-8) promoter activity. The expression levels of GFP induced by zinc reflected the endogenous IL-8 expression levels and the pro-inflammatory responses. Next, we found that fibrin hydrogel can reproduce permeability to zinc ion of a human skin equivalent model and is therefore a promising material to assess skin permeability to zinc ion. Then, we constructed a fibrin hydrogel–based in vitro bioassay system for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and a fibrin hydrogel layer. This bioassay system is a promising in vitro permeation test due to its technical simplicity and good predictability. Overall, we believe that our bioassay system can be widely used in the cosmetics and pharmaceutical industries.
A nanoparticle (NP) is defined as a material having at least one dimension in a range of 1–100 nm  and unique physicochemical properties, such as high catalytic activity and a distinct absorbance spectrum, compared with micron-scale particles . These unique properties are beneficial for industrial use, and many NPs are employed in consumer products. Among them, titanium dioxide (TiO2) NPs and zinc oxide (ZnO) NPs have been most extensively used for years. As TiO2 and ZnO both absorb and reflect ultraviolet (UV) radiation, these are incorporated into commercial sunscreens or skin care products in order to prevent sunburn, skin cancer, and photo aging. As the ability of these particles to protect from UV exposure is directly related to their size , the particles used in sunscreens have small sizes and large specific surface areas that lead to high chemical reactivity, such as the generation of free radicals and reactive oxygen species . As the production and use of NPs in sunscreen have increased over the past few years, concerns have risen regarding their potential risks to human health and the environment.
Several studies have shown that ZnO NPs have potential adverse effects, such as decrease of cell viability, loss of membrane integrity, and induction of cell apoptosis . In addition, ZnO NPs can potentially induce pro-inflammatory responses both in vitro and in vivo [6,7,8,9]. ZnO NP–mediated toxicity may be divided into two categories on the basis of the mode of action: the first category is related to the physicochemical properties of ZnO NPs, such as size, shape, and surface charge. For example, Hsiao and Huang  concluded that both shape and size of ZnO NPs influence cell viability and interleukin-8 (IL-8) production in human lung epithelial cells. The second category is related to the solubility of ZnO NPs. Toxic zinc ions released from ZnO NPs induce oxidative stress, DNA damage, and inflammation [11,12,13]. Based on the results of these studies, the Scientific Committee on Consumer Safety (SCCS) of the European Commission concluded that ZnO NPs contained in sunscreens do not have any adverse effects on human after application on healthy, intact, or sunburned skin . However, as in vitro, ex vivo, or in vivo methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the safety or toxicity of ZnO NPs toward humans and the environment.
The major possible route of entry of NPs into the human body is absorption through the trachea, gastrointestinal tract, and skin . In particular, the skin is the largest organ of the body and plays an important role in protecting organs from chemical substances and biological agents. As sunscreen cosmetics containing ZnO NPs are applied daily on skin, the skin penetration and adverse effects of these NPs have emerged as a major cause of concern. Several in vitro and in vivo studies suggested that ZnO NPs do not penetrate the viable epidermis but remain on the stratum corneum (SC) [16,17,18,19,20,21,22]. On the other hand, Gulson and co-workers reported the detection of 68Zn in blood and urine after the application of sunscreen containing ZnO NPs on healthy human skin, although it was not clear if 68Zn penetrated as ZnO NPs or zinc ion [23, 24]. Similarly, it was reported that 68Zn levels in mouse internal organs were increased by the topical application of sunscreen containing ZnO NPs . In addition, an in vivo study on human volunteers carried out by Leite-Silva et al.  demonstrated that ZnO NPs penetrated the stratum granulosum of the epidermis. Therefore, it is currently impossible to confirm the presence or absence of skin penetration of ZnO NPs.
The permeation and safety assessment are important for the development of new cosmetic or pharmaceutic products before human application. To estimate the harmfulness of a substance to humans, conventionally, toxicological data are obtained by investigating the toxicological profiles of the substance on animals. Although animal experiments have been one of the main approaches for permeation and safety assessment, those have been controversial because of ethical considerations. In addition, as in vivo animal tests of finished cosmetic products or cosmetic ingredients are now prohibited, in vitro methods are preferred. Human skin models and porcine skin models are mainly recommended by the Organisation for Economic Cooperation and Development (OECD) for skin permeation studies of chemicals in OECD TG 428 . Permeability is assessed by quantifying concentration in the acceptor compartment after permeation through the skin layer. However, these models have not yet been validated for NPs and are expensive and time-consuming . In addition, some studies suggested that the porcine skin is a poor model of human skin for studies of transdermal permeability to NPs [29, 30]. The three-dimensional (3D) skin models are highly valuable and effective tools to evaluate the permeation or safety of cosmetic ingredients or drug formulations . Currently, several 3D skin models, such as EpiSkin™, SkinEthic™, and EpiDerm™, have been developed and become commercially available. These models are useful tools for testing of corrosivity and irritancy as well as in drug permeability studies . However, to our knowledge, there are no standardized and validated in vitro methods to simultaneously evaluate skin permeability to and pro-inflammatory activity mediated by ZnO NPs.
In this study, armed with the objective of developing a cell-based bioassay system for the detection of pro-inflammatory response induced by zinc ion, we established a stable IL-8 reporter cell line, IL-8-HaCaT, by transfecting plasmid vector into HaCaT cells. In the plasmid vector, green fluorescent protein (GFP) gene expression is regulated by IL-8 promoter activity. We examined endogenous IL-8 mRNA levels and IL-8 promoter activity in IL-8-HaCaT cells, after treating the cells with zinc chloride (ZnCl2), ZnO NPs, or silica-coated ZnO (ZnO-Si) NPs, and confirmed the correlation between their induction of IL-8 expression and IL-8 promoter activity. In addition, we found that the permeability to zinc ion of the human skin equivalent model (HSEM) can be reproduced by using fibrin hydrogel as the permeable layer. This bioassay system offers an accurate, convenient, and high-throughput method for the identification of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs.
Materials and methods
Preparation of collagen, alginate, and fibrin hydrogels
To prepare collagen hydrogels, various volumes of 2 mg/mL collagen gel solution (Nitta Gelatin, Osaka, Japan) were added to each well of 24-well plates, and the solution was allowed the gel at 37 °C for 30 min. To prepare alginate hydrogels, various volumes of 10 mg/mL sodium alginate solution were added to each well of 24-well plates. Calcium chloride solution (100 mM, Kimica, Tokyo, Japan) was used to cross-link the alginate solution to form the hydrogels. To prepare fibrin hydrogels, thrombin solution containing 50 units/mL thrombin (Sigma-Aldrich, St. Louis, MO) and 80 mM CaCl2 (Wako Pure Chemical Industries, Osaka, Japan), and fibrinogen solution containing 60 mg/mL fibrinogen (Sigma-Aldrich) were prepared in phosphate-buffered saline (PBS) prior to use . Fibrin formation was initiated by mixing the thrombin solution and the fibrinogen solution in the ratio of 1:5 before allowing the mixture to gel at 37 °C in an atmosphere of 5% CO2 for 30 min.
Scanning electron microscopy
The hydrogels were fixed in acetone for 1 h at − 80 °C and dehydrated with a series of increasing concentrations of ethanol solutions (70%, 80%, 90%, 95%, and 100%). After the ethanol dehydration, the samples were freeze-dried in t-butyl alcohol and coated with osmium tetroxide. The coated samples were observed under a scanning electron microscope (SEM, Zeiss Merlin; Carl Zeiss, Oberkochen, Germany).
Measurement of zinc concentration
Fibrin hydrogels were incubated with ZnCl2 (Wako Pure Chemical Industries) for 24 and 48 h. After that, the concentrations of zinc ion released into the underlying culture well were measured by an inductively coupled plasma optical emission spectrometer (ICP-OES, Agilent 5110 VDV; Agilent Technologies, Tokyo, Japan).
Cell culture and treatment
Human keratinocyte HaCaT cells were purchased from the German Cancer Research Center (DKFZ, Heidelberg, Germany). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; HyClone, Thermo Fisher Scientific, Inc., Waltham, MA) at 37 °C in an atmosphere of 5% CO2. For experiments, the cells were seeded in multi-well plates (Thermo Fisher Scientific, Inc.) of different sizes at the density of 5 × 105 cells/mL and incubated for 24 h. Subsequently, the culture medium was replaced with freshly prepared DMEM containing 10% FBS, 25 mM HEPES (pH 7.0), and ZnCl2 or ZnO NPs at various concentrations and incubated at 37 °C in an atmosphere of 5% CO2.
Generation of stable cell line
The IL-8 promoter region was amplified from the human genome by PCR using the following primer set: forward, 5′-GACTCTCGAGCCTCAAGTCTTAGGTTGGTTG-3′; and reverse, 5′-CTGAGGATCCGTGTGCTCTGCTGTCTCTG-3′ . The resulting DNA fragment was digested with XhoI and BamHI, gel-purified, and ligated into similarly treated pAcGFP1-1 vector (Clontech, Mountain View, CA) by using T4 DNA ligase (Takara Bio), and then cloned into ECOS competent Escherichia coli DH5 alpha (Nippon Gene, Toyama, Japan). Stable transfection of the HaCaT cells was carried out using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. The stably transfected cells were selected by using culture medium containing 800 μg/mL G418 (Nacalai Tesque, Kyoto, Japan). After approximately 4 weeks, G418-resistant cell clones were established.
Fluorescence microscopy and flow cytometry analysis
To determine GFP expression levels in the IL-8-HaCaT cells, the cells were seeded in 96- or 12-well plates at 5 × 105 cells/mL. After 24-h incubation, the medium was replaced with DMEM containing 10% FBS, 25 mM HEPES (pH 7.0), and TNF-α or ZnCl2 at various concentrations. For the visualization of GFP fluorescence in the IL-8-HaCaT cells, the cells were observed under a BZ-X710 all-in-one fluorescence microscope (Keyence, Osaka, Japan). For the measurement of GFP fluorescence levels, the cells were detached with trypsin, washed with PBS, and analyzed with a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) at the excitation wavelength of 488 nm and the emission wavelength of 525 nm. Data were collected from at least 10,000 gated events.
Preparation of ZnO or ZnO-Si dispersions
ZnO NPs and ZnO-Si NPs were purchased from Ishihara Sangyo Kaisha, Ltd. (Osaka, Japan) and Showa Denko K.K. (Tokyo, Japan), respectively. According to the previous report, ZnO NP has a diameter of 20–50 nm and a specific surface area of 31.5 m2/g, and ZnO-Si NP has a diameter of 10–100 nm and a specific surface area of 23.2 m2/g . The NP-medium dispersions were prepared as described previously . In brief, ZnO NPs or ZnO-Si NPs were dispersed in 1% bovine serum albumin at the concentration of 10 mg/mL by sonication prior to use. The solutions were diluted with culture medium or PBS and applied to the cells.
Cells were cultured with various concentrations of ZnCl2, ZnO NPs, or ZnO-Si NPs for 24 h. Cell viability was determined using a Premix WST-1 Cell Proliferation Assay System (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Cell membrane damage was determined with a Cytotoxicity Detection KitPLUS (lactate dehydrogenase (LDH) release assay; Roche Diagnostics GmbH, Mannheim, Germany).
IL-8 enzyme-linked immunosorbent assay
Cells were cultured with various concentrations of ZnCl2, ZnO NPs, or ZnO-Si NPs for 24 or 48 h. IL-8 protein levels in the cell supernatants were measured using a Human IL-8 ELISA Ready-SET-Go! Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions.
Isolation of total RNA and quantitative real-time PCR
IL-8-HaCaT cells under the fibrin hydrogel were harvested by using TRIzol reagent (Invitrogen, Carlsbad, CA), and total RNA was purified with an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. IL-8 mRNA levels were analyzed using the TaqMan gene expression assay (ID: Hs00174103_m1, Applied Biosystems). The housekeeping gene β-actin (ID: Hs99999903_m1) was used as the internal control. Target mRNA levels were measured using an Applied Biosystems 7300 real-time PCR system. The IL-8 expression levels in each sample were normalized to β-actin and then compared with controls. The results are reported as fold change relative to control.
3D human keratinocyte skin equivalent model
The human reconstituted epidermis 3D EpiDerm™ model (EPI-200) was purchased from MatTek Corporation (Ashland, MA) and prepared according to the protocol provided by the manufacturer. EPI-200 was treated with ZnCl2 (2 or 5 mM), ZnO NPs (1 mg/mL), or ZnO-Si NPs (1 mg/mL) and incubated at 37 °C in an atmosphere of 5% CO2 for 24 and 48 h. Cell viability was measured by the MTT assay. Total RNA was extracted by using TRIzol reagent and the RNeasy Mini Kit, as described above. The concentrations of zinc ion released into the underlying culture well were measured by ICP-OES.
All assays were conducted in triplicate at the very least. Data are expressed as means and standard deviation (SD). Statistical analyses were performed by analysis of variance (ANOVA) using the Dunnett test for multiple comparisons. The Student’s t test was used to determine the statistical significance of differences between two groups.
Results and discussion
Evaluation of permeability of natural hydrogel polymers
We first investigated the permeability of three kinds of natural hydrogel polymers, namely, collagen, alginate, and fibrin, to blue dextran dye. These hydrogels are widely used as biomaterials for cell culture, tissue engineering, and drug delivery . For experiments, the hydrogels were cast in Transwell insert and incubated with 100 μL of 1% blue dextran solution for 1 week. After that, the concentrations of blue dextran dye released into the underlying culture well were determined by measuring the absorbance at 620 nm. As shown in Fig. 1a, the blue dextran dye permeated the fibrin hydrogel in a thickness-dependent manner. On the other hand, the collagen and alginate hydrogels showed high and low permeability, respectively, with little thickness dependency. SEM images suggested that the permeability of these hydrogel polymers was dependent on pore size (Fig. 1b). Recently, Hou et al.  generated an artificial skin model by stacking human dermal fibroblast and collagen layer using 3D bioprinting technology, and used this to screen for transdermal permeability to NPs. Although they clearly revealed that positively charged NPs deeply penetrated their skin model, the collagen hydrogel used in our study is not suitable for the assessment of permeability to NPs because of its large pore size. In addition, we investigated the permeability of fibrin hydrogel to ZnCl2 and found that zinc ion permeated the fibrin hydrogel in a time-dependent manner (Fig. 1c). These results indicated that the fibrin hydrogel is a candidate material for the permeable layer which gradually permeates zinc ion.
Effects of zinc on IL-8 expression in HaCaT cells
To verify the cytotoxic and pro-inflammatory potential of zinc ion on HaCaT cells, the cellular damages and IL-8 productions were analyzed (Fig. 2). Cytotoxicity of ZnCl2 on HaCaT cells was assessed in terms of cell viability and cell membrane damage by the WST-1 assay and the LDH release assay, respectively (Fig. 2a and b). When HaCaT cells were treated with ZnCl2 at various concentrations for 24 h, no cytotoxicity was observed up to 100 μM. ZnCl2 showed significant inhibitory effect against HaCaT cells with the 50% inhibitory concentration (IC50) value of 225.5 ± 1.2 μM (Fig. 2a, inset). Interestingly, although cell membrane damage was detected at the concentration of 200 μM ZnCl2, cell viability was increased by approximately twofold at this concentration. This phenomenon is similar to a previous report that HaCaT cells treated with nontoxic concentrations of ZnCl2 showed increased proliferation and survival . To investigate inflammatory response in HaCaT cells, IL-8 release into culture medium was measured after 24- and 48-h exposure to ZnCl2 (Fig. 2c). IL-8 is a chemokine that is produced by many types of cells in response to inflammatory stimuli, and its transcription is upregulated by zinc . IL-8 protein levels were significantly and dose-dependently increased in ZnCl2-exposed cells. In addition, we investigated IL-8 mRNA levels in HaCaT cells exposed to ZnCl2 (Fig. 2d). Consistent with the IL-8 protein level data, ZnCl2 exposure increased IL-8 mRNA levels in HaCaT cells, indicating that the measurement of IL-8 expression levels is a reliable indicator of zinc-mediated pro-inflammatory response. At high ZnCl2 dose (200 μM), we observed IL-8 mRNA level was increased at 24 h, and that was decreased at 48 h (Fig. 2d). These results are consistent with previous report that investigated the effect of ZnCl2 on IL-8 mRNA levels using A549 cells . Recently, new test guidelines, including the IL-8 Luc assay (OECD TG442E), were released by OECD for skin sensitization tests . The IL-8 Luc assay evaluates the effect of substances on IL-8 promoter activity by measuring luciferase gene activity under the control of the IL-8 promoter [43, 44]. Taken together, our results indicate that IL-8 protein and mRNA expression levels are upregulated in HaCaT cells exposed to ZnCl2, and are a suitable parameter for the evaluation of zinc-dependent induction of pro-inflammatory response without significant cytotoxicity.
Construction of IL-8-HaCaT stable cell line
Reporter assay systems are widely used to study promoter activities and beneficial for many fields of research, including cell biology, in vivo imaging, and chemical assessment [45,46,47]. GFP is one of the most frequently used reporter genes and does not require substrates or reagents to emit quantifiable light signal. Stoehr et al.  demonstrated that the IL-8 reporter assay system using lung epithelial cell lines is valuable for evaluating the pro-inflammatory response induced by ZnO NPs. HaCaT cells were stably transfected with plasmid that allows for GFP expression under the control of the IL-8 promoter (hereinafter referred to IL-8-HaCaT cells). It was reported that the IL-8 promoter activity is stimulated by tumor necrosis factor alpha (TNF-α) via activation of the nuclear factor kappa B (NF-κB) signaling pathway . Then, to confirm whether IL-8-HaCaT cells expressed GFP gene in an IL-8 promoter–dependent manner, GFP reporter gene activity was assessed by fluorescence microscopy and flow cytometry in IL-8-HaCaT cells treated with TNF-α (Fig. 3). Representative fluorescence microscopy images shown in Fig. 3a indicate that treatment with TNF-α caused an increase of GFP fluorescence intensity. To perform a quantitative analysis of GFP fluorescence, we assessed the GFP fluorescence intensity of TNF-α-treated cells by flow cytometry (Fig. 3b). Consistent with the observation by fluorescence microscopy, GFP fluorescence intensity was increased by the treatment with TNF-α in a dose-dependent manner. Similar results were obtained from IL-8-HaCaT cells treated with ZnCl2 (Fig. 3c and d). These results suggested that IL-8-HaCaT cells may be used to evaluate agents affecting IL-8 promoter activity.
Response of IL-8-HaCaT cells laminated with fibrin hydrogel to TNF-α and ZnCl2
To evaluate whether a combination of fibrin hydrogel and IL-8-HaCaT cells is useful for permeability assays, we investigated the GFP and endogenous IL-8 expression levels in IL-8-HaCaT cells treated with TNF-α and ZnCl2. The IL-8-HaCaT cells were seeded into Transwell insert and incubated for 24 h. After that, the cells were laminated with fibrin hydrogel, and then TNF-α or ZnCl2 was applied on the fibrin hydrogel. As shown in Fig. 4a, although TNF-α and ZnCl2 at very high concentrations were required, they penetrated the fibrin hydrogel and induced GFP expression via IL-8 promoter–dependent activation. Since apically applied zinc gradually permeated fibrin hydrogel (Fig. 1c), the GFP fluorescence from IL-8-HaCaT cells treated with ZnCl2 was not observed at 24 h, but that was significantly induced at 48 h (Fig. 4a). In addition, to verify whether the induction of GFP fluorescence by TNF-α and ZnCl2 reflects the expression of endogenous IL-8, we performed quantitative real-time PCR analysis of IL-8 expression levels. As shown in Fig. 4b, the IL-8 mRNA expression level was markedly increased by the treatment with TNF-α. The treatment with ZnCl2 also induced IL-8 mRNA expression after 48 h. The GFP expression induced by ZnCl2 treatment was reduced with the increase of fibrin hydrogel volume (Fig. 4c), indicating that the permeability of fibrin hydrogel to zinc ion could be controlled by the hydrogel thickness. As zinc ion could easily permeate 50 μL of fibrin hydrogel, a significant cell death and low fluorescence level were observed. These results suggested that low molecular weight proteins and metallic ions, such as TNF-α and Zn2+, permeate the fibrin hydrogel and trigger the IL-8 promoter–dependent activation of GFP expression.
Comparison of permeability of 3D human skin equivalent model (EPI-200) and fibrin hydrogel to zinc ion
To investigate the permeability of human skin to zinc ion, the human reconstituted epidermis 3D EpiDerm model (EPI-200) was used as HSEM. The HSEM consisted of human-derived epidermal keratinocytes and developed a SC with barrier function. It was also suggested that the HSEM is suitable for the assessment of skin permeability to NPs [50, 51]. EPI-200 was incubated with ZnCl2 solution for 24 and 48 h. After that, the concentrations of zinc ion released into the underlying culture well were measured by ICP-OES. As shown in Fig. 5a and b, zinc ion permeated EPI-200 in a dose- and time-dependent manner without any cytotoxicity. Then, we investigated whether the permeability of EPI-200 to zinc ion could be reproduced by controlling fibrin hydrogel thickness (Fig. 5c). We found that the permeability to zinc ion of 200–225 μL of fibrin hydrogel is similar to that of EPI-200. In this condition, although the GFP expression induced by zinc ion was almost abolished (Fig. 5d), the endogenous IL-8 expression levels in IL-8-HaCaT cells (Fig. 5e) were similar to those in EPI-200 (Fig. 5f). These results suggested that fibrin hydrogel could reproduce permeability to zinc ion of HSEM, and is useful as a permeable layer to assay for skin permeability to zinc ion.
Effects of ZnO NPs on IL-8 expression in HaCaT cells
To investigate permeability to zinc ion released from NPs, we used two kinds of ZnO NPs, uncoated ZnO NPs and silica-coated ZnO (ZnO-Si) NPs. Previously, it was reported that the cytotoxicity of ZnO NPs was reduced by coating with a silica layer because the silica layer reduced the release of zinc ion and decreased surface contact with cells . The zinc ion release was suppressed by silica coating in 20 mM citrate buffer (pH 4.5), 20 mM sodium phosphate buffer (pH 7.2), and culture medium (Table 1). When HaCaT cells were treated with ZnO or ZnO-Si NPs at various concentrations for 24 h, the cytotoxicity of the NPs, assessed by WST-1 (Fig. 6a) and LDH release (Fig. 6b) assays, was reduced by the silica coating modification, similar to a previous study . The viability of cells treated with 35 μg/mL or higher concentration of ZnO NPs was decreased even after 4-h treatment (Fig. 6a, inset). In addition, we investigated inflammatory responses in HaCaT cells by evaluating IL-8 mRNA levels in the cells exposed ZnO or ZnO-Si NPs (Fig. 6c). Previously, Wu et al.  demonstrated that ZnO NPs caused an inflammatory response by increasing IL-8 production through the activation of the NF-κB signaling pathway. The IL-8 levels were significantly and dose-dependently increased in the ZnO NP–exposed cells. The inflammatory response caused by ZnO NPs was also reduced by the silica coating. To confirm whether IL-8-HaCaT cells expressed GFP gene in response to ZnO or ZnO-Si NPs, GFP reporter gene activity was assessed by fluorescence microscopy and flow cytometry. Representative fluorescence microscopy images are shown in Fig. 6d. Treatment with 20 μg/mL ZnO NPs caused an increase of GFP fluorescence intensity. The GFP fluorescence in IL-8-HaCaT cells treated with 35 μg/mL or higher concentration of ZnO NPs could not be detected because of cell death (Fig. 6a and d). In the case of ZnO-Si NPs, on the other hand, 35 μg/mL or higher concentration of ZnO-Si NPs was required to visualize GFP expression. To perform a quantitative analysis, we assessed the GFP fluorescence intensities of ZnO or ZnO-Si NP–treated cells by flow cytometry (Fig. 6e). Consistent with the results of endogenous IL-8 expression levels (Fig. 6c) and the fluorescence microscopic observations (Fig. 6d), the increase of GFP fluorescence intensity was observed by the treatment with ZnO NPs, and was suppressed by silica coating. These results indicated that ZnO and ZnO-Si NPs are suitable model NPs for the validation study of fibrin hydrogel–based skin permeation assay for zinc ion released from NPs.
Validation of fibrin hydrogel–based in vitro bioassay system to determine skin permeability to zinc ion released from NPs
To investigate the permeability of human skin to zinc ion, EPI-200 was incubated with ZnO or ZnO-Si NPs for 24 and 48 h. After that, cell viability and the concentrations of zinc ion released into the underlying culture well were measured by the MTT assay and ICP-OES, respectively. As shown in Fig. 7a and b, zinc ion released from NPs permeated EPI-200 in a time-dependent manner without any cytotoxicity. A high concentration of zinc ion from ZnO NPs permeated EPI-200 compared with that from ZnO-Si NPs, and this corroborated the increase in endogenous IL-8 expression (Fig. 7c). The levels of IL-8 expression were correlated with the concentrations of zinc ion released into underling culture well in the 3D skin model (EPI-200) after exposure to ZnCl2, ZnO, or ZnO-Si (Figs. 5a, f and 7b, c). Then, to investigate the permeability of the fibrin hydrogel–based in vitro bioassay system to zinc ion, the fibrin hydrogel–laminated IL-8-HaCaT cells were incubated with ZnO NPs or ZnO-Si NPs for 48 h, and then the concentrations of zinc ion that was released into the underlying culture well were measured. As shown in Fig. 7d, zinc ion released from the NPs permeated the fibrin hydrogel–laminated IL-8-HaCaT cells, but the permeation was suppressed by silica coating, consistent with the results obtained from EPI-200 (Fig. 7b). In addition, we investigated the endogenous IL-8 expression levels in the fibrin hydrogel–laminated IL-8-HaCaT cells and found that the IL-8 mRNA levels were significantly increased by the treatment with ZnO NPs but reduced by the silica coating (Fig. 7e). Interestingly, the sensitivity of the fibrin hydrogel–laminated IL-8-HaCaT cells to permeated zinc ion is higher than that of EPI-200. As shown in Fig. 7f, the observation of GFP expression by fluorescence microscopy corroborates the endogenous IL-8 expression levels. However, the GFP expression induced by ZnO NPs and ZnO-Si NPs was not observed by dispersing the NPs in PBS, indicating that the solubility of NPs plays an important role in the induction of inflammatory response because ZnO NPs and ZnO-Si NPs are hardly soluble in PBS (Table 1). These results are consistent with previous reports indicating that although ZnO NPs are not able to diffuse through the intact skin, zinc ion released from NPs permeates the SC, diffuses into the viable epidermis, and finally reaches the systemic circulation [54, 55]. Taken together, these results revealed that the fibrin hydrogel–based in vitro bioassay system developed in this study is valuable to estimate skin permeability to and pro-inflammatory activity mediated by zinc ions released from NPs.
In this study, we constructed a stable reporter cell line expressing GFP under the control of IL-8 promoter activity. The GFP fluorescence intensities induced by the treatment with ZnCl2, ZnO NPs, or ZnO-Si NPs accurately corroborated endogenous IL-8 expression. In addition, we found that fibrin hydrogel can reproduce HSEM permeability to zinc ion and is useful as a permeable layer to assay for skin permeability to zinc ion. Finally, we developed an accurate, convenient, and high-throughput platform for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and fibrin hydrogel. We believe that this platform is a promising in vitro permeability test that could potentially be used in the cosmetics and pharmaceutical industries due to its technical simplicity, good predictability, and high-throughput format.
Dulbecco’s modified Eagle’s medium
Enzyme-linked immunosorbent assay
Fetal bovine serum
Green fluorescent protein
Human skin equivalent model
Inductively coupled plasma optical emission spectrometer
Nuclear factor kappa B
Organisation for Economic Cooperation and Development
Scientific Committee on Consumer Safety
- TiO2 :
Tumor necrosis factor alpha
Silica-coated zinc oxide
ISO. ISO/TS 80004-2:2015, Nanotechnologies – vocabulary. Part 2: nano-objects. 2015.
Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect. 2005;113:823–39.
Smijs TG, Pavel S. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness. Nanotechnol Sci Appl. 2011;4:95–112.
Nel A, Xia T, Mädler L, Lin N. Toxic potential of materials at the nanolevel. Science. 2006;311:622–7.
Król A, Pomastowski P, Rafińska K, Railean-Plugaru V, Buszewski B. Zinc oxide nanoparticles: synthesis, antiseptic activity and toxicity mechanism. Adv Colloid Interf Sci. 2017;249:37–52.
Heng BC, Zhao X, Tan EC, Khamis N, Assodani A, Xiong S, et al. Evaluation of the cytotoxic and inflammatory potential of differentially shaped zinc oxide nanoparticles. Arch Toxicol. 2011;85:1517–28.
Babin K, Antoine F, Goncalves DM, Girard D. TiO2, CeO2 and ZnO nanoparticles and modulation of the degranulation process in human neutrophils. Toxicol Lett. 2013;221:57–63.
Roy R, Kumar S, Verna AK, Sharma A, Chaudhari BP, Tripathi A, et al. Zinc oxide nanoparticles provide an adjuvant effect to ovalbumin via a Th2 response in Balb/c mice. Int Immunol. 2014;26:159–72.
Silva LR, Girard D. Human eosinophils are direct targets to nanoparticles: zinc oxide nanoparticles (ZnO) delay apoptosis and increase the production of the pro-inflammatory cytokines IL-1β and IL-8. Toxicol Lett. 2016;259:11–20.
Hsiao IL, Huang Y. Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells. Sci Total Environ. 2011;409:1219–28.
Deng X, Luan Q, Chen W, Wang Y, Wu M, Zhang H, et al. Nanosized zinc oxide particles induce neural stem cell apoptosis. Nanotechnology. 2009;20:115101.
Hackenberg S, Scherzed A, Technau A, Kessler M, Froelich K, Ginzkey C, et al. Cytotoxic, genotoxic and pro-inflammatory effects of zinc oxide nanoparticles in human nasal mucosa cells in vitro. Toxicol in Vitro. 2011;25:657–63.
Fukui H, Horie M, Endoh S, Kato H, Fujita K, Nishio K, et al. Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung. Chem Biol Interact. 2012;198:29–37.
SCCS. Opinion on zinc oxide (nano form). SCCS/1489/12. 2012.
De Matteis V. Exposure to inorganic nanoparticles: routes of entry, immune response, biodistribution and in vitro/in vivo toxicity evaluation. Toxics. 2017;5:29.
Gamer AD, Leibolt E, van Ravenzwaay B. The in vitro absorption of microfine zinc oxide and titanium dioxide through porcine skin. Toxicol in Vitro. 2006;20:301–7.
Cross SE, Innes B, Roberts M, Tsuzuki T, Robertson TA, McCormick P. Human skin penetration of sunscreen nanoparticles: in-vitro assessment of a novel micronized zinc oxide formulation. Skin Pharmacol Physiol. 2007;20:148–54.
Filipe P, Silva JN, Silva R, Cirne de Castro JL, Marques Gomes M, Alves LC, et al. Stratum corneum is an effective barrier to TiO2 and ZnO nanoparticle percutaneous absorption. Skin Pharmacol Physiol. 2009;22:266–75.
Szikszai Z, Kertész Z, Bodnár E, Majora I, Borbíróc I, Kissa AZ, et al. Nuclear microprobe investigation of the penetration of ultrafine zinc oxide into intact and tape-stripped human skin. Nucl Instrum Meth B. 2010;268:2160–3.
Monteiro-Riviere NA, Wiench K, Landsiedel R, Schulte S, Inman AO, Riviere JE. Safety evaluation of sunscreen formulations containing titanium dioxide and zinc oxide nanoparticles in UVB sunburned skin: an in vitro and in vivo study. Toxicol Sci. 2011;123:264–80.
Detoni CB, Coradini K, Back P, Oliveira CM, Andrade DF, Beck RC, et al. Penetration, photo-reactivity and photoprotective properties of nanosized ZnO. Photochem Photobiol Sci. 2014;13:1253–60.
Ryu HJ, Seo MY, Jung SK, Maeng EH, Lee SY, Jang DH, et al. Zinc oxide nanoparticles: a 90-day repeated-dose dermal toxicity study in rats. Int J Nanomed. 2014;9:137–44.
Gulson B, McCall M, Korsch M, Gomez L, Casey P, Oytam Y, et al. Small amounts of zinc from zinc oxide particles in sunscreens applied outdoors are absorbed through human skin. Toxicol Sci. 2010;118:140–9.
Gulson B, Wong H, Korsch M, Gomez L, Casey P, McCall M, et al. Comparison of dermal absorption of zinc from different sunscreen formulation and differing UV exposure based on stable isotope tracing. Sci Total Environ. 2012;420:313–8.
Osmond-McLeod MJ, Oytam Y, Kirby JK, Gomez-Fernandez L, Baxter B, McCall MJ. Dermal absorption and short-term biological impact in hairless mice from sunscreens containing zinc oxide nano- or larger particles. Nanotoxicology. 2014;8:72–84.
Leite-Silva VR, Le Lamer M, Sanchez WY, Liu DC, Sanchez WH, Morrow I, et al. The effect of formulation on the penetration of coated and uncoated zinc oxide nanoparticles into the viable epidermis of human skin in vivo. Eur J Pharm Biopharm. 2013;84:297–308.
OECD. OECD test guideline for testing of chemicals. Test no. 428: dermal delivery and percutaneous absorption: in vitro method. 2004.
Elliott NT, Yuan F. A review of three-dimensional in vitro tissue models for drug discovery and transport studies. J Pharm Sci. 2011;100:59–74.
Magnusson BM, Walters KA, Roberts MS. Veterinary drug delivery: potential for skin penetration enhancement. Adv Drug Deliv Rev. 2001;50:205–27.
Zvyagin AV, Zhao X, Gierden A, Sanchez W, Ross JA, Roberts MS. Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo. J Biomed Opt. 2008;13:064031.
Yun YE, Jung YJ, Choi YJ, Choi JS, Cho YW. Artificial skin models for animal-free testing. J Pharm Investig. 2018;48:215–23.
Flaten GE, Palac Z, Engesland A, Filipović-Grčić J, Vanić Ž, Škalko-Basnet N. In vitro skin models as a tool in optimization of drug formulation. Eur J Pharm Sci. 2015;75:10–24.
Cui X, Boland T. Human microvasculature fabrication using thermal inkjet printing technology. Biomaterials. 2009;31:6221–7.
De Larco JE, Wuertz BR, Yee D, Rickert BL, Furcht LT. Atypical methylation of interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells. Proc Natl Acad Sci U S A. 2003;100:13988–93.
Horie M, Fujita K, Kato H, Endoh S, Nishio K, Komaba LK, et al. Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area. Metallomics. 2012;4:350–60.
Tabei Y, Sugino S, Eguchi K, Tajika M, Abe H, Nakajima Y, et al. Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages. Biochem Biophys Res Commun. 2017;490:499–505.
Caliari SR, Burdick JA. A practical guide to hydrogels for cell culture. Nat Methods. 2016;13:405–14.
Hou X, Liu S, Wang M, Wiraja C, Huang W, Chan P, et al. Layer-by-layer 3D constructs of fibroblasts in hydrogel for examining transdermal penetration capability of nanoparticles. SLAS Technol. 2017;22:447–53.
Emri E, Miko E, Bai P, Boros G, Nagy G, Rózsa D, et al. Effects of non-toxic zinc exposure on human epidermal keratinocytes. Metallomics. 2015;7:499–507.
Kim YM, Reed W, Wu W, Bromberg PA, Graves LM, Samet JM. Zn2+-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells. Am J Phys Lung Cell Mol Phys. 2006;290:L1028–35.
Saptarshi SR, Feltis BN, Wright PF, Lopata AL. Investigating the immunomodulatory nature of zinc oxide nanoparticles at sub-cytotoxic levels in vitro and after intranasal instillation in vivo. J Nanobiotechnol. 2015;13:6.
OECD. OECD test guideline for testing of chemicals. Test no. 442E: in vitro skin sensitization assays addressing the key event on activation of dendritic cells on the adverse outcome pathway for skin sensitization. 2018.
Takahashi T, Kimura Y, Saito R, Nakajima Y, Ohmiya Y, Yamasaki K, et al. An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8. Toxicol Sci. 2011;124:359–69.
Kimura Y, Fujimura C, Ito Y, Takahashi T, Nakajima Y, Ohmiya Y, et al. Optimization of the IL-8 Luc assay as an in vitro test for skin sensitization. Toxicol In Vitro. 2015;29:1816–30.
Daunert S, Barrett G, Felciano JS, Shetty RS, Shrestha S, Smith-Spencer W. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev. 2000;100:2705–38.
Nakajima Y, Ohmiya Y. Bioluminescence assays: multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay. Expert Opin Drug Discov. 2010;5:835–49.
Tabei Y, Murotomi K, Umeno A, Horie M, Tsujino Y, Masutani B, et al. Antioxidant properties of 5-hydroxy-4-phenyl-butenolide via activation of Nrf2/ARE signaling pathway. Food Chem Toxicol. 2017;107:129–37.
Stoehr LC, Endes C, Radauer-Preiml I, Boyles MS, Casals E, Balog S, et al. Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions. Part Fibre Toxicol. 2015;12:29.
Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol. 2001;11:372–7.
Jeong SH, Kim JH, Yi SM, Lee JP, Kim JH, Sohn KH, et al. Assessment of penetration of quantum dots through in vitro and in vivo human skin using the human skin equivalent model and the tape stripping method. Biochem Biophys Res Commun. 2010;394:612–5.
Lee O, Lee SH, Jeong SH, Kim J, Ryu HJ, Oh C, et al. A quantitative study of nanoparticle skin penetration with interactive segmentation. Med Biol Eng Comput. 2016;54:1469–79.
Ramasamy M, Das M, An SS, Yi DK. Role of surface modification in zinc oxide nanoparticles and its toxicity assessment toward human dermal fibroblast cells. Int J Nanomed. 2014;9:3707–18.
Wu W, Samet JM, Peden DB, Bromberg PA. Phosphorylation of p65 is required for zinc oxide nanoparticle-induced interleukin 8 expression in human bronchial epithelial cells. Environ Health Perspect. 2010;118:982–7.
Holmes AM, Song Z, Moghimi HR, Roberts MS. Relative penetration of zinc oxide and zinc ions into human skin after application of different zinc oxide formulations. ACS Nano. 2016;10:1810–9.
Mohammed YH, Holmes A, Haridass IN, Sanchez WY, Studier H, Grice JE, et al. Support for the safe use of zinc oxide nanoparticle sunscreens: lack of skin penetration or cellular toxicity after repeated application in volunteers. J Invest Dermatol. 2019;139:308–15.
We thank S. Sugino and A. Tada of AIST for excellent technical assistance.
Conflict of interest
W. Lin, S. Shiomoto, and T. Nakayama are employees of the sponsor of this study. However, this did not influence the objectivity of the study. The authors declare that they have no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Tabei, Y., Lin, W., Shiomoto, S. et al. Development of fibrin hydrogel–based in vitro bioassay system for assessment of skin permeability to and pro-inflammatory activity mediated by zinc ion released from nanoparticles. Anal Bioanal Chem 412, 8269–8282 (2020). https://doi.org/10.1007/s00216-020-02970-5
- Zinc oxide
- Fibrin hydrogel
- Skin permeation