s2fieldsofstudy: Biology Chemistry

text:
Interplay between Nrf2 and NF-κB in Neuroinflammatory Diseases Journal of Clinical & Cellular Immunology

Many neurodegenerative conditions involve redox imbalance and neuroinflammation. Nuclear factor erythroid 2- related factor 2 (Nrf2) and nuclear factor-κB (NF-κB) are key transcription factors regulating antioxidant and inflammatory pathways, respectively. These two opposing factors are inversely regulated, with activity of one most often accompanied by diminished activity of the other, giving rise to the oxidative stress and neuroinflammation evident in neurodegeneration. Emerging evidence is uncovering a complex interplay between Nrf2 and NF-κB, involving extensive interaction of regulatory mechanisms including cytosolic activators and repressors, transactivation partners and direct transcriptional crosstalk. Understanding these interactions may provide insight into the pathophysiology of neuroinflammatory diseases and facilitate discovery of novel therapeutic targets.

Nrf2 Function and Activation
Nrf2 is the master regulator of antioxidant pathways, responsible for promoting the transcription of hundreds of antioxidant and cytoprotective genes, and in the brain appears to be repressed in neurons and predominantly restricted to glia [1,2]. Nrf2 is a cap'n' collar basic leucine zipper transcription factor. Under normal conditions, Nrf2 is bound in the cytosol by its negative regulator Kelch-like ECHassociated protein (Keap1), a substrate adaptor protein of a Cullin3dependent ubiquitin E3 ligase complex. Keap1 constitutively targets Nrf2 for proteasomal degradation. When activated, Nrf2 dissociates from Keap1 and can translocate to the nucleus where it forms heterodimers with small Maf proteins and binds to antioxidant response elements (AREs), promoting the transcription of a battery of antioxidant and cytoprotective genes [3]. Nrf2 targets include genes for glutathione synthesis and utilization (e.g. GCLM, GCLC, GSTs), the thioredoxin and peroxiredoxin systems, NAPDH generation, iron metabolism (HMOX1, FTL, FTH) and quinone detoxification (NQO1) [4]. Nrf2 can be activated in response to oxidative stress and electrophiles via Keap1. Keap1 contains several highly sensitive cysteine residues that when oxidised cause Nrf2 to dissociate from Keap1, allowing Nrf2 to translocate to the nucleus. Thus Keap1 acts as a redox sensor [3]. Nrf2 activity is also regulated by kinases such as GSK3β [5].

Inverse Regulation of Nrf2 and NF-κB in Neurodegeneration
Inflammation, with elevated pro-inflammatory mediators and oxidative stress, is common to many neurodegenerative diseases, as evidenced by the presence of activated astrocytes and microglia in the affected regions of the brain and/or spinal cord in diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD), generally associated with degenerating neurons. Activated microglia are also found within and around amyloid plaques in AD, while activated astrocytes surround the plaques. Multiple sclerosis (MS), particularly the relapsing-remitting form, presents with transient localized sites of inflammation, whereby infiltrating peripheral T cells and microglia attack axonal myelin sheaths and oligodendrocytes. Stroke and traumatic brain injury (TBI) involve glial and peripheral immune cell activation at the site of injury that varies as the injury progresses. This extensive neuroinflammation and oxidative stress is consistent with elevated NF-κB activity concomitant with impaired Nrf2 activity [10,11], hence there is abundant evidence for inverse regulation of Nrf2 and NF-κB in neurodegenerative disease. Indeed, Nrf2 expression is diminished in human post-mortem tissue of AD and ALS patients [12,13]. Further evidence for the interplay between Nrf2 and NF-κB comes from experimental induction of inflammation. LPS-induced inflammation in the hippocampus is exacerbated in Nrf2-deficient mice [14], while induction of inflammation via α-synuclein in BV2 microglia is enhanced in the absence of Nrf2 [15]. Deletion of Nrf2 enhances inflammation and is associated with worsened outcomes in animal models of AD [16,17], PD [18], MS [19], and TBI [20]. These studies show that the absence of Nrf2 enhances NF-κB activity.
In humans, dimethyl fumarate is approved for the treatment of relapsing-remitting MS under the trade name Tecfidera following two successful Phase III trials [36], while the Nrf2 inducer DL-3-nbutylphthalide has produced positive outcomes in phase II clinical trials for acute ischemic stroke (administered within 48h of stroke onset) and vascular cognitive impairment without dementia [37,38]. However, none of these trials specifically interrogated Nrf2 or NF-κB signalling. Interestingly, NF-κB is inhibited in B cells isolated from MS patients administered Tecfidera [39], and this inhibition seems to occur in cultured lymphocytes in a manner independent of Nrf2 [40]. Clearly Nrf2 and NF-κB activity are inversely related in neuroinflammatory diseases. The molecular mechanisms governing the interplay between Nrf2 and NF-κB will now be discussed ( Figure  1).

Activation of Nrf2 and NF-κB Signalling by Oxidative Stress
Nrf2 is well known to be regulated by oxidative stress via oxidation of sensitive cysteine residues on Keap1, causing release of Nrf2 and subsequent nuclear translocation [3]. In addition to proinflammatory mediators, NF-κB is also believed to be regulated by oxidative stress. This stems from early investigations showing activation of NF-κB signalling by oxidants and inhibition of NF-κB signalling in the presence of antioxidants occurring at the level of NF-κB release from IκB [41][42][43]. This led to speculations that Nrf2 may impede NF-κB activity by eliminating oxidative stress. However, more subtle and specific interactions between oxidants/antioxidants and NF-κB are emerging [44]. For instance, inhibition of NF-κB signalling by antioxidants has been shown to occur independent of their antioxidant activity, such as N-acetylcysteine inhibiting TNFα stimulation of NF-κB via interference with the TNFα receptor. In the case of pyrrolidine dithiocarbamate (PDTC), a well-known inhibitor of NF-κB that prevents the dissociation of NF-κB from IκB [45], its inhibitory action was originally ascribed to its antioxidant actions [42]. More recently, the mechanism by which PDTC inhibits NF-κB has been shown to be via disruption of IκB ubiquitin ligase activity [46]. However, PDTC also activates Nrf2 in brain and cultured astrocytes but not neurons [2], and improves cognition in the APP/PS1 AD mouse model [47]. Whether Nrf2 activation by PDTC is driven by NF-κB inhibition, or by other independent mechanisms remains to be elucidated.

Promiscuity of Nrf2 and NF-κB Activators and Repressors
There is considerable interaction between Nrf2 and NF-κB activators and repressors. For instance, Nrf2 binds to Keap1 via a conserved ETGE motif in Nrf2. IKK also contains an ETGE motif and can bind to Keap1 [48][49][50]. Keap1-mediated degradation of IKK is demonstrated by the elevation of IKK and NF-κB signalling when Keap1 is knocked down [48,50]. Furthermore, while Keap1 knockdown is well-known to induce Nrf2 signalling (due to lack of repression) [3], the influence of Nrf2 per se can be excluded as coknockdown of Nrf2 has no effect [48]. In contrast to the proteasomal degradation of Nrf2, Keap1-dependent degradation of IKK likely occurs via an autophagy-dependent pathway [50], as per the degradation of Keap1 itself [51]. Importantly, the ETGE motif in IKK is conserved across human, chimpanzee, chicken and dog, but not mouse, rat, xenopus or medaka fish [48,50], which may influence investigations of this relationship in many in vitro and preclinical animal models. The relative extent and conditions governing the binding of Keap1 to IKK and Nrf2 remains unclear.
Conversely, there is also crosstalk between the NF-κB-related E3 ligase adapter protein β-TrCP and Nrf2. Following phosphorylation by IKK, IκB releases NF-κB and is targeted for proteasomal degradation via the β-TrCP-Skp1-Cullin1 ubiquitin ligase complex in manner analogous to Nrf2 degradation via the Keap1-Cul3-Rbx1 complex [8]. Nrf2 can also be targeted for degradation in a Keap1-independent manner by β-TrCP, in a process mediated by GSK3β-induced phosphorylation of activated Nrf2, thereby shutting down Nrf2 activity [52,53]. As per IKK/Nrf2 interactions with Keap1, the relative competition of Nrf2 and IκB for β-TrCP requires further investigation. Figure 1: Interplay between Nrf2 and NF-κB regulation. NF-κB (depicted as p65/p50 heterodimer) is bound in the cytoplasm by IκB. In response to external stimuli or possibly oxidative stress, IKK is activated and phosphorylates IκB, causing dissociation of NF-κB which subsequently translocates to the nucleus where it promotes the transcription of proinflammatory genes by binding to κB motifs. Phosphorylated IκB binds to β-TrCP and is targeted for proteasomal degradation. Under normal conditions, Nrf2 is bound by Keap1 in the cytosol and also targeted for proteasomal degradation. Upon activation by oxidative stress or electrophiles, Nrf2 is release from Keap1 and can translocate to the nucleus where it forms a heterodimer with small Maf proteins (sMaf) and binds to antioxidant response elements (ARE) to promote the transcription of antioxidant genes. Nrf2 can also bind to NF-κB target genes and repress their transcription, in a process possibly involving binding partners and not influenced by presence or absence of the ARE sequence or NF-κB binding. Both NF-κB and Nrf2 compete for CBP, which promotes DNA binding. Activated Nrf2 can be phosphorylated by GSK3β, which facilitates binding by β-TrCP and targeting for proteasomal degradation. Keap1 is degraded by autophagy, and can also bind and degrade IKK via autophagy.

Interactions between Nrf2 and NF-κB Transactivational Activity
There is also extensive crosstalk between Nrf2 and NF-κB in the nucleus. NF-κB can directly promote Nrf2 transcription via the κB motif in the promoter region of the NFE2L2 gene encoding Nrf2 [60].
Both transcription factors also compete with CREB binding protein (CBP), which assists in binding of the transcription factors to their cognate DNA motifs [61,62]. Phosphorylated p65 appears to have a higher affinity for CBP than Nrf2, outcompeting the latter and diminishing Nrf2 target transcription [62]. On the other hand, Nrf2 activation in microglial-like BV2 cells by ethyl pyruvate competes with p65 for CBP, suppressing p65 transcriptional activity [63]. Activated p65 can also repress Nrf2 signalling by promoting hypoacetylation of histones via HDAC3, which also sequesters CBP and/or the small Maf protein MafK, all repressing Nrf2 activity [62].
In addition to these many and varied mechanisms of co-regulation, direct transcriptional repression of NF-κB proinflammatory targets by Nrf2 has recently been described. Kobayashi et al. [64] find that Nrf2 binds to the promoter region of proinflammatory targets of NF-κB, repressing their transcription. This appears to occur regardless of NF-κB binding, and does not require the ARE sequence, to which Nrf2 binding typically results in promotion of transcription [64]. Nrf2 binding to NF-κB targets may involve binding partners, although their identity remains to be determined. The mechanism by which Nrf2 represses transcription appears to involve inhibition of RNA Pol II recruitment, thus preventing transcription [64]. This landmark study identifies for the first time a mechanism by which Nrf2 transcriptional activity directly represses proinflammatory cytokine production. The study focuses on peripheral macrophages, and it remains to be determined whether this mechanism is maintained in inflammatory cells of the central nervous system.

Conclusions
Nrf2 and NF-κB control the transcription of redox balance and inflammatory pathways, respectively. Given the opposing nature of these pathways, it is not surprising that there is extensive crosstalk between almost every level of Nrf2 and NF-κB regulation. The idea that NF-κB is regulated by redox status, which in turn is controlled by Nrf2, is too simplistic and a more complex picture is emerging. It is likely that the balance of Nrf2 and NF-κB signalling is dynamically governed by a combination of external stimuli and the prevailing cellular and environmental conditions, specific to given cell types and tissues. The mechanisms modulating Nrf2 and NF-κB in neuroinflammatory diseases require further investigation, which could uncover new therapeutic targets for their treatment.


extfieldsofstudy:
Materials Science
Medicine

text:
Epidermal Penetration of Gold Nanoparticles and Its Underlying Mechanism Based on Human Reconstructed 3D Episkin Model

: Nanomaterials are widely used in diverse aspects, and their translocation behavior through the skin would be helpful in understanding the corresponding exposure risks. To reveal how surface functionalization of nanoparticles in ﬂ uences the skin penetration, three kinds of gold nanoparticles (GNPs) with negatively, neutrally, and positively charged surfaces, that is, cetyltrimethylammonium bromide-coated GNPs (CTAB@ GNP), polyvinylpyrrolidone-coated GNPs (PVP@GNP), and citrate-coated GNPs (Citrate@GNP), were studied using human reconstructed 3D Episkin model. The measurement of Au distribution in diverse layers of the Episkin model indicated that all three GNPs could penetrate through the epidermis, wherein CTAB@GNP with positive surface charges exhibited the highest e ﬃ ciency in skin penetration. The upward osmosis of the medium proteins con ﬁ rmed the occurrence of skin permeation induced by GNP treatment, and the lipid network in the stratum corneum was also altered as the consequence of GNP exposure. When compared to Citrate@GNP and PVP@GNP, CTAB@GNP signi ﬁ cantly compromised the tight junction of keratinocytes, causing paracellular penetration of nanoparticles. The existence of cytoplasmic gold showed the transcytosis pathway through endocytosis and exocytosis processes was the main epidermic penetration behavior of the tested GNPs. The study on GNP penetration process through the 3D Episkin model has, on one hand, o ﬀ ered a promising approach to evaluate the translocation process of nanoparticles across the skin, and, on the other hand, provided mechanism explanation for diverse penetration behaviors of GNPs with di ﬀ erent surface charges. The ﬁ ndings herein would be of great help in nanotechnology improvement and nanosafety evaluation.

INTRODUCTION
The application of nanomaterials has been expanding in multiple aspects, such as medical vehicle, imaging probe, and daily supply additives, etc., thus causing potential exposure risk of the environment and human beings. 1−4 Both in vivo and in vitro experiments have indicated that nanomaterials may cause diverse toxicities, 5−10 which make nanosafety an important issue, and nanoimpact is now being hotly debated. Understanding how nanomaterials enter into bodies would be of great importance in explaining their potential toxicological effects.
The skin is the largest organ in the human body, 11 which offers the most extensive exposure interface for the exogenous substances from diverse sources. Among its main constituting layers, that is, epidermis, dermis, and subsidiary organs, 12 the epidermis is the outer layer of the skin, which provides the substantial barrier function and keeps the epidermal homeostasis. 13 As shown in Figure S1, the epidermal layer contains four types of sublayers, that is, stratum corneum, stratum granulosum, spinous layer, and basal layer from top to the bottom, and the maintenance of the epidermis depends on differentiation and simultaneous migration of keratinocytes from the bottom to the top surface. 11 Considering the potential usage of nanomaterials in some specific products like skin care pharmaceuticals, etc., and other unintended exposure routes, the study on their permeation behavior through the epidermis would be helpful in guiding their design and risk assessment.
Previous studies have indicated that different nanomaterials with distinctive physiochemical characteristics could exhibit unique biological behaviors, including skin penetration. 11,14,15 Some nanoparticles (NPs) hardly penetrate the skin barrier, 16−18 while others were found to exert transdermal delivery capacities based on both in vitro and in vivo studies, 19−21 indicating the controversial findings on the penetration behaviors of NPs through the skin barrier. Because of the high stability of gold nanoparticles (GNPs), they are considered as an excellent fine particle model for skin penetration, and have been extensively studied in this area. For example, Fernandes et al. 15 compared the penetration behavior of gold nanomaterials with different shapes, and found GNPs were less efficient in epidermal penetration than gold nanorods. The size effect of GNPs on skin penetration was also discussed on the basis of the excised epidermis model, 22 and small GNPs were found to penetrate more easily into the deep layer of epidermis through paracellular pathway than large ones. The computer simulation studies also showed the important distinction in nanoparticle size-related translocation and the permeation of GNPs through model skin lipid membrane. 23 Apparently, substantial data have confirmed GNPs could penetrate the epidermis through diverse routes. Surface modification with diverse ligands could regulate different properties of NPs, like surface charges, hydrophobicity− hydrophilicity, etc., for multiple purposes, and how surface charges influence their penetration behavior still remains blur. What is more, although ex vivo experiments based on excised epidermis model could help to provide useful information on nanomaterial induced biological processes, their feasibility in high throughput assay still faces lots of challenges, and alternatives to animal studies are now mandatory in dermocosmetic and other related industries. Three-dimensional (3D) cell cultures at tissue or organ levels, which could provide more solid data on explaining the real scenario in vivo than two-dimensional (2D) monolayer cultures, are now being extensively explored and urgently encouraged.
A novel commercial available human reconstructed 3D Episkin model is an in vitro epidermis model with gas−liquid culture ( Figure S2), which has been recommended as a uniform alternative by the Organization for Economic Co-operation and Development (OECD) for testing in vitro permeability. 24 Pharmaceutical and dermocosmetic industries are now using this model as an efficient prediction tool for diverse research and development tests, such as skin-penetration rates of various agents, inflammatory reactions, effects of UV irradiation, and hormone metabolism. 25 In toxicological studies, this Episkin model is now being increasingly investigated as well. For example, Abdallah et al. 26 found log K ow was the main cofactor for the percutaneous penetration of hexabromocyclododecanes (HBCD) and tetrabromobisphenol A (TBBPA) in the Episkin model, showing its feasibility and relevance as a biologically active barrier for in vitro assays. Therefore, this human reconstructed 3D Episkin model is a very promising approach, and has high potentials in nanotoxicological studies.
In this Article, we used a human reconstructed 3D Episkin model and tested the epidermic permeation for three kinds of GNPs with different surface charges. The downward penetration of GNPs through the epidermis and the upward osmosis of the medium protein were influenced by the surface functionalization of GNPs, wherein cetyltrimethylammonium bromide coated GNPs (CTAB@GNP) with the positively charged surface exhibited the most efficient skin penetration behavior among all. GNP exposure could influence lipid structure, and penetrate the epidermis by both paracellular route and transcellular pathway. The research on surface functionalization-related penetration process of GNPs through the 3D Episkin model has provided a promising approach to assess the translocation of nanoparticles through the skin, and revealed the underlying mechanisms during this biological process.

MATERIALS AND METHODS
2.1. Reagents. The stock solution of Citrate@GNP (5 nm, 63 μg/ mL) was purchased from BBI Solutions (UK). The other two coating materials, including polyvinylpyrrolidone (PVP 10, >99%) and cetyltrimethylammonium bromide (CTAB, >99%), were obtained from Sigma-Aldrich. All other chemicals or reagents used in this study were specified below.
2.2. Surface Modification and Characterization of GNPs. Surface functionalization of GNPs was performed on the basis of the method of ligand exchange. First, 0.5 mL of 10 mM CTAB was slowly added to 6 mL of 21 μg/mL Citrate@GNP aqueous solution under vigorous stirring during 90 s to obtain CTAB@GNP. As for PVP@ GNP, 10 mL of 63 μg/mL Citrate@GNP was boiled for 5 min with drop addition of 1 mL of 40 g/L PVP 10. The synthesized reaction mixtures were purified by ultrafiltration (5 KD ultrafilter, Millipore, U.S.) at 4°C (3500 rpm, 20 min) to remove the free ligands. The concentrations of the prepared GNP solutions were determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 8800, U.S.). The working solutions were diluted to 10 μg/mL and stored at 4°C until use.
The morphologies of three GNPs were observed by transmission electron microscopy (TEM, JEOL, Japan) using 230 mesh copper grid coated with carbon membrane. Their sizes based on TEM characterization were evaluated and fitted with Gauss Distribution. The UV−vis spectra of GNPs were analyzed by scanning the range of 450−650 nm at room temperature using a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The surface charges and hydrodynamic sizes were measured by Zeta Sizer Nano ZS (Malvern, UK), and illustrated by zeta potentials and hydrated particle sizes, which were re-evaluated after 7 days storage to confirm the stabilities of the prepared GNPs.
2.3. Configuration and Culture of Human Reconstructed 3D Episkin Model. The kit of 3D Episkin reconstructed human epidermis model was purchased from Shanghai Episkin Biotechnology Co., Ltd. (China), and the experimental setup was shown in Figure S2. It was based on the transwell-like chamber, consisting with the inset and the 12-well plate. The insert contained two layers, that is, the bottom collagen layer as the support material and the upper epidermis layer as the tested target. In each well, 1.5 mL of the commercially supplied culture medium (Shanghai Episkin Biotechnology Co., Ltd., China) was added to maintain the normal proliferation and differentiation of the keratinocytes. The cell condition during the transportation was ensured by checking the attached temperature tag (less than 40°C), and the cell culture was performed overnight at 37°C in 5% CO 2 and 100% humidity. The epidermis cell cultures could be maintained for at least 1 week according to the supplier's instruction. The quality control of the product was strictly guaranteed, and necessary characterization for morphology, half inhibitory concentration (IC 50 ), and histological scores were performed and provided along each batch of the kits by the supplier (Table S1).
2.4. GNP Exposure to 3D Episkin Model. Before exposure, the insets containing the collagen and epidermis were switched to the commercially available test medium (Shanghai Episkin Biotechnology Co., Ltd., China), and maintained for 2 h. The top interface of epidermis was evenly exposed to 10 μL of 10 μg/mL GNP solutions for 2 h, and the negative control was set by adding 10 μL of Milli-Q water. After exposure, the top interface of epidermis was washed by 100 μL of Dulbecco's phosphate buffered saline (DPBS, Gibco), and the surface solution was transferred and stored at −80°C for subsequent protein analysis. Five-time wash with DPBS was performed to completely remove excessive GNPs on the epidermis surface. The bottom medium (0.5 mL) was also collected for daily monitoring of Au contents. The Episkin culture was continued by transferring the insets back to the culture medium for 22 h. The exposure, sample collection, and subsequent culture were carried out in a similar way for a second and third time. After the third time 22 h culture, the exposure experiments were terminated, and the tissue samples were punched off from inserts, and submitted to direct analysis using attenuated total reflection Flourier transformation infrared spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC), or snap frozen in liquid nitrogen and subsequently stored at −80°C for ICP-MS analysis, SDS-PAGE separation, RT-PCR, and Western blot assays, or fixed in 10% buffered formalin (Sinopharm, China) for imaging mass spectrometry analysis, histopathological observation, and immunofluorescence assay, or fixed in 2.5% glutaraldehyde (pH 7.3, Sinopharm, China) containing 0.1 M sodium cacodylate (Sinopharm, China) for scanning transmission electron microscopy coupled with energy dispersive system (STEM-EDS) analysis. As for medium samples, 0.5 mL of the test medium was kept at −20°C until Au analysis.
2.5. ICP-MS Analysis for GNP Distribution. The tissue samples, including the epidermis and the collagen layers collected from the inset, were shortly warmed at 56°C, and the collagen matrix was torn off from the epidermis layer. After weighing, these two kinds of samples were dissolved in 1 mL of aqua regia, digested at room temperature for 12 h, and then heated at 95°C until the solution volumes were less than 0.5 mL. The residue was diluted with Milli-Q water to the final volume of 20 mL. The as-prepared samples were submitted to the analysis of Au contents using Agilent 8800 ICP-MS (U.S.), and the results were expressed as ng/g in epidermis and collagen matrix samples. As for the analysis of Au contents in the test medium, 0.5 mL of samples was processed and analyzed in a way similar to that described above.
The decrease ratio in the test medium was calculated using the following equation: where C day1 means Au concentration in the test medium collected on exposure day 1, and C day2or3 means Au concentration in the test medium collected on exposure day 2 or 3. 2.6. Imaging Mass Spectrometry Analysis for GNP Penetration in Epidermis. The epidermis samples from different exposure groups were fixed in 10% buffered formalin for at least 24 h, and dehydrated with a series of sucrose solutions (10%, 20%, and 30%) for 12 h in each step. The samples were then immersed in OCT embedding medium (SAKURA, U.S.), and placed in liquid nitrogen for 1 min. The as-prepared tissue samples were frozen sectioned to 40 μm at −20°C using a Leica HM 525 cryostat microtome (Germany), and submitted to laser ablation ICP-MS (LA-ICP-MS) analysis. GNP penetration through epidermis was evaluated using a NWR 213 laser ablation system (NEW WAVE, U.S.) coupled with a NexION 300D ICP-MS instrument (Perkin Elemer, U.S.). Ablation carrier gas was helium, and the flow rate was 0.8 L/min. Argon was introduced through a Y-piece at the typical flow rate of 0.6 L/min. The optical operating conditions were given in Table S2. 2.7. Protein Analysis Based on SDS-PAGE and LC−MS/MS. The solution samples daily collected on the top of epidermis after GNP exposure were quantitatively analyzed for protein concentrations using BCA assay (Thermo Fisher, U.S.). After concentration normalization, the protein profiles from different exposure groups were characterized by SDS-PAGE separation (Biorad, U.S.) coupled with silver staining (Beyotime Biotechnology, China). The main protein bands in SDS-PAGE were cut off, and the protein structures were unfolded with dithiothreitol (DTT) and iodacetamide (IAM) solution (Sigma, U.S.). After dehydration with acetonitrile, the gel samples were overnight digested by trypsin. The digested samples were submitted to LC−MS/MS analysis (Bruker Daltonics, U.S.) for protein identification. The software used for protein search was MASCOT (version 2.3.01), and the parameters were listed in Table  S3.
The exosmosis protein amounts in surface solutions were compared in different exposure groups after the first time GNP exposure based on protein concentrations measured by BCA kit (Thermo Fisher, U.S.).
2.8. Histopathological Analysis. The epidermis samples from different exposure groups were fixed and dehydrated in a way similar to the sample pretreatment for LA-ICP-MS analysis. The tissues were embedded in paraffin, sectioned to 4 μm, and submitted to hematoxylin and eosin staining (H&E, Sinopharm, China). The as-prepared sections were observed and pictured under a microscope (Olympus IX73, Japan) at 20× magnification.
2.9. Lipid Network Analysis Using ATR-FTIR. The fresh epidermis samples were carefully placed on the slides and wiped by filter paper, which were dried overnight in a desiccator. The complete stretched samples were then placed on an ATR diamond crystal for the subsequent measurements. The spectra were scanned using a Thermo spectrophotometer (Nicolet 5700, U.S.) with the wavenumber ranging from 500 to 3400 cm −1 , the scan resolution was 4 cm −1 , and a total 128 scans were controlled. The raw data were processed using Savitsky−Golay function.
2.10. Crystal Transition Analysis Using DSC. The specimens from ATR-FTIR experiments were folded into pellets, which were subsequently placed into aluminum pans and crimped, and then added in a test calorimeter. DSC heat flow curves were measured on a DSC 8000 system (Perkin Elemer, U.S.) from 0 to 120°C, and the reverse progress was controlled from 120 to 0°C. The heating or cooling rate was 10°C/min. The baseline was scanned using two empty pans to eliminate any thermo interferences. The data were finally analyzed by Pyris software.
2.11. RT-PCR for Transcriptional Levels of Tight Junction and Exocytosis-Related Proteins. Total RNA was extracted from epidermis samples by Trizol reagent (Invitrogen, U.S.) and purified following the commercially recommended protocol. The reverse transcription of mRNA to cDNA was performed by one-step iScript cDNA Synthesis Kit (Biorad, U.S.). The cDNA samples were diluted by 10-fold, submitted to RT-PCR analysis using SYBR Green Supermix kit on a Roche 480 system (Roche, UK). The tested genes included occludin, zonula occludens protein 1 (ZO-1), claudin, ATP-binding cassette transporter subfamily member C1 (ABCC1), vesicle secretion-related TRAPP, divalent metal transporter 1 (DMT1), copper transporting ATPase (ATP7A, ATP7B), and glyceraldehyde-phosphate dehydrogenase (GAPDH). The related forward and reverse primers were listed in Table S4. The relative transcriptional levels of the target genes were normalized to GAPDH mRNA expression and calculated by the 2 −ΔΔCT method.
2.12. Western Blot Analysis. The epidermis samples were homogenized in liquid nitrogen and lysed with 200 μL of lysis buffer (10 mM Tris-HCl buffer, containing 2 mM NaF, 1% cocktail, 1 mM Na 3 VO 4 , and 0.6 mM H 2 O 2 , pH 7.4). After 3 h extraction, the samples were centrifuged (12 000g) at 0°C for 15 min. The supernatants were quantitatively analyzed for protein normalization, and processed for SDS-PAGE analysis. Equal amount of protein sample from each exposure group was loaded and separated on the denaturing gradient gel (BioRad, U.S.) for 1 h, and transferred to a 0.22 μm nitrocellulose membrane (Millipore, U.S.). The membrane was blocked for 2 h, and separately incubated with different primary antibodies overnight at 4°C . The primary antibodies used in this study were antioccludin (1:1000, Abcam, U.S.), anticlathrin (1:1000, Abcam, U.S.), anticaveolin-1 (1:1000, Abcam, U.S.), and anti-GAPDH antibodies (1:1000, Abcam, U.S.). The subsequent processes included the incubation with the corresponding second antibodies (peroxidaseconjugated goat antirabbit second antibodies from ZSGB-BIO, 1:2500; China) and the development for the target protein expressions (Pierce ECL, Thermo Scientific, U.S.). The pixel densities on the developed X-ray film were evaluated by Quantity One software for the target protein expression levels.
2.13. Immunofluorescence Assay for Occludin. The fixed epidermis samples were processed in a way similar to the sample pretreatment for LA-ICP-MS analysis. The frozen sections (5 μm) were washed with PBS for 5 min under slight rotatory shaking. After a brief air-dry, the sections were immersed into 3% hydrogen peroxide for 15 min, and washed with PBS three times (5 min each). The antigen repair was performed by incubating the section samples with trypsin at 37°C for 5 min. After PBS wash, 10% goat serum (VWR Shanghai Co., Ltd., China) was applied to block the nonspecific binding sites. The samples were subsequently incubated with the primary antioccludin antibody (1:50, Abcam, U.S.) at 4°C overnight, followed by 1 h incubation of Alexa 488 labeled antirabbit second antibody (1:100, ZSGB-BIO, China). The slides loaded with section  samples were sealed with antifade mounting medium containing DAPI (VECTOR, U.S.), and stored in the dark at 4°C. The samples were observed and photographed using a Leica TCS-SP5 confocal laser scanning microscope (Germany).
2.14. Elemental Au Distribution Based on STEM-EDS Analysis. The epidermis samples from different exposure groups were cut into 1−2 mm 3 pieces and immediately immersed into 2.5% glutaraldehyde containing 0.1 M sodium cacodylate (pH 7.3). After 24 h, the specimens were post fixed in osmium tetroxide buffer (Sigma, U.S.) for 1 h. After being washed, the tissue samples were immersed into uranyl acetate for 20 min, and subsequently dehydrated in a series of ethanol solutions. After embedding into epoxy resin, 100 nm ultrathin sections were prepared using a slicing machine (Leica UC7, Germany). The ultrastructure of the specimen and elemental Au distribution were photographed and analyzed under a F20 fieldemission STEM coupled with EDS (FEI, U.S.).
2.15. Statistical Analysis. Each experiment condition was performed in triplicate, and all assays were independently performed three times or more. The data were represented as mean ± standard deviation. The significant difference between the negative control and the exposure groups was denoted by a p value less than 0.05 (*) or 0.01 (**) using t-test.

RESULTS AND DISCUSSION
3.1. Characterization of GNPs. Citrate@GNP was used to prepare PVP-and CTAB-coated GNPs (i.e., PVP@GNP, CTAB@GNP) by surface charge modification as revealed by the synthesis strategy in Figure 1A. TEM observation indicated that the initial Citrate@GNP was spherical, and the particle shape did not obviously change during the subsequent ligand exchanging reactions ( Figure 1B). The results from TEM images fitted with Gauss function showed that the particle sizes of both Citrate@GNP and PVP@GNP were around 5 nm, while that of CTAB@GNP was around 7.68 nm ( Figure 1C). Considering CTAB molecular was 2 nm in length and its bilayer was about 3 nm, this result was consistent with the theoretical calculation. Similar findings on surface ligands influencing particle sizes were previously reported, 27−30 and CTAB was found to have a relatively shorter carbon chain and lower molecule weight, when compared to the other positive charge regulating ligands, like polyethylenimine (PEI), polyethylene glycol (PEG), and poly(diallyldimethylammonium chloride) (PDADMAC). It was thus selected herein to regulate the positive surface charge of GNPs, avoiding too much change in particle sizes. The zeta potentials of Citrate@GNP, PVP@ GNP, and CTAB@GNP were −25.70, −5.09, and 26.50 mV, respectively (Table S5), representing they had negatively, slightly negatively (regarding as neutral herein), and positively charged surfaces. The dynamic light scattering (DLS) measurement showed that the hydrated sizes were 5.10, 5.85, and 8.76 nm for Citrate@GNP, PVP@GNP, and CTAB@GNP, separately (Table S5), which were well consistent with TEM results ( Figure 1C). Both zeta potentials and hydrated sizes of these three GNPs were remeasured after 7-day storage to evaluate their stabilities, and the results remained unchanged (Table S6). The surface charges of nanoparticles could guarantee the high stabilities of their aquatic dispersions, 11 and the as-prepared GNPs in this study were quite stable. The analysis of surface plasmon resonance (SPR) showed all GNPs had the maximum absorption peak at 520 nm ( Figure 1D), showing that surface functionalization did not alter the particle characteristics and their dispersities. 22 Three kinds of ligands used herein, that is, citrate, PVP, and CTAB, were all hydrophilic, which also endowed GNPs with high aquatic dispersity and good stability in water suspensions.

Surface Functionalization
Influences GNP Penetration. The penetration of GNPs through the Episkin model was evaluated by the analysis of elemental gold distribution in diverse layers, including the epidermis, collagen matrix, and culture medium (Figure 2), using ICP-MS (Agilent 8800). The results showed Au concentrations in epidermis ranged from 600 to 3000 ng/g (Figure 2A), and those in collagen were in the range of 15−65 ng/g ( Figure 2B). This was reasonable as the epidermis was the upper layer of the Episkin model closely adjacent to the surface where GNPs were daily administered. When three GNPs were compared, CTAB@GNP with the positive surface charges had the most efficient penetration through the Episkin model, as demonstrated by the relatively high Au concentrations in both epidermis and collagen layers. As for PVP@GNP and Citrate@GNP, comparable penetration was observed with the former one exhibiting a little bit more efficiency. Consistent with the previous finding, 15 this could be explained by the fact that the surface charge of skin was physiologically negative, and would easily adsorb the particles with positively charged surface. Considering the potential contribution of cytotoxic CTAB to the skin penetration of GNPs, CTAB@GNP was purified by ultrafiltration to remove free CTAB molecules, and the observed effect was believed to be attributed to CTAB@GNP itself. A similar particulatespecific hemolytic effect was observed for CTAB coated gold nanorods by comparing to that of the equivalent amount of CTAB small molecular. 31 On the basis of the calculation of the maximum number of the ligand molecular adsorbed on spherical particle, 32 the total CTAB coated on GNP was about 0.6 μM when the exposure concentration of CTAB@ GNP was 10 μg/mL, which was far less than the cytotoxic level of CTAB (100 μM) to human skin keratinocytes and fibroblast cells, 28,29 further revealing the skin penetration of CTAB@ GNP was free from the potential cytotoxicity of the surface ligand. In view of the surface charge-relevant effect, similar findings on the skin penetration of positively charged GNPs were observed when the surface ligands were polyethylene glycol (PEG) 15 and poly dimethyl diallyl ammonium chloride (PDADMAC), 30 which had relatively high biocompatibilities, showing positive surface charges did matter.
To reveal the diffusion behavior of GNPs through the epidermis, the cross section from each exposure groups was submitted to LA-ICP-MS (PerkinElmer, U.S.) analysis, and the results were indicated in Figure S3. CTAB@GNP showed apparent permeation through epidermis, which exhibited in a passive diffusion mode from the top to the bottom due to the concentration gradient caused by three-time treatments performed daily ( Figure S3A). As for Citrate@GNP with negative surface charges, their penetration seemed rather random and unevenly, with several scattered spots containing relatively high Au levels ( Figure S3B). Considering the repelling force between negatively charged particles and epidermic cells, Citrate@GNP was not readily absorbed, and the limited partial penetration through epidermis could be possibly contributed by the compromised defense function of the skin model. The imaging mass spectrometry for PVP@ GNP exposed samples showed low levels of Au uniformly distributed in epidermis, indicating that limited but even permeation occurred for this kind of nanoparticle ( Figure  S3C).
To test if GNPs could penetrate through the epidermis and collagen layer, Au concentrations in bottom medium were daily monitored, and the results in Figure 2C show that the highest Au level (80 ng/mL) was observed in CTAB@GNP exposure group on day 1, confirming its most efficient penetration through the Episkin model when compared to the other two GNPs. This finding was consistent with what was observed by LA-ICP-MS ( Figure S3). As the effect of exposure time was explored, an obvious decreasing trend was observed in medium Au concentration of CTAB@GNP exposure group, while a slight decrease occurred in those of the other two groups. This could be ascribed to the fact that the keratinocytes of epidermis gradually aged, which could be well characterized by the rapid growth of the epidermis, and the stacked differentiated keratinocytes formed the functionally improved skin barrier as the time prolonged. The upper layer of stratum corneum became thicker as revealed previously, 33 and the blockage of GNP penetration thus increased.
3.3. GNP Exposure Causes Upward Osmosis through the Episkin. As demonstrated above, GNPs could penetrate through the Episkin model, showing the permeability of the epidermic barrier. Therefore, whether the medium or epidermic contents could effuse to the top surface under concentration gradient force was worthy of investigation. The exposure solutions were collected 2 h after GNP exposure, and submitted to protein analysis. As indicated by SDS-PAGE analysis in Figure 3A, apparent protein bands with similar profile appeared in all samples on day 1, while protein levels were nearly undetectable on day 2. This result was consistent with the finding that decreased Au levels were observed in bottom mediums ( Figure 2C When the exosmosis protein in exposure solutions collected on day 1 was quantitatively measured, it was found that the concentrations varied with different GNP treatments ( Figure  3C). Apparently, the relative protein concentration in CTAB@ GNP exposure group was significantly higher than that in the control (p < 0.05), while PVP@GNP and Citrate@GNP exposure groups showed no difference from the control. This result demonstrated that GNPs with positively charged surfaces induced relatively higher permeability of the Episkin model than the others, which was consistent with the results of epidermic and medium Au concentrations on day 1 ( Figure  2C). Considering there was no cytoplasmic protein observed in exposure solutions as revealed by Figure 3A and B, the exosmosis medium protein detected on exposure day 1 could penetrate the epidermis through the paracellular routes.
3.4. GNP Exposure Induces Structural Changes in Lipid Network of Epidermis. As shown in Figure S1, the layer of stratum corneum lies on the top surface of the epidermis, which is mainly composed of lipid and enucleated keratinocytes from the complete differentiation of keratinocytes. The lipid network is essential in inhibiting the deep diffusion of exogenous chemicals or particles, and controlling material exchange out of the skin. 34 As GNPs encountered the epidermis, they first contacted the stratum corneum, and the investigation on how NPs passed through the stratum corneum would help to reveal the penetration behavior of GNPs in the epidermis. Histopathological observation revealed that no obvious structural changes occurred in the epidermis, including the stratum corneum layer, upon GNP exposure ( Figure S4), suggesting the penetration process of GNPs was independent of histopathological injury or cytotoxicities from surface ligands, as both citrate and PVP had relatively high biocompatibilities, 35,36 and the CTAB amount coated on GNP was far less than its cytotoxic levels to either keratinocytes 29 or fibroblasts. 28 Nevertheless, cell normal function could be compromised by GNP exposure, even if no obvious structural changes were apparently observable. As indicated by Schneider et al., 37 there were two ways for nanoparticle penetration: (1) the paracellular lipid-stack space of corneocytes and (2) transport into and out of the keratin-rich corneocytes. To study the effects of GNPs on the stratum corneum, its lipid network was characterized by ATR-FTIR and DSC.
As the penetration depth of the evanescent wave from ATR-IR was typically between 0.5 and 2 μm, it could help to capture the spatial organization information on the lipid functional groups in the stratum corneum layer. 38 As indicated in Figure  4A, the band of 2920 cm −1 in the control, which designated the lipid network in stratum corneum, 39 was shifted to 2925, 2927, and 2937 cm −1 upon the treatments of CTAB@GNP, PVP@ GNP, and Citrate@GNP, respectively. Considering this band was attributed to the stretching vibrations of ceramides, fatty acid, and cholesterol in stratum corneum, 40 the band shifts indicated GNPs caused the alterations of lipid lamellae from trans to gauche formation. This transformation was associated with the lipid fluidization of stratum corneum, 41 and the interaction between GNPs and acyl side chains was the main cause for the transformation in lipid network. Comparatively, Citrate@GNP with negatively charged surface exerted more influences on wavenumber shift than did the other two GNPs, showing their potentially higher interactions with lipid network. Similarly, Wang et al. 42 reported that the negatively charged nanoparticles could induce the transformation of lipid bilayer interface from semifluidization bilayer to gelled membrane. In contrast, the lipid network could be turned to fluid bilayer by positively charged nanoparticles, 43 resulting in easier penetration of the positively charged nanoparticles through the bilayer as compared to negatively charged nanoparticles as observed in Figure 2.
The stretching vibration of amide indicated by 1640 cm −1 is mainly from the keratin component in stratum corneum layer. 39 As shown in Figure 4B, PVP@GNP induced the left shift of this band, representing their interaction with keratin due to the cluster formation between the biocompatible coating of PVP and keratin. Nevertheless, CTAB@GNP and Citrate@GNP did not show any effects.
DSC is a thermoanalytical technique commonly used in the detection of phase transitions, 44 and typical thermal transitions were previously characterized for stratum corneum membranes from diverse species. 45 On the basis of monitoring the heat flowing to the epidermis to increase its temperature at the same rate as the reference, the thermal transitions were investigated for both the control and the GNP exposure groups, and the results were depicted in Figure 4C. The DSC thermogram of the untreated control epidermis showed two endothermic transitions at 46−49°C (I) and 65−73°C (II). Endothermic transition I was associated with the transformation of lipid crystal from orthorhombic form to hexagonal system, and II was attributed to the phase change of hexagonal system to liquid state. 46 As for CTAB@GNP and Citrate@GNP exposed skin, only endothermic transition II was observed, indicating the original lipid network of epidermis became disorderly. 47 It was surprising that a new endothermic transition at 74−75°C was formed in PVP@GNP exposed samples, which suggested the potential cluster formation due to the interaction of PVP coating with keratin. This result was consistent with the wavenumber shift at 1640 cm −1 in ATR-FTIR analysis ( Figure  4B). Altogether, the tested three GNP species apparently exhibited unique characteristics in their interactions with the lipid network and keratin in the stratum corneum layer of epidermis, regarding their distinctive influences characterized by ATR-FTIR and DSC.
3.5. GNP Penetration through Paracellular Route. The stratum granulosum layer lies beneath the stratum corneum in the 3D Episkin model ( Figure S1), which provides the substantial barrier function through the cell junctions, 48 wherein the tight junctions seal up cellular gaps, preventing exogenous pathogen or chemicals from deep invasion. 49 The compromised cell junctions would possibly offer direct penetration route for NPs. As the main components of the tight junction, 50 the transcriptional levels of three biomarkers, including ZO-1, occludin, and claudin-1, were evaluated in different exposure groups. The results in Figure 5A illustrate that CTAB@GNP exposure causes significant elevation in the transcriptional level of occludin, but does not influence the other two (i.e., ZO-1 and claudin-1). As indicated, the claudins were corresponding to the pore way through which ions at the sizes of around 0.4 nm could diffuse, while occludin consisted of the leak way, regulating the macromolecular penetration. 51 ZO-1 indirectly mediated tight junction by anchoring claudins and occludin to the cytoskeleton with PDZ and GUK domains. 50 The increase in occludin mRNA suggested that CTAB@GNP influenced the leak way, which could contribute to the penetration of the NPs. In contrast to CTAB@GNP, neither PVP@GNP nor Citrate@GNP changed the transcriptional levels of the tested biomarkers ( Figure 5A), revealing that they could have little effect on the tight junction of keratinocytes. To further confirm GNP induced effects on the expression of occludin, the exposed epidermis samples were submitted to Western blot analysis, and the results confirmed that CTAB@GNP notably increased the occludin protein level, while the other two GNPs had no effects ( Figure 5B), which was well consistent with the results in Figure 5A.
The immunofluorescence experiment was further performed to characterize occludin location in keratinocytes. As indicated in Figure 5C, occludin stained with green fluorescence was mainly lined in the cytomembrane of the control, forming the tight layered keratinocytes with clear nuclei stained with DAPI locating in the center. In contrast to the hollow structure probed by occludin in the control, the widespread of this protein was observed in the cytoplasm of CTAB@GNP exposed keratinocytes, showing cellular internalization of occludin occurred in this group. 52 Interspaces marked by red frame appeared in the adjacent cytomembranes. Nevertheless, this effect was not obvious in the other two exposure groups, and their occludin distribution was mainly located in cytomembrane, which was similar to the control. The intercellular space revealed by immunostaining of occludin absence in cell junction area in CTAB@GNP exposure group was not necessary detectable in normal histopathological observation due to its noncytotoxic stimulation and the intact tissue structure. According to the previously established knowledge, 53 occludin in tight junction contained two extracellular helix domains, and they were connected with those of adjacent occludin molecular. The C-terminal of occludin, as the intracellular domain, was linked with ZO-1 in a phosphorylation-dependent pathway, through which occludin was anchored in cell membrane, sealing the paracellular gap of keratinocytes. Loss of the binding of occludin with ZO-1 would cause its dispersion in cytoplasm and open of paracellular space. CTAB@GNP induced the disintegration of occludin in cytomembrane, and the subsequent internalization would explain their penetration pathway through the Episkin model. The high regulation of occludin at both transcriptional and protein levels could be contributed by the intricate cellular regulatory network. The stable distribution of membrane occludin in PVP@GNP and Citrate@GNP exposure groups suggested normal function of the tight junction in keratinocytes, 54,55 indicating little paracellular route appeared for NPs penetration. This finding was consistent with the trend of protein upward osmosis observed in Figure 3, showing CTAB@GNP induced the strongest effect on Episkin permeability among the three tested GNPs.
3.6. GNP Penetration through Transcytosis Pathway. Besides the paracellular route, NPs could possibly realize the penetration of the Episkin model through transcytosis pathway. To confirm if cellular internalization of GNPs occurred, STEM-EDS was used to determine Au distribution in keratinocytes. As indicated in Figure 6A, the cytoplasm was qualitatively analyzed for elemental gold contents, and the results were adjusted by elemental carbon contents. It was obvious that GNP exposure significantly induced elevation in cytoplasmic gold contents when compared to the control (p < 0.05), showing the existence of endocytosis process. Gold levels in CTAB@GNP exposure groups were comparatively higher than those in PVP@GNP and Citrate@GNP exposure groups. This trend was consistent with the results in Figure 2A. Nevertheless, the difference of gold contents between CTAB@GNP and the other two exposure groups in Figure 6A was not as big as that in Figure 2A, which suggested the paracellular penetration of CTAB@GNP observed in Figure 5 also greatly contributed to its permeation through the Episkin. Although GNP images at the single-particle resolution would give more solid evidence on the penetration behavior of the tested nanoparticles, the rigidity of the specimen failed to tolerate the electron beam of STEM with higher energy under large magnification, suggesting the challenges in further studies on the improvement of the related analytical technique.
As for the process of endocytosis, three manners, including clathrin-mediated endocytosis, caveolae-mediated internalization, and clathrin or caveolea-independent internalization, were mainly involved. 56 The endocytosis based on the use of chemical inhibitors and low temperature control could trigger some other cellular responses, besides the blockage of endocytosis, 57 thus complicating the biological processes. The expressions of clathrin and caveolin have been extensively studied, 58−61 and the high potentials of these protein biomarkers have been implicated in the explanation of cellular endocytosis. Herein, the investigation on both clathrin and caveolin-1 expression showed that no significant changes occurred in most exposure groups (p > 0.05, Figure 6B), except a little increase of caveolin-1 in the CTAB@GNP exposure group. Apparently, only caveolin-1 might play a slight role in endocytosis of CTAB@GNP, and the potential cellular internalization of GNPs as indicated by increased Au levels in Episkin tissues (Figures 2A and 6A) could be mainly regulated by a clathrin or caveolea-independent pathway. As indicated previously, 62 the surface curvature of cells mediated clathrin or caveolae-dependent cellular internalization of NPs, via which the allowable penetration particle sizes were around 5 nm or more. Those smaller than 5 nm may be transported into cells through other pathways. 63 Although the particle sizes of three GNPs tested herein were qualified (≥5 nm, Figure 1), the critical sizes of both Citrate@GNP and PVP@GNP (5.10 and 5.85 nm) could induce less effects on clathrin or caveolaedependent cellular internalization than did CTAB@GNP (8.76 nm). In addition, their surface functionalization with different charges could greatly influence their contact with cellular biomembrane. 64 As compared to negatively or neutrally charged NPs, positively charged NPs could break through the cytomembrane more easily, 65−67 and some negatively charged NPs could be transferred into cells through the interaction with some positive areas in membranes. 57 The exocytosis of GNPs could also be an important process, influencing their penetration behaviors through the Episkin. In general, ABCC1, TRAPP, ATP7A, ATP7B, and DMT1 are responsible for transporting and exporting metal ions or particles. 68,69 The evaluation of the transcriptional levels of these five biomarkers showed CTAB@GNP significantly increased TRAPP, DMT1, ATP7A, and STP7B mRNA expressions (p < 0.05 or 0.01, Figure 6C), but did not affect ABCC1 expression. In contrast to CTAB@GNP, the PVP@ GNP only significantly increased the transcriptional levels of ATP7A and ATP7B (p < 0.05), while Citrate@GNP slightly increased ATP7A mRNA level (p < 0.05), but significantly decreased ATP7A mRNA level (p < 0.01, Figure 6C). This result suggested that positively charged GNPs could be exported from cells with the help of vesicle secretion-related protein and metal transporters, and those with neutrally or negatively charged surfaces could experience exocytosis process with metal transporters involved. The distinctive responses due to the treatments of GNPs with unique characteristics indicated intricate cellular exocytosis processes were induced in keratinocytes.

CONCLUSION
In summary, this research revealed the distinctive penetration behaviors of three GNPs based on human reconstructed 3D Episkin model due to their different surface functionalization. Although all GNPs could induce the phase change of lipid lamella and pass through the epidermis, positively charged GNPs exhibited the most efficient skin penetration through both the paracellular routes and the transcellular pathway, when compared to neutrally or negatively charged GNPs. The surface functionalization-regulated GNP penetration in Episkin has provided substantial evidence on the biosafety or bioefficiency evaluation for NPs with different coatings in cosmetics and some other transdermal pharmaceutical development, and the 3D epidermis platform has also demonstrated very promising in skin-related studies on emerging nanomaterial penetration. The information on the biological effects regulated by a systematic variation of the physiochemical factors, like surface charge or particle size, would be highly encouraged for the perspective of nanotechnology improvement in future studies.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13700. Figure S1, schematic diagram of human epidermis; Figure S2, outline of 3D Episkin model; Figure S3, laser ablation ICP-MS results; Figure S4, histopathological section graphs; Table S1, quality control of the Episkin model obtained from the vendor; Table S2, instrumental parameters for laser ablation ICP-MS analysis; Table S3, parameters for protein identification; Table S4, primer sequences; Table S5, zeta potentials and hydrated particle sizes of three GNPs; and Table S6, zeta potentials and hydrated particle sizes of three GNPs after 7-day storage (PDF)