Analysis of PM2.5-induced cytotoxicity in human HaCaT cells based on a microfluidic system

Accepted Manuscript Analysis of PM2.5-induced cytotoxicity in human HaCaT cells based on a microfluidic system

Yuxiao Zhang, Lulu Zheng, Jiang Tuo, Qi Liu, Xinlian Zhang, Zhixuan Xu, Sixiu Liu, Guodong Sui PII: DOI: Reference:

S0887-2333(17)30102-9 doi: 10.1016/j.tiv.2017.04.018 TIV 3982

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

16 July 2016 11 February 2017 12 April 2017

Please cite this article as: Yuxiao Zhang, Lulu Zheng, Jiang Tuo, Qi Liu, Xinlian Zhang, Zhixuan Xu, Sixiu Liu, Guodong Sui , Analysis of PM2.5-induced cytotoxicity in human HaCaT cells based on a microfluidic system. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tiv(2017), doi: 10.1016/ j.tiv.2017.04.018

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ACCEPTED MANUSCRIPT Analysis of PM2.5-induced cytotoxicity in human HaCaT cells based on a microfluidic system Yuxiao Zhang

a,1

, Lulu Zheng

a,1

, Jiang Tuo b, Qi Liu a, Xinlian Zhang a, Zhixuan Xu a, Sixiu Liu

* , Guodong Sui a,*

a

Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of

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a,

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Environmental Science & Engineering, Fudan University, 220 Handan Road, Shanghai 200433,

Department of Dermatology, Huashan Hospital, Fudan University, 12 Middle Urumqi Road,

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b

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PR China.

Shanghai 200040, PR China.

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*Corresponding authors.

E-mail addresses: [email protected] (S. Liu), [email protected] (G. Sui); Tel:

These authors contributed equally.

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1

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+86-21-55665623; Fax: +86-21-65642080.

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Abstract: Human exposure to PM2.5 causes several adverse health effects. Skin is the first barrier

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against harmful environmental substances and can has direct contact with PM2.5, but there is no study about PM2.5-induced cytotoxicity in human skin cells on the molecular level partially due to the shortcomings of traditional research methods. In present study, we established a microfluidic system including a cell culture chip integrated with a high-throughput protein microarray chip to investigate the mechanism of PM2.5-mediated cytotoxicity in human HaCaT cells. We found that PM2.5 was lodged inside the cytoplasm, mitochondria and nucleus of HaCaT cells by TEM. Flow cytometry analysis indicated that the cell apoptosis rate increased from 0.49% to 53.4%. The 1

ACCEPTED MANUSCRIPT results of protein microarray showed that NF-κB and NALP3 signal transductions were activated in HaCaT cells after PM2.5 stimulations, up-regulating the expression of IL-1β and IL-6, which resulted in inflammatory response in HaCaT cells. Our findings provide a molecular insight into PM2.5-induced skin injury.

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Keywords: PM2.5, HaCaT, Cytotoxicity, Inflammation, Microfluidics

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1. Introduction

Over the past decades, China has experienced rapid urban and economic development. This

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has exacerbated air pollution and increased the occurrence of haze, especially in the most

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developed cities, such as Shanghai [1]. According to the 2012 Asian Development Bank report, less than 1% of China’s 500 largest cities met the air quality standards recommended by the World

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Health Organization (WHO) [2]. Particulate matter (PM) is a major component of air pollution. It

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is a complex mixture of extremely fine particles composed of metals, organic pollutants, acids, allergens, endotoxin and so on [3]. Epidemiological studies suggest that exposure to PM can

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induce several adverse health effects [4], such as respiratory [5-7] and cardiovascular [8-10]

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diseases. These diseases are primarily caused by PM2.5, which is composed of particulates with an aerodynamic diameter of less than 2.5 μm [11]. For example, PM2.5 has been shown to increase lung cancer mortality [12]. The WHO specialized cancer agency, the International Agency for Research on Cancer, has announced that PM is classified as carcinogenic to humans (Group 1) [13]. Skin is one of the major routes for external substances [14]. Keratinocytes are the main cell of the human skin epidermis [15] and form an interface between the body and 2

ACCEPTED MANUSCRIPT environment [16], such that they have direct contact with PM2.5. Studies have shown that keratinocytes can sense danger signals and conduct immune responses [17]. It has been reported that Mycobacterium leprae increased the expression of cathelicidin and TNF-α in keratinocytes [18]. ZnO nanoparticles induces generation of ROS to initiate apoptosis

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processes of keratinocytes [19]. ERK1/2 and p38 pathways are involved in cellular

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responses to ultraviolet B (UVB) exposure in keratinocytes [20]. Particularly, Asian dust

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storm increased the expression of inflammatory cytokines such as IL-6 and IL-8 in human keratinocytes [21]. PM2.5 contains many harmful substances such as allergens, bacteria,

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viruses and so on. And there are some reports that suggest PM2.5 causes inflammatory skin

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diseases, such as Jean Krutmann et al. identified that ambient particle pollution might lead to oxidative stress causing skin inflammation and aggravate atopic dermatitis [22]. Kyung

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Eun Kim et al. have reported that PM are involved in acne and psoriasis [23]. Magnani et

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al. showed that PM2.5 caused inflammation to human epidermis tissue [24]. This indicate that PM2.5 have adverse health effect on skin. However, there is little study about

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PM2.5-induced cytotoxicity in human keratinocytes on the molecular level.

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Traditional protein analysis techniques such as enzyme-linked immunosorbent assay (ELISA) arise problems when the sample volume is limited and analysis of multiple proteins are required [25]. Microfluidic devices have the advantage of low reagent consumption and high-throughput format [26]. In present study, we established a microfluidic system which integrated a cell culture chip with a protein microarray chip to investigate the mechanism of PM2.5-mediated cytotoxicity in keratinocyte HaCaT cells. 2. Material and methods 3

ACCEPTED MANUSCRIPT 2.1. Chemicals and reagents AZ-50XT photoresist was purchased from AZ Electronic Materials, USA. Polydimethylsiloxane (PDMS) (RTV-615) was purchased from Momentive Specialty Chemicals (NY). DMEM, fetal bovine serum, penicillin, trypsin-EDTA, and PBS (PH 7.4)

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were purchased from Gibco, USA. All antibodies were from Abcam, Hong Kong. The

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Annexin V-FITC kit and cell lysis buffer were from Beyotime Biotechnology, China.

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Bovine serum albumin (BSA) and Tween20 were purchased from Genebase Gene-Tech Co., China. ELISA kits were purchased from Xi Tang Bioscience Technology Co., China.

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All other reagents were purchased from Sigma-Aldrich.

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2.2. PM2.5 collection and process

We collected PM2.5 from November 2011 to January 2012 at Fudan University in

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Shanghai. The fine particles were sampled using a PM 2.5 high-throughput air sampler

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(Thermo Andersen, Thermo Fisher, MA, USA) fitted with a quartz filter membrane (Japan Toyo filter paper co., Ltd) that was replaced daily. The membranes were cut into small

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pieces, suspended in ultrapure water (18.2 MΩ resistance) and sonicated for 1 h. The

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suspension was filtered through six layers of a sterilized gauze filter and dried in a vacuum freeze drier [27]. The membranes were then sonicated with methanol for 3 h. After that, the suspension was dried under nitrogen. All PM 2.5 powder was stored at −20℃. Before use, PM2.5 was suspended in cell culture medium at 1 mg/ml and sonicated for 10 min for sterilization and then diluted to the needed concentration. Then PM2.5 suspensions were filtered with a 300-meshes sterile filter membrane to remove the quartz fibre which may affect the experimental results. 4

ACCEPTED MANUSCRIPT 2.3. Microfluidic system fabrication The microfluidic system was fabricated by soft lithography [28]. To form molds, AZ-50XT photoresist was coated on a silicon wafer and patterned by exposure to UV light through a mask. The cell culture chip included the fluid layer and the control layer. The

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fluid layer was made of PDMS (RTV 615A and B in a 5:1 ratio) and the control layer was

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made of PDMS (RTV 615A and B in a 20:1 ratio). After curing at 80℃ for 1 h, the two

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layers were glued together and bonded onto a PDMS (RTV 615A and B in a 20:1 ratio) coated glass slide. For the microarray chip, PolymerSlide™ G (CapitalBio, China) was

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used for the protein microarrays. The slide surface was covered by a thin three-dimensional

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polymer layer with aldehyde modification. Protein molecules were bound to the polymer layer after spotting. The printed slide was used as a substrate and the PDMS layers were

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precisely covered on the printed areas of the microarray slide. The screws at the edges of

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the acrylic plates were used to hold the PDMS chip and microarray slide together. 2.4. Modification of cell culture chip

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For the cell culture, the chip surface was modified from hydrophobic to hydrophilic

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according to the protocol reported previously [29]. The culture chambers were pre-treated with a HCl (1% v/v)-H2O2 (1% v/v) ethanol solution for 10 min at room temperature, followed by 12 h incubation with a 0.01% (w/v) poly-L-lysine solution. Then, the excess solution was removed and the chips were dried at room temperature. Coated chips were sterilized at 121℃ for 30 min and then dried at 60℃ before utilization. 2.5. Cell culture and exposure to PM 2.5

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ACCEPTED MANUSCRIPT The HaCaT cells (ATCC) were maintained in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin at 37℃ under 5% CO2 in a humidified chamber. Subconfluent HaCaT cells were dissociated with an EDTA-trypsin solution. After centrifugation, the cells were re-suspended with culture medium to 10 6/ml and then plated on the modified

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cell culture chip. After a 24 h incubation period, the culture medium was removed and then

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PM2.5 suspensions were added to the culture chamber to stimulate the HaCaT cells.

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2.6. Transmission electron microscopy (TEM)

HaCaT cells cultured on microfluidic chip were treated with PM 2.5 at a concentration

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of 150 μg/ml for 24 h. After being harvested into a centrifugal tube, the cells were fixed

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with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) containing 2% sucroseat for at least 1.5 h at 4℃. Next, the cells underwent postfixation with 1% OsO 4 in 0.1 M

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cacodylate buffer (pH 7.3) for 1.5 h at room temperature. Cells were dehydrated with

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graded ethanol concentrations. The cells were embedded in EPON 812 and heated at 60 ℃ for 48 h. Following this, the cells were cut with a FC7-UC7 ultracut (Leica, Germany).

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Ultrathin sections were stained with lead citrate and uranyl acetate and analyzed by Tecnai

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G220 TWIN (FEI, America). 2.7. Microarray assay To evaluate the effect of PM 2.5 on HaCaT cells related to real life situations, we selected PM2.5 samples in the light pollution, moderate pollution and severe pollution weather respectively. The daily average concentration of the samples were 53.8, 94.8 and 164.8 μg/m3. According to the latest index of PM 2.5 standard from the U.S.EPA in 2012 (35.5~55.4 μg/m3, unhealthy for sensitive groups; 55.5~150.4 μg/m3, unhealthy; 6

ACCEPTED MANUSCRIPT 150.5~250.4 μg/m3, very unhealthy), the AQI Category of the chosen days were unhealthy for sensitive groups, unhealthy and very unhealthy respectively. Then HaCaT cells on culture chips were stimulated with PM 2.5 at a concentration of 0, 50, 100, 150 μg/ml for 24 h. For the time-course experiment, HaCaT cells were treated with 50 μg/ml PM 2.5. The cell

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lysis solution was loaded into the culture chambers and the chips were maintained at 4℃

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for 30 min. Then, the mixture of the culture medium and cell lysate of two parallel

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chambers were pumped into the microarray chambers for protein detection though tubing connecting the culture and microarray chips.

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Schematic illustration of the immunoassay on microarray chips was shown in Figure 1.

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Unoccupied microarray chamber areas were blocked with 3% BSA at 37℃ for 60min. This reduced the background value for the final analysis. Next, the lysate was pumped into the

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microarray chambers separately. The regular valves were closed to prevent cross-contamination of

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different samples. The chip was incubated at 37℃ for 60 min, and PBST washing buffer was used to rinse the microarray chambers. Mouse antihuman NF-κB, IL-6, IL-1β, NALP3 and

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caspase-1 antibodies were mixed together and pumped into the microarray chambers. All of the

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antibodies were diluted in PBS at a final concentration of 2μg/ml. After incubation at 37℃ for 60 min, the chambers were washed as previously described. The secondary antibody goat anti-mouse was diluted in PBS at 1:1000. Alexa Fluor 555 was used as tracer for signal generation. The secondary antibody was loaded and incubated at 37℃ for 60 min. Finally, the microarray chambers were washed with PBST buffer and double-distilled H2O. The acrylic plates were removed and the microarray slide was separated from the PDMS layer. The slide was dried by centrifugation at 200 rpm for 5 min and detected by LuxScan Dx24 (Beijing Capital Bio Co., Ltd., 7

ACCEPTED MANUSCRIPT China). Data were analyzed using in-house analysis software. 2.8. ELISA assay HaCaT cells were stimulated with PM2.5 and lysed as described in 2.7. Microarray assay. The mixture of the culture medium and cell lysate were pumped out of the cell culture chip, collected

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into centrifugal tubes and stored at -20℃. NF-κB, IL-6, IL-1β, NALP3 and caspase-1 were

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measured using sandwich ELISA kits according to the manufacturer’s instructions. The

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absorbance of each well was read on an ELISA-Reader (Molecular Devices, America) at 450 nm. In addition, HaCaT cells cultured in 24-well-plate were also stimulated with PM2.5. After that,

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the culture medium was used for the detection of IL-6 and IL-1β and the cell lysate was used for

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the detection of NF-κB, NALP3 and caspase-1 by traditional ELISA for further comparison. 2.9. Apoptosis detection

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HaCaT cells on chips were stimulated with PM2.5 at a concentration of 150 μg/ml for 24 h

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and then harvested into a flow cytometry tube. The cells were stained with an Annexin V-FITC kit according to the manufacturer’s instructions. FITC and propidium iodide (PI) were immediately

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analyzed with a Gallios (Beckman Coulter, America) flow cytometer. Both fluorescence was

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excited by a 488 nm argon laser. Emissions were measured at 525 nm for FITC (FL-1 channel) and 620 nm for PI (FL-3 channel). 3. Results and discussion 3.1. Design of microfluidic system The microfluidic system included two parts: a cell culture chip and a microarray chip (Figure 2A). The cell culture chip had four parallel chambers (length 4 mm, width 3 mm, depth 25 μm) (Figure 2B), and the area of one chamber is about 0.19 cm 2. All the chambers 8

ACCEPTED MANUSCRIPT were modified with poly-L-lysine solution then HaCaT cells could attach to the cell culture chamber. The chip was automatically perfused with slowly flowing culture medium (50 μL/h) by the syringe pump. After 24h, HaCaT cells could spread through the whole bottom (Figure 2D). By this time, the number of cells are 4 ~ 5×104 so that two chambers are

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equivalent to the size of one well of the 96-well-plate. The regular valves were designed to

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produce a closed system. The microarray chip was able to respond to four different

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samples at the same time. One inlet was shared by these chambers for the wash buffer and antibodies. Meanwhile, each chamber had a unique outlet and another unique inlet for

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samples (Figure 2C). Regular valves were used to control the fluids, thus the

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immune-reactions were carried out in a parallel or independent way. Traditionally, cells are grown in a macroscale and static culture environment in vitro,

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which is very different from the complex microenvironment in vivo [30]. Microfluidic

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chips has a high surface-area to volume ratio, which give a more efficient oxygen supply to cells for maintaining metabolism through diffusion [31]. Moreover, perfusion culture on

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microfluidic chips provide cells continuous nutrient supply and waste removal, giving cells

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a stable environment [32]. In addition, the continuous flow can provide cells shear stress [33]. Hence, the microfluidic chips simulate better than traditional cell culture platforms on the native cellular microenvironment. 3.2. TEM analysis of HaCaT cells stimulation with PM2.5 The results of TEM analysis were shown in Figure 3. Untreated HaCaT cells were used as a control. We can see that the control cells remained a smooth surface, while also exhibiting the typical structural features of this cell line (Figure 3A, D). After treatment 9

ACCEPTED MANUSCRIPT with PM2.5, the cell shape was changed (Figure 3B, C). Free particles in the cytoplasm, mitochondria and nucleus were visible (Figure 3E, F). Mitochondria were destroyed, with the ridge obscured (Figure 3E). Damage to mitochondria can generate additional ROS, which play an important role in the activation of inflammatory pathways [34]. Nucleus is

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the control center of the cell, when PM 2.5 enters the cell nucleus, DNA damage and

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apoptosis in HaCaT cells may result [35].

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3.3. Effects of PM2.5 on signal transductions

The expression of NF-κB, IL-6, IL-1β, NALP3 and caspase-1 in HaCaT cells were

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shown in Figures 4. The results indicated the mean of three repeating spots (Figure 4A(i)).

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As shown in Figures 4A(ii)-(vi), the signal to noise ratio of all the studied cytokines increased as the concentration of PM 2.5 rose. In the time-course experiment, cytokines were

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detected at 3, 6, 9, 12 and 24 h of treatment with PM 2.5. Compared with the control groups,

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NF-κB and IL-6 peaked at 6 h after treatment and gradually decreased afterwards (Figure 4B(i), (ii)). The expression of NALP3 and caspase-1 reached a maximum at 12 h after

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stimulation and then decreased at 24 h (Figure 4B(iv), (v)). As the common product of

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these two transductions, the first peak of IL-1β was at 6 h, which was consistent with NF-κB. Another peak was at 12 h, similar to caspase-1. PM2.5 is a complex mixture of harmful substances, such as acids, organic chemicals, metals, and so on. In addition, bacteria and viruses that adhere to PM 2.5 may act as pathogens, causing cellular immune response. Keratinocytes are nonprofessional immune cells, which can conduct immune response through producing antimicrobial peptides (AMPs), chemokines and secreting numerous cytokines, such as IL-1, IL-6 and TNF-α 10

ACCEPTED MANUSCRIPT [36]. However, a dysregulated immune response can cause inflammatory skin diseases [17], such as allergic dermatitis, atopic dermatitis solar and solar dermatitis. Studies have shown that inflammatory signal transductions were activated in the epidermis of pa tients with inflammatory skin disease [17]. As a key regulator of inflammation in keratinocytes,

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NALP3 inflammasomes can be activated by bacterial RNA, bacterial toxins or ROS

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generation [37]. NF-κB can be activated by a wide range of stimuli, such as stress signals

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or pathogens [38]. Therefore, the effect of PM 2.5 on the skin is likely to be complicated. Our data indicated that PM2.5 activated both NF-κB and NALP3 transductions. Caspase-1

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was activated through NALP3 and then cleaved pro-IL-1β to matured IL-1β. Meanwhile,

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NF-κB entered the nucleus and up-regulated the expression of IL-1β and IL-6. All of these induced the inflammatory response in HaCaT cells (Figure 5).

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Inflammation is a cancer-causing biological factor [39]. It is believed that NF-κB is

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closely involved in the process of various tumors, such as lymphoma [40], liver cancer [41] and lung cancer [42]. NF-κB controls the expression of the genes linked with the

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proliferation, invasion and metastasis of cancer [43]. As PM2.5 may increase lung cancer,

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the mechanism by which PM 2.5 may be responsible for related diseases, such as skin cancer, requires further research. 3.4. Comparison between protein microarray and ELISA When HaCaT cells were treated with PM2.5 for protein microarray detection, they were simultaneously treated in the same way for traditional ELISA as a comparison. The mixture of the culture medium and cell lysate was pumped out of the cell culture chip for ELISA detection. Linear regression analysis showed good consistency between the microarray assay and ELISA. 11

ACCEPTED MANUSCRIPT For the concentration gradient experiment, the R2 values of NF-κB, IL-6, IL-1β, NALP3 and caspase-1 were 0.87, 0.86, 0.82, 0.82 and 0.84, respectively (Figure 6A). For the time-course experiment, R2 values of NF-κB, IL-6, IL-1β, NALP3 and caspase-1 were 0.85, 0.87, 0.89, 0.80 and 0.86, respectively (Figure 6B). This suggested that the microarray chip was capable of protein

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detection. To further validated the proposed methodology, we confirm our results using HaCaT

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cells cultured on classical cell culture 24-well-plate. As shown in Figure 7, linear regression

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analysis indicated a significant correlation between the two systems. The R2 values of NF-κB, IL-6, IL-1β, NALP3 and caspase-1 were 0.86, 0.84, 0.80, 0.79 and 0.80 for the concentration

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gradient experiment (Figure 7A) and 0.82, 0.86, 0.87, 0.80 and 0.85 for the time-course

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experiment (Figure 7B). Moreover, it can be seen that the R2 values between the microarray assay and ELISA results of proteins from cells cultured on chips were better than that between

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microarray assay and ELISA results of proteins from cells cultured on 24-well-plate. These

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demonstrated that HaCaT cells cultured on chips performed better than that cultured on classical cell culture support and our microfluidic system is suitable for our study.

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Compared with ELISA, five different proteins were able to be detected in one chamber on

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microarray chips at the same time. The immune reaction was able to be conducted in the same manner, thereby improving the efficiency and reducing errors. The consumption of PM2.5 was decreased to approximately 1 μL on chips, while requiring 1 ml for 24-well-plates. Because the PM2.5 sample was not sufficient for all of the studies, including component chemical analysis and bio-analysis of damage to cells, reduction of PM2.5 consumptions on chips was particularly important. 3.5. PM2.5 raised the apoptosis rate of HaCaT cells 12

ACCEPTED MANUSCRIPT As shown in Figure 7, HaCaT cells responded with significantly increased apoptosis rate after exposure to 150 μg/ml PM2.5. The apoptosis rate of control cells was 0.49% (Figure 8A), while cells treated with PM2.5 was 53.4% (Figure 8B). This indicated that PM2.5 caused an adverse effect on the growth of HaCaT cells.

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4. Conclusions

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We developed a high-throughput microfluidic system that integrated a cell culture and protein

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microarray chip together. Using this platform, we demonstrated that PM2.5 entered into keratinocyte HaCaT cells and increased the cell apoptosis rate. NF-κB and NALP3 transductions

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were activated and induced inflammatory response in HaCaT cells. Our findings provide an

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insight into the skin cytotoxicity induced by PM2.5. Acknowledgments

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This work was supported by the National Natural Science Foundation of China

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(NSFC, grant number: 21577019, 21577020, 21377027 and 21527814) and Opening Project of Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention

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(LAP3) (grant number: FDLAP16003).

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ACCEPTED MANUSCRIPT Figure captions Figure 1. Illustration of the microarray chip operations for immunoassay. The states of the valves were distinguished by different colors: blue for close and red for open. Gray indicated that the channels were empty.

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Figure 2. (A) Photograph of the (a) microfluidic system, (b) cell culture chip and (c)

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microarray chip, (B) Schematic diagram of the cell culture chip, (C) Schematic diagram of

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microarray chip used for protein detection, (D) HaCaT cells in modified cell culture chip. Figure 3. TEM images of (A) and (D) control HaCaT cells, (B), (C), (E) and (F) HaCaT

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cells exposure to 150 μg/ml PM 2.5. Abbreviations: N, nucleus; M, mitochondria; P,

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Particle. Bars = 2μm for (A), (B) and (C); 1 μm for (F); and 0.5 μm for (D) and (E). Figure 4. (A) HaCaT cells were treated with different concentration of PM 2.5, and the

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cytokines were detected by the microarray chip, the five row of protein spots in white

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rectangle were IL-1β，NF-κB，IL-6，NALP3 and caspase1 respectively, (B) HaCaT cells were treated with 50μg/ml PM 2.5 for different times, and the cytokines were detected by the

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microarray chip. *p < 0.05; PM 2.5/controls were compared for each concentration or time.

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Figure 5. Model of PM 2.5-induced immune response in HaCaT cells. PM 2.5 entered into HaCaT cells, which activated the NALP3 inflammasome and resulted in the processing of IL-1β. NF-κB was also activated and regulated the expression of IL-1β and IL-6 after PM2.5 exposure. up-regulated;

Protein detected in our research, the expressions of which were Protein in related signal transductions.

Figure 6. Linear regression analysis between microarray and ELISA results of proteins from HaCaT cells cultured on chips. (A) HaCaT cells were treated with different concentration of PM2.5, 19

ACCEPTED MANUSCRIPT (B) HaCaT cells were treated with 50 μg/ml PM2.5 for different times. Figure 7. Linear regression analysis between microarray and ELISA results of proteins from HaCaT cells cultured on 24-well-plate. (A) HaCaT cells were treated with different concentration of PM2.5, (B) HaCaT cells were treated with 50 μg/ml PM2.5 for different times.

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Figure 8. Flow cytometry detection of (A) control HaCaT cells, and (B) HaCaT cells exposure to

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apoptosis); Annexin V+/PI+ (late apoptosis and necrosis).

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ACCEPTED MANUSCRIPT Highlights

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PM2.5-induced cytotoxicity in HaCaT cells was clarified on the molecular level.

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NF-κB and NALP3 were activated, resulting in inflammatory response in HaCaT cells.

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The platform allowed for 3D cell culture and high-throughput protein detection.

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