Investigation of Cell-to-cell Transfer of Polystyrene Microplastics Through Extracellular Vesicle-mediated Communication

doi:10.21203/rs.3.rs-4639504/v1

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Investigation of Cell-to-cell Transfer of Polystyrene Microplastics Through Extracellular Vesicle-mediated Communication

https://doi.org/10.21203/rs.3.rs-4639504/v1

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Plastics have become an integral part of human life, and their production is increasing annually. Plastics are broken down into small particles known as microplastics (MPs) with particle size of < 5 mm in the environment because of a variety of factors. MPs are prevalent in the environment and all living organisms are exposed to their effects. In this study, we investigated whether polystyrene (PS)-MPs were transferred from cell-to-cell via extracellular vesicles (EVs). This study showed that cell-derived EVs could transport plastic particles. In addition, using a real-time imaging device, we confirmed that PS-MPs were transported by EVs that accumulated in the cells. This study provides an understanding of the potential effects of PS-MPs on living organisms via EVs and suggests directions for future research.

Microplastics

Extracellular vesicles

Transfer

Environmental pollution

Ishikawa cells

Plastics have become essential to human life, with global production reaching approximately 367 million tons in 2020, and is estimated to increase by 29% by the year 2028 (Aslani et al., 2021; Tiseo, 2021). These plastics can break down over time into microplastice (MPs) and nanoplastics (NPs) with particle sizes of < 5 mm and 1 µm, respectively (Devi et al., 2023). These are fragmented by UV light, environmental conditions, and other factors (Bhatia et al., 2024; Gigault et al., 2018). MPs are common in water, sea salt, and food crops; therefore, they can easily enter and accumulate in the human body via food and drinking water (Aslani et al., 2021; Bradney et al., 2019; Yuan et al., 2022). Thus, the widespread exposure to MPs requires immediate attention to understand the effects of plastics on human health. Chronic ingestion of MPs causes oxidative stress and inflammatory responses via the generation of reactive oxygen species (ROS) (K. et al., 2022; Peng et al., 2023; Schmidt et al., 2023; Yang et al., 2022). This induces toxicity in vital human and animal organs owing to the ability of MPs to translocate across cell membranes (Hu et al., 2021; Peng et al., 2023). Furthermore, studies have reported that an increase in the concentration of polystyrene (PS)-MPs within cells can lead to the accumulation of these particles, ultimately resulting in cell death and other adverse effects (Banerjee et al., 2022; Kwon et al., 2022). This indicates that further research is needed to elucidate the intracellular movement of MPs to prevent their potential impact on human health.

Extracellular vesicles (EVs) are small membrane particles surrounded by a lipid bilayer that are released into the environment by various cell types (Doyle & Wang, 2019; Hao et al., 2024; Sarkar & Patranabis, 2024). EVs are significantly involved in cell-to-cell and intercellular communication via the exchange of proteins, nucleic acids, and lipids between cells (Reseco et al., 2024; Y. Wang et al., 2024). Exosomes, a type of EVs, play an important role in cell-to-cell communication by transporting microRNAs (miRNAs/miRs), messenger RNAs (mRNAs), and proteins between cells (Tkach & Théry, 2016; Zhu et al., 2024). These exosome properties have been used for the development of diagnostics for various diseases; however, more research is still needed.

A recent study reported that serum-derived EVs can transport polyethylene terephthalate (PET)-MPs and alter the miRNA content of EVs (Mierzejewski et al., 2023). Moreover, plastics treatment increases the number of EVs released from the cells (Yan et al., 2023). Although numerous reports have revealed adverse effects related to the accumulation of MPs in human and animal cells, the mechanisms by which MPs accumulate and move between cells remain unclear.

Our study aimed to elucidate the mechanism by which intracellularly accumulated PS-MPs move out of the cell and the process by which they are transferred to other cells. we investigated the presence of PS-MPs in EVs by extracting EVs from Ishikawa cells that had accumulated fluorescent PS-MPs ((FL)PS-MPs) and analyzed them using Flow cytometry. Additionally, we used confocal microscopy to detect PS-MPs in cells treated with extracted EVs and IncuCyte live-cell imaging to observe PS-MPs entry into cells from stained EVs.This study provides a fundamental basis to understand the potential effects of MPs on cells.

2.1 Ishikawa Cell Culture

Ishikawa cells were cultured in minimum essential medium (MEM)/ Earle's Balanced Salt Solution (EBSS) (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 2% penicillin-streptomycin (P/S; Cytiva, Marlborough, MA, USA) at 37°C in a 5% CO₂ incubator.

2.2 Isolation of EVs After Treatment of Cells with Polystyrene Microplastics

Ishikawa cells were seeded at 3 × 10⁵ cells/well in T25 flasks (SPL, Pochen, Korea) and cultured to approximately 60% confluency. The medium was then removed, and the cells were treated with MEM/EBSS supplemented with 1% FBS containing 1 µm PS-MPs (Merck, Darmstadt, Germany) or 1 µm green fluorescent unmodified microplastic beads (CD Bioparticles, NY, USA) at a concentration of 1 mg/mL for 24 hours at 37°C in a 5% CO2 incubator. After 24 h, the cells were washed three times with cold phosphate-buffered saline (Welgene, Gyeongsan, Korea), trypsinized, and seeded in fresh T25 flasks for 24 h in a 5% CO2 incubator to facilitate the isolation of EVs. EVs were isolated using ExoQuick-TC (SBI, Palo Alto, CA, USA) according to the manufacturer's specifications. Briefly, the culture medium was centrifuged at 1200 × g for 20 min and 5 mL of the resulting supernatant was combined with 1 mL of ExoQuick-TC. The mixture was then incubated at 4°C for 24 h. Subsequently, the medium-supernatant complex was centrifuged at 1500 × g for 30 min, followed by the removal of supernatant and centrifuged at 1500 × g for 5 min. The precipitated EVs pellets were used for subsequent cell treatment and flow cytometry analysis. The remaining cells were fixed with 4% paraformaldehyde (Forbio) and subjected to flow cytometry.

2.3 Flow cytometry assay

Flow cytometric analysis was conducted using FACSCanto II (BD Biosciences, San Jose, CA, USA) with FCSalyzer software (version 1.8.0) to analyze the presence of intracellular PS-MPs or PS-MPs in EVs. Extracted EVs and fixed cells were excited for forward scattering (FSC) and side scattering (SSC) using a 488 nm (blue) laser, whereas the excitation for the green (FL)PS-MPs was a 508 nm (blue-green) laser. Green fluorescence was recorded using an emission filter at 530/30 nm. The settings were optimized for the detection of small particles. The acquisition time was kept constant for all samples and a log scale was used throughout. All the experiments were performed with four replicates.

2.4 Staining the Extracted EVs

The 500X stock solution was diluted to a final concentration of 50X ExoBrite™ 560/585 cholera toxin subunit B (CTB) EV staining solution (Biotium, San Francisco, USA) and subsequently added to the precipitated EVs pellet. The solution was gently mixed and incubated for 30 min in the dark at room temperature.

2.5 Live-Cell imaging Assay

The IncuCyte® Live-Cell Analysis System (Sartorius, Göttingen, Germany) was used to capture real-time images of live cells at specific time intervals over 24 h. Subsequently, the cells were analyzed based on those images and compiled into a video. Ishikawa cells were seeded at 105 cells/well in 24-well plates coated with poly-L-lysine (Merck, Darmstadt, Germany) and grown until they reached approximately 60% confluency. The cells were then treated with stained EVs and incubated for 10 min at 37°C in a 5% CO2 incubator with NucSpot Live 650 nuclear stain (Biotium, San Francisco, CA, USA) at a final concentration of 1 µM for nuclear staining. The samples were then incubated for 24 h in an incubator equipped with an IncuCyte Live-Cell Analysis System for live-cell imaging.

2.6 Confocal Fluorescence Microscopy for Fixed Cell Imaging.

Ishikawa cells were seeded on poly-l-lysine (Merck, Darmstadt, Germany)- coated coverslips. Extracted EVs were added to cells when they reached 60% confluence. After 24 h, the medium was removed, and the cells were fixed with 4% paraformaldehyde on a shaker at 40 rpm for 15 min in the dark at room temperature. Cells were mounted with mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Thermo Scientific, Waltham, MA, USA) and examined using a confocal microscope (LSM 980, Carl Zeiss, Oberkochen, Germany) with an oil-immersion objective lens (Plan-Apochromat 40x/1.4 oil DIC M27; Carl Zeiss Microscopy, GmbH).

2.7 Statistical Analysis

Graphs and statistical analyses were performed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). Data collected from at least three independent replicates were analyzed and expressed as the mean ± SEM. One-way ANOVA was used to analyze the differences between the control and PS-MPs treated groups. Differences were considered statistically significant at P < 0.05. The levels of statistical significance are as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

3.1 Detection of PS-MPs in the cells

The results show the morphology of subcultured cells after treatment with 1 µm PS-MP and 1 µm (FL)PS-MPs at a concentration of 1 mg/mL (Fig. 1a). The analysis of fluorescence intensity in (FL)PS-MP-treated cells using flow cytometry showed that 46.3 ± 8.4% of the cells in the (FL)PS-MP treatment group exhibited fluorescence (Fig. 1b & 1c). These results indicate that (FL)PS-MPs were internalized by the cells.

3.2 Detection of PS-MPs in cell-derived EVs

To determine the presence of MPs in cell-derived EVs, EVs were isolated from the medium of PS-MPs (PS-MPs_EVs)- or ((FL)PS-MPs_EVs)-treated cells and analyzed for fluorescence using flow cytometry. The results showed that green fluorescence was detected in cell-derived EVs treated with (FL)PS-MPs (Fig. 2a & 2b). The fluorescence intensity was 51.6 ± 6.2% in (FL)PS-MPs_EVs compared to that in the control group (Fig. 2c). This suggests that EVs contain microplastics and contribute to their intercellular transfer.

3.3 Visualization of EV-derived PS-MPs Entering the Cell.

To investigate the entry of EV-derived PS-MPs into cells, cells were treated with stained (FL)PS-MPs_EVs and imaged for 24 h using an IncuCyte live-cell imaging system. When cells were treated with stained (FL)PS-MPs_EVs, vesicles containing PS-MPs, which appeared yellow, gradually fused with the cells at 6 h 50 min, and microplastics were eventually observed within the cells (Fig. 3a). These results suggested that EVs may be involved in the cell-to-cell migration of PS-MPs.

3.4 Investigation of Intracellular Accumulation of PS-MPs After Treatment with EVs.

To investigate the intracellular accumulation of EV-delivered PS-MPs, the cells were treated with EVs for 24 h and examined for the presence of PS-MPs using confocal microscopy. The results showed that when cells were exposed to EVs derived from PS-MP-treated cells, MPs were observed within the cells (Fig. 4a). The internalization of the PS-MPs is shown in the Z-stack images of the cells (Fig. 4b). The ortho-XZ plane is indicated by a yellow box on the top side of the 2-D image, and the YZ plane is indicated by a green box on the right side of the image (Fig. 4b-1). The 3-D Z-stack axis of the multiple image overlays in Fig. 4b-1 shows the internalization of PS-MPs. The position of the (FL)PS-MPs relative to the red nucleus was determined using the XYZ dimensions. For image 4b-1, the XZ and YZ planes are vertical sections on the paper for the best visualization and are shown at the bottom and right sides of each of the main XY images. These results suggested that MPs can be transferred intracellularly via EVs.

In this study, we investigated whether PS-MPs are transferred from cell-to-cell via EVs. To confirm the presence of PS-MPs in EVs, (FL)PS-MPs were purchased, EVs were fluorescently stained, and double staining was confirmed by flow cytometry (Fig. S1). The (FL)PS-MPs_EVs identified by double staining were then added to the cells to track whether the PS-MPs within the EVs entered the cells in real-time. To the best of our knowledge, our results are the first to demonstrate the involvement of EVs in cell-to-cell transfer of MPs.

Recent studies have shown that MPs are involved in the production and transformation of EVs in cells. According to Wang et al., the production of EVs increased by approximately 4.3-fold in cells treated with PS-MPs compared to untreated cells (Wang et al., 2024). The size of EVs also varied depending on the concentration MPs. These reports suggest that MPs induce increased production of EVs and are involved in the variation in EVs size.

Furthermore, it has been reported that miRNAs in EVs, whose expression is altered by MPs, are associated with the development of metabolic syndrome and diabetes (Gondaliya et al., 2020; Mierzejewski et al., 2023; Russo et al., 2018). Although recent studies have revealed the effects of altered EV expression, there has been limited research on the mechanisms by which accumulated MPs are transferred to other organs.

Mierzejewski et al. found that PET-MPs are transported in serum-derived EVs in an immature gilt model. Their findings also revealed that PET-MPs negatively affect physical function through differentially regulated miRNAs. This study suggests that MPs are transported by EVs, and that MPs within EVs can move to other organs. Furthermore, in previous studies, we observed that MPs that accumulate intracellularly migrate out of the cell, as shown in Fig. S2. These results indicated that EVs are involved in the migration of intracellularly accumulated MPs to other cells and tissues. This study clearly demonstrated the mechanism of cell-to-cell transfer of MPs by EVs.

To remove as much of the remaining MPs as possible when extracting EVs from MPs-treated cells, cells were treated with PS-MPs for 24 h, washed three times, and EVs were extracted from the medium of the passaged cells. To determine whether PS-MPs were present within the cell-derived EVs or were aggregated, the cell-derived EVs were stained, and the IncuCyte liv-cell imaging system was employed to detect the color-merged vesicles. The resulting temporal sequence of the images, in which the yellow vesicles surrounding the cell gradually fused with the cell and intracellular microplastics accumulated, indicated that the PS-MPs present in the EVs were internalized by the cell (Fig. 3). Although the low magnification of the IncuCyte live-cell imaging system made it difficult to closely observe PS-MPs internalization in the cells, the 3D Z-stack capability of the confocal microscope enabled us to observe the precise internalization of PS-MPs in the cells (Fig. 4). Studies have reported that MPs affect the size of EVs, suggesting that the accumulation of MPs in EVs may be related to size variation. In future studies, we will investigate whether an increase in EVs size and the induction of EVs production by MPs play a role in the accumulation of MPs. Further studies are needed to identify the defense mechanisms that cells utilize to eliminate EV-delivered MPs and assess the potential effects of EV-delivered MPs on cells.

In conclusion, our results showed that PS-MPs are transferred from cell-to-cell via cell-derived EVs. These results, supported by real-time imaging and detailed analysis, contribute to the understanding of the dynamics of EV-derived PS-MPs internalization into cell. Our findings highlight the importance of further research on the potential health effects of plastic-containing EVs in humans.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00239426 and 2023R1A2C2006225).

Author Contribution

NK participated in the design of the study, carried out all experiments, analyzed the data, and drafted the manuscript. JHP and IL contributed to the investigation related to this study, helped in data analysis, and drafted the manuscript. Both GSJ and MJL helped the investigation related to this study. JHL contributed to the conception and helped to design the study. YSC contributed to the conception of this study. WI and SC designed this study and wrote the manuscript with comments from coauthors, and all authors collaborated on the work.

Data Availability

Data is provided within the manuscript or supplementary information files. Data will be made available on request.

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No competing interests reported.

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Investigation of Cell-to-cell Transfer of Polystyrene Microplastics Through Extracellular Vesicle-mediated Communication

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Ishikawa Cell Culture

2.2 Isolation of EVs After Treatment of Cells with Polystyrene Microplastics

2.3 Flow cytometry assay

2.4 Staining the Extracted EVs

2.5 Live-Cell imaging Assay

2.6 Confocal Fluorescence Microscopy for Fixed Cell Imaging.

2.7 Statistical Analysis

3. Results

3.1 Detection of PS-MPs in the cells

3.2 Detection of PS-MPs in cell-derived EVs

3.3 Visualization of EV-derived PS-MPs Entering the Cell.

3.4 Investigation of Intracellular Accumulation of PS-MPs After Treatment with EVs.

4. Discussion

Declarations

Declaration of competing interest

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

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