Nanoplastic toxicity and uptake in kidney cells: differential effects of concentration, particle size, and polymer type

Nanoplastic toxicity and uptake in kidney cells: differential effects of concentration, particle size, and polymer type | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nanoplastic toxicity and uptake in kidney cells: differential effects of concentration, particle size, and polymer type Hayden Louis Gillings, Darling M Rojas-Canales, Soon Wei Wong, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7588029/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Cell Biology and Toxicology → Version 1 posted 36 You are reading this latest preprint version Abstract Nanoplastics (NPs, <1 µm) are emerging environmental contaminants capable of crossing biological barriers and interacting at the cellular and subcellular level. Despite evidence of microplastics in human kidney tissue and urine, the renal effects of NPs remain poorly understood. This study investigated the short-term effects of NP polymer type, size, and concentration on human proximal tubule cells (HK-2). Cells were exposed for 24 h to carboxylated polystyrene (PS), poly(methyl methacrylate) (PMMA), and polyethylene (PE) NPs (15–100 nm) at concentrations from 0.2 to 200 µg/mL. NP size, charge, and morphology were characterised by scanning electron microscopy, dynamic light scattering, and zeta potential. Cell morphology, viability, cell cycle distribution, and NP internalisation were assessed by microscopy and flow cytometry. Low-concentration exposures had minimal effects, whereas 100 and 200 µg/mL induced marked morphological changes, including cytoplasmic granularity. Viability decreased significantly at 200 µg/mL for several NP types, with PE NPs causing the largest reduction (79.4%). Polymer type influenced outcomes, with PE and PMMA NPs causing greater morphological disruption than PS. Size effects were most evident in cell cycle analysis: 15 nm and 20 nm PS NPs and 100 nm PMMA NPs induced phase arrest without major viability loss. NP internalisation increased with concentration but varied with polymer type, with PE NPs showing preferential perinuclear localisation. These findings demonstrate that NP effects on kidney cells depend on polymer chemistry, particle size, concentration, and highlight the need for long-term studies using environmentally relevant NPs to better assess kidney toxicity risk. nanoplastics kidney cytotoxicity polystyrene poly(methyl methacrylate) polyethylene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In recent years, there has been increasing concern regarding the health risks and the potential toxicity associated with nanoplastic (NP) exposure. NPs are typically defined as any plastic particles below 1 µm in diameter, either intentionally manufactured (e.g. latex nanoparticles) or the byproduct of plastic degradation in the environment (International Organization for Standardization 2020 ). They represent the smallest size fraction of microplastics (MPs) which are defined as plastic fragments smaller than 5 mm. It is important to note that particles that are 1 µm in diameter differ significantly from 100 nm NPs in their behaviour and properties due to factors such as Brownian motion, diffraction limits and enhanced ability to cross cell membrane (Atugoda et al. 2023 ; Gigault et al. 2021 ; Masseroni et al. 2022 ). While the majority of reports of the effects of plastic particle exposure have focused on larger size fractions, there are compelling reasons to investigate the effects of nanoparticles below 100 nm — particularly regarding their behaviour in biological matrices and potential health impacts — as particles in this size range can more readily cross biological barriers, interact at the cellular and subcellular level, and exhibit unique surface reactivity. So far research into the toxicity and potential health effects of NPs has been predominantly focused on the lungs, liver and gastrointestinal system (Kihara et al. 2021 ; Lin et al. 2022 ; Schwarzfischer et al. 2022 ) with minimal examination of the impact and toxicity of NPs on the renal system (Goodman et al. 2022 ). This is particularly concerning as MPs have recently been detected in healthy human blood, kidney tissue and urine samples (Leslie et al. 2022 ; Marfella et al. 2024 ; Massardo et al. 2024 ; Nihart et al. 2025 ). Massardo et al. ( 2024 ) found 26 MPs between 1 and 29 µm in kidney tissue, and between 3 and 13 µm in urine samples. Nihart et al. ( 2025 ) assessed kidney tissue samples from 2016 and 2024 and reported a total plastic mass of 402.5 and 404.8 µg per gram of tissue, respectively. This study also noted an increased incidence of MPs within the kidney tissue localised to the glomeruli and tubules. Given these findings, it is important to understand how this accumulation may be occurring within the renal system. The kidney filters about 180 litres of blood each day, removing waste while reclaiming essential substances like electrolytes, glucose, and proteins (Molitoris et al. 2022 ; Zhuo and Li 2013 ). This process begins in the glomerulus, where blood is filtered. The glomerular filtrate then continues into the renal tubules, where most solutes are reabsorbed, particularly in the proximal tubule, which handles up to 80% of this workload (Adhipandito et al. 2021 ; Zhuo and Li 2013 ). The glomerular filtration barrier (GFB) is composed of podocytes, glomerular endothelial cells and the glomerular basement membrane (Nielsen et al. 2016 ). The GFB is theorised to have a pore size of 4 nm (Adhipandito et al. 2021 ), and yet a known filtration limit of 6–8 nm, while albumin, the most common protein within blood, has a diameter of 14 nm at its widest point and 3.8 nm at its smallest point (Gburek et al. 2021 ; Molitoris et al. 2022 ). As a result, only small amounts of albumin pass through the GFB under normal conditions, but through the use of specialised endocytic pathways facilitated by podocytes, they are then reabsorbed by tubular cells and recycled (Gburek et al. 2021 ). Importantly, these same pathways may also allow nano-sized particles, such as NPs, to cross the barrier and be reabsorbed by tubular cells, providing a possible explanation for their accumulation within the kidney. Previous nanoparticle exposure studies have observed accumulation of particles in both the GFB and proximal tubule, and in some cases, complete filtration of the particles into urine (Fan et al. 2021 ; Lawrence et al. 2017 ; Naumenko et al. 2019 ; Williams et al. 2018 ; Zuckerman and Davis 2013 ). In these studies, the charge of the particles was also addressed, concluding that positively and negatively charged particles differed in their accumulation behaviour. Specifically, due to the negative charge of the GFB, positively charged nanoparticles had a higher propensity to accumulate, compared to their negatively charged counterparts (Naumenko et al. 2019 ; Zuckerman and Davis 2013 ). Other studies have highlighted differences in cytotoxicity, inflammation and cell response following exposure to various nanoparticles, such as silver (Kennedy et al. 2019 ), zinc oxide (Shehata et al. 2022 ), and silica (Passagne et al. 2012 ; Rafieepour et al. 2019 ; Wang et al. 2009 ). This indicates that biological outcomes may be driven not only by the particle physical property (nanosize and shape) but also by their chemical properties, such as surface charge, atomic composition and functional groups, which influences interactions with cell membranes, organelles and biomolecules. With this knowledge, it could be expected that the extent of NPs cytotoxicity and accumulation within the kidney also differ depending on their sizes, surface chemistry and charges. Yet, most studies evaluating the toxicity of NPs on mammalian cells only look at 1 type of polymer: polystyrene. Across current NP studies, polystyrene (PS) NPs are overrepresented – despite accounting for only ~ 6% of global plastic waste - while other polymer types remain largely understudied (Pradel et al. 2023 ). This narrow focus limits both environmental relevance and toxicological insight, as different polymers may engage in substantially different interactions with biological systems. Expanding NP research to include a broader range of polymer types and sizes is therefore necessary to better reflect real-world exposures and uncover polymer-specific toxicological effects. Given the potential for accumulation, recirculation and filtration of NPs via the kidney, testing the fate and effects of small NPs, particularly those less than 100 nm, is crucial to understanding renal impact. In this study, the proximal tubule cell line, Human Kidney 2 (HK-2), was exposed to polystyrene (PS), poly(methyl methacrylate) (PMMA), and polyethylene (PE) NPs, from 15 to 100 nm in size, at concentrations ranging from 0.1 µg/mL to 200 µg/mL. It is hypothesised that exposure to higher NP concentrations will result in increased cell response, while cell response will vary dependent on the polymer type. The toxicity and biological effects of NPs with varying sizes, polymers and concentrations on HK-2 cells was assessed, with the aim of evaluating how these factors impact cell viability, cell cycles and internalisation and to provide insight into the potential implications of short-term NP exposure on kidney function. Materials and Methods Nanoplastics Carboxylated (-COOH) NPs of various sizes and polymer types were obtained from multiple suppliers. Specifically, PS NPs 20 nm and 100 nm in diameter were purchased from Thermo Fisher Scientific (Manufacturer A). PS NPs 15 nm and 100 nm in diameter, 50 nm PMMA and PE NPs were purchased from Lab261 (Manufacturer B). PMMA NPs 50 nm and 100 nm in diameter were purchased from Phosphorex (Manufacturer C). For NP internalisation experiments, different types of fluorescent NPs were used. An initial experiment was performed to test for potential fluorophore leaching effects, using both internally and externally labelled particles: externally labelled red fluorescent (RF) 100 nm PS NPs (Magsphere Inc), and 100 nm Fluoro-Max PS NPs (Thermo Fisher Scientific) internally labelled with Europium (Eu). Europium, as a fluorophore, can be beneficial for bioaccumulation studies due to its large Stokes shift, and reduced susceptibility to photobleaching, which in turn can reduce the effects of autofluorescence and increase visibility of NPs (Cheignon et al. 2022 ; Crawford et al. 2015 ). For subsequent internalisation experiments, fluorescently labelled PS NPs 15 nm and 100 nm in diameter as well as 50 nm PMMA and PE NPs (Lab261) were used to test the effect of polymer type on internalisation. The 20 nm and 100 nm NPs supplied by Manufacturer A were provided respectively as 41 mg/mL and 45 mg/mL stock solutions in deionised water. All NPs from manufacturers B and C were supplied as 10 mg/mL stock solutions in deionised water containing surfactant and 2 mM sodium azide (NaN 3 ). The volume and type of surfactants used in the stock solutions were unknown aside from those supplied by Lab261, which were specified to be 0.1% Tween-20. Dilutions Immediately prior to dilution, all NP stocks were briefly vortexed and then sonicated in a water bath sonicator (Soniclean Digital Benchtop Ultrasonic Cleaner 80TD) for 15 minutes to facilitate homogenisation of the NP suspensions. All non-fluorescent NPs were diluted to concentrations of 2 mg/mL (2,000 µg/mL), 200 µg/mL, 20 µg/mL and 2 µg/mL in PBS. All fluorescent NPs were diluted in PBS to concentrations of 1 mg/mL (1,000 µg/mL), 100 µg/mL, 10 µg/mL and 1 µg/mL, or in the case of the RF and Eu PS NPs, these were diluted to the same number of particles per cell as the non-fluorescent 100 nm PS NPs to ensure consistency with the NP exposure experiments. Prior to cell treatment, all NP dilutions were further diluted at a ratio of 1:10 into cell culture media to final concentrations of 200 µg/mL, 20 µg/mL, 2 µg/mL and 0.2 µg/mL for NP exposure experiments, or 100 µg/mL, 10 µg/mL, 1 µg/mL and 0.1 µg/mL for NP internalisation experiments. The particles per mL concentrations of the NP stock solutions were either provided by the manufacturer upon receipt of the stocks, or calculated using the following equation: Equation 1: \(\:particles\:per\:mL=\frac{grams\:per\:mL}{density\:\times\:\:(4/3\times\:\pi\:{r}^{3})}\) To determine the number of particles per cell during the exposure period, the following calculation was used: Equation 2: \(\:particles\:per\:cell=\frac{(particles\:per\:mL\times\:well\:volume)}{number\:of\:cells}\) A full summary of the NP suspensions used in this study can be found in Supplementary Tables 1 and 2. Characterisation The shape, size, polydispersity index and surface charge of all NPs used was assessed via scanning electron microscopy (SEM), dynamic light scattering (DLS) and zeta potential measurement, respectively. In both cases NP suspension at 100 µg/mL were prepared in Milli-Q water. For SEM, a 5 µL drop was placed on a silicon wafer and allowed to air dry before coating the substrate with a thin layer of platinum to prevent beam damage. Imaging was conducted with a Gemini II (Bruker), equipped with a field emission gun and operated at an accelerating voltage of 5 kV and a working distance of 3 mm. DLS measurements were carried out on a Zetasizer Nano ZS (Malvern Instruments, UK) with a 4 mW He-Ne laser (633 nm) as a light source and a non-invasive backscatter (NIBS) detection angle of 173°. The analysis was performed at 25°C in disposable polystyrene cuvettes with 2 minutes sample equilibration. The same equipment in conjunction with a folded capillary cell under Smoluchowski approximation was used for zeta potential measurements. Each sample was measured in triplicate. Cell Culture and Nanoplastics Exposure HK-2 cells (ATCC CRL-2190) were cultured in 1:1 Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 (Gibco), and supplemented with 10 mM HEPES (Gibco), 10% fetal calf serum (FCS; Bovogen Biologicals) and 1X penicillin, streptomycin and glutamine mix (Gibco) at 37°C with 5% CO 2 . Cells were seeded at 0.1 × 10 6 cells/mL in either 12-well cell culture plates or 96-well clear bottom cell culture plates and incubated for 24-hours before replenishing the cells with control or NP treatment media at volumes of 500 µL for 12-well plates, or 100 µL for 96-well plates. The NP concentrations in different well volumes utilised across the experiments were set to ensure consistent nanoplastic particle-per-cell numbers within the experiments and are summarised in Supplementary Tables 1 and 2. The HK-2 cells were incubated with the control or NP treatment media for 24-hours before proceeding with the intended assay. Cell Morphology Assessment For experiments using standard NPs, cells were imaged after the 24-hour NP incubation period, immediately prior to cell collection. Images were taken using a Thermo Fisher EVOS M5000 Imaging System, or a ZEISS Primovert inverted cell culture microscope with a ZEISS Axiocam 208 color/202 mono microscope camera. Micrograph images were taken of cells at the centre of the plate wells, with a 40x objective. Cell segmentation and granularity were determined using QuPath (v0.5.1) (Bankhead et al. 2017 ). Live/Dead Cell Viability Assay The flow cytometry viability assay was performed with a modified version of the protocol provided by Thermo Fisher Scientific ( 2016 ). The HK-2 cells were washed with PBS and detached using TrypLE (Gibco) as per the manufacturer’s instructions. The detached cells, along with the supernatant and PBS washes, were collected into Fluorescence-Activated Cell Sorting (FACS) tubes and centrifuged for 5 minutes at 300 × g, then washed and resuspended in 100 µL of PBS. A positive control containing live and dead cells (50:50) was generated by heat-treating 50 µL of the cell suspension at 65°C for 5 minutes and then cooled on ice for an additional 5 minutes, before being transferred back to the FACS tube. All cells were washed and resuspended with PBS once again before a 30-minute incubation with 0.5 µL of LIVE/DEAD Aqua dye in 500 µL of PBS, followed by a 15-minute fixation with 4% formaldehyde. After fixation, the cells were centrifuged for 5 minutes at 300 × g before resuspension in 200 µL of PBS with 1% FCS. The stained and fixed cells were stored at 4°C for a maximum 1–2 days prior to analysis via flow cytometry (CytoFlex-S, Beckman Coulter). Acquired flow cytometry data was processed using FlowJo (v10.8.1; BD Biosciences). Cell Cycle Analysis HK-2 cells were detached using TrypLE as per manufacturer's instructions. All cells were centrifuged for 5 minutes at 300 × g, cell pellet was washed in and resuspended in 200 µL of PBS. The cells were then fixed with a final concentration of 80% EtOH and stored at − 20°C for a minimum of 24-hours. Post fixation, cells were centrifuged for 5 minutes at 300 × g, and the cells were permeabilised and stained with propidium iodide (PI) staining solution. The PI staining solution consists of 200 µg/mL of RNASE-A, 50 µg/mL of PI, and 0.1% Triton X-100 diluted in PBS. Cells were incubated in the dark 30 minutes, and then immediately analysed via flow cytometry (CytoFlex-S, Beckman Coulter). Acquired flow cytometry data was processed using FlowJo (v10.8.1; BD Biosciences). Nanoplastic Internalisation Nanoplastic internalisation in HK-2 cells was performed using fluorescently labelled NPs, following a modified version of the methods outlined in studies by Liu et al. ( 2022 ) and Wang et al. ( 2022 ). A full summary of the concentrations and NPs used can be found in Supplementary Table 2. Microscopy Analysis Post-exposure, the cells were washed 3 times with PBS to remove as many non-internalised NPs as possible. The cells were fixed with 4% formaldehyde for 15 minutes, permeabilised with 0.1% Triton X-100 for 5 minutes, then stained with ActinGreen 488 ReadyProbes (Alexa Fluor 488, Invitrogen) and NucBlue Live ReadyProbes (Hoechst 33342, Invitrogen) for 30 minutes prior to visualisation via microscopy (Nikon Eclipse Ti2; 40x objective). All wells were imaged with the DAPI, FITC and TexasRed filter cubes, with the exception of the Eu PS NPs, which was additionally imaged with a modified Cy-5 filter cube, containing the DAPI emission filter, to ensure correct excitation, emission and visualisation of the NPs. A minimum of 3 wells were used as replicates of the cell exposure conditions, with at least 1 image per well, corresponding to an area of 192,466 µm 2 . After capture, each image was processed via NIS-Elements AR (v5.30.07; Nikon) to remove any background fluorescence, then analysed to collect the mean fluorescence index (MFI) of each filter capture. Flow Cytometry Analysis Post-exposure, the cells were washed 3 times with PBS to remove as many non-internalised NPs as possible, then detached using TrypLE as per the manufacturer's instructions. All cells were centrifuged for 5 minutes at 300 × g, the cell pellet was washed in PBS and fixed with 4% formaldehyde for 15 minutes. Following fixation, all cells were centrifuged for 5 minutes at 300 × g, cell pellet was washed in and resuspended in 200 µL of PBS for storage at 4°C for a maximum 1–2 days prior to analysis via flow cytometry (CytoFlex-S, Beckman Coulter). The internalised NPs were detected using the APC channel to detect RF PS NPs, and the Violet660 channel to detect the Eu PS NPs. Acquired flow cytometry data was processed using FlowJo (v10.8.1; BD Biosciences). Statistical Analysis Statistical analysis of cell morphology data was performed using R (v.4.4.2) (R Core Team 2024 ). All other data was analysed using GraphPad Prism (v10.4.0; GraphPad Software). Results Nanoplastic Characterisation The shape, size and surface charge of the NPs was determined through SEM and DLS analysis. Visualisation of the NPs under SEM confirmed consistent size and sphericity of all NP types investigated (Fig. 1 a-h). DLS analysis confirmed that the measured sizes were comparable to the expected particle sizes, with most NPs assessed as being within 3 to 6 nm of the size indicated on the product certificate of analysis provided by the manufacturer (Table 1 ). All particles displayed negative zeta potentials, as expected for NPs with -COOH surface modifications in Milli-Q water (Table 1 ). Table 1 Characterisation of size and zeta potential of NPs via DLS analysis. *Expected size and mean diameter as provided by manufacturers. Supplier Polymer Expected Size* (nm) Mean Diameter* (nm) Measured Size (nm) Zeta Potential (mV) Thermo Fisher Scientific PS 20 28 48.0 ± 2.6 -41.9 ± 7.4 100 110 131.8 ± 0.6 -35.2 ± 3.2 Lab261 PS 15 12.5 15.6 ± 0.4 -26.9 ± 6.4 100 89 82.9 ± 1.0 -40.6 ± 3.0 PMMA 50 53 46.9 ± 0.1 -27.4 ± 2.4 PE 50 68.2 64.1 ± 0.5 -39.8 ± 4.4 Phosphorex PMMA 50 50 74.9 ± 0.5 -34.9 ± 0.6 PMMA 100 100 79.4 ± 0.9 -36.0 ± 0.8 Cell Morphology Assessment Following the 24-hour NP exposure, changes to the morphology of the cells was assessed by microscopy analysis. When comparing cell exposure to NPs of varying sizes, polymers and concentrations, these factors could be attributed to identifiable morphological changes (Fig. 2 and Supplementary Figs. 1 and 2). Commonly, incidences of multinucleation increased in cells exposed to NPs across all types and sizes as compared to the control group and more so in the lower 0.2 µg/mL and 2 µg/mL concentrations. This is coupled with misshapen or irregular cells, primarily in the higher 20 µg/mL and 200 µg/mL concentrations, which made it difficult to distinguish individual cells. In contrast, untreated cells maintained regular cell shape, consistent cell sizing and defined cell borders. A cell segmentation analysis was performed to quantify changes to cell granularity and morphology following NP exposure (Supplementary Fig. 3). When compared to the control group, the cells exposed to NP treatments systematically expressed a significant reduction (Adjusted P-value < 0.0001) in the mean pixel intensity (Supplementary Fig. 4). While there appears to be no visual morphological differences across all concentrations of the 20 nm NPs (Fig. 2 and Supplementary Fig. 1), increases in the exposure concentration from 0.2 µg/mL to 200 µg/mL was associated with significant increases in the entropy and heterogeneity of the cells, 34.4% and 78.8% respectively (Fig. 3 a and b). In contrast, the 100 nm treatment group displayed noticeable granulation within the cytoplasm of cells exposed to the two highest NP concentrations, localised around the nuclei of the cells (Fig. 2 and Supplementary Fig. 1), associated with a 10.2% increase in cell heterogeneity (Fig. 3 d). While the source of the granulation is unclear, this could be a result of aggregation and accumulation of the PS NPs within the cytoplasm. This granulation is also visible and significantly increased in the 200 µg/mL PE and PMMA NP treatment groups (P-value < 0.0001) (Fig. 2 , Supplementary Fig. 5). Both sizes of PMMA NPs from manufacturer C aggregated so significantly at 200 µg/mL that the cells became difficult to visualise, thus no real difference between the two sizes could be identified (Fig. 2 ). This aggregation also impacted the cell segmentation analysis and were excluded from the granularity analysis as a result. Live/Dead Analysis Cell viability was assessed by live/dead flow cytometry assay. In the flow cytometry plots, the live cell population shifts away from the live cell gate, towards the dead cell zone as the concentration of NPs increases from 0.2 µg/mL to 200 µg/mL (Fig. 4 a and Supplementary Figs. 6 and 7). Despite these noticeable shifts in the scatterplots, statistical significance was typically seen only at the highest NP concentration. Specifically, after 24-hour exposure to the 20 nm and 100 nm PS NPs from Manufacturer A, there was no significant reduction in cell viability when comparing concentrations, aside from the 20 µg/mL concentration of the 100 nm treatment group (P-value = 0.017). This is in direct contrast to the 15 nm and 100 nm PS NPs from Manufacturer B which, following exposure at 200 µg/mL, resulted in a significant reduction in cell viability of 12.5% (P-value = < 0.0001) and 7.6% (P-value = 0.0001) respectively, when compared to the control (Fig. 4 b and c). This concentration dependent population shift can also be seen in the PE and PMMA treatment groups (Fig. 4 a). Cell viability was significantly reduced by 16% (P-value < 0.0001) and 4.7% (P-value 0.0008), after exposure to 200 µg/mL of the respective PE and PMMA NPs (Fig. 4 d). When comparing the two sizes and manufacturers of PMMA, the reduced viability was similar between the three treatment groups. The difference in viability between the two groups of 50 nm PMMA NPs from manufacturer B and C is 0.87% and non-significant. Similarly, the difference in viability between the two sizes (50 and 100 nm) from Manufacturer C was 0.7% and non-significant. Interestingly, across all treatment groups, the largest reduction in cell viability was seen after treatment with the PE NPs at a concentration 200 µg/mL, reducing viability to 79.46% (P-value < 0.0001). This could imply a link between the type of polymer and cell viability. An additional experiment was completed with both sizes of PS NPs from Manufacturer A in which the NPs were spiked with NaN 3 and Tween-20 to determine if the presence of these additives in NP solutions was contributing to the cell death seen at the higher concentrations. After exposure to the spiked NPs for 24-hours, the cells treated with 100 nm PS NPs saw increased cell death at 20 µg/mL and 200 µg/mL (Supplementary Fig. 8). The positive controls of cells treated with NaN 3 and Tween-20 without NPs showed no change to cell viability on their own. Cell Cycle Analysis Following exposure to varying size and concentrations of PS, PE and PMMA NPs, a cell cycle assay was performed to determine the proportion of cells in three distinct cell cycle phases, namely the G0/G1, S and G2/M phase (Supplementary Figs. 9 and 10). Significant cell cycle arrest was seen in the cells treated with the smallest 15 nm and 20 nm PS NPs (Fig. 5 a and c), with no significant changes observed in the larger 100 nm PS NPs from either manufacturer (Fig. 5 b and d). Cells treated with the 15 nm PS NPs exhibited a dose-dependent increase in the percentage of cells in the G0/G1 phase (5.8% to 11.6%), with a significant increase in the 20 µg/mL and 200 µg/mL treatment groups (Fig. 5 c). In the 20 nm PS NP treatment group, the opposite was seen, with significant decreases of the percentage of cells in the G0/G1 phase observed in a dose-dependent manner (Fig. 5 a). A similar shift was seen in the percentage of cells in the S phase (2.4% to 3.4%), with significant increases in the cells of the 0.2, 20 and 200 µg/mL treatment groups, and significant decreases in the percentage of cells in the G2/M phase of the 20 µg/mL group (Fig. 5 a). Interestingly, when assessing the effect of NPs of other polymer types, the opposite is seen when compared to PS NPs. No significant effect to the cell cycle was seen in the cells exposed to 50 nm PE and PMMA NPs (Fig. 5 e-g), rather the cells exposed to 100 nm PMMA NPs observed significant arrest at the S phase, with significant reduction in the percentage of cells in the G0/G1 phase at a concentration of 200 µg/mL (Fig. 5 h). Nanoplastic Internalisation The NP internalisation study was initially conducted using two different 100 nm PS NPs to assess for potential fluorophore leaching: externally labelled RF PS NPs, and internally labelled Eu PS NPs. In both cases, the fluorescent NPs accumulated within the cytoplasm of the cell and often close to the nucleus (Fig. 6 ). Uptake of fluorescent NPs were only visible via microscopy at the highest NPs concentrations investigated, namely 7 µg/mL (Supplementary Fig. 11) and 70 µg/mL concentrations (Fig. 6 ). After the 24-hour exposure period, the cells exposed to the RF PS NPs typically displayed brighter aggregates, with apparent accumulation outside of the cell membrane. In contrast, cells exposed to the Eu PS NPs shone more dimly, but with clear accumulation around the nucleus (Fig. 6 i and n). Analysis of the mean fluorescence of the microscopy images indicated uptake of both the RF and Eu PS NPs (Fig. 6 o). A significant increase was identified in the MFI of cells treated with concentrations of 7 µg/mL and 70 µg/mL of RF PS NPs, suggesting a dose dependent uptake of NPs. In comparison, all concentrations of Eu PS NPs expressed significant MFI increases when compared to the control group, with the largest proportion of uptake seen in the 70 µg/mL treatment group (Fig. 6 o). Internalisation of both fluorescent PS NPs was also assessed via flow cytometry as an alternate way to assess NP uptake (Fig. 6 p). Much like the microscopy images, the MFI as expressed from the flow cytometry data indicated significant increases in NP uptake in the 7 µg/mL (P-value 0.0005) and 70 µg/mL (P-value 0.0185) concentrations of the RF PS NPs. In the Eu PS NP treatment group, the MFI of the 70 µg/mL concentration was increased when compared to the 7 µg/mL concentration, although this increase was not significant (Fig. 6 p). Further analysis of NP internalisation was performed to assess for differences in uptake between different polymers and sizes of NPs (Fig. 7 ). To evaluate the effect of NPs size, two populations of PS NPs were used, one with particles 15 nm in diameter, the other with 100 nm NPs. When visualising the cells treated with 15 nm PS NPs, internalisation of the NPs within the cytoplasm was not visible at this magnification, although significant particle aggregation was seen to accumulate on the exterior of the cell (Fig. 7 h and i). The cells exposed to the 100 nm PS NPs presented similarly to the initial internalisation experiment, with brighter aggregates of the particles that appear to sit outside of the cell membrane (Fig. 7 m and n). To evaluate the effect of polymer type, two population of 50 nm NPs were used, one made of PE, and another one made of PMMA particles. The 50 nm PMMA treatment group showed some similarities to the 100 nm PS NP group, presenting a mix of bright aggregates on the outer membrane of the cell, with dimmer NP populations that appear to be accumulated within the cell membrane (Fig. 7 r and s). Most interestingly, the cells exposed to the 50 nm PE NPs almost exclusively express dimmer NP populations that had been internalised within the cell membrane. Within these populations, a clear point of accumulation can be seen within each cell, which often overlaps within or immediately adjacent to the cell’s nucleus (Fig. 7 w and x). The difference in uptake between the different NP polymer types, particularly with the PE NPs, may indicate that some polymers are able to be more readily internalised by cells. Analysis of the MFI from the microscopy images also indicates a dose-dependent uptake of the NPs within a 24-hour exposure period (Fig. 7 y). Increased MFIs are seen at all concentrations for all NP treatment groups, with a significant increase in the 100 µg/mL groups. The largest increase in MFI was seen in the cells treated with PMMA, which is likely to be attributed to the combination of internalised NPs and aggregates adhered to the outer cell membrane. With the PE NP treated cells having the lowest increase in MFI at 100 µg/mL could be attributed to the strict internalisation of the particles (Fig. 7 z). Discussion The impact of short-term exposure to NPs of different polymers, sizes and concentrations was tested on HK-2 proximal tubule cells. Microscopy and flow cytometry was used to evaluate physical and biological effects. Effect of concentration : The HK-2 kidney cell line is largely unaffected by short-term exposure to low NPs concentrations. In contrast, effects were evident at the highest concentrations tested (100 µg/mL and 200 µg/mL). At these levels, morphological changes were pronounced, including misshapen cells, and increased granularity – features absent in the control groups (Figs. 2 and 3 ). Flow cytometry viability assessment supported these observations: all NPs from Manufacturers B and C caused a significant reduction in viability at 200 µg/mL, with an overall reduction between 5% to 17% relative to controls (Fig. 4 ). Furthermore, given that the viability assay detects dye uptake only when membrane integrity is compromised, the flow cytometry results also imply increased membrane permeability at higher NP concentrations. Cell cycle analysis also revealed a dose-dependent deregulation, with significance increasing at higher NP concentrations (Fig. 5 ). Internalisation of NP within cells also correlated with increasing exposure concentrations. Fluorescence microscopy and flow cytometry analysis confirmed greater uptake at 100 µg/mL and significant MFI increases at these concentrations for all polymer types and particle sizes (Figs. 6 and 7 ). Effect of polymer : Cell responses were also influenced by the polymeric composition of the NP particles, with variation in morphology, functional changes and internalisation patterns, seen between polymers of comparable sizes and concentration. The most pronounced effect on viability resulted from exposure to 50 nm PE NPs. Compared with PS NPs, which generally maintained cell shape and morphology, PE and PMMA NPs induced irregular cell shape with multinucleation and increased granularity (Fig. 2 ). Flow cytometry further highlighted polymer-dependant effects: a lthough significant reductions in viability occurred with all NPs from Manufacturers B and C at 200 µg/mL (Fig. 4 ), the greatest decrease was observed following exposure to the PE NPs (Fig. 5 c). Despite the similarity in size, the 50 nm PMMA NPs from Manufacturer B and C both had a lesser impact on viability when compared to the 50 nm PE NPs. Notably, the 100 nm PMMA NPs also caused significant S-phase arrest at 200 µg/mL (Fig. 5 h), whereas 100 nm PS NPs did not—neither for manufacturer A nor for manufacturer B, which expressed a similar change in viability, as well as containing the same concentration of sodium azide as the 100 nm PMMA NPs from manufacturer C. In this case, the size, additive content, and surface charge were equivalent, yet the cellular responses differed, supporting the conclusion that the NP polymer composition alone can drive differential biological effects. Previous studies have shown that surface functionalisation influences NP toxicity, with carboxylic acid (COOH) or amine (NH 2 ) modified PS NPs generally found to be more harmful than pristine particles, and COOH- less toxic than -NH 2 modification (Chen et al. 2023 ; González-Fernández et al. 2021 ; Wang et al. 2023 ). Here all NPs were functionalised with COOH, and all had negative surface charge (Fig. 1 q-x). Yet polymer-dependent differences persisted, indicating that the chemical composition of the polymer itself may also be a determinant of NP toxicity, not just surface charge. These polymer-dependent differences in viability and cell cycle arrest suggest that environmental mixtures of NPs may exert heterogeneous renal effects, underscoring the need to assess multiple polymer types in toxicology studies. Effect of size : NP size was associated with differences in cell response, with smaller NPs (15 nm, 20 nm and 50 nm) often showing greater effects than their 100 nm counterparts. The most significant size-related effects were observed in cell cycle analysis: the two smallest sizes of PS (15 nm and 20 nm) caused dysregulation after 24 hours of exposure, even at concentrations as low as 0.2 µg/mL in one instance – S-phase arrest for the 20 nm PS NPs (Fig. 5 a and b). This was the only case in which such a low concentration produced a statistically significant impact. These results could suggest that smaller sizes of PS NPs are more readily able to interfere with cell growth and proliferation. As S-phase arrest is commonly associated with DNA damage (Chao et al. 2017 ), this could indicate potential interaction with the nucleus and have implications in increased cell stress. These effects were observed in azide-free particles (Manufacturer A), ruling out attribution to this toxic additive. Taken together, the cell cycle findings may be indicative that smaller NP sizes cause DNA damage, warranting investigation in longer-term exposure studies. Unfortunately, these findings were not strongly mirrored in the viability assays, where responses remained largely unchanged across all NP sizes at all concentrations below 200 µg/mL. A notable exception was the 50 nm PMMA NPs, which reduced viability significantly at 20 µg/mL, whereas the 100 nm PMMA NPs from the same manufacturer only caused a reduction at ten times that concentration (200 µg/mL). These results differ to the study performed by Li et al. ( 2023 ), in which comparable sizes of PS NPs (20 nm, 60 nm and 100 nm) were exposed to human embryonic kidney cells, where it was seen that the 20 nm PS NPs were significantly more toxic than the larger NPs, expressing near complete cell death at the 75 µg/mL and 125 µg/mL concentrations tested, compared to ~ 80% viability for the cells exposed to the 60 nm and 100 nm PS NPs (Li et al. 2023 ). Previous studies assessing exposure to PS NPs above 100 nm in size, including MPs, consistently showed no impact on kidney cell viability, regardless of concentration or exposure time (Goodman et al. 2022 ; Li et al. 2023 ; Wang et al. 2022 ). No clear size-related differences were observed in the uptake assays, with fluorescence intensity remaining comparable between small and large particles (Fig. 7 z). The one difference observed was the size of the NP aggregates, with the small NPs only visible as larger, more diffuse, aggregates, likely due to resolution limits. It is worth noting here, that single 15 or 20 nm NPs or small aggregates of them would not be detectable via microscopy. In terms of cell morphology, cells exposed to the smaller NPs generally appeared more consistent than those treated with their larger equivalents, although this too may be due to limits in imaging resolution. Other considerations The presence of additives, or lack thereof, within NP suspensions also appeared to impact viability and cell response, particularly for PS NPs from Manufacturer B which contained NaN 3 and Tween-20. These NPs significantly reduced HK-2 cell viability compared to additive-free PS-NPs from manufacturer A, where viability was largely unchanged (Fig. 4 c). When PS NPs from Manufacturer A were spiked with NaN 3 and Tween-20, cell viability decreased, despite the additives on their own showing no effects at equivalent concentrations (Supplementary Fig. 8). This result could infer some level of synergistic biological effect where exposure to the additives makes the cells more susceptible to NP-induced toxicity. Given the known cytotoxicity of NaN 3 even at low concentrations (Heinlaan et al. 2020 ; Petersen et al. 2022 ; Pikuda et al. 2019 ), the presence of additives should be addressed and controlled in NP studies. Whether the observed effects of additives result from membrane degradation, altered permeability, or increased endocytic/phagocytic activity within the cell, is yet to be determined. It is important to note that the 15 nm PS NPs from Manufacturer B, as well as the 50 and 100 nm PMMA NPs from Manufacturer C exhibited a tendency to aggregate within the treatment period. Aggregation, often visible in suspension within the cell media, may have an impact on the cell’s response (Fig. 2 , Supplementary Figs. 1 and 2). Outlook and future work This study used an immortalised proximal tubule cell line, commercially produced spherical NPs, and a short 24-hour exposure period, which cannot fully replicate the complexity of human kidney tissue or long-term exposure scenarios. Primary kidney cells or 3-dimensional spheroid models could provide additional physiological relevance in future works. Commercial NPs also differ from environmentally derived particles, which are typically more heterogeneous in shape, size and composition. This is a significant and common limitation across NP research into cellular and systemic effects and toxicity. The NPs used in this study were of a consistent size (Fig. 1 ), sphericity and composed of a single polymer type, and while not mimicking environmentally relevant scenarios, the controlled particle properties used here allowed us to isolate and directly compare the effects of polymer type, size, and concentration on kidney cell responses. While the lower concentrations tested in this study may be indicative of short-term environmental exposure, real-world contact with NPs is more likely to occur at even lower concentrations than the ones tested in this study, but over an extended period of time. For instance, a recent study by ten Hietbrink et al. ( 2025 ) evaluated concentrations of NPs smaller than 1 µm within the North Atlantic Ocean, identifying an average NP concentration of 15.1 mg/m 3 (0.015 µg/mL equivalent), with PS equating to 4.06 mg/m 3 (0.004 µg/mL equivalent). These concentrations are only 1 order of magnitude smaller than the lowest concentration tested here. The potential cumulative effects and delayed biological impacts cannot be reflected in short-term, acute exposure models. However, a recent study by Nihart et al. ( 2025 ) saw a 2.3 µg per gram increase of plastic in kidney tissue between 2016 and 2024, which seems to indicate that bioaccumulation does occur, and may do so in a more pronounced fashion as environmental exposure increases. Hence the concentrations used here may become more and more biologically relevant as plastic pollution increases over time. For now, short-term studies like this one are instead useful for assessing the immediate impacts, cellular responses and toxicity of specific NP types, and offer a framework for designing longer term experiments with environmentally relevant exposure thresholds necessary to further understanding of possible toxicity, effect and health impacts. Conclusion The findings of the study demonstrate that while lower concentrations of NPs may not result in immediate toxicity to the HK-2 cell line, particularly in terms of short-term exposure, higher NP burdens can compromise overall cell health and function, affecting morphology, viability and cell cycle regulation. The results also indicate that NP effects are influenced not only by concentration but also by polymer composition and particle size, with some combinations inducing significant cellular changes even at relatively low doses. Given that proximal tubule cells play a critical role in reabsorption and overall kidney filtration efficiency, sustained or repeated damage to these cells could impair kidney function, potentially leading to NP accumulation in kidney tissue, reduced clearance capacity, and increased NP recirculation in the bloodstream over time. This highlights the importance of investigating more realistic, environmentally relevant NP exposure, that reflect chronic, low-level contact over extended periods, and that account for the diversity of NP characteristics in terms of concentration, size, polymer types and chemical additives likely to be encountered in real-world settings. Such studies should also explore mechanistic endpoints, including potential DNA damage and long-term functional consequences, to fully assess the risks posed by environmental NPs to kidney health and systemic exposure. Declarations Competing Interests None. Funding This work was supported by the Australian Research Council Future Fellowship Grant (FT200100301), The Flinders Foundation and the Flinders Medical Centre Renal Research Fund. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hayden Louis Gillings, Iliana Delcheva and Soon Wei Wong. The first draft of the manuscript was written by Hayden Louis Gillings and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement This work used the NCRIS and Government of South Australia enabled Australian National Fabrication Facility - South Australian Node (ANFF-SA) and Microscopy Australia at the University of South Australia. We would also like to acknowledge and thank Dr. Giles Best at the Flow Cytometry Facility at Flinders University. These facilities provided vital infrastructure and services to complete this study. References Adhipandito CF, Cheung SH, Lin YH, Wu SH. Atypical Renal Clearance of Nanoparticles Larger Than the Kidney Filtration Threshold. Int J Mol Sci. 2021;22. https://doi.org/10.3390/ijms222011182 Atugoda T, Piyumali H, Wijesekara H, Sonne C, Lam SS, Mahatantila K, Vithanage M. Nanoplastic occurrence, transformation and toxicity: a review. 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18:37:00","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163762,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/8529503f2fa207a533dedbe8.html"},{"id":94221364,"identity":"64bf4e9b-4d9f-46a0-a96b-22279e9d427c","added_by":"auto","created_at":"2025-10-23 18:37:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":686267,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterisation of NPs via scanning electron microscopy (SEM). a) 20 nm PS, b) 100 nm PS, c) 15 nm PS, d) 100 nm PS, e) 50 nm PMMA, f) 50 nm PE, g) 50 nm PMMA, h) 100 nm PMMA. Scale bar = 100 nm. i-p) Size confirmation of NPs via dynamic light scattering (DLS). q-x) Zeta potential of NPs via DLS\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/3b33fd65f9f603f6441521ad.png"},{"id":94221908,"identity":"90e331f5-c86e-43e4-ae47-bd9462f11f5e","added_by":"auto","created_at":"2025-10-23 18:45:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":885938,"visible":true,"origin":"","legend":"\u003cp\u003eMicrographs of HK-2 cells following 24-hour exposure to NPs of various sizes and polymers from three different manufacturers. All cells were treated with either 15 nm, 20 nm or 100 nm PS NPs, 50 nm PE NPs, and 50 nm or 100 nm PMMA NPs at increasing concentrations from 0.2 µg/mL to 200 µg/mL. 40x magnification. Scale bar = 25 µm. N=3. Data is representative of individual replicates tested. Representative micrographs of cells exposed to intermediate NP concentrations are provided in Supplementary Fig. 1 and 2\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/fbd00862f20155ce3c05a125.png"},{"id":94221362,"identity":"e8ccb37f-647a-4ca2-8ac4-b93822d21e36","added_by":"auto","created_at":"2025-10-23 18:37:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":421980,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of cell morphology via Haralick Entropy and Difference of Variance in HK-2 cells following 24-hours of NP exposure. Cell morphology was assessed via QuPath cell segmentation and analysis comparing 0.2 µg/mL and 200 µg/mL NP concentrations. a) Entropy and b) Difference of Variance of 20 nm PS NPs. c) Entropy and d) Difference of Variance of 100 nm PS NPs. Data is representative of individual replicates tested and normalised to the ROI area. Adjusted P-value = *** ≤0.001, **** ≤0.0001\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/b95a1d770ae1d863034a319c.png"},{"id":94221909,"identity":"0f705fb3-afef-4c7a-aa5b-d9cbc9f4f992","added_by":"auto","created_at":"2025-10-23 18:45:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":679310,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of HK-2 cells exposed to various sizes and concentrations of PS, PMMA and PE NPs for 24 hours. Viability was assessed via flow cytometry using Live/Dead Aqua to fluorescently label dead cells. Cells were treated with increasing concentrations from 0.2 µg/mL to 200 µg/mL. a) Flow cytometry assessment of viability comparing NPs from Manufacturer B at 0.2 µg/mL and 200 µg/mL. b) Comparison of cell viability after exposure 200 µg/mL concentrations of various NP polymers, sizes and manufacturers. c) Comparison of cell viability after exposure to PS NPs from Manufacturer A and B. d) Comparison of cell viability after exposure to PE and PMMA NPs from Manufacturer B and C. N=3. Data is representative of replicate experiments and is expressed as mean with SEM. Significance was determined via two-way ANOVA with multiple comparisons Uncorrected Fisher’s least-significant difference test. P-value = *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/3d4cd00b8f87a4ce5094ec18.png"},{"id":94221368,"identity":"37bd8e48-904d-4176-b369-71828d6b3378","added_by":"auto","created_at":"2025-10-23 18:37:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1859879,"visible":true,"origin":"","legend":"\u003cp\u003eExposure of HK-2 cells to varying sizes and concentrations of NPs resulted in dysregulation of the cell cycle. Cells were treated for 24-hours with NPs at concentrations from 0.2 µg/mL to 200 µg/mL. Cell cycle was assessed via flow cytometry utilising propidium iodide (PI) staining. a) Proportion of cells in each phase after treatment with 20 nm PS NPs from Manufacturer A. b) Proportion of cells in each phase after treatment with 100 nm PS NPs from Manufacturer A. c) Proportion of cells in each phase after treatment with 15 nm PS NPs from Manufacturer B. d) Proportion of cells in each phase after treatment with 100 nm PS NPs from Manufacturer B. e) Proportion of cells in each phase after treatment with 50 nm PMMA NPs from Manufacturer B. f) Proportion of cells in each phase after treatment with 50 nm PE NPs from Manufacturer B. g) Proportion of cells in each phase after treatment with 50 nm PMMA NPs from Manufacturer C. h) Proportion of cells in each phase after treatment with 100 nm PMMA NPs from Manufacturer C. Data is representative of replicate experiments and is expressed as SEM. N=3. Significance was determined via two-way ANOVA with multiple comparisons Uncorrected Fisher’s least-significant difference test. P-value = * ≤0.05, ** ≤0.01, **** ≤0.0001\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/d49650d2f38f7842573b67dd.png"},{"id":94221370,"identity":"e175da66-d56f-47d2-8aa9-e427ad7e77c2","added_by":"auto","created_at":"2025-10-23 18:37:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":697464,"visible":true,"origin":"","legend":"\u003cp\u003eInternalisation of RF and Eu 100 nm PS NPs after 24-hours of exposure. a-n) Fluorescence microscopy images of HK-2 cells following NP exposure at 70 µg/mL. Cytoskeleton stained with Actin Green (Phalloidin), Nucleus stained with NucBlue (Hoescht 33342). Image taken at 400x (10x magnification with 40x objective) on Nikon Ti2 inverted microscope. Scale bar = 50 µm. Scale bar on zoomed images = 10 µm. o) MFI of red fluorescent (RF) and Europium-labelled (Eu) 100 nm PS NPs at 0.07 to 70 µg/mL (1.36 × 10\u003csup\u003e8 \u003c/sup\u003eto 1.36 × 10\u003csup\u003e11\u003c/sup\u003e particles/mL). MFI generated within the NIS program from cell images taken with the Nikon Ti-2 microscope. Cells treated with RF PS NPs were imaged using the TexRed filter, while cells treated with Eu PS NPs were imaged with a modified filter cube. p) Flow cytometry assessment of RF and Eu NP internalisation. Cells were analysed via flow cytometer using the APC and Violet660 filters to detect the fluorescence of both NPs. Data is representative of technical replicates. N=3. Significance was determined via unpaired T-test with multiple comparisons Holm-Šídák method. P-value = * ≤0.05\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/f99bf0ba6bbf7825feabd571.png"},{"id":94221373,"identity":"a5b00472-1390-43a9-8739-fd92a57060c6","added_by":"auto","created_at":"2025-10-23 18:37:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":899637,"visible":true,"origin":"","legend":"\u003cp\u003eInternalisation of RF NPs after 24-hours of exposure. 100 µg/mL. A-X) Fluorescence microscopy images of HK-2 cells following NP exposure. Cytoskeleton stained with Actin Green (Phalloidin). Nucleus stained with NucBlue (Hoescht 33342). Image taken at 400x (10x magnification with 40x objective) on Nikon Ti2 inverted microscope. Scale bar = 50 µm. Scale bar on zoomed images = 10 µm. Y) MFI of fluorescent PS, PE and PMMA NPs at concentrations of 0.1, 1, 10 and 100 µg/mL. Z) MFI of fluorescent PS, PE and PMMA NPs at 100 µg/mL. MFI generated within the NIS program from cell images taken with the Nikon Ti-2 microscope. Cells imaged using the TexRed filter after staining and fixation of the cells. Data is representative of technical replicates. N=3. Significance was determined via two-way ANOVA with multiple comparisons Uncorrected Fisher’s least-significant difference test. P-value = * ≤0.05, ** ≤0.01, **** ≤0.0001\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/02d95fe7f3c1035e3184397f.png"},{"id":100614607,"identity":"a1300af3-308b-4145-93e8-c93e4c6d3ce9","added_by":"auto","created_at":"2026-01-19 17:22:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6928456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/ac567db1-6d3e-4440-bdd0-0aed4395a36d.pdf"},{"id":94221917,"identity":"822185a2-3794-4984-b6b8-f2bc776bf990","added_by":"auto","created_at":"2025-10-23 18:45:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7717084,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationNanoplastictoxicityanduptakeinkidneycellsGillingsetal..docx","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/09f04a860688e2e971d4d6c5.docx"},{"id":94221910,"identity":"62f35e09-621d-4be8-b8d2-0533cdb24c85","added_by":"auto","created_at":"2025-10-23 18:45:00","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":624402,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7588029/v1/dbdb0ed34ef1197ac3e287a8.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanoplastic toxicity and uptake in kidney cells: differential effects of concentration, particle size, and polymer type","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, there has been increasing concern regarding the health risks and the potential toxicity associated with nanoplastic (NP) exposure. NPs are typically defined as any plastic particles below 1 \u0026micro;m in diameter, either intentionally manufactured (e.g. latex nanoparticles) or the byproduct of plastic degradation in the environment (International Organization for Standardization \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They represent the smallest size fraction of microplastics (MPs) which are defined as plastic fragments smaller than 5 mm. It is important to note that particles that are 1 \u0026micro;m in diameter differ significantly from 100 nm NPs in their behaviour and properties due to factors such as Brownian motion, diffraction limits and enhanced ability to cross cell membrane (Atugoda et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gigault et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Masseroni et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile the majority of reports of the effects of plastic particle exposure have focused on larger size fractions, there are compelling reasons to investigate the effects of nanoparticles below 100 nm \u0026mdash; particularly regarding their behaviour in biological matrices and potential health impacts \u0026mdash; as particles in this size range can more readily cross biological barriers, interact at the cellular and subcellular level, and exhibit unique surface reactivity. So far research into the toxicity and potential health effects of NPs has been predominantly focused on the lungs, liver and gastrointestinal system (Kihara et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schwarzfischer et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with minimal examination of the impact and toxicity of NPs on the renal system (Goodman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This is particularly concerning as MPs have recently been detected in healthy human blood, kidney tissue and urine samples (Leslie et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Marfella et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Massardo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nihart et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Massardo et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found 26 MPs between 1 and 29 \u0026micro;m in kidney tissue, and between 3 and 13 \u0026micro;m in urine samples. Nihart et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) assessed kidney tissue samples from 2016 and 2024 and reported a total plastic mass of 402.5 and 404.8 \u0026micro;g per gram of tissue, respectively. This study also noted an increased incidence of MPs within the kidney tissue localised to the glomeruli and tubules.\u003c/p\u003e\u003cp\u003eGiven these findings, it is important to understand how this accumulation may be occurring within the renal system. The kidney filters about 180 litres of blood each day, removing waste while reclaiming essential substances like electrolytes, glucose, and proteins (Molitoris et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhuo and Li \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This process begins in the glomerulus, where blood is filtered. The glomerular filtrate then continues into the renal tubules, where most solutes are reabsorbed, particularly in the proximal tubule, which handles up to 80% of this workload (Adhipandito et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhuo and Li \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The glomerular filtration barrier (GFB) is composed of podocytes, glomerular endothelial cells and the glomerular basement membrane (Nielsen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The GFB is theorised to have a pore size of 4 nm (Adhipandito et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and yet a known filtration limit of 6\u0026ndash;8 nm, while albumin, the most common protein within blood, has a diameter of 14 nm at its widest point and 3.8 nm at its smallest point (Gburek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Molitoris et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As a result, only small amounts of albumin pass through the GFB under normal conditions, but through the use of specialised endocytic pathways facilitated by podocytes, they are then reabsorbed by tubular cells and recycled (Gburek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Importantly, these same pathways may also allow nano-sized particles, such as NPs, to cross the barrier and be reabsorbed by tubular cells, providing a possible explanation for their accumulation within the kidney.\u003c/p\u003e\u003cp\u003ePrevious nanoparticle exposure studies have observed accumulation of particles in both the GFB and proximal tubule, and in some cases, complete filtration of the particles into urine (Fan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lawrence et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Naumenko et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zuckerman and Davis \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In these studies, the charge of the particles was also addressed, concluding that positively and negatively charged particles differed in their accumulation behaviour. Specifically, due to the negative charge of the GFB, positively charged nanoparticles had a higher propensity to accumulate, compared to their negatively charged counterparts (Naumenko et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zuckerman and Davis \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Other studies have highlighted differences in cytotoxicity, inflammation and cell response following exposure to various nanoparticles, such as silver (Kennedy et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), zinc oxide (Shehata et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and silica (Passagne et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rafieepour et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This indicates that biological outcomes may be driven not only by the particle physical property (nanosize and shape) but also by their chemical properties, such as surface charge, atomic composition and functional groups, which influences interactions with cell membranes, organelles and biomolecules. With this knowledge, it could be expected that the extent of NPs cytotoxicity and accumulation within the kidney also differ depending on their sizes, surface chemistry and charges. Yet, most studies evaluating the toxicity of NPs on mammalian cells only look at 1 type of polymer: polystyrene. Across current NP studies, polystyrene (PS) NPs are overrepresented \u0026ndash; despite accounting for only\u0026thinsp;~\u0026thinsp;6% of global plastic waste - while other polymer types remain largely understudied (Pradel et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This narrow focus limits both environmental relevance and toxicological insight, as different polymers may engage in substantially different interactions with biological systems. Expanding NP research to include a broader range of polymer types and sizes is therefore necessary to better reflect real-world exposures and uncover polymer-specific toxicological effects.\u003c/p\u003e\u003cp\u003eGiven the potential for accumulation, recirculation and filtration of NPs via the kidney, testing the fate and effects of small NPs, particularly those less than 100 nm, is crucial to understanding renal impact. In this study, the proximal tubule cell line, Human Kidney 2 (HK-2), was exposed to polystyrene (PS), poly(methyl methacrylate) (PMMA), and polyethylene (PE) NPs, from 15 to 100 nm in size, at concentrations ranging from 0.1 \u0026micro;g/mL to 200 \u0026micro;g/mL. It is hypothesised that exposure to higher NP concentrations will result in increased cell response, while cell response will vary dependent on the polymer type.\u003c/p\u003e\u003cp\u003eThe toxicity and biological effects of NPs with varying sizes, polymers and concentrations on HK-2 cells was assessed, with the aim of evaluating how these factors impact cell viability, cell cycles and internalisation and to provide insight into the potential implications of short-term NP exposure on kidney function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eNanoplastics\u003c/h2\u003e\u003cp\u003eCarboxylated (-COOH) NPs of various sizes and polymer types were obtained from multiple suppliers. Specifically, PS NPs 20 nm and 100 nm in diameter were purchased from Thermo Fisher Scientific (Manufacturer A). PS NPs 15 nm and 100 nm in diameter, 50 nm PMMA and PE NPs were purchased from Lab261 (Manufacturer B). PMMA NPs 50 nm and 100 nm in diameter were purchased from Phosphorex (Manufacturer C). For NP internalisation experiments, different types of fluorescent NPs were used. An initial experiment was performed to test for potential fluorophore leaching effects, using both internally and externally labelled particles: externally labelled red fluorescent (RF) 100 nm PS NPs (Magsphere Inc), and 100 nm Fluoro-Max PS NPs (Thermo Fisher Scientific) internally labelled with Europium (Eu). Europium, as a fluorophore, can be beneficial for bioaccumulation studies due to its large Stokes shift, and reduced susceptibility to photobleaching, which in turn can reduce the effects of autofluorescence and increase visibility of NPs (Cheignon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Crawford et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For subsequent internalisation experiments, fluorescently labelled PS NPs 15 nm and 100 nm in diameter as well as 50 nm PMMA and PE NPs (Lab261) were used to test the effect of polymer type on internalisation.\u003c/p\u003e\u003cp\u003eThe 20 nm and 100 nm NPs supplied by Manufacturer A were provided respectively as 41 mg/mL and 45 mg/mL stock solutions in deionised water. All NPs from manufacturers B and C were supplied as 10 mg/mL stock solutions in deionised water containing surfactant and 2 mM sodium azide (NaN\u003csub\u003e3\u003c/sub\u003e). The volume and type of surfactants used in the stock solutions were unknown aside from those supplied by Lab261, which were specified to be 0.1% Tween-20.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDilutions\u003c/h3\u003e\n\u003cp\u003eImmediately prior to dilution, all NP stocks were briefly vortexed and then sonicated in a water bath sonicator (Soniclean Digital Benchtop Ultrasonic Cleaner 80TD) for 15 minutes to facilitate homogenisation of the NP suspensions. All non-fluorescent NPs were diluted to concentrations of 2 mg/mL (2,000 \u0026micro;g/mL), 200 \u0026micro;g/mL, 20 \u0026micro;g/mL and 2 \u0026micro;g/mL in PBS. All fluorescent NPs were diluted in PBS to concentrations of 1 mg/mL (1,000 \u0026micro;g/mL), 100 \u0026micro;g/mL, 10 \u0026micro;g/mL and 1 \u0026micro;g/mL, or in the case of the RF and Eu PS NPs, these were diluted to the same number of particles per cell as the non-fluorescent 100 nm PS NPs to ensure consistency with the NP exposure experiments. Prior to cell treatment, all NP dilutions were further diluted at a ratio of 1:10 into cell culture media to final concentrations of 200 \u0026micro;g/mL, 20 \u0026micro;g/mL, 2 \u0026micro;g/mL and 0.2 \u0026micro;g/mL for NP exposure experiments, or 100 \u0026micro;g/mL, 10 \u0026micro;g/mL, 1 \u0026micro;g/mL and 0.1 \u0026micro;g/mL for NP internalisation experiments. The particles per mL concentrations of the NP stock solutions were either provided by the manufacturer upon receipt of the stocks, or calculated using the following equation:\u003c/p\u003e\u003cp\u003eEquation 1: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:particles\\:per\\:mL=\\frac{grams\\:per\\:mL}{density\\:\\times\\:\\:(4/3\\times\\:\\pi\\:{r}^{3})}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eTo determine the number of particles per cell during the exposure period, the following calculation was used:\u003c/p\u003e\u003cp\u003eEquation 2: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:particles\\:per\\:cell=\\frac{(particles\\:per\\:mL\\times\\:well\\:volume)}{number\\:of\\:cells}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eA full summary of the NP suspensions used in this study can be found in Supplementary Tables\u0026nbsp;1 and 2.\u003c/p\u003e\n\u003ch3\u003eCharacterisation\u003c/h3\u003e\n\u003cp\u003eThe shape, size, polydispersity index and surface charge of all NPs used was assessed via scanning electron microscopy (SEM), dynamic light scattering (DLS) and zeta potential measurement, respectively. In both cases NP suspension at 100 \u0026micro;g/mL were prepared in Milli-Q water. For SEM, a 5 \u0026micro;L drop was placed on a silicon wafer and allowed to air dry before coating the substrate with a thin layer of platinum to prevent beam damage. Imaging was conducted with a Gemini II (Bruker), equipped with a field emission gun and operated at an accelerating voltage of 5 kV and a working distance of 3 mm. DLS measurements were carried out on a Zetasizer Nano ZS (Malvern Instruments, UK) with a 4 mW He-Ne laser (633 nm) as a light source and a non-invasive backscatter (NIBS) detection angle of 173\u0026deg;. The analysis was performed at 25\u0026deg;C in disposable polystyrene cuvettes with 2 minutes sample equilibration. The same equipment in conjunction with a folded capillary cell under Smoluchowski approximation was used for zeta potential measurements. Each sample was measured in triplicate.\u003c/p\u003e\n\u003ch3\u003eCell Culture and Nanoplastics Exposure\u003c/h3\u003e\n\u003cp\u003eHK-2 cells (ATCC CRL-2190) were cultured in 1:1 Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) and Ham\u0026rsquo;s F-12 (Gibco), and supplemented with 10 mM HEPES (Gibco), 10% fetal calf serum (FCS; Bovogen Biologicals) and 1X penicillin, streptomycin and glutamine mix (Gibco) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were seeded at 0.1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL in either 12-well cell culture plates or 96-well clear bottom cell culture plates and incubated for 24-hours before replenishing the cells with control or NP treatment media at volumes of 500 \u0026micro;L for 12-well plates, or 100 \u0026micro;L for 96-well plates. The NP concentrations in different well volumes utilised across the experiments were set to ensure consistent nanoplastic particle-per-cell numbers within the experiments and are summarised in Supplementary Tables\u0026nbsp;1 and 2. The HK-2 cells were incubated with the control or NP treatment media for 24-hours before proceeding with the intended assay.\u003c/p\u003e\n\u003ch3\u003eCell Morphology Assessment\u003c/h3\u003e\n\u003cp\u003eFor experiments using standard NPs, cells were imaged after the 24-hour NP incubation period, immediately prior to cell collection. Images were taken using a Thermo Fisher EVOS M5000 Imaging System, or a ZEISS Primovert inverted cell culture microscope with a ZEISS Axiocam 208 color/202 mono microscope camera. Micrograph images were taken of cells at the centre of the plate wells, with a 40x objective. Cell segmentation and granularity were determined using QuPath (v0.5.1) (Bankhead et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLive/Dead Cell Viability Assay\u003c/h2\u003e\u003cp\u003eThe flow cytometry viability assay was performed with a modified version of the protocol provided by Thermo Fisher Scientific (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The HK-2 cells were washed with PBS and detached using TrypLE (Gibco) as per the manufacturer\u0026rsquo;s instructions. The detached cells, along with the supernatant and PBS washes, were collected into Fluorescence-Activated Cell Sorting (FACS) tubes and centrifuged for 5 minutes at 300 \u0026times; g, then washed and resuspended in 100 \u0026micro;L of PBS. A positive control containing live and dead cells (50:50) was generated by heat-treating 50 \u0026micro;L of the cell suspension at 65\u0026deg;C for 5 minutes and then cooled on ice for an additional 5 minutes, before being transferred back to the FACS tube. All cells were washed and resuspended with PBS once again before a 30-minute incubation with 0.5 \u0026micro;L of LIVE/DEAD Aqua dye in 500 \u0026micro;L of PBS, followed by a 15-minute fixation with 4% formaldehyde. After fixation, the cells were centrifuged for 5 minutes at 300 \u0026times; g before resuspension in 200 \u0026micro;L of PBS with 1% FCS. The stained and fixed cells were stored at 4\u0026deg;C for a maximum 1\u0026ndash;2 days prior to analysis via flow cytometry (CytoFlex-S, Beckman Coulter). Acquired flow cytometry data was processed using FlowJo (v10.8.1; BD Biosciences).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell Cycle Analysis\u003c/h3\u003e\n\u003cp\u003eHK-2 cells were detached using TrypLE as per manufacturer's instructions. All cells were centrifuged for 5 minutes at 300 \u0026times; g, cell pellet was washed in and resuspended in 200 \u0026micro;L of PBS. The cells were then fixed with a final concentration of 80% EtOH and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for a minimum of 24-hours. Post fixation, cells were centrifuged for 5 minutes at 300 \u0026times; g, and the cells were permeabilised and stained with propidium iodide (PI) staining solution. The PI staining solution consists of 200 \u0026micro;g/mL of RNASE-A, 50 \u0026micro;g/mL of PI, and 0.1% Triton X-100 diluted in PBS. Cells were incubated in the dark 30 minutes, and then immediately analysed via flow cytometry (CytoFlex-S, Beckman Coulter). Acquired flow cytometry data was processed using FlowJo (v10.8.1; BD Biosciences).\u003c/p\u003e\n\u003ch3\u003eNanoplastic Internalisation\u003c/h3\u003e\n\u003cp\u003eNanoplastic internalisation in HK-2 cells was performed using fluorescently labelled NPs, following a modified version of the methods outlined in studies by Liu et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Wang et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A full summary of the concentrations and NPs used can be found in Supplementary Table\u0026nbsp;2.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMicroscopy Analysis\u003c/h2\u003e\u003cp\u003ePost-exposure, the cells were washed 3 times with PBS to remove as many non-internalised NPs as possible. The cells were fixed with 4% formaldehyde for 15 minutes, permeabilised with 0.1% Triton X-100 for 5 minutes, then stained with ActinGreen 488 ReadyProbes (Alexa Fluor 488, Invitrogen) and NucBlue Live ReadyProbes (Hoechst 33342, Invitrogen) for 30 minutes prior to visualisation via microscopy (Nikon Eclipse Ti2; 40x objective). All wells were imaged with the DAPI, FITC and TexasRed filter cubes, with the exception of the Eu PS NPs, which was additionally imaged with a modified Cy-5 filter cube, containing the DAPI emission filter, to ensure correct excitation, emission and visualisation of the NPs. A minimum of 3 wells were used as replicates of the cell exposure conditions, with at least 1 image per well, corresponding to an area of 192,466 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e. After capture, each image was processed via NIS-Elements AR (v5.30.07; Nikon) to remove any background fluorescence, then analysed to collect the mean fluorescence index (MFI) of each filter capture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eFlow Cytometry Analysis\u003c/h2\u003e\u003cp\u003ePost-exposure, the cells were washed 3 times with PBS to remove as many non-internalised NPs as possible, then detached using TrypLE as per the manufacturer's instructions. All cells were centrifuged for 5 minutes at 300 \u0026times; g, the cell pellet was washed in PBS and fixed with 4% formaldehyde for 15 minutes. Following fixation, all cells were centrifuged for 5 minutes at 300 \u0026times; g, cell pellet was washed in and resuspended in 200 \u0026micro;L of PBS for storage at 4\u0026deg;C for a maximum 1\u0026ndash;2 days prior to analysis via flow cytometry (CytoFlex-S, Beckman Coulter). The internalised NPs were detected using the APC channel to detect RF PS NPs, and the Violet660 channel to detect the Eu PS NPs. Acquired flow cytometry data was processed using FlowJo (v10.8.1; BD Biosciences).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis of cell morphology data was performed using R (v.4.4.2) (R Core Team \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). All other data was analysed using GraphPad Prism (v10.4.0; GraphPad Software).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eNanoplastic Characterisation\u003c/h2\u003e\u003cp\u003eThe shape, size and surface charge of the NPs was determined through SEM and DLS analysis. Visualisation of the NPs under SEM confirmed consistent size and sphericity of all NP types investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-h). DLS analysis confirmed that the measured sizes were comparable to the expected particle sizes, with most NPs assessed as being within 3 to 6 nm of the size indicated on the product certificate of analysis provided by the manufacturer (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All particles displayed negative zeta potentials, as expected for NPs with -COOH surface modifications in Milli-Q water (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacterisation of size and zeta potential of NPs via DLS analysis. *Expected size and mean diameter as provided by manufacturers.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSupplier\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePolymer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExpected Size*\u003c/p\u003e\u003cp\u003e(nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMean Diameter*\u003c/p\u003e\u003cp\u003e(nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMeasured Size\u003c/p\u003e\u003cp\u003e(nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZeta Potential (mV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eThermo Fisher Scientific\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e48.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e131.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-35.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eLab261\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e82.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-40.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePMMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e64.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePhosphorex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePMMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e74.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePMMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e79.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-36.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCell Morphology Assessment\u003c/h2\u003e\u003cp\u003eFollowing the 24-hour NP exposure, changes to the morphology of the cells was assessed by microscopy analysis. When comparing cell exposure to NPs of varying sizes, polymers and concentrations, these factors could be attributed to identifiable morphological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Figs.\u0026nbsp;1 and 2). Commonly, incidences of multinucleation increased in cells exposed to NPs across all types and sizes as compared to the control group and more so in the lower 0.2 \u0026micro;g/mL and 2 \u0026micro;g/mL concentrations. This is coupled with misshapen or irregular cells, primarily in the higher 20 \u0026micro;g/mL and 200 \u0026micro;g/mL concentrations, which made it difficult to distinguish individual cells. In contrast, untreated cells maintained regular cell shape, consistent cell sizing and defined cell borders.\u003c/p\u003e\u003cp\u003eA cell segmentation analysis was performed to quantify changes to cell granularity and morphology following NP exposure (Supplementary Fig.\u0026nbsp;3). When compared to the control group, the cells exposed to NP treatments systematically expressed a significant reduction (Adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the mean pixel intensity (Supplementary Fig.\u0026nbsp;4). While there appears to be no visual morphological differences across all concentrations of the 20 nm NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1), increases in the exposure concentration from 0.2 \u0026micro;g/mL to 200 \u0026micro;g/mL was associated with significant increases in the entropy and heterogeneity of the cells, 34.4% and 78.8% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). In contrast, the 100 nm treatment group displayed noticeable granulation within the cytoplasm of cells exposed to the two highest NP concentrations, localised around the nuclei of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1), associated with a 10.2% increase in cell heterogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). While the source of the granulation is unclear, this could be a result of aggregation and accumulation of the PS NPs within the cytoplasm. This granulation is also visible and significantly increased in the 200 \u0026micro;g/mL PE and PMMA NP treatment groups (P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig.\u0026nbsp;5). Both sizes of PMMA NPs from manufacturer C aggregated so significantly at 200 \u0026micro;g/mL that the cells became difficult to visualise, thus no real difference between the two sizes could be identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This aggregation also impacted the cell segmentation analysis and were excluded from the granularity analysis as a result.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLive/Dead Analysis\u003c/h2\u003e\u003cp\u003eCell viability was assessed by live/dead flow cytometry assay. In the flow cytometry plots, the live cell population shifts away from the live cell gate, towards the dead cell zone as the concentration of NPs increases from 0.2 \u0026micro;g/mL to 200 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Supplementary Figs.\u0026nbsp;6 and 7). Despite these noticeable shifts in the scatterplots, statistical significance was typically seen only at the highest NP concentration. Specifically, after 24-hour exposure to the 20 nm and 100 nm PS NPs from Manufacturer A, there was no significant reduction in cell viability when comparing concentrations, aside from the 20 \u0026micro;g/mL concentration of the 100 nm treatment group (P-value\u0026thinsp;=\u0026thinsp;0.017). This is in direct contrast to the 15 nm and 100 nm PS NPs from Manufacturer B which, following exposure at 200 \u0026micro;g/mL, resulted in a significant reduction in cell viability of 12.5% (P-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and 7.6% (P-value\u0026thinsp;=\u0026thinsp;0.0001) respectively, when compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and c).\u003c/p\u003e\u003cp\u003eThis concentration dependent population shift can also be seen in the PE and PMMA treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Cell viability was significantly reduced by 16% (P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and 4.7% (P-value 0.0008), after exposure to 200 \u0026micro;g/mL of the respective PE and PMMA NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). When comparing the two sizes and manufacturers of PMMA, the reduced viability was similar between the three treatment groups. The difference in viability between the two groups of 50 nm PMMA NPs from manufacturer B and C is 0.87% and non-significant. Similarly, the difference in viability between the two sizes (50 and 100 nm) from Manufacturer C was 0.7% and non-significant. Interestingly, across all treatment groups, the largest reduction in cell viability was seen after treatment with the PE NPs at a concentration 200 \u0026micro;g/mL, reducing viability to 79.46% (P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This could imply a link between the type of polymer and cell viability.\u003c/p\u003e\u003cp\u003eAn additional experiment was completed with both sizes of PS NPs from Manufacturer A in which the NPs were spiked with NaN\u003csub\u003e3\u003c/sub\u003e and Tween-20 to determine if the presence of these additives in NP solutions was contributing to the cell death seen at the higher concentrations. After exposure to the spiked NPs for 24-hours, the cells treated with 100 nm PS NPs saw increased cell death at 20 \u0026micro;g/mL and 200 \u0026micro;g/mL (Supplementary Fig.\u0026nbsp;8). The positive controls of cells treated with NaN\u003csub\u003e3\u003c/sub\u003e and Tween-20 without NPs showed no change to cell viability on their own.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCell Cycle Analysis\u003c/h2\u003e\u003cp\u003eFollowing exposure to varying size and concentrations of PS, PE and PMMA NPs, a cell cycle assay was performed to determine the proportion of cells in three distinct cell cycle phases, namely the G0/G1, S and G2/M phase (Supplementary Figs.\u0026nbsp;9 and 10). Significant cell cycle arrest was seen in the cells treated with the smallest 15 nm and 20 nm PS NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and c), with no significant changes observed in the larger 100 nm PS NPs from either manufacturer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and d). Cells treated with the 15 nm PS NPs exhibited a dose-dependent increase in the percentage of cells in the G0/G1 phase (5.8% to 11.6%), with a significant increase in the 20 \u0026micro;g/mL and 200 \u0026micro;g/mL treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In the 20 nm PS NP treatment group, the opposite was seen, with significant decreases of the percentage of cells in the G0/G1 phase observed in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). A similar shift was seen in the percentage of cells in the S phase (2.4% to 3.4%), with significant increases in the cells of the 0.2, 20 and 200 \u0026micro;g/mL treatment groups, and significant decreases in the percentage of cells in the G2/M phase of the 20 \u0026micro;g/mL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInterestingly, when assessing the effect of NPs of other polymer types, the opposite is seen when compared to PS NPs. No significant effect to the cell cycle was seen in the cells exposed to 50 nm PE and PMMA NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-g), rather the cells exposed to 100 nm PMMA NPs observed significant arrest at the S phase, with significant reduction in the percentage of cells in the G0/G1 phase at a concentration of 200 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eNanoplastic Internalisation\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe NP internalisation study was initially conducted using two different 100 nm PS NPs to assess for potential fluorophore leaching: externally labelled RF PS NPs, and internally labelled Eu PS NPs. In both cases, the fluorescent NPs accumulated within the cytoplasm of the cell and often close to the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Uptake of fluorescent NPs were only visible via microscopy at the highest NPs concentrations investigated, namely 7 \u0026micro;g/mL (Supplementary Fig.\u0026nbsp;11) and 70 \u0026micro;g/mL concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After the 24-hour exposure period, the cells exposed to the RF PS NPs typically displayed brighter aggregates, with apparent accumulation outside of the cell membrane. In contrast, cells exposed to the Eu PS NPs shone more dimly, but with clear accumulation around the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei and n).\u003c/p\u003e\u003cp\u003eAnalysis of the mean fluorescence of the microscopy images indicated uptake of both the RF and Eu PS NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eo). A significant increase was identified in the MFI of cells treated with concentrations of 7 \u0026micro;g/mL and 70 \u0026micro;g/mL of RF PS NPs, suggesting a dose dependent uptake of NPs. In comparison, all concentrations of Eu PS NPs expressed significant MFI increases when compared to the control group, with the largest proportion of uptake seen in the 70 \u0026micro;g/mL treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eo). Internalisation of both fluorescent PS NPs was also assessed via flow cytometry as an alternate way to assess NP uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ep). Much like the microscopy images, the MFI as expressed from the flow cytometry data indicated significant increases in NP uptake in the 7 \u0026micro;g/mL (P-value 0.0005) and 70 \u0026micro;g/mL (P-value 0.0185) concentrations of the RF PS NPs. In the Eu PS NP treatment group, the MFI of the 70 \u0026micro;g/mL concentration was increased when compared to the 7 \u0026micro;g/mL concentration, although this increase was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ep).\u003c/p\u003e\u003cp\u003eFurther analysis of NP internalisation was performed to assess for differences in uptake between different polymers and sizes of NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). To evaluate the effect of NPs size, two populations of PS NPs were used, one with particles 15 nm in diameter, the other with 100 nm NPs. When visualising the cells treated with 15 nm PS NPs, internalisation of the NPs within the cytoplasm was not visible at this magnification, although significant particle aggregation was seen to accumulate on the exterior of the cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh and i). The cells exposed to the 100 nm PS NPs presented similarly to the initial internalisation experiment, with brighter aggregates of the particles that appear to sit outside of the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003em and n). To evaluate the effect of polymer type, two population of 50 nm NPs were used, one made of PE, and another one made of PMMA particles. The 50 nm PMMA treatment group showed some similarities to the 100 nm PS NP group, presenting a mix of bright aggregates on the outer membrane of the cell, with dimmer NP populations that appear to be accumulated within the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003er and s). Most interestingly, the cells exposed to the 50 nm PE NPs almost exclusively express dimmer NP populations that had been internalised within the cell membrane. Within these populations, a clear point of accumulation can be seen within each cell, which often overlaps within or immediately adjacent to the cell\u0026rsquo;s nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ew and x). The difference in uptake between the different NP polymer types, particularly with the PE NPs, may indicate that some polymers are able to be more readily internalised by cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of the MFI from the microscopy images also indicates a dose-dependent uptake of the NPs within a 24-hour exposure period (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ey). Increased MFIs are seen at all concentrations for all NP treatment groups, with a significant increase in the 100 \u0026micro;g/mL groups. The largest increase in MFI was seen in the cells treated with PMMA, which is likely to be attributed to the combination of internalised NPs and aggregates adhered to the outer cell membrane. With the PE NP treated cells having the lowest increase in MFI at 100 \u0026micro;g/mL could be attributed to the strict internalisation of the particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ez).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe impact of short-term exposure to NPs of different polymers, sizes and concentrations was tested on HK-2 proximal tubule cells. Microscopy and flow cytometry was used to evaluate physical and biological effects.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffect of concentration\u003c/em\u003e: The HK-2 kidney cell line is largely unaffected by short-term exposure to low NPs concentrations. In contrast, effects were evident at the highest concentrations tested (100 \u0026micro;g/mL and 200 \u0026micro;g/mL). At these levels, morphological changes were pronounced, including misshapen cells, and increased granularity \u0026ndash; features absent in the control groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Flow cytometry viability assessment supported these observations: all NPs from Manufacturers B and C caused a significant reduction in viability at 200 \u0026micro;g/mL, with an overall reduction between 5% to 17% relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, given that the viability assay detects dye uptake only when membrane integrity is compromised, the flow cytometry results also imply increased membrane permeability at higher NP concentrations. Cell cycle analysis also revealed a dose-dependent deregulation, with significance increasing at higher NP concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Internalisation of NP within cells also correlated with increasing exposure concentrations. Fluorescence microscopy and flow cytometry analysis confirmed greater uptake at 100 \u0026micro;g/mL and significant MFI increases at these concentrations for all polymer types and particle sizes (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffect of polymer\u003c/em\u003e: Cell responses were also influenced by the polymeric composition of the NP particles, with variation in morphology, functional changes and internalisation patterns, seen between polymers of comparable sizes and concentration. The most pronounced effect on viability resulted from exposure to 50 nm PE NPs. Compared with PS NPs, which generally maintained cell shape and morphology, PE and PMMA NPs induced irregular cell shape with multinucleation and increased granularity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Flow cytometry further highlighted polymer-dependant effects: \u003cem\u003ea\u003c/em\u003elthough significant reductions in viability occurred with all NPs from Manufacturers B and C at 200 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the greatest decrease was observed following exposure to the PE NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Despite the similarity in size, the 50 nm PMMA NPs from Manufacturer B and C both had a lesser impact on viability when compared to the 50 nm PE NPs. Notably, the 100 nm PMMA NPs also caused significant S-phase arrest at 200 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), whereas 100 nm PS NPs did not\u0026mdash;neither for manufacturer A nor for manufacturer B, which expressed a similar change in viability, as well as containing the same concentration of sodium azide as the 100 nm PMMA NPs from manufacturer C. In this case, the size, additive content, and surface charge were equivalent, yet the cellular responses differed, supporting the conclusion that the NP polymer composition alone can drive differential biological effects.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that surface functionalisation influences NP toxicity, with carboxylic acid (COOH) or amine (NH\u003csub\u003e2\u003c/sub\u003e) modified PS NPs generally found to be more harmful than pristine particles, and COOH- less toxic than -NH\u003csub\u003e2\u003c/sub\u003e modification (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gonz\u0026aacute;lez-Fern\u0026aacute;ndez et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Here all NPs were functionalised with COOH, and all had negative surface charge (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eq-x). Yet polymer-dependent differences persisted, indicating that the chemical composition of the polymer itself may also be a determinant of NP toxicity, not just surface charge. These polymer-dependent differences in viability and cell cycle arrest suggest that environmental mixtures of NPs may exert heterogeneous renal effects, underscoring the need to assess multiple polymer types in toxicology studies.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffect of size\u003c/em\u003e: NP size was associated with differences in cell response, with smaller NPs (15 nm, 20 nm and 50 nm) often showing greater effects than their 100 nm counterparts. The most significant size-related effects were observed in cell cycle analysis: the two smallest sizes of PS (15 nm and 20 nm) caused dysregulation after 24 hours of exposure, even at concentrations as low as 0.2 \u0026micro;g/mL in one instance \u0026ndash; S-phase arrest for the 20 nm PS NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b). This was the only case in which such a low concentration produced a statistically significant impact. These results could suggest that smaller sizes of PS NPs are more readily able to interfere with cell growth and proliferation. As S-phase arrest is commonly associated with DNA damage (Chao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), this could indicate potential interaction with the nucleus and have implications in increased cell stress. These effects were observed in azide-free particles (Manufacturer A), ruling out attribution to this toxic additive. Taken together, the cell cycle findings may be indicative that smaller NP sizes cause DNA damage, warranting investigation in longer-term exposure studies.\u003c/p\u003e\u003cp\u003eUnfortunately, these findings were not strongly mirrored in the viability assays, where responses remained largely unchanged across all NP sizes at all concentrations below 200 \u0026micro;g/mL. A notable exception was the 50 nm PMMA NPs, which reduced viability significantly at 20 \u0026micro;g/mL, whereas the 100 nm PMMA NPs from the same manufacturer only caused a reduction at ten times that concentration (200 \u0026micro;g/mL). These results differ to the study performed by Li et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), in which comparable sizes of PS NPs (20 nm, 60 nm and 100 nm) were exposed to human embryonic kidney cells, where it was seen that the 20 nm PS NPs were significantly more toxic than the larger NPs, expressing near complete cell death at the 75 \u0026micro;g/mL and 125 \u0026micro;g/mL concentrations tested, compared to ~\u0026thinsp;80% viability for the cells exposed to the 60 nm and 100 nm PS NPs (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies assessing exposure to PS NPs above 100 nm in size, including MPs, consistently showed no impact on kidney cell viability, regardless of concentration or exposure time (Goodman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNo clear size-related differences were observed in the uptake assays, with fluorescence intensity remaining comparable between small and large particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ez). The one difference observed was the size of the NP aggregates, with the small NPs only visible as larger, more diffuse, aggregates, likely due to resolution limits. It is worth noting here, that single 15 or 20 nm NPs or small aggregates of them would not be detectable via microscopy. In terms of cell morphology, cells exposed to the smaller NPs generally appeared more consistent than those treated with their larger equivalents, although this too may be due to limits in imaging resolution.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eOther considerations\u003c/strong\u003e\u003cp\u003eThe presence of additives, or lack thereof, within NP suspensions also appeared to impact viability and cell response, particularly for PS NPs from Manufacturer B which contained NaN\u003csub\u003e3\u003c/sub\u003e and Tween-20. These NPs significantly reduced HK-2 cell viability compared to additive-free PS-NPs from manufacturer A, where viability was largely unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003c/p\u003e\u003cp\u003eWhen PS NPs from Manufacturer A were spiked with NaN\u003csub\u003e3\u003c/sub\u003e and Tween-20, cell viability decreased, despite the additives on their own showing no effects at equivalent concentrations (Supplementary Fig.\u0026nbsp;8). This result could infer some level of synergistic biological effect where exposure to the additives makes the cells more susceptible to NP-induced toxicity. Given the known cytotoxicity of NaN\u003csub\u003e3\u003c/sub\u003e even at low concentrations (Heinlaan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Petersen et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pikuda et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the presence of additives should be addressed and controlled in NP studies. Whether the observed effects of additives result from membrane degradation, altered permeability, or increased endocytic/phagocytic activity within the cell, is yet to be determined.\u003c/p\u003e\u003cp\u003eIt is important to note that the 15 nm PS NPs from Manufacturer B, as well as the 50 and 100 nm PMMA NPs from Manufacturer C exhibited a tendency to aggregate within the treatment period. Aggregation, often visible in suspension within the cell media, may have an impact on the cell\u0026rsquo;s response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Figs.\u0026nbsp;1 and 2).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eOutlook and future work\u003c/strong\u003e\u003cp\u003eThis study used an immortalised proximal tubule cell line, commercially produced spherical NPs, and a short 24-hour exposure period, which cannot fully replicate the complexity of human kidney tissue or long-term exposure scenarios. Primary kidney cells or 3-dimensional spheroid models could provide additional physiological relevance in future works. Commercial NPs also differ from environmentally derived particles, which are typically more heterogeneous in shape, size and composition. This is a significant and common limitation across NP research into cellular and systemic effects and toxicity. The NPs used in this study were of a consistent size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), sphericity and composed of a single polymer type, and while not mimicking environmentally relevant scenarios, the controlled particle properties used here allowed us to isolate and directly compare the effects of polymer type, size, and concentration on kidney cell responses.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eWhile the lower concentrations tested in this study may be indicative of short-term environmental exposure, real-world contact with NPs is more likely to occur at even lower concentrations than the ones tested in this study, but over an extended period of time. For instance, a recent study by ten Hietbrink et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) evaluated concentrations of NPs smaller than 1 \u0026micro;m within the North Atlantic Ocean, identifying an average NP concentration of 15.1 mg/m\u003csup\u003e3\u003c/sup\u003e (0.015 \u0026micro;g/mL equivalent), with PS equating to 4.06 mg/m\u003csup\u003e3\u003c/sup\u003e (0.004 \u0026micro;g/mL equivalent). These concentrations are only 1 order of magnitude smaller than the lowest concentration tested here. The potential cumulative effects and delayed biological impacts cannot be reflected in short-term, acute exposure models. However, a recent study by Nihart et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) saw a 2.3 \u0026micro;g per gram increase of plastic in kidney tissue between 2016 and 2024, which seems to indicate that bioaccumulation does occur, and may do so in a more pronounced fashion as environmental exposure increases. Hence the concentrations used here may become more and more biologically relevant as plastic pollution increases over time. For now, short-term studies like this one are instead useful for assessing the immediate impacts, cellular responses and toxicity of specific NP types, and offer a framework for designing longer term experiments with environmentally relevant exposure thresholds necessary to further understanding of possible toxicity, effect and health impacts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings of the study demonstrate that while lower concentrations of NPs may not result in immediate toxicity to the HK-2 cell line, particularly in terms of short-term exposure, higher NP burdens can compromise overall cell health and function, affecting morphology, viability and cell cycle regulation. The results also indicate that NP effects are influenced not only by concentration but also by polymer composition and particle size, with some combinations inducing significant cellular changes even at relatively low doses. Given that proximal tubule cells play a critical role in reabsorption and overall kidney filtration efficiency, sustained or repeated damage to these cells could impair kidney function, potentially leading to NP accumulation in kidney tissue, reduced clearance capacity, and increased NP recirculation in the bloodstream over time. This highlights the importance of investigating more realistic, environmentally relevant NP exposure, that reflect chronic, low-level contact over extended periods, and that account for the diversity of NP characteristics in terms of concentration, size, polymer types and chemical additives likely to be encountered in real-world settings. Such studies should also explore mechanistic endpoints, including potential DNA damage and long-term functional consequences, to fully assess the risks posed by environmental NPs to kidney health and systemic exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eNone.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Australian Research Council Future Fellowship Grant (FT200100301), The Flinders Foundation and the Flinders Medical Centre Renal Research Fund.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hayden Louis Gillings, Iliana Delcheva and Soon Wei Wong. The first draft of the manuscript was written by Hayden Louis Gillings and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work used the NCRIS and Government of South Australia enabled Australian National Fabrication Facility - South Australian Node (ANFF-SA) and Microscopy Australia at the University of South Australia. We would also like to acknowledge and thank Dr. Giles Best at the Flow Cytometry Facility at Flinders University. These facilities provided vital infrastructure and services to complete this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdhipandito CF, Cheung SH, Lin YH, Wu SH. Atypical Renal Clearance of Nanoparticles Larger Than the Kidney Filtration Threshold. 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Adv Chronic Kidney D. 2013;20:500\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.ackd.2013.06.003\u003c/span\u003e\u003cspan address=\"10.1053/j.ackd.2013.06.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-biology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbto","sideBox":"Learn more about [Cell Biology and Toxicology](https://www.springer.com/journal/10565)","snPcode":"10565","submissionUrl":"https://submission.nature.com/new-submission/10565/3","title":"Cell Biology and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"nanoplastics, kidney, cytotoxicity, polystyrene, poly(methyl methacrylate), polyethylene","lastPublishedDoi":"10.21203/rs.3.rs-7588029/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7588029/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanoplastics (NPs, \u0026lt;1 µm) are emerging environmental contaminants capable of crossing biological barriers and interacting at the cellular and subcellular level. Despite evidence of microplastics in human kidney tissue and urine, the renal effects of NPs remain poorly understood. This study investigated the short-term effects of NP polymer type, size, and concentration on human proximal tubule cells (HK-2). Cells were exposed for 24 h to carboxylated polystyrene (PS), poly(methyl methacrylate) (PMMA), and polyethylene (PE) NPs (15–100 nm) at concentrations from 0.2 to 200 µg/mL. NP size, charge, and morphology were characterised by scanning electron microscopy, dynamic light scattering, and zeta potential. Cell morphology, viability, cell cycle distribution, and NP internalisation were assessed by microscopy and flow cytometry. Low-concentration exposures had minimal effects, whereas 100 and 200 µg/mL induced marked morphological changes, including cytoplasmic granularity. Viability decreased significantly at 200 µg/mL for several NP types, with PE NPs causing the largest reduction (79.4%). Polymer type influenced outcomes, with PE and PMMA NPs causing greater morphological disruption than PS. Size effects were most evident in cell cycle analysis: 15 nm and 20 nm PS NPs and 100 nm PMMA NPs induced phase arrest without major viability loss. NP internalisation increased with concentration but varied with polymer type, with PE NPs showing preferential perinuclear localisation. These findings demonstrate that NP effects on kidney cells depend on polymer chemistry, particle size, concentration, and highlight the need for long-term studies using environmentally relevant NPs to better assess kidney toxicity risk.\u003c/p\u003e","manuscriptTitle":"Nanoplastic toxicity and uptake in kidney cells: differential effects of concentration, particle size, and polymer type","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 18:36:55","doi":"10.21203/rs.3.rs-7588029/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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