Research Article

Cellular Uptake and Nuclear Accumulation of Polystyrene Nanoplastics in 3T3 Fibroblasts and Hepatocytes of Rattus norvegicus

Febriyansyah Saputra1, Alfiah Hayati1*, Adiibtia Septiani1, Manikya Pramudya1, Raden Joko Kuncoroningrat Susilo2, Vuanghao Lim3, Bayyinatul Muchtaromah4

1Department of Biology, Faculty of Science and Technology, University of Airlangga, Indonesia; 2Department of Engineering, Faculty of Advanced Technology and Multidiscipline, Universitas Airlangga; 3Department of Toxicology, Advanced Medical and Dental Institute Universiti Sains Malaysia, Malaysia; 4Biology Department, Maulana Malik Ibrahim Islamic University, Malang, Indonesia.

Received | March 06, 2025; Accepted | April 08, 2025; Published | June 05, 2025

*Correspondence | Alfiah Hayati, Department of Biology, Faculty of Science and Technology, University of Airlangga, Indonesia; Email: [email protected]

Citation | Saputra F, Hayati A, Septiani A, Pramudya M, Susilo RJK, Lim V, Muchtaromah B (2025). Cellular uptake and nuclear accumulation of polystyrene nanoplastics in 3T3 fibroblasts and hepatocytes of Rattus norvegicus. Adv. Anim. Vet. Sci. 13(7): 1491-1501.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.7.1491.1501

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Plastic pollution has emerged as a critical environmental challenge, with macro- and microplastics fragmenting into even smaller particles, known as nanoplastics, through physical, chemical, and biological degradation (Allen et al., 2022). These nanoscale fragments have infiltrated ecosystems and food chains, raising growing concerns about their potential health risks (Hayati et al., 2024). Nanoplastics exist as both homogeneous and heterogeneous aggregates, exhibiting unique physicochemical properties that enhance their interactions with biological systems (Liu et al., 2019).

The International Organization for Standardization (ISO) defines nanoparticles, including nanoplastics, as materials with external dimensions between 1 and 100 nm (Kiran et al., 2022; Nguyen et al., 2019). However, the International System of Units (SI) extends the nanoplastic definition to include plastic fragments up to 1 μm (1000 nm). Due to their high surface area, nanoplastics can adsorb environmental contaminants and migrate through biological membranes. Once inside living organisms, they may accumulate in cells, potentially triggering toxic effects (Triwahyudi et al., 2023).

The ability of nanoplastics to penetrate cellular structures poses a growing health concern. Studies suggest that these particles enter cells via endocytosis or passive diffusion, allowing them to reach intracellular compartments and interact with critical biomolecules (Hayati et al., 2024). Their presence within the body has been linked to oxidative stress, inflammation, and disruptions in cellular function, which could contribute to liver dysfunction, respiratory distress, skin disorders, and reproductive health issues (Pitt et al., 2018; Zhang et al., 2020). However, despite increasing concerns, research on nanoplastics’ long-term health effects remains in its early stages (Saputra et al., 2025).

To understand the biological risks, both in vitro and in vivo models are essential in toxicological studies (Bijukumar et al., 2023). In vitro models, such as 3T3 fibroblast cell cultures, are widely employed due to their sensitivity, reproducibility, and relevance in assessing cellular stress responses, morphological changes, and nanoparticle uptake (Bhardwaj and Webster, 2015), while Rattus norvegicus provides a relevant physiological model due to its biological similarities to humans (Khan et al., 2019). Due to their widespread use and slow degradation, polystyrene contributes significantly to global plastic waste, ultimately fragmenting into micro- and nano-sized particles that are highly persistent in the environment (Gilani et al., 2023; Lv et al., 2024; Zhang et al., 2024).

PSNPs, with a nanoscale size of <100 nm, can directly interact with lipid membranes and cellular proteins, modulating biological processes at the molecular level (Lv et al., 2024). Upon internalization, their excretion is limited, potentially leading to accumulation in the digestive tract. Some particles may traverse the intestinal epithelium, enter the bloodstream, and distribute across various tissues and organs (Li et al., 2024). However, their intracellular dynamics and long-term biological impacts remain largely unexplored, underscoring the need for further research. Understanding the mechanisms of PSNP-cell interactions is crucial for assessing their toxicological impact.

This nanotoxicology study aims to provide deeper insights into the biological impacts of nanoplastics, particularly PSNPs, on cellular systems and their potential to disrupt physiological functions. Research on PSNP transport mechanisms into cells has identified key pathways, including endocytosis, protein corona formation, and direct lipid membrane interactions. However, significant knowledge gaps remain in comprehensively understanding these processes and their cellular consequences, necessitating further investigation into PSNP-related health risks. This study investigates the transport mechanisms of PSNPs into cells, focusing on primary pathways such as endocytosis and membrane interactions. Experimental analyses explore the biological effects of PSNP exposure, including oxidative stress induction, structural disruptions, and inflammation, which may lead to cellular dysfunction. By examining cellular uptake dynamics and assessing the potential long-term impacts of PSNPs on biological systems, this research aims to provide critical insights into their broader implications for overall health.

MATERIALS AND METHODS

PSNP Characterization

PSNPs used in this study were obtained from Sigma-Aldrich (USA). As specified by the manufacturer, the PSNPs are carboxylate-modified, monodisperse polystyrene spheres with an average diameter of 100 nm. The particle size distribution was determined by dynamic light scattering (DLS), with a polydispersity index (PDI) of less than 0.1, indicating a narrow size distribution and uniformity. The zeta potential of the PSNPs is approximately –40 mV in aqueous suspension, reflecting a stable surface charge that promotes dispersion and prevents aggregation in cell culture medium. This characterization The PSNPs were stored and handled according to the supplier’s guidelines, and no visible signs of agglomeration were observed during experimental procedures. The PSNPs were stored and handled in accordance with the supplier’s guidelines, and no visible agglomeration was observed during experimental procedures. These physicochemical characteristics have also been confirmed in previous independent studies, which reported similar size, charge, and dispersion profiles (Shorny et al., 2023).

Cell Culture (In Vitro Experiment)

Mouse embryo fibroblast cells (3T3) were obtained from the Parasitology Laboratory, Universitas Gadjah Mada, Indonesia. The cells were cultured in T225 flasks using Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin-streptomycin solution, and 0.5% fungizone to maintain optimal growth conditions. The cell cultures were maintained at 37°C in a 5% CO₂ atmosphere. For the experiment, the 3T3 cells were divided into two groups: a control group without PSNP and a treatment group exposed to PSNP at a concentration of 100 µL/mL. After 48 hours of treatment, the cells were collected and prepared for confocal microscopy analysis to assess PSNP uptake and intracellular distribution. All experiments were conducted in triplicate.

Animal Study (In Vivo Experiment)

The in vivo study was conducted using adult Wistar rats (Rattus norvegicus L.), weighing 200 g, obtained from the Animal Laboratory, Faculty of Pharmacy, Universitas Airlangga. The experimental animals were maintained under controlled conditions at a temperature of 25°C ± 2°C with a 12-hour light-dark cycle. Rats were provided ad libitum access to drinking water and were fed 80%–90% of their standard daily food intake. All experimental procedures, including animal handling and treatment protocols, were conducted in accordance with ethical guidelines and were approved by the Research Ethics Committee, Faculty of Veterinary Medicine, Universitas Airlangga (1157/HRECC.FODM/X/2023). Each experimental group was conducted in triplicate.

Experimental Design

The rats were randomly assigned to two groups: a control group and a treatment group. The control group received distilled water, while the treatment group was administered PSNPs (Sigma-Aldrich, USA) orally at a dose of 0.5 mL (10 µL/kg) twice daily for 30 consecutive days. The selected PSNP concentration was based on previous dose-response studies and our own preliminary experiments, which demonstrated that this dosage consistently induced measurable biological effects without causing excessive cytotoxicity. The PSNP suspension was freshly prepared and thoroughly mixed before each administration to ensure uniformity. At the end of the 30-day treatment period, the rats were euthanized following ethical guidelines, and liver samples were carefully excised under sterile conditions. The collected liver tissues were immediately fixed in 10% neutral-buffered formalin for histological analysis.

Preparation of Confocal Microscopy Slides

The preparation of confocal microscopy slides involved several steps. Cultured cells were fixed with 4% formaldehyde. A 5 µL suspension of the fixed cells was placed onto a glass slide and evenly spread to create a uniform monolayer. Following fixation, the slides were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) and Nile Red (Sigma-Aldrich, USA). After staining, the slides were thoroughly rinsed to remove excess dye and then examined using a confocal microscope for high-resolution imaging (Tarafdar et al., 2022).

Histological and Confocal Microscopy Analysis

Histological evaluation was conducted to observe liver tissue morphology, detect structural changes, and assess the toxic effects of PSNP. The liver tissue was fixed in 10% formalin solution for 48 hours. Following fixation, the samples underwent a series of graded washing steps to remove residual fixative and prepare the tissue for staining. The processed liver sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei and Nile Red to highlight lipid components, allowing for a detailed assessment of cellular architecture and potential lipid accumulation. Imaging was conducted using a laser scanning confocal microscope (Olympus) to obtain high-resolution fluorescence images. Additionally, 3D image analysis was performed using Thermo Scientific HCS Studio 2.0 to enhance visualization and quantification of structural changes within the liver tissue. The cell diameter was calculated by averaging the longest and shortest diameters of cells. The accumulation of PSNPs was quantified by measuring red lipid fluorescence intensity using ImageJ software, providing a precise assessment of PSNP uptake and distribution within the liver tissue. Measurements were conducted on 100 cells per sample, with each analysis performed in triplicate.

Statistical Analysis

Data were analyzed using GraphPad Prism 10.3.1 with one-way ANOVA (p < 0.05) to compare control and NP-treated groups. Normality and variance were checked with Shapiro-Wilk and Levene’s tests. Quantitative histological data were processed with ImageJ, and results were expressed as mean ± SD, with Tukey’s post hoc test for significant differences. Descriptive analysis was used for qualitative observations.

RESULTS AND DISCUSSION

PSNP Accumulation in 3T3 Fibroblasts and Rattus norvegicus Hepatocytes

Exposure to PSNPs significantly increased their accumulation in both 3T3 fibroblast cells and Rattus norvegicus hepatocytes (Figure 1). Fluorescence imaging demonstrated minimal red fluorescence in the control groups, indicating negligible PSNP presence (Figure 1A). In contrast, PSNP-exposed cells exhibited a substantial increase in red fluorescence, suggesting pronounced nanoparticle accumulation. Quantitative analysis (Figure 1B) reinforced these findings, showing significantly higher PSNP accumulation in treated groups compared to controls (p < 0.05), as indicated by different letter annotations. These results highlight the capacity of PSNPs to accumulate within both cell types, suggesting their potential for intracellular retention and associated biological effects.

 

The observed uptake of PSNPs is consistent with previous studies demonstrating nanoparticle internalization in various cell lines, including fibroblasts and hepatocytes (Deng et al., 2017; Wang et al., 2022). Nanoparticles can enter cells through diverse pathways such as clathrin-mediated endocytosis, caveolae-dependent uptake, and macropinocytosis, influenced by factors including size, surface charge, and composition (Mazumdar et al., 2021). The interaction of PSNPs with 3T3 fibroblasts is particularly noteworthy, as fibroblasts are pivotal in tissue maintenance and repair. Disruptions to fibroblast function could potentially impair wound healing and compromise extracellular matrix integrity (Dong et al., 2024). Meanwhile, the accumulation of PSNPs in hepatocytes raises additional concerns, given the liver’s critical role in detoxification and metabolic homeostasis. The increased PSNP retention in hepatocytes suggests a potential risk of nanoparticle-induced hepatotoxicity, emphasizing the need for further investigation into the mechanisms of PSNP-mediated liver toxicity (Das et al., 2024).

Morphological Changes in Cells Due to PSNP Exposure

Exposure to PSNPs led to significant morphological and structural changes in both 3T3 fibroblasts and Rattus norvegicus hepatocytes. Confocal microscopy imaging (Figures 2A and 2B) revealed marked differences between control and PSNP-exposed cells. In the control group (Figure 2A), cells displayed a uniform structure with well-defined nuclei (white arrows) and minimal red fluorescence, indicating normal lipid distribution. In contrast, PSNP-exposed cells (Figure 2B) exhibited a substantial increase in red fluorescence (yellow arrows), suggesting enhanced lipid accumulation or alterations in membrane composition. Additionally, the cytoplasmic distribution appeared irregular, with significant variations in cell shape and size, indicative of potential cellular stress responses. Quantitative analysis (Figures 2C and 2D) corroborated these observations, showing that both cell and nucleus diameters were significantly increased in PSNP-treated groups compared to controls (p < 0.05), and a substantial increase in necrotic cells was observed, highlighting the cytotoxic effects of PSNP exposure.

Our findings offer strong indirect evidence suggesting that PSNPs likely interact with both cellular proteins and lipids. Confocal microscopy revealed pronounced cytoplasmic disorganization, enlarged cell and nuclear diameters, irregular morphology, and intense Nile Red fluorescence localized predominantly in the cytoplasm and near the nuclear envelope. These fluorescence patterns strongly suggest PSNP accumulation, as well as lipid remodeling or droplet formation. Such changes align with established literature indicating lipid perturbation, membrane destabilization, and oxidative stress induced by nanoplastic exposure (Hayati et al., 2023). Such disruptions may compromise membrane fluidity, affect signaling pathways, and initiate inflammatory responses (Itri et al., 2014). Together, these observations provide sufficient evidence to support our current conclusions while also highlighting directions for deeper mechanistic exploration in future studies.

 

Our results also align with previous reports showing that micro- and nanoplastics can interfere with lipid homeostasis, induce oxidative damage, and destabilize cytoskeletal structures (Hayati et al., 2023). The irregular cytoplasmic distribution observed in PSNP-treated cells implies potential disruptions in actin filament dynamics and cytoskeletal integrity. Prior research has shown that nanoplastics can destabilize the cytoskeleton, resulting in altered cell morphology, impaired intracellular transport, and compromised cellular function (Schirinzi et al., 2017). The increase in cell and nucleus size may reflect cytoskeletal reorganization or swelling due to osmotic imbalance and stress signaling, indicating that PSNPs affect not only structural stability but also intracellular homeostasis and fibroblast function.

Importantly, the enhanced red fluorescence and visible changes in membrane structure suggest that PSNPs may interact directly with cellular lipids. Nanoplastics are known to integrate into lipid bilayers or alter membrane fluidity, potentially impairing membrane integrity, signal transduction, and lipid metabolism (Khan and Jia, 2023). The intensified Nile Red signal in PSNP-exposed cells may reflect not only the presence of the nanoparticles themselves but also a PSNP-induced accumulation of lipids or formation of lipid-rich domains. These disruptions may promote lipid peroxidation, further exacerbating oxidative stress and cytotoxicity (Nurbani et al., 2025).

Furthermore, intracellular accumulation of PSNPs could initiate inflammatory responses and activate apoptotic signaling pathways, ultimately compromising cell viability and proliferation (Khan and Jia, 2023). The significant rise in necrotic cells in PSNP-treated samples supports the hypothesis of PSNP-induced cytotoxicity. Previous studies have linked nanoplastic exposure to oxidative stress, mitochondrial dysfunction, and the activation of apoptosis, which may progress to necrosis if cellular damage is irreversible (Chen et al., 2025; Lee et al., 2022; Mahmud et al., 2024).

While this study did not include direct biochemical assays to confirm specific PSNP interactions with cellular proteins or lipids, the combined morphological, quantitative, and fluorescence-based evidence strongly supports the likelihood of such interactions. These observations are in agreement with a growing body of research on nanoplastic-induced cellular damage through mechanisms involving protein corona formation, membrane destabilization, and oxidative stress (Wang et al., 2023). Future studies incorporating proteomic and lipidomic analyses, as well as targeted assays for oxidative markers and cytoskeletal dynamics, will be essential to fully characterize the molecular mechanisms underlying PSNP-induced toxicity.

High-Resolution Analysis of PSNP Localization in 3T3 Cells

To further investigate PSNP accumulation within 3T3 cells, confocal microscopy analysis at 400x magnification was performed. Figure 3 illustrates the distinct morphological differences between control and PSNP-exposed cells, stained with DAPI (blue) to highlight nuclei and Nile Red (red) to visualize PSNP presence. In the control group (Figure 3A), 3T3 cells exhibited normal morphology, with a round, well-defined nucleus and a uniformly distributed cytoplasm, indicative of healthy cellular conditions. In contrast, PSNP-exposed cells (Figure 3B) exhibited intense red fluorescence signals, predominantly localized in the cytoplasm and around the nuclear envelope (highlighted by red asterisks), indicating considerable nanoparticle internalization and accumulation.

 

The intensified red fluorescence and its perinuclear localization suggest that PSNPs are not only taken up by cells but also trafficked through the cytoplasm, potentially accumulating in vesicular structures such as endosomes or lysosomes. This distribution pattern aligns with earlier studies showing that nanoplastics can enter eukaryotic cells via endocytic pathways, including clathrin-mediated endocytosis and micropinocytosis (Viršek et al., 2017). Once internalized, PSNPs may evade lysosomal degradation due to their chemical stability and accumulate in intracellular compartments, leading to functional disruptions.

The close proximity of PSNPs to the nuclear envelope further raises concerns regarding possible interactions with nuclear components. Such interactions may compromise nuclear membrane integrity, interfere with nucleocytoplasmic transport, and influence processes such as DNA replication, transcription, and ribosome biogenesis. Disruption in these regulatory pathways could ultimately impact gene expression, promote genomic instability, or trigger stress-induced signaling cascades. Although nuclear entry was not directly confirmed in this specific image set, the fluorescence pattern observed is suggestive of near-nuclear or intranuclear localization, warranting further investigation.

Furthermore, the altered fluorescence intensity and distribution patterns in PSNP-treated cells may reflect changes in lipid metabolism or membrane composition. Nile Red, a lipophilic dye, preferentially binds to lipid-rich regions; therefore, increased fluorescence may result from lipid droplet accumulation, membrane remodeling, or PSNP association with lipid bilayers. This interpretation is consistent with previous studies demonstrating that nanoplastics can disturb membrane dynamics, alter lipid raft organization, and impair membrane-associated signaling pathways (Ji et al., 2024; Kumah et al., 2023; Lai et al., 2022). Collectively, these observations suggest that PSNP exposure may induce changes in lipid composition and membrane integrity, contributing to cytotoxic effects through both physical accumulation and biochemical disruption.

Structural Changes in Hepatocytes Following PSNP Exposure

The animal study using Rattus norvegicus revealed significant structural changes in hepatocytes following exposure to PSNPs, as illustrated in Figure 4. Fluorescent staining with DAPI (blue) and Nile Red (red) was utilized to assess hepatocyte morphology and PSNP distribution. In the control group (Figure 4A), hepatocytes displayed normal structural characteristics, with well-defined, round nuclei (white arrows) and uniform cytoplasmic distribution (yellow arrows). The absence of red fluorescence indicated no PSNP accumulation, suggesting healthy cellular conditions. With early-stage PSNP exposure (Figure 4B), red fluorescence began to appear within specific cytoplasmic regions, indicating initial PSNP infiltration. Although the nuclei remained intact, mild PSNP presence in the cytoplasm suggested the onset of cellular exposure to nanoplastics. As PSNP exposure progressed (Figure 4C), more pronounced red fluorescence was observed, demonstrating increased PSNP accumulation within both the cytoplasm and perinuclear regions. The red-stained spots were more abundant, highlighting the extensive uptake of PSNPs by hepatocytes. The appearance of red signals near the nuclei also suggested a potential translocation of particles toward the nuclear envelope.

In the advanced PSNP exposure stage (Figure 4D), red fluorescence was highly intensified, indicating significant PSNP accumulation within the hepatocyte cytoplasm and nucleus. The nuclear structural changes and the widespread red fluorescence suggest considerable cellular stress and possible functional disruptions due to PSNP exposure. The presence of PSNPs in the nucleus raises concerns about potential impacts on cellular processes, including gene expression regulation and genomic stability. PSNPs can penetrate biological membranes, including the plasma and nuclear membranes (Tarafdar et al., 2022). The fluorescence patterns observed in this study show a gradual infiltration of PSNPs into hepatocytes, starting from the cytoplasm (Figures 4B and 4C) and eventually reaching the nucleus (Figure 4D). Nanoplastics typically enter cells through endocytosis mechanisms, particularly macropinocytosis and clathrin-mediated endocytosis (Liu et al., 2021). The detection of PSNPs within the nucleus suggests either disruption of the nuclear envelope or active transport through the nuclear pore complex. The accumulation of PSNPs within the nucleus poses a significant risk, as it may induce DNA damage and increase the likelihood of genetic mutations, potentially disrupting hepatocyte function and liver metabolism (Haldar et al., 2023). Previous studies have shown that prolonged PSNP exposure can trigger oxidative stress, promote inflammatory responses, and impair organelle function within liver cells (Hu and Palić, 2020).

 

Given the liver’s critical role in detoxification and blood filtration, it is a primary target for nanoparticle accumulation (Campanale et al., 2020). PSNP-induced hepatotoxicity can manifest through alterations in hepatocyte morphology, increased intracellular vacuole formation, nuclear deformation, and enhanced apoptotic activity. Additionally, PSNP exposure can elevate reactive oxygen species (ROS) production, leading to oxidative stress, lipid peroxidation, and mitochondrial dysfunction (Deng et al., 2017; Saputra et al., 2025). Another critical observation is the disruption of liver metabolism, evidenced by changes in detoxification enzyme expression, which could impair hepatocyte function and overall liver health. The findings of this study confirm that PSNPs can infiltrate the cytoplasm and nucleus of Rattus norvegicus hepatocytes, as demonstrated by fluorescence staining with Nile Red and DAPI. The ability of PSNPs to reach the nucleus underscores their potential to cause cellular damage, disrupt liver function, and pose a substantial toxicological risk to the health of exposed organisms.

Intracellular Distribution of PSNPs and Potential Cellular Impact

Figure 5 illustrates the structural characteristics of Rattus norvegicus hepatocytes following PSNP exposure, highlighting the intracellular distribution of PSNP within the cytoplasm and nucleus. DAPI staining (blue) reveals round or oval hepatocyte nuclei with bright blue fluorescence, while Nile Red staining (red) indicates PSNP accumulation as red fluorescent dots. The presence of PSNP in both the cytoplasm and nucleus suggests successful internalization and potential for subcellular disruption.

The detection of PSNP within the nucleus is particularly concerning, as it indicates that nanoplastics can penetrate the nuclear membrane, likely through endocytosis or active transport via the nuclear pore complex (Dehghanian et al., 2023). This raises the possibility of direct interactions with nuclear components such as chromatin, transcription factors, or DNA repair machinery. Such interactions may impair gene regulation, disrupt nucleolar activity, or induce genomic instability (Misteli and Soutoglou, 2009). Although our study did not directly assess gene expression or DNA integrity, the close proximity of PSNPs to the nucleus and nucleolus strongly suggests a potential risk of DNA damage, altered transcription, and impaired ribosomal biogenesis. In addition to nuclear effects, the accumulation of PSNPs in the cytoplasm confirms active cellular uptake, likely through macropinocytosis or clathrin-mediated endocytosis. This internalization can trigger oxidative stress, inflammation, and mitochondrial dysfunction-factors known to indirectly influence gene expression by activating stress-responsive transcription pathways or epigenetic modifications (Mahmud et al., 2024). Furthermore, the presence of PSNPs in the extracellular space suggests their ability to migrate through tissues and enter systemic circulation, which could facilitate distribution to other vital organs. This observation aligns with previous reports that nanoplastics can cross biological barriers and accumulate in metabolically active tissues (Lai et al., 2022).

 

Given the liver’s critical role in detoxification and metabolic regulation, PSNP accumulation could significantly impair normal liver function, leading to hepatotoxic effects, including oxidative stress, lipid peroxidation, and mitochondrial dysfunction (Allameh et al., 2023). The progressive stages of PSNP infiltration (steps 1 to 6 in Figure 5) indicate not only physical accumulation within cells but also potential biochemical interactions with nuclear components. The proximity of PSNP to the nucleolus (step 6) raises concerns about possible disruptions to ribosomal biogenesis and alterations in cellular protein synthesis. Moreover, PSNP-induced oxidative stress may impair gene transcription, lead to cell cycle arrest, and activate pro-inflammatory pathways, potentially contributing to hepatocyte dysfunction and liver tissue necrosis (Shi et al., 2021). The ability of PSNPs to reach the hepatocyte nucleus has significant implications for human health and ecosystems. Accumulation in critical organs such as the liver and penetration into the nucleus suggest that PSNPs could similarly affect other vital organs. The liver’s primary role in metabolism and detoxification makes it especially vulnerable to metabolic disturbances caused by PSNP exposure. These particles could lead to hepatotoxicity, disrupt energy homeostasis, and interfere with normal liver function (Wang et al., 2024).

The long-term effects of PSNP accumulation in the body could contribute to chronic cellular damage and increase the risk of liver disease, cancer, and endocrine disorders (Amereh et al., 2020; Chiang et al., 2024). This study demonstrates that PSNPs can enter hepatocytes and infiltrate the nucleus, potentially causing oxidative stress, disrupting gene expression, and inducing other hepatotoxic effects. The mechanisms for PSNP entry include endocytosis and passive diffusion, with biological impacts linked to DNA damage, inflammation, and metabolic disturbances (Mahmud et al., 2024; Rajendran and Chandrasekaran, 2023).

CONCLUSIONS AND RECOMMENDATIONS

PSNPs can effectively penetrate cell membranes, accumulate in the cytoplasm, and reach the nucleus in both 3T3 fibroblast cells and Rattus norvegicus hepatocytes. Their intracellular presence is associated with pronounced morphological alterations, cytoplasmic disorganization, and evidence of cellular stress, suggesting interference with vital metabolic and structural functions. These effects likely occur via active endocytic pathways and possible nuclear transport mechanisms. The accumulation of PSNPs within critical subcellular compartments highlights their potential to disrupt membrane integrity, induce oxidative stress, and interfere with nuclear processes, raising important concerns about genotoxicity and long-term cellular health. These findings underscore the urgent need for further research into the chronic impacts of nanoplastic exposure, particularly studies that investigate molecular toxicity mechanisms, including inflammation, oxidative damage, and genetic instability.

ACKNOWLEDGEMENTS

This study was funded by Airlangga Research Fund Batch 2 from Universitas Airlangga at 2023 (907/UN3/2023).

NOVELTY STATEMENTS

This study provides new insights into the intracellular behavior of PSNPs, demonstrating their ability not only to accumulate within the cytoplasm but also to penetrate the nuclear membrane of both 3T3 fibroblast cells and Rattus norvegicus hepatocytes. This nuclear localization of PSNPs, particularly in hepatocytes, is a novel finding that highlights potential risks to cellular and genetic stability.

AUTHOR’S CONTRIBUTIONS

Febriyansyah Saputra, Alfiah Hayati, Adiibtia Septiani, Manikya Pramudya, Raden Joko Kuncoroningrat Susilo, Vuanghao Lim and Bayyinatul Muchtaromah: Conceptualized and designed the study and participated in the investigation and the drafting of the paper as well as, analyzed and interpreted the data and revised the paper critically for intellectual content and approved the final version of the paper to be published. All authors agree to be accountable for all aspects of the work.

Conflict of Interest

This study has no conflict interest between all of authors

REFERENCES

Ali N, Katsouli J, Marczylo EL, Gant TW, Wright S, Bernardino de la Serna J (2024). The potential impacts of micro-and-nano plastics on various organ systems in humans. eBioMedicine 99: 104901. https://doi.org/10.1016/j.ebiom.2023.104901

Allameh A, Niayesh-Mehr R, Aliarab A, Sebastiani G, Pantopoulos K (2023). Oxidative Stress in Liver Pathophysiology and Disease. Antioxidants (Basel), 12. https://doi.org/10.3390/antiox12091653

Allen S, Allen D, Karbalaei S, Maselli V, Walker TR (2022). Micro(nano)plastics sources, fate, and effects: What we know after ten years of research. J. Hazard. Mater. Adv., 6:, 100057. https://doi.org/10.1016/j.hazadv.2022.100057

Amereh F, Babaei M, Eslami A, Fazelipour S, Rafiee M (2020). The emerging risk of exposure to nano(micro)plastics on endocrine disturbance and reproductive toxicity: From a hypothetical scenario to a global public health challenge. Environ. Poll., 261: 114158. https://doi.org/10.1016/j.envpol.2020.114158

Bhardwaj G, Webster TJ (2015). Increased NIH 3T3 fibroblast functions on cell culture dishes which mimic the nanometer fibers of natural tissues. Int. J. Nanomed., 10: 5293-5299. https://doi.org/10.2147/IJN.S83007

Cai H, Xu EG, Du F, Li R, Liu J, Shi H (2021). Analysis of environmental nanoplastics: Progress and challenges. Chem. Eng. J., 410: 128208. https://doi.org/10.1016/j.cej.2020.128208

Campanale C, Massarelli C, Savino I, Locaputo V, Uricchio VF (2020). A Detailed Review Study on Potential Effects of Microplastics and Additives of Concern on Human Health. Int. J. Environ. Res. Public Health, 17. https://doi.org/10.3390/ijerph17041212

Chen X, Wang L, Liu K, Wang Q, Li R, Niu L, Wu H (2025). Maternal exposure to polystyrene nanoplastics induces sex-specific kidney injury in offspring. Ecotoxicol. Environ. Saf., 293: 118006. https://doi.org/10.1016/j.ecoenv.2025.118006

Chiang CC, Yeh H, Shiu RF, Chin WC, Yen TH (2024). Impact of microplastics and nanoplastics on liver health: Current understanding and future research directions. World J. Gastroenterol., 30: 1011-1017. https://doi.org/10.3748/wjg.v30.i9.1011

Das SK, Sen K, Ghosh B, Ghosh N, Sinha K, Sil PC (2024). Molecular mechanism of nanomaterials induced liver injury: A review. World J. Hepatol., 16: 566-600. https://doi.org/10.4254/wjh.v16.i4.566

Dehghanian Z, Asgari Lajayer B, Biglari Z, Nayeri S, Ahmadabadi M, Taghipour L, Senapathi V, Astatkie T, Price GW (2023). Micro (nano) plastics uptake, toxicity and detoxification in plants: Challenges and prospects. Ecotoxicol. Environ. Saf., 268: 115676. https://doi.org/10.1016/j.ecoenv.2023.115676

Deng Y, Zhang Y, Lemos B, Ren H (2017). Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Sci. Rep., 7: 46687. https://doi.org/10.1038/srep46687

Dong Y, Johnson BA, Ruan L, Zeineldin M, Bi T, Liu AZ, Raychaudhuri S, Chiu I, Zhu J, Smith B, Zhao N, Searson P, Watanabe S, Donowitz M, Larman TC, Li R (2024). Disruption of epithelium integrity by inflammation-associated fibroblasts through prostaglandin signaling. Sci. Adv., 10: eadj7666. https://doi.org/10.1126/sciadv.adj7666

Gilani IE, Sayadi S, Zouari N, Al-Ghouti MA (2023). Plastic waste impact and biotechnology: Exploring polymer degradation, microbial role, and sustainable development implications. Bioresour. Technol. Rep., 24: 101606. https://doi.org/10.1016/j.biteb.2023.101606

Haldar S, Yhome N, Muralidaran Y, Rajagopal S, Mishra P (2023). Nanoplastics Toxicity Specific to Liver in Inducing Metabolic Dysfunction-A Comprehensive Review. Genes (Basel), 14. https://doi.org/10.3390/genes14030590

Hayati A, Rohmatika AU, Nurbani FA, Safitri M, Pramudya M, Susilo RJK (2024a). Nanoplastik dan Bahan Alam: Dampak dan Solusi. Airlangga University Press, Surabaya. https://omp.unair.ac.id/aup/catalog/download/1264/684/2831?inline=1.

Hayati A, Wilujeng WP, Pramudya M, Dewi FRP, Muchtaromah B, Raden JK (2024b). The Effect of Exposure to Polystyrene Nanoplastics on Cytokine Levels and Reproductive System of Male Tilapia. Trop J Nat Prod Res. 8:6300–3. https://doi.org/10.26538/tjnpr/v8i2.30

Hayati A, Pramudya M, Soepriandono H, Wahyuni AD, Dewi FRP (2023). Effects of Medicinal Plants Rhizome on Growth Performance of Tilapia (Oreochromis niloticus) Exposed to Micro Plastics. AIP Conference Proceeding. 2554, 090012. https://doi.org/10.1063/5.0105225

Hayati A, Pramudya M, Soepriandono H, Adriansyah W, Dewi FRP (2022). Assessing the recovery of steroid levels and gonadal histopathology of tilapia exposed to polystyrene particle pollution by supplementary feed. Veterinary World, 15(2), 517–523. https://doi.org/10.14202/vetworld.2022.517-523

Hu M, Palić D (2020). Micro- and nano-plastics activation of oxidative and inflammatory adverse outcome pathways. Redox Biol., 37: 101620. https://doi.org/10.1016/j.redox.2020.101620

Itri R, Junqueira HC, Mertins O, Baptista MS (2014). Membrane changes under oxidative stress: the impact of oxidized lipids. Biophys. Rev., 6: 47-61. https://doi.org/10.1007/s12551-013-0128-9

Janani R, Bhuvana S, Geethalakshmi V, Jeyachitra R, Sathishkumar K, Balu R, Ayyamperumal R (2024). Micro and nano plastics in food: A review on the strategies for identification, isolation, and mitigation through photocatalysis, and health risk assessment. Environ. Res., 241: 117666. https://doi.org/10.1016/j.envres.2023.117666

Ji Y, Wang Y, Wang X, Lv C, Zhou Q, Jiang G, Yan B, Chen L (2024). Beyond the promise: Exploring the complex interactions of nanoparticles within biological systems. J. Hazard. Mater., 468: 133800. https://doi.org/10.1016/j.jhazmat.2024.133800

Khan A, Jia Z (2023). Recent insights into uptake, toxicity, and molecular targets of microplastics and nanoplastics relevant to human health impacts. Science, 26: 106061. https://doi.org/10.1016/j.isci.2023.106061

Khan AH, Zou Z, Xiang Y, Chen S, Tian XL (2019). Conserved signaling pathways genetically associated with longevity across the species. Biochimica et Biophysica Acta (BBA) – Mol. Basis Dis., 1865: 1745-1755. https://doi.org/10.1016/j.bbadis.2018.09.001

Kiran BR, Kopperi H, Venkata Mohan S (2022). Micro/nano-plastics occurrence, identification, risk analysis and mitigation: challenges and perspectives. Rev. Environ. Sci. Bio. Technol., 21: 169-203. https://doi.org/10.1007/s11157-021-09609-6

Kumah EA, Fopa RD, Harati S, Boadu P, Zohoori FV, Pak T (2023). Human and environmental impacts of nanoparticles: a scoping review of the current literature. BMC Public Health, 23: 1059. https://doi.org/10.1186/s12889-023-15958-4

Lai H, Liu X, Qu M (2022). Nanoplastics and Human Health: Hazard Identification and Biointerface. Nanomaterials (Basel), 12. https://doi.org/10.3390/nano12081298

Lee SE, Yi Y, Moon S, Yoon H, Park YS (2022). Impact of Micro- and Nanoplastics on Mitochondria. Metabolites, 12. https://doi.org/10.3390/metabo12100897

Li Y, Chen L, Zhou N, Chen Y, Ling Z, Xiang P (2024). Microplastics in the human body: A comprehensive review of exposure, distribution, migration mechanisms, and toxicity. Sci. Total Environ., 946: 174215. https://doi.org/10.1016/j.scitotenv.2024.174215

Liu L, Xu K, Zhang B, Ye Y, Zhang Q, Jiang W (2021). Cellular internalization and release of polystyrene microplastics and nanoplastics. Sci. Total Environ., 779: 146523. https://doi.org/10.1016/j.scitotenv.2021.146523

Liu Y, Hu Y, Yang C, Chen C, Huang W, Dang Z (2019). Aggregation kinetics of UV irradiated nanoplastics in aquatic environments. Water Res., 163: 114870. https://doi.org/10.1016/j.watres.2019.114870

Lv S, Wang Q, Li Y, Gu L, Hu R, Chen Z, Shao Z (2024). Biodegradation of polystyrene (PS) and polypropylene (PP) by deep-sea psychrophilic bacteria of Pseudoalteromonas in accompany with simultaneous release of microplastics and nanoplastics. Sci. Total Environ., 948: 174857. https://doi.org/10.1016/j.scitotenv.2024.174857

Mahmud F, Sarker DB, Jocelyn JA, Sang QA (2024). Molecular and Cellular Effects of Microplastics and Nanoplastics: Focus on Inflammation and Senescence. Cells, 13. https://doi.org/10.3390/cells13211788

Mazumdar S, Chitkara D, Mittal A (2021). Exploration and insights into the cellular internalization and intracellular fate of amphiphilic polymeric nanocarriers. Acta Pharm. Sin., B 11: 903-924. https://doi.org/10.1016/j.apsb.2021.02.019

Misteli T, Soutoglou E (2009). The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat. Rev. Mol. Cell. Biol., 10: 243-254. https://doi.org/10.1038/nrm2651

Nguyen B, Claveau-Mallet D, Hernandez LM, Xu EG, Farner JM, Tufenkji N (2019). Separation and Analysis of Microplastics and Nanoplastics in Complex Environmental Samples. Acc. Chem. Res., 52: 858-866. https://doi.org/10.1021/acs.accounts.8b00602

Nurbani FA, Pramudya M, Safitri M, Sugiharto, Ainurohim, A. Hayati (2025). Potential of Cinnamomum burmanni Leaf Extract as an Exogenous Antioxidant and Spermatoprotective for Rattus norvegicus L. Exposed to Polystyrene Nanoplastics. HAYATI (Journal Biosci). 32(3):693–9. https://doi.org/10.4308/hjb.32.3.693-699

Pitt JA, Trevisan R, Massarsky A, Kozal JS, Levin ED, Di Giulio RT (2018). Maternal transfer of nanoplastics to offspring in zebrafish (Danio rerio): A case study with nanopolystyrene. Sci. Total Environ., 643: 324-334. https://doi.org/10.1016/j.scitotenv.2018.06.186

Rajendran D, Chandrasekaran N (2023). Journey of micronanoplastics with blood components. RSC Adv., 13: 31435-31459. https://doi.org/10.1039/D3RA05620A

Saputra F, Pramata AD, Soegianto A, Hu SY (2025). Vitamin E Mitigates Polystyrene-Nanoplastic-Induced Visual Dysfunction in Zebrafish Larvae. Int. J. Mol. Sci., 26: 1216. https://doi.org/10.3390/ijms26031216

Saputra F, Pramata AD, Soegianto A, Hu S-Y (2025). Polystyrene nanoplastics cause developmental abnormalities, oxidative damage and immune toxicity in early zebrafish development. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 295, 110216. https://doi.org/10.1016/j.cbpc.2025.110216

Schirinzi GF, Pérez-Pomeda I, Sanchís J, Rossini C, Farré M, Barceló D (2017). Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environ. Res., 159: 579-587. https://doi.org/10.1016/j.envres.2017.08.043

Shi S, Wang L, van der Laan LJW, Pan Q, Verstegen MMA (2021). Mitochondrial Dysfunction and Oxidative Stress in Liver Transplantation and Underlying Diseases: New Insights and Therapeutics. Transplantation, 105: 2362-2373. https://doi.org/10.1097/TP.0000000000003691

Shorny A, Steiner F, Hörner H, Skoff SM (2023). Imaging and identification of single nanoplastic particles and agglomerates. Scientific Reports 13, 10275. https://doi.org/10.1038/s41598-023-37290-y

Tarafdar A, Choi SH, Kwon JH (2022). Differential staining lowers the false positive detection in a novel volumetric measurement technique of microplastics. J. Hazard. Mater., 432: 128755. https://doi.org/10.1016/j.jhazmat.2022.128755

Triwahyudi H, Soehargo L, Muniroh L, Qolbi RN, Aini TQ, Kurnia RFZ, Hayati A (2023). Potential of Red Seaweed ( Dichotomania obtusata ) on Immune Response and Histopathology of Rat Testis Exposed to Nanoplastics. Trop J Nat Prod Res. 7:2969–73. http://www.doi.org/10.26538/tjnpr/v7i5.20

Viršek MK, Lovšin MN, Koren Š, Kržan A, Peterlin M (2017). Microplastics as a vector for the transport of the bacterial fish pathogen species Aeromonas salmonicida. Mar. Pollut. Bull., 125: 301-309. https://doi.org/10.1016/j.marpolbul.2017.08.024

Wang H, Shi X, Gao Y, Zhang X, Zhao H, Wang L, Zhang X, Chen R (2022). Polystyrene nanoplastics induce profound metabolic shift in human cells as revealed by integrated proteomic and metabolomic analysis. Environ. Int., 166: 107349. https://doi.org/10.1016/j.envint.2022.107349

Wang J, Cong J, Wu J, Chen Y, Fan H, Wang X, Duan Z, Wang L (2023). Nanoplastic-protein corona interactions and their biological effects: A review of recent advances and trends. TrAC Trends Anal. Chem., 166: 117206. https://doi.org/10.1016/j.trac.2023.117206

Wang X, Deng K, Zhang P, Chen Q, Magnuson JT, Qiu W, Zhou Y (2024). Microplastic-mediated new mechanism of liver damage: From the perspective of the gut-liver axis. Sci. Total Environ., 919: 170962. https://doi.org/10.1016/j.scitotenv.2024.170962

Xu M, Halimu G, Zhang Q, Song Y, Fu X, Li Y, Li Y, Zhang H (2019). Internalization and toxicity: A preliminary study of effects of nanoplastic particles on human lung epithelial cell. Sci. Total Environ., 694: 133794. https://doi.org/10.1016/j.scitotenv.2019.133794

Yong CQY, Valiyaveettil S, Tang BL (2020). Toxicity of Microplastics and Nanoplastics in Mammalian Systems. Int. J. Environ. Res. Public Health, 17: 1509. https://doi.org/10.3390/ijerph17051509

Zhang X, Yin Z, Xiang S, Yan H, Tian H (2024). Degradation of Polymer Materials in the Environment and Its Impact on the Health of Experimental Animals: A Review. Polymers, 16: 2807. https://doi.org/10.3390/polym16192807

Zhang Y, Kang S, Allen S, Allen D, Gao T, Sillanpää M (2020). Atmospheric microplastics: A review on current status and perspectives. Earth-Sci. Rev., 203: 103118. https://doi.org/10.1016/j.earscirev.2020.103118

Zheng Y, Sun J, Luo Z, Li Y, Huang Y (2024). Emerging mechanisms of lipid peroxidation in regulated cell death and its physiological implications. Cell Death Dis., 15: 859. https://doi.org/10.1038/s41419-024-07244-x