Pulmonary Toxicity of Polymethyl methacrylate nanoplastics via Intratracheal Intubation in Mice

Pulmonary Toxicity of Polymethyl methacrylate nanoplastics via Intratracheal Intubation in Mice | 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 Article Pulmonary Toxicity of Polymethyl methacrylate nanoplastics via Intratracheal Intubation in Mice Changsic Youn, Yu-Jin Jo, Jeongwoo Kwon, Seung-Bin Yoon, Hyeong-Ju You, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7466865/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Plastics, ubiquitous in daily life and industry, are released into the environment in substantial quantities. Instead of complete biodegradation, plastic waste fragments into smaller particles, accumulating as nanoplastics (NPs; <1 μm). Humans are exposed to NPs through inhalation and ingestion of contaminated water and food, which can induce cytotoxicity through physical and chemical pathways. Polymethyl methacrylate (PMMA), commonly used in implants and artificial bones, has been identified in human lungs and associated with pulmonary embolism. While PMMA NP toxicity has been reported in vitro, their in vivo effects, as well as the underlying mechanism, remain poorly understood. In this study, we investigated the pulmonary effects of inhaled PMMA NPs in mice. Mice received 20 or 100 μg of PMMA NPs (25 nm) via intratracheal intubation for 28 days. PMMA-NP preparation and characterization are described in the Methods section. Exposed mice exhibited body weight loss and pulmonary accumulation of PMMA NPs. Bronchoalveolar lavage fluid (BALF) analysis revealed increased cell count and elevated inflammatory cytokines in serum and BALF. Histopathology (H&E staining) revealed abnormalities in lung tissue and alterations in protein and RNA expression. The findings demonstrate that respiratory exposure to PMMA NPs induces lung inflammation, tissue damage, and molecular dysregulation. Biological sciences/Biotechnology Health sciences/Diseases Health sciences/Medical research Nanoplastic Lung toxicity Intratracheal intubation Inflammation PMMA Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In modern society, plastics are extensively used, encompassing daily life, industry, medicine, and agriculture. Global plastic consumption is projected to increase annually from 464 Mt in 2020 to 884 Mt by 2050 1 . This increase will lead to a proportional rise in plastic waste, which is becoming a serious environmental concern. If current production and waste management trends persist, approximately 12,000 Mt of plastic waste will accumulate in landfills or leak into the environment by 2050, with severe consequences for the ecosystem 2 . Among plastic waste, microplastics (MPs; 1 µm to 1 mm) and nanoplastics (NPs; <1 µm), which are not visible to the naked eye, have emerged as contaminants of increasing ecological and public health concern. These MPs and NPs exist in various shapes (Spheres, fibers, and particles) and polymer types (e.g., polyethylene [PE], polystyrene [PS], polypropylene [PP], polyethylene terephthalate [PET], and polymethyl methacrylate [PMMA]) 3 – 5 . NPs generally accumulate in the environment through various degradation of larger plastics and MPs via mechanical abrasion, chemical oxidation, biodegradation, and photooxidation 6 – 13 . Due to their small size, NPs can easily cross cellular membranes, potentially exerting more severe impacts than MPs 14 . In marine environment, NPs can bioaccumulate and biomagnify in marine organisms ranging from phytoplankton, zooplankton, crustaceans, mollusks, and fish, resulting in impaired growth, altered development and reproduction, behavioral changes, and increased mortality 13 , 15 – 17 . This can disrupt the balance of marine ecosystems. NPs are also ubiquitous in the soil, where they can be ingested by soil microorganisms and animals, accumulate, disrupt plant physiology and induce oxidative stress, thereby affecting plant growth and biochemistry 18 . Humans can be exposed to MPs and NPs primarily through ingestion (contaminated food and water) and inhalation. Airborne particles can also penetrate the respiratory tract, reaching the lungs 19 . These plastic particles can accumulate in humans, and have been detected in human feces, lungs, brain, blood, placenta, and saliva 20 – 25 . Their presence in various tissues and organs of the human body poses potential health risks. Multiple toxicological studies have been conducted on MP and NP. In vitro experiments showed that exposure to NPs causes various adverse effects, including disruption of intestinal epithelial tight junctions, oxidative stress, granulosa cell apoptosis, and hepatocyte fibrosis 26 – 30 . In vivo studies in mammals demonstrate that exposure can cause neurological dysfunction, liver and kidney damage, sperm quality deterioration, testicular and ovarian injury, and transplacental transfer of particles 26 , 28 , 29 , 31 – 34 . However, existing studies have limitations. First, in vitro experiments are limited because they cannot fully replicate the complex in vivo environment. Second, even during in vivo experiments, most studies simulate oral, dermal, or waterborne exposure, and only limited models directly exposed NPs through the respiratory system. Third, research tends to be biased toward certain types of plastics. For example, it has been shown that PMMA, which is widely used in medical procedures, such as vertebroplasty, has been implicated in postoperative pulmonary embolism and lung damage 35 – 37 . Although PMMA NPs are detected in the lungs of humans, in vivo studies on their pulmonary effects remain scarce. Therefore, investigating the pulmonary toxicity of PMMA-NPs is crucial, given their widespread medical use and their confirmed presence in human lungs. This study aims to address this gap by directly exposing animal models to NPs via the respiratory tract, thereby overcoming the limitations of previous studies. To assess the subacute effects of repeated exposure, we implemented a 28-day exposure regimen, consistent with standard toxicological assessment guidelines. Mice were exposed daily to PMMA NPs through intratracheal intubation and pulmonary toxicity was identified by monitoring diseases and symptoms that occurred in the tissues. Results NPs inhaled through the airways accumulate in the lungs To investigate the effects of NP inhalation on the respiratory system, six-week-old mice were exposed to NPs daily via intratracheal intubation under respiratory anesthesia for 28 days (Fig. 1 a). To confirm that the exposure to NPs via the respiratory system resulted in accumulation in the lungs, we performed in vivo fluorescence imaging (Fig. 1 b). We observed that the fluorescence intensity significantly increased in the 100 µg group compared to the control group (Fig. 1 c). To determine the effects on mice, we measured their body weight. Body weight gain was significantly reduced in the treatment group compared to that in the control group (Supplementary Data S1A and S1B), although no significant difference was observed in the lengths of the mice. However, the Lee index (length from the head to the anus relative to the body weight) and the body width of the mice were both significantly reduced in the treatment group (Supplementary Data S1B). These results confirm that respiratory exposure to NPs leads to pulmonary accumulation and physiological changes. Inhalation of NPs causes lung diseases Bronchoalveolar lavage fluid (BALF) is typically used to determine the effect of NPs on the lungs and to evaluate lung injury 38 , 39 . We measured protein levels and changes in cell count in the BALF. Cell counts were significantly higher in the treatment group than that in the control group (Fig. 2 a), and the bicinchoninic acid (BCA) assay also indicates a significant increase in total protein level in the treated groups (Fig. 2 b). Next, H&E staining was performed to confirm lung disease. The invasion of immune and foam cells was confirmed in the treated group (Fig. 2 c). Additionally, immunofluorescence imaging further confirmed NPs accumulation not only within lung tissue but also within inflammatory cells (Supplementary Data S2A). These results indicate that the respiratory exposure to NPs can result in lung disease. Inhalation of NPs causes lung inflammation NP exposure increased total cell counts, protein levels, and inflammatory cell infiltration in BALF. To confirm inflammation in the mice, serum levels of proinflammatory cytokines (IL-6, IL-1β, TNF-α) were measured. The results confirm that the levels of inflammatory cytokines were significantly increased in the 100 µg group (Fig. 3 a). Next, we measured the levels of inflammatory cytokines in the BALF to confirm the occurrence of lung inflammation. The levels of IL-6, IL-1β, and TNF-α were significantly increased in the 100 µg group and TNF-α was significantly increased in the 20 µg group (Fig. 3 b). To confirm which inflammatory cell changes in the lungs caused the inflammation, we then measured the number of inflammatory cells (macrophages, lymphocytes, eosinophils, neutrophils, and basophils) using H&E staining in BALF. In the NP treatment group, the number of macrophages significantly decreased, whereas the numbers of lymphocytes and neutrophils significantly increased in the 100 µg group (Fig. 3 c, 3 d). Foamy macrophages were identified and their area ratio was significantly increased in the NPs treatment group (Fig. 3 e). Additionally, immunofluorescence revealed that the intensity of foamy macrophages and lung damage CD36, as well as their colocalization with lung tissue, were significantly increased in the 100 µg group (Supplementary Data S2A, S2B and S2C) 40 , 41 . To determine whether the levels of inflammatory cytokines were elevated in the lung tissue, we examined both protein and mRNA levels. A significant increase in both protein and mRNA levels of inflammatory cytokines was observed in the 100 µg group (Fig. 3 f, 3 g, and 3 h). These results confirm that respiratory exposure to NPs induces the formation of foamy macrophages and promotes lung inflammation. Inhalation of NPs causes lung fibrosis The results indicate abnormalities in the lung tissue. To investigate the potential induction of fibrosis, lung tissues exposed to NP were subjected to Sirius Red and Masson's trichrome staining (Fig. 4 a and 4 b). The results show that the percentage of Sirius Red-positive areas and Masson's trichrome-positive lung tissue significantly increased in the NP group (Fig. 4 c and 4 d). Correspondingly, protein levels of collagen type 1 (COL-1) and α-smooth muscle actin (α-SMA), key genes associated with fibrosis, were significantly elevated in the NP group (Fig. 4 e and 4 f). Collectively, these results suggest that NP exposure induced fibrotic changes in the lung. Discussion In this study, we investigated the effects of PMMA NPs on the respiratory system of mice. Our results reveal that mice exposed to PMMA NPs exhibit a significant decrease in body weight gain, accompanied by reductions in the obesity index, Lee's index, and body width. Serum analysis further confirmed an increase in inflammatory cytokines, specifically IL-6, IL-1β, and TNF-α. These systemic inflammatory responses likely contribute to the reduced weight gain because IL-6 and TNF-α are known mediators of inflammatory-induced anorexia 42 – 45 . Additionally, increased energy expenditure due to lung injury and systemic inflammation may further contribute to weight loss. These results suggest that respiratory exposure to PMMA NPs can lead to local pulmonary toxicity, accompanied by systemic inflammatory responses and potential metabolic changes. BALF analysis is widely used for evaluating lung disease in clinical settings. In this study, we observed a significant increase in both total cell counts and protein levels in the BALF. Elevated protein in the BALF can result from serum protein leakage across the air–blood barrier, local production by pulmonary immune and epithelial cells, including T cells, alveolar macrophages, bronchial epithelial cells, and alveolar epithelial cells type I and II 46 . Total cell counts in BALF commonly increases due to increase in immune cells during inflammation, pulmonary fibrosis, smoking, and lung cancer 47 – 49 . Our results are consistent with these findings, showing a significant increase in BALF inflammatory cytokines, marked inflammatory cell infiltration in the lung tissue, and the formation of foamy macrophages, thus confirming the induction of lung inflammation. Notably, the results of this study indicate the formation of foamy macrophages (foam cells, characterized by numerous cytoplasmic vacuoles) in the lung tissue and immune cells in the BALF. Foam cells arise when macrophages engulf excessive amounts of altered lipids (e.g., oxidized lipids) or particulates and accumulate them as lipid droplets in their cytoplasm. Beyond lipid storage, foam cells influence immune responses by secreting inflammatory cytokines and altering antigen presentation 50 . Their presence has been consistently documented in various pulmonary pathological conditions, including inflammation, fibrosis, Chronic Obstructive Pulmonary Disease (COPD), tuberculosis, lung granuloma, and COVID-19 51–55 . Previous research has shown that exposure to cigarette smoke, particulate matter, and nanosilica can induce lung diseases, such as inflammation, and cause macrophage dysfunction, leading to foam cell formation 56 – 59 . Consistent with this, our supplementary data confirm that PMMA NPs are primarily present in alveolar macrophages. This suggests that PMMA NPs may alter the lung environment through inflammation and promote differentiation into foam cells, potentially via lipid dysregulation within macrophages that internalize the NPs or direct cytotoxicity of the particles. Immunofluorescence and further studies confirmed PMMA-NP uptake by macrophages and revealed a significant increase in the expression of the scavenging receptor CD36. Since CD36 is known to mediate the uptake of oxidized lipids and particulate matter, this suggests that macrophages can actively internalize PMMA NPs through a CD36-mediated pathway 60 . This mechanism is likely a critical first step in the cascade leading to foam cell formation, altered lipid metabolism, and inflammatory cytokine secretion. The increased CD36 expression suggests a potential molecular link between PMMA-NP exposure and the observed macrophage dysfunction and lung pathology. Further studies are needed to elucidate this direct interaction and its signaling pathways. The results of the BALF analysis confirm that the proportions of neutrophils and lymphocytes increase following PMMA-NP exposure. This increase in the number of inflammatory cells in the lungs suggests that PMMA-NP exposure triggers an inflammatory response in the lungs. Neutrophils play an important role in innate immune responses to pathogens, such as bacteria and fungi, as well as to non-infectious stimuli. However, if inappropriately or excessively activated in the absence of infection, they secrete proteolytic enzymes and reactive oxygen species (ROS), leading to tissue injury and chronic inflammation 61 – 64 . Lymphocytes, comprising T cells, B cells, and natural killer (NK) cells, are vital components of the immune system. These specialized cells function in antibody production, direct cytotoxicity against virus-infected and tumor cells, and regulation of overall immune responses. When the immune system is activated, there's a noticeable increase in lymphocyte proliferation, which signals a robust and effective immune response 65 . Recruitment and activation of these immune cells in the lungs is regulated by proinflammatory cytokine. TNF-α and IL-1, activate vascular endothelial cells, facilitate cellular organization, and promote chemotaxis of macrophages and neutrophils to the lungs 66 – 70 . IL-6 is also a key player in immune responses and inflammation, and its expression and release are induced by various stimuli, including lipopolysaccharides (LPS), poly (I), poly (C), IL-1, tumor necrosis factor (TNF), and platelet-derived growth factor 71 – 73 . In this inflammatory environment, alveolar macrophages can differentiate into foam cells by internalizing PMMA NPs. Foam cells are associated with chronic inflammation, and their formation may interfere with the immune function of macrophages 74 . Foamy macrophages can also secrete inflammatory cytokines (like IL-1 and IL-6), further promoting inflammation 50 , 75 . This suggests a cascade: PMMA-NP exposure damages and irritates the lung tissue, leading to an increased secretion of proinflammatory cytokines such as TNF-α and IL-1. This, in turn, recruits immune cells, such as neutrophils and lymphocytes, from the blood into the lungs. During this process, macrophages undergo lipid-induced differentiation into foam cells. It has been hypothesized that these foam cells further amplify the pulmonary inflammatory response by secreting inflammatory cytokines, such as IL-1 and IL-6, creating a self-perpetuating inflammatory cycle. This study also confirmed an increased expression of collagen and α-SMA in lung tissue following PMMA-NP exposure, both of which are major indicators of fibrosis. This increase in proteins suggests that the lung tissue accumulates excessive extracellular matrix (ECM) in response to damage, activating fibroblasts and differentiating them into myofibroblasts 76 , 77 . Furthermore, Sirius Red and Masson's trichrome staining confirmed increased collagen deposition, indicating the progression of lung fibrosis. Inflammatory cytokines of the IL-1 family (e.g., IL-1β, IL-18) are well-known major mediators of fibrosis 78 . In this study, both IL-1β and IL-18 in the PMMA NPs exposure group were significantly elevated compared to the control group, suggesting that exposure to PMMA NPs can induce an inflammatory response and promote fibrosis. In addition, foam cells may also exacerbate fibrosis by secreting IL-1 family cytokines, stimulating surrounding cells, and reinforcing a fibrotic environment 50 , 79 . In this study, foamy macrophages were examined after exposure to PMMA NPs, suggesting that fibrosis was induced by the secretion of fibrotic cytokines (IL-1 family) from damaged and inflamed tissues and foamy macrophages. Although this study provides valuable insights into the pulmonary toxicity of PMMA NPs, it has several limitations. First, systemic effects were not evaluated. As shown in Supplementary Data S1, the weight loss observed after the exposure to PMMA NPs suggests possible systemic involvement. While such systemic effects have been predicted, impacting other organs as well, this study did not confirm them. Further studies are needed to confirm whether inhaled NPs translocate to other organs and induce pathological changes. Second, the conditions are limited. In atmospheric environments, MPs and NPs occur in various sizes, shapes, and chemical compositions exist in a complex manner 80 , 81 . However, only spherical PMMA NPs with a specific size (25 nm) were used in this study. Therefore, it is difficult to generalize the results of this study to human exposure scenarios and other types of plastics. Further comparative toxicological studies across a range of plastic type and size are required. Third, a detailed mechanistic study is required. This study confirms that PMMA NPs cause lung inflammation and fibrosis; however, detailed mechanisms, such as the specific cellular and molecular signaling pathways that cause these pathological phenomena, have not been identified and in-depth research is required. Fourth, the focus of this study was placed on elucidating the toxicity of PMMA NPs. Approaches using rescue agents to alleviate or prevent their toxic effects (inflammation and fibrosis) were not explored. Follow-up rescue studies are required to reduce the health risks associated with NPs exposure. Fifth, this study was conducted using a single-sex cohort (female mice). Sex-specific differences can significantly influence the toxicological consequences of NP exposure. Therefore, while valuable, the results of this study may not be fully generalizable to all mice, regardless of gender. Future studies should include comparative studies between male and female mice to assess potential sex-specific differences in PMMA-NP toxicity and provide a more comprehensive risk assessment. Addressing these limitations will provide insights into the potential health risks associated with NPs and facilitate the development of effective countermeasures. In conclusion, this study evaluated the potential respiratory toxicity of PMMA NPs in humans through environmental inhalation exposure. Our findings confirm that inhaled PMMA NPs accumulate in the lung tissues, particularly within the cytoplasm of alveolar macrophages, and induce several pathological changes. Specifically, PMMA exposure triggers a pulmonary inflammatory response characterized by increased infiltration of inflammatory cells (e.g., neutrophils, lymphocytes) into the alveolar space and an increase in the secretion of inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α). Furthermore, structural lung damage and pathological cellular changes, including pulmonary fibrosis with excessive collagen deposition and foam cell formation, reflecting disrupted lipid metabolism and morphological alterations in macrophages. These findings suggest that PMMA NPs accumulate in the lungs and induce pulmonary inflammation, fibrosis, and changes in the distribution and differentiation of inflammatory cells, posing a significant threat to respiratory health. Methods Animals All animal experiments were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-24311). All animal procedures were performed in compliance with relevant institutional guidelines and regulations. This study’s animal experiments are reported in accordance with the ARRIVE guidelines ( http://arriveguidelines.org ) to ensure transparency and reproducibility. Six- to seven-week-old female ICR mice were housed at 23 ± 1°C under a 12-hour light/dark cycle, with food and water ad libitum. Nanoparticle intratracheal intubation PMMA NPs with a size of 25 nm (Cat No: 01-00-251), and the same particles conjugated with green fluorescent dye (Cat No: 29-00-251) were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). Six-week-old mice weighing an average of 24–26 g were exposed to NPs via daily intratracheal intubation for 28 days. Mice were were anesthetized using 1.5% isoflurane inhalation in an induction chamber. Isoflurane was mixed with oxygen, and all anesthetic procedures were controlled using a anesthetic machine (ROYAL-77S; Elpis Medical, Gangwon-do, Korea), and NPs were administered via a catheter. The control group received 50 µL of saline, and the treatment groups were exposed to 20 µg/50 µL and 100 µg/50 µL of NPs. Body weight was measured daily shortly before NP administration. Mice were sacrificed 24 h after the final exposure and samples were collected. Preparation and analysis of bronchoalveolar lavage fluid After 28 days of NP treatment, BALF was obtained by washing the lungs three times with 1 mL of saline. BALF was centrifuged at 300 × g for 7 min at 4°C, and the supernatant was collected for Enzyme-Linked Immunosorbent Assay (ELISA) and BCA analysis. The pellet was resuspended in 200 µL of saline, and the saline was dried onto slides. Each slide was stained with hematoxylin and eosin (H&E), and 400 cells were selected. The slides were examined using a light microscope (ICC50 E, Leica Microsystems, Wetzlar, Germany). Individual cells were classified according to cell and nuclear morphology (basophils, characterized by the presence of numerous small granules; neutrophils, identified by 3–5 segmented nuclei; eosinophils, distinguished by pink-stained cytoplasm and bilobed nuclei; monocytes, identified by large nuclei; and macrophages, identified by irregular shape and smaller nuclei compared to monocytes). Fluorescence measurements of the lungs After 28 days of NP treatment, fluorescence imaging was performed using an in vivo imaging system (Uinv-420; Davinch-K, Seoul, Korea) to determine the accumulation of NPs in the lungs. Histological analysis Fixed lung tissues were processed for paraffin block production using an automated tissue processor (HistoCore PEARL; Leica Microsystems, Wetzlar, Germany) and paraffin embedding station (HistoCore Arcadia H; Leica Microsystems, Germany). Paraffin blocks were sectioned at a thickness of 5 µm to produce slides, which were stained with H&E for histological analysis. Stained sections were visualized using a slide scanner (Aperio VERSA 8, Leica Microsystems, Germany) to determine the presence or absence of lung disease. BALF and ELISA analysis To measure protein levels in BALF, the supernatants were analyzed using the BCA Assay (Pierce™ BCA Protein Assay Kits, Thermo Fisher Scientific, Waltham, MA, USA). To assess systemic and pulmonary inflammatory cytokine levels (IL-6, IL-1β, TNF-α), serum and BALF samples were tested using ELISA kits (M6000B-1, MLB00C-1, MTA00B-1, R&D Systems, Minneapolis, USA) according to the manufacturer's instructions. BCA and ELISA signals were measured using a microplate reader (EPOCH2, BioTek, Vermont, USA). Picosirius red staining Paraffin-embedded lung tissue slides were deparaffinized three times with xylene for 15 min each, rehydrated twice with 100% ethanol for 1 min each and twice with 95% ethanol for 1 min each, and finally rehydrated in distilled water for 5 min. Lung tissue sections were stained with picrosirius red solution (ab150681; Abcam, Cambridge, UK) for 1 h and washed with 5% acetic acid. Finally, sections were dehydrated several times with ethanol, washed with xylene, and fixed with toluene. Lung tissue sections were stained with picrosirius red and visualized using a slide scanner (Aperio VERSA 8; Leica Microsystems, Germany). Masson’s trichrome staining deparaffinized lung tissue slides were stained using a trichrome staining kit (ab150686, Abcam, UK) according to the manufacturer's instructions. Lung tissue sections were stained with Masson's trichrome and visualized using a slide scanner (Aperio VERSA 8; Leica Microsystems, Germany). Western blotting Lung tissues were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Pierce RIPA Buffer, 89901, Thermo Fisher Scientific, USA), and 1 mL PhosSTOP (PhosSTOP EASYpack; Roche, Chugai, Switzerland) was used for protein extraction. The tissue lysates were prepared using beads and incubated on ice for 10 min. The lysed tissues were centrifuged at 12,000 RPM for 30 min at 4°C, and 500 µL of the supernatant was collected. Protein concentrations were measured using the BCA assay. Laemmli buffer containing beta-mercaptoethanol (#1610747, BIO RAD, California, USA) was added to the samples and boiled at 98°C for 5 min. Proteins were separated using 10% SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)(10% Mini-PROTEIN TGX Precast Protein Gels, BIO RAD) and transferred onto PVDF (Polyvinylidene difluoride) membranes. After blocking in Tris-buffered saline containing 0.25% Tween 20 (TBST) and 5% BSA for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (SuperSignal West Pico Plus; Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Densitometric analysis for semi-quantification was performed using ImageJ software (version 1.47; NIH, Bethesda, MD, USA; http://imagej.nih.gov/ij ). Real-time quantitative polymerase chain reaction Lung tissues were lysed with RIPA lysis buffer (89901; Pierce RIPA Buffer, Thermo Fisher Scientific, USA). Total RNA was then recovered from lung tissues using the RNeasy Mini Kit (74106, Qiagen, Venlo, Netherlands). Poly(A) mRNA was reverse-transcribed in a 20 µL reaction mixture containing oligo(dT)20 primer, 5X RT buffer, 10 U of RNase inhibitor ReverTra Ace (Toyobo, Osaka, Japan), and 10 mM dNTP (Deoxyribonucleoside triphosphate) mixture. Secondary RNA structures were denatured by reacting at 42°C for 60 min to facilitate cDNA synthesis. The reaction was terminated by incubation at 99°C for 5 min. The resulting cDNA was used as a template for polymerase chain reaction (PCR) amplification using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA). The PCR cycling conditions were 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C for 40 cycles, followed by an extended incubation at 72°C for 5 min. The housekeeping gene H2A.Z RNA was used as the internal standard for each group. The mouse primers were designed using Primer3 ( http://bioinfo.ebc.ee/mprimer3 ). The primer sequences are listed in Supplementary Table S1 . Statistical analysis All experiments were performed in triplicate for each treatment or imaging study. To evaluate the differences in fluorescence intensity, total cell counts, and protein levels in BALF, ELISA, immune cell analysis, macrophage size measurement, Western blot, and fibrosis analysis between the control and NP-treated groups, Dunnett’s one-way ANOVA was employed. Data are presented as mean ± standard error of the mean (SEM). Declaration of generative AI in scientific writing This declaration does not apply to the use of AI for purposes beyond grammar and spell checking. Declarations Acknowledgements This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program [grant number KGM5162524]. Author contributions Changsic Youn: Writing – original draft, Methodology, Investigation, Formal analysis, and Data curation. Yu-Jin Jo: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, and Conceptualization. Jeongwoo Kwon:Writing – review and editing, Resources, Data curation. Seung-Bin Yoon: Writing – review and editing, Resources, Data curation. Hyeong-Ju You: Resources, Data curation. Ji-Su Kim:Writing – review and editing, Visualization, Supervision, Project administration, Funding acquisition, and Conceptualization. Data availability The data supporting the findings of this study are available from the corresponding author upon request. Funding declaration This research was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5162524). Competing interests The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study. References Dokl, M. et al. 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Micro- and nano-plastics in the atmosphere: A review of occurrence, properties and human health risks. Journal of Hazardous Materials 465 , 133412, doi:https://doi.org/10.1016/j.jhazmat.2023.133412 (2024). Additional Declarations No competing interests reported. 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encompassing daily life, industry, medicine, and agriculture. Global plastic consumption is projected to increase annually from 464 Mt in 2020 to 884 Mt by 2050 \u003csup\u003e1\u003c/sup\u003e. This increase will lead to a proportional rise in plastic waste, which is becoming a serious environmental concern. If current production and waste management trends persist, approximately 12,000 Mt of plastic waste will accumulate in landfills or leak into the environment by 2050, with severe consequences for the ecosystem \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among plastic waste, microplastics (MPs; 1 \u0026micro;m to 1 mm) and nanoplastics (NPs; \u0026lt;1 \u0026micro;m), which are not visible to the naked eye, have emerged as contaminants of increasing ecological and public health concern. These MPs and NPs exist in various shapes (Spheres, fibers, and particles) and polymer types (e.g., polyethylene [PE], polystyrene [PS], polypropylene [PP], polyethylene terephthalate [PET], and polymethyl methacrylate [PMMA]) \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. NPs generally accumulate in the environment through various degradation of larger plastics and MPs via mechanical abrasion, chemical oxidation, biodegradation, and photooxidation \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDue to their small size, NPs can easily cross cellular membranes, potentially exerting more severe impacts than MPs \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In marine environment, NPs can bioaccumulate and biomagnify in marine organisms ranging from phytoplankton, zooplankton, crustaceans, mollusks, and fish, resulting in impaired growth, altered development and reproduction, behavioral changes, and increased mortality \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This can disrupt the balance of marine ecosystems. NPs are also ubiquitous in the soil, where they can be ingested by soil microorganisms and animals, accumulate, disrupt plant physiology and induce oxidative stress, thereby affecting plant growth and biochemistry \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Humans can be exposed to MPs and NPs primarily through ingestion (contaminated food and water) and inhalation. Airborne particles can also penetrate the respiratory tract, reaching the lungs \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These plastic particles can accumulate in humans, and have been detected in human feces, lungs, brain, blood, placenta, and saliva \u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Their presence in various tissues and organs of the human body poses potential health risks. Multiple toxicological studies have been conducted on MP and NP. \u003cem\u003eIn vitro\u003c/em\u003e experiments showed that exposure to NPs causes various adverse effects, including disruption of intestinal epithelial tight junctions, oxidative stress, granulosa cell apoptosis, and hepatocyte fibrosis \u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e studies in mammals demonstrate that exposure can cause neurological dysfunction, liver and kidney damage, sperm quality deterioration, testicular and ovarian injury, and transplacental transfer of particles \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, existing studies have limitations. First, \u003cem\u003ein vitro\u003c/em\u003e experiments are limited because they cannot fully replicate the complex \u003cem\u003ein vivo\u003c/em\u003e environment. Second, even during \u003cem\u003ein vivo\u003c/em\u003e experiments, most studies simulate oral, dermal, or waterborne exposure, and only limited models directly exposed NPs through the respiratory system. Third, research tends to be biased toward certain types of plastics. For example, it has been shown that PMMA, which is widely used in medical procedures, such as vertebroplasty, has been implicated in postoperative pulmonary embolism and lung damage \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Although PMMA NPs are detected in the lungs of humans, \u003cem\u003ein vivo\u003c/em\u003e studies on their pulmonary effects remain scarce. Therefore, investigating the pulmonary toxicity of PMMA-NPs is crucial, given their widespread medical use and their confirmed presence in human lungs. This study aims to address this gap by directly exposing animal models to NPs via the respiratory tract, thereby overcoming the limitations of previous studies. To assess the subacute effects of repeated exposure, we implemented a 28-day exposure regimen, consistent with standard toxicological assessment guidelines. Mice were exposed daily to PMMA NPs through intratracheal intubation and pulmonary toxicity was identified by monitoring diseases and symptoms that occurred in the tissues.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eNPs inhaled through the airways accumulate in the lungs\u003c/h2\u003e\u003cp\u003eTo investigate the effects of NP inhalation on the respiratory system, six-week-old mice were exposed to NPs daily via intratracheal intubation under respiratory anesthesia for 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To confirm that the exposure to NPs via the respiratory system resulted in accumulation in the lungs, we performed \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We observed that the fluorescence intensity significantly increased in the 100 \u0026micro;g group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). To determine the effects on mice, we measured their body weight. Body weight gain was significantly reduced in the treatment group compared to that in the control group (Supplementary Data S1A and S1B), although no significant difference was observed in the lengths of the mice. However, the Lee index (length from the head to the anus relative to the body weight) and the body width of the mice were both significantly reduced in the treatment group (Supplementary Data S1B). These results confirm that respiratory exposure to NPs leads to pulmonary accumulation and physiological changes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInhalation of NPs causes lung diseases\u003c/h3\u003e\n\u003cp\u003eBronchoalveolar lavage fluid (BALF) is typically used to determine the effect of NPs on the lungs and to evaluate lung injury \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. We measured protein levels and changes in cell count in the BALF. Cell counts were significantly higher in the treatment group than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), and the bicinchoninic acid (BCA) assay also indicates a significant increase in total protein level in the treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Next, H\u0026amp;E staining was performed to confirm lung disease. The invasion of immune and foam cells was confirmed in the treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Additionally, immunofluorescence imaging further confirmed NPs accumulation not only within lung tissue but also within inflammatory cells (Supplementary Data S2A). These results indicate that the respiratory exposure to NPs can result in lung disease.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInhalation of NPs causes lung inflammation\u003c/h3\u003e\n\u003cp\u003eNP exposure increased total cell counts, protein levels, and inflammatory cell infiltration in BALF. To confirm inflammation in the mice, serum levels of proinflammatory cytokines (IL-6, IL-1β, TNF-α) were measured. The results confirm that the levels of inflammatory cytokines were significantly increased in the 100 \u0026micro;g group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Next, we measured the levels of inflammatory cytokines in the BALF to confirm the occurrence of lung inflammation. The levels of IL-6, IL-1β, and TNF-α were significantly increased in the 100 \u0026micro;g group and TNF-α was significantly increased in the 20 \u0026micro;g group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). To confirm which inflammatory cell changes in the lungs caused the inflammation, we then measured the number of inflammatory cells (macrophages, lymphocytes, eosinophils, neutrophils, and basophils) using H\u0026amp;E staining in BALF. In the NP treatment group, the number of macrophages significantly decreased, whereas the numbers of lymphocytes and neutrophils significantly increased in the 100 \u0026micro;g group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Foamy macrophages were identified and their area ratio was significantly increased in the NPs treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Additionally, immunofluorescence revealed that the intensity of foamy macrophages and lung damage CD36, as well as their colocalization with lung tissue, were significantly increased in the 100 \u0026micro;g group (Supplementary Data S2A, S2B and S2C)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To determine whether the levels of inflammatory cytokines were elevated in the lung tissue, we examined both protein and mRNA levels. A significant increase in both protein and mRNA levels of inflammatory cytokines was observed in the 100 \u0026micro;g group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). These results confirm that respiratory exposure to NPs induces the formation of foamy macrophages and promotes lung inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInhalation of NPs causes lung fibrosis\u003c/h3\u003e\n\u003cp\u003eThe results indicate abnormalities in the lung tissue. To investigate the potential induction of fibrosis, lung tissues exposed to NP were subjected to Sirius Red and Masson's trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The results show that the percentage of Sirius Red-positive areas and Masson's trichrome-positive lung tissue significantly increased in the NP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Correspondingly, protein levels of collagen type 1 (COL-1) and α-smooth muscle actin (α-SMA), key genes associated with fibrosis, were significantly elevated in the NP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Collectively, these results suggest that NP exposure induced fibrotic changes in the lung.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the effects of PMMA NPs on the respiratory system of mice. Our results reveal that mice exposed to PMMA NPs exhibit a significant decrease in body weight gain, accompanied by reductions in the obesity index, Lee's index, and body width. Serum analysis further confirmed an increase in inflammatory cytokines, specifically IL-6, IL-1β, and TNF-α. These systemic inflammatory responses likely contribute to the reduced weight gain because IL-6 and TNF-α are known mediators of inflammatory-induced anorexia \u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e–\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Additionally, increased energy expenditure due to lung injury and systemic inflammation may further contribute to weight loss. These results suggest that respiratory exposure to PMMA NPs can lead to local pulmonary toxicity, accompanied by systemic inflammatory responses and potential metabolic changes.\u003c/p\u003e\u003cp\u003eBALF analysis is widely used for evaluating lung disease in clinical settings. In this study, we observed a significant increase in both total cell counts and protein levels in the BALF. Elevated protein in the BALF can result from serum protein leakage across the air–blood barrier, local production by pulmonary immune and epithelial cells, including T cells, alveolar macrophages, bronchial epithelial cells, and alveolar epithelial cells type I and II \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Total cell counts in BALF commonly increases due to increase in immune cells during inflammation, pulmonary fibrosis, smoking, and lung cancer \u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e–\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Our results are consistent with these findings, showing a significant increase in BALF inflammatory cytokines, marked inflammatory cell infiltration in the lung tissue, and the formation of foamy macrophages, thus confirming the induction of lung inflammation.\u003c/p\u003e\u003cp\u003eNotably, the results of this study indicate the formation of foamy macrophages (foam cells, characterized by numerous cytoplasmic vacuoles) in the lung tissue and immune cells in the BALF. Foam cells arise when macrophages engulf excessive amounts of altered lipids (e.g., oxidized lipids) or particulates and accumulate them as lipid droplets in their cytoplasm. Beyond lipid storage, foam cells influence immune responses by secreting inflammatory cytokines and altering antigen presentation \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Their presence has been consistently documented in various pulmonary pathological conditions, including inflammation, fibrosis, Chronic Obstructive Pulmonary Disease (COPD), tuberculosis, lung granuloma, and COVID-19 \u003csup\u003e51–55\u003c/sup\u003e. Previous research has shown that exposure to cigarette smoke, particulate matter, and nanosilica can induce lung diseases, such as inflammation, and cause macrophage dysfunction, leading to foam cell formation \u003csup\u003e\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e–\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Consistent with this, our supplementary data confirm that PMMA NPs are primarily present in alveolar macrophages. This suggests that PMMA NPs may alter the lung environment through inflammation and promote differentiation into foam cells, potentially via lipid dysregulation within macrophages that internalize the NPs or direct cytotoxicity of the particles.\u003c/p\u003e\u003cp\u003eImmunofluorescence and further studies confirmed PMMA-NP uptake by macrophages and revealed a significant increase in the expression of the scavenging receptor CD36. Since CD36 is known to mediate the uptake of oxidized lipids and particulate matter, this suggests that macrophages can actively internalize PMMA NPs through a CD36-mediated pathway \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. This mechanism is likely a critical first step in the cascade leading to foam cell formation, altered lipid metabolism, and inflammatory cytokine secretion. The increased CD36 expression suggests a potential molecular link between PMMA-NP exposure and the observed macrophage dysfunction and lung pathology. Further studies are needed to elucidate this direct interaction and its signaling pathways.\u003c/p\u003e\u003cp\u003eThe results of the BALF analysis confirm that the proportions of neutrophils and lymphocytes increase following PMMA-NP exposure. This increase in the number of inflammatory cells in the lungs suggests that PMMA-NP exposure triggers an inflammatory response in the lungs. Neutrophils play an important role in innate immune responses to pathogens, such as bacteria and fungi, as well as to non-infectious stimuli. However, if inappropriately or excessively activated in the absence of infection, they secrete proteolytic enzymes and reactive oxygen species (ROS), leading to tissue injury and chronic inflammation \u003csup\u003e\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e–\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Lymphocytes, comprising T cells, B cells, and natural killer (NK) cells, are vital components of the immune system. These specialized cells function in antibody production, direct cytotoxicity against virus-infected and tumor cells, and regulation of overall immune responses. When the immune system is activated, there's a noticeable increase in lymphocyte proliferation, which signals a robust and effective immune response \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Recruitment and activation of these immune cells in the lungs is regulated by proinflammatory cytokine. TNF-α and IL-1, activate vascular endothelial cells, facilitate cellular organization, and promote chemotaxis of macrophages and neutrophils to the lungs \u003csup\u003e\u003cspan additionalcitationids=\"CR67 CR68 CR69\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e–\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. IL-6 is also a key player in immune responses and inflammation, and its expression and release are induced by various stimuli, including lipopolysaccharides (LPS), poly (I), poly (C), IL-1, tumor necrosis factor (TNF), and platelet-derived growth factor \u003csup\u003e\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e–\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. In this inflammatory environment, alveolar macrophages can differentiate into foam cells by internalizing PMMA NPs. Foam cells are associated with chronic inflammation, and their formation may interfere with the immune function of macrophages \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Foamy macrophages can also secrete inflammatory cytokines (like IL-1 and IL-6), further promoting inflammation \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. This suggests a cascade: PMMA-NP exposure damages and irritates the lung tissue, leading to an increased secretion of proinflammatory cytokines such as TNF-α and IL-1. This, in turn, recruits immune cells, such as neutrophils and lymphocytes, from the blood into the lungs. During this process, macrophages undergo lipid-induced differentiation into foam cells. It has been hypothesized that these foam cells further amplify the pulmonary inflammatory response by secreting inflammatory cytokines, such as IL-1 and IL-6, creating a self-perpetuating inflammatory cycle.\u003c/p\u003e\u003cp\u003eThis study also confirmed an increased expression of collagen and α-SMA in lung tissue following PMMA-NP exposure, both of which are major indicators of fibrosis. This increase in proteins suggests that the lung tissue accumulates excessive extracellular matrix (ECM) in response to damage, activating fibroblasts and differentiating them into myofibroblasts \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Furthermore, Sirius Red and Masson's trichrome staining confirmed increased collagen deposition, indicating the progression of lung fibrosis. Inflammatory cytokines of the IL-1 family (e.g., IL-1β, IL-18) are well-known major mediators of fibrosis \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. In this study, both IL-1β and IL-18 in the PMMA NPs exposure group were significantly elevated compared to the control group, suggesting that exposure to PMMA NPs can induce an inflammatory response and promote fibrosis. In addition, foam cells may also exacerbate fibrosis by secreting IL-1 family cytokines, stimulating surrounding cells, and reinforcing a fibrotic environment \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. In this study, foamy macrophages were examined after exposure to PMMA NPs, suggesting that fibrosis was induced by the secretion of fibrotic cytokines (IL-1 family) from damaged and inflamed tissues and foamy macrophages.\u003c/p\u003e\u003cp\u003eAlthough this study provides valuable insights into the pulmonary toxicity of PMMA NPs, it has several limitations. First, systemic effects were not evaluated. As shown in Supplementary Data S1, the weight loss observed after the exposure to PMMA NPs suggests possible systemic involvement. While such systemic effects have been predicted, impacting other organs as well, this study did not confirm them. Further studies are needed to confirm whether inhaled NPs translocate to other organs and induce pathological changes. Second, the conditions are limited. In atmospheric environments, MPs and NPs occur in various sizes, shapes, and chemical compositions exist in a complex manner \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. However, only spherical PMMA NPs with a specific size (25 nm) were used in this study. Therefore, it is difficult to generalize the results of this study to human exposure scenarios and other types of plastics. Further comparative toxicological studies across a range of plastic type and size are required. Third, a detailed mechanistic study is required. This study confirms that PMMA NPs cause lung inflammation and fibrosis; however, detailed mechanisms, such as the specific cellular and molecular signaling pathways that cause these pathological phenomena, have not been identified and in-depth research is required. Fourth, the focus of this study was placed on elucidating the toxicity of PMMA NPs. Approaches using rescue agents to alleviate or prevent their toxic effects (inflammation and fibrosis) were not explored. Follow-up rescue studies are required to reduce the health risks associated with NPs exposure. Fifth, this study was conducted using a single-sex cohort (female mice). Sex-specific differences can significantly influence the toxicological consequences of NP exposure. Therefore, while valuable, the results of this study may not be fully generalizable to all mice, regardless of gender. Future studies should include comparative studies between male and female mice to assess potential sex-specific differences in PMMA-NP toxicity and provide a more comprehensive risk assessment. Addressing these limitations will provide insights into the potential health risks associated with NPs and facilitate the development of effective countermeasures.\u003c/p\u003e\u003cp\u003eIn conclusion, this study evaluated the potential respiratory toxicity of PMMA NPs in humans through environmental inhalation exposure. Our findings confirm that inhaled PMMA NPs accumulate in the lung tissues, particularly within the cytoplasm of alveolar macrophages, and induce several pathological changes. Specifically, PMMA exposure triggers a pulmonary inflammatory response characterized by increased infiltration of inflammatory cells (e.g., neutrophils, lymphocytes) into the alveolar space and an increase in the secretion of inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α). Furthermore, structural lung damage and pathological cellular changes, including pulmonary fibrosis with excessive collagen deposition and foam cell formation, reflecting disrupted lipid metabolism and morphological alterations in macrophages. These findings suggest that PMMA NPs accumulate in the lungs and induce pulmonary inflammation, fibrosis, and changes in the distribution and differentiation of inflammatory cells, posing a significant threat to respiratory health.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\n\n"},{"header":"Methods","content":"\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e All animal experiments were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-24311). All animal procedures were performed in compliance with relevant institutional guidelines and regulations. This study’s animal experiments are reported in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"http://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to ensure transparency and reproducibility. Six- to seven-week-old female ICR mice were housed at 23 ± 1°C under a 12-hour light/dark cycle, with food and water ad libitum.\u003c/p\u003e\u003ch3\u003eNanoparticle intratracheal intubation\u003c/h3\u003e\u003cp\u003ePMMA NPs with a size of 25 nm (Cat No: 01-00-251), and the same particles conjugated with green fluorescent dye (Cat No: 29-00-251) were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). Six-week-old mice weighing an average of 24–26 g were exposed to NPs via daily intratracheal intubation for 28 days. Mice were were anesthetized using 1.5% isoflurane inhalation in an induction chamber. Isoflurane was mixed with oxygen, and all anesthetic procedures were controlled using a anesthetic machine (ROYAL-77S; Elpis Medical, Gangwon-do, Korea), and NPs were administered via a catheter. The control group received 50 µL of saline, and the treatment groups were exposed to 20 µg/50 µL and 100 µg/50 µL of NPs. Body weight was measured daily shortly before NP administration. Mice were sacrificed 24 h after the final exposure and samples were collected.\u003c/p\u003e\u003ch2\u003ePreparation and analysis of bronchoalveolar lavage fluid\u003c/h2\u003e\u003cp\u003eAfter 28 days of NP treatment, BALF was obtained by washing the lungs three times with 1 mL of saline. BALF was centrifuged at 300 × g for 7 min at 4°C, and the supernatant was collected for Enzyme-Linked Immunosorbent Assay (ELISA) and BCA analysis. The pellet was resuspended in 200 µL of saline, and the saline was dried onto slides. Each slide was stained with hematoxylin and eosin (H\u0026amp;E), and 400 cells were selected. The slides were examined using a light microscope (ICC50 E, Leica Microsystems, Wetzlar, Germany). Individual cells were classified according to cell and nuclear morphology (basophils, characterized by the presence of numerous small granules; neutrophils, identified by 3–5 segmented nuclei; eosinophils, distinguished by pink-stained cytoplasm and bilobed nuclei; monocytes, identified by large nuclei; and macrophages, identified by irregular shape and smaller nuclei compared to monocytes).\u003c/p\u003e\u003ch2\u003eFluorescence measurements of the lungs\u003c/h2\u003e\u003cp\u003eAfter 28 days of NP treatment, fluorescence imaging was performed using an \u003cem\u003ein vivo\u003c/em\u003e imaging system (Uinv-420; Davinch-K, Seoul, Korea) to determine the accumulation of NPs in the lungs.\u003c/p\u003e\u003ch2\u003eHistological analysis\u003c/h2\u003e\u003cp\u003eFixed lung tissues were processed for paraffin block production using an automated tissue processor (HistoCore PEARL; Leica Microsystems, Wetzlar, Germany) and paraffin embedding station (HistoCore Arcadia H; Leica Microsystems, Germany). Paraffin blocks were sectioned at a thickness of 5 µm to produce slides, which were stained with H\u0026amp;E for histological analysis. Stained sections were visualized using a slide scanner (Aperio VERSA 8, Leica Microsystems, Germany) to determine the presence or absence of lung disease.\u003c/p\u003e\u003ch2\u003eBALF and ELISA analysis\u003c/h2\u003e\u003cp\u003eTo measure protein levels in BALF, the supernatants were analyzed using the BCA Assay (Pierce™ BCA Protein Assay Kits, Thermo Fisher Scientific, Waltham, MA, USA). To assess systemic and pulmonary inflammatory cytokine levels (IL-6, IL-1β, TNF-α), serum and BALF samples were tested using ELISA kits (M6000B-1, MLB00C-1, MTA00B-1, R\u0026amp;D Systems, Minneapolis, USA) according to the manufacturer's instructions. BCA and ELISA signals were measured using a microplate reader (EPOCH2, BioTek, Vermont, USA).\u003c/p\u003e\u003ch2\u003ePicosirius red staining\u003c/h2\u003e\u003cp\u003eParaffin-embedded lung tissue slides were deparaffinized three times with xylene for 15 min each, rehydrated twice with 100% ethanol for 1 min each and twice with 95% ethanol for 1 min each, and finally rehydrated in distilled water for 5 min. Lung tissue sections were stained with picrosirius red solution (ab150681; Abcam, Cambridge, UK) for 1 h and washed with 5% acetic acid. Finally, sections were dehydrated several times with ethanol, washed with xylene, and fixed with toluene. Lung tissue sections were stained with picrosirius red and visualized using a slide scanner (Aperio VERSA 8; Leica Microsystems, Germany).\u003c/p\u003e\u003ch2\u003eMasson’s trichrome staining\u003c/h2\u003e\u003cp\u003edeparaffinized lung tissue slides were stained using a trichrome staining kit (ab150686, Abcam, UK) according to the manufacturer's instructions. Lung tissue sections were stained with Masson's trichrome and visualized using a slide scanner (Aperio VERSA 8; Leica Microsystems, Germany).\u003c/p\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eLung tissues were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Pierce RIPA Buffer, 89901, Thermo Fisher Scientific, USA), and 1 mL PhosSTOP (PhosSTOP EASYpack; Roche, Chugai, Switzerland) was used for protein extraction. The tissue lysates were prepared using beads and incubated on ice for 10 min. The lysed tissues were centrifuged at 12,000 RPM for 30 min at 4°C, and 500 µL of the supernatant was collected. Protein concentrations were measured using the BCA assay. Laemmli buffer containing beta-mercaptoethanol (#1610747, BIO RAD, California, USA) was added to the samples and boiled at 98°C for 5 min. Proteins were separated using 10% SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)(10% Mini-PROTEIN TGX Precast Protein Gels, BIO RAD) and transferred onto PVDF (Polyvinylidene difluoride) membranes. After blocking in Tris-buffered saline containing 0.25% Tween 20 (TBST) and 5% BSA for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (SuperSignal West Pico Plus; Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Densitometric analysis for semi-quantification was performed using ImageJ software (version 1.47; NIH, Bethesda, MD, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imagej.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://imagej.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eReal-time quantitative polymerase chain reaction\u003c/h2\u003e\u003cp\u003eLung tissues were lysed with RIPA lysis buffer (89901; Pierce RIPA Buffer, Thermo Fisher Scientific, USA). Total RNA was then recovered from lung tissues using the RNeasy Mini Kit (74106, Qiagen, Venlo, Netherlands). Poly(A) mRNA was reverse-transcribed in a 20 µL reaction mixture containing oligo(dT)20 primer, 5X RT buffer, 10 U of RNase inhibitor ReverTra Ace (Toyobo, Osaka, Japan), and 10 mM dNTP (Deoxyribonucleoside triphosphate) mixture. Secondary RNA structures were denatured by reacting at 42°C for 60 min to facilitate cDNA synthesis. The reaction was terminated by incubation at 99°C for 5 min. The resulting cDNA was used as a template for polymerase chain reaction (PCR) amplification using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA). The PCR cycling conditions were 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C for 40 cycles, followed by an extended incubation at 72°C for 5 min. The housekeeping gene H2A.Z RNA was used as the internal standard for each group. The mouse primers were designed using Primer3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfo.ebc.ee/mprimer3\u003c/span\u003e\u003cspan address=\"http://bioinfo.ebc.ee/mprimer3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The primer sequences are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate for each treatment or imaging study. To evaluate the differences in fluorescence intensity, total cell counts, and protein levels in BALF, ELISA, immune cell analysis, macrophage size measurement, Western blot, and fibrosis analysis between the control and NP-treated groups, Dunnett’s one-way ANOVA was employed. Data are presented as mean ± standard error of the mean (SEM).\u003c/p\u003e\u003ch2\u003eDeclaration of generative AI in scientific writing\u003c/h2\u003e\u003cp\u003eThis declaration does not apply to the use of AI for purposes beyond grammar and spell checking.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program [grant number KGM5162524].\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eChangsic Youn: Writing \u0026ndash; original draft, Methodology, Investigation, Formal analysis, and Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYu-Jin Jo: Writing \u0026ndash; original draft, Methodology, Investigation, Formal analysis, Data curation, and Conceptualization.\u003c/p\u003e\n\u003cp\u003eJeongwoo Kwon:Writing \u0026ndash; review and editing, Resources, Data curation.\u003c/p\u003e\n\u003cp\u003eSeung-Bin Yoon: Writing \u0026ndash; review and editing, Resources, Data curation.\u003c/p\u003e\n\u003cp\u003eHyeong-Ju You: Resources, Data curation.\u003c/p\u003e\n\u003cp\u003eJi-Su Kim:Writing \u0026ndash; review and editing, Visualization, Supervision, Project administration, Funding acquisition, and Conceptualization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003eFunding declaration\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5162524).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDokl, M.\u003cem\u003e et al.\u003c/em\u003e Global projections of plastic use, end-of-life fate and potential changes in consumption, reduction, recycling and replacement with bioplastics to 2050. \u003cem\u003eSustainable Production and Consumption\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 498-518, doi:https://doi.org/10.1016/j.spc.2024.09.025 (2024).\u003c/li\u003e\n\u003cli\u003eGeyer, R., Jambeck, J. 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Instead of complete biodegradation, plastic waste fragments into smaller particles, accumulating as nanoplastics (NPs; \u0026lt;1 μm). Humans are exposed to NPs through inhalation and ingestion of contaminated water and food, which can induce cytotoxicity through physical and chemical pathways. Polymethyl methacrylate (PMMA), commonly used in implants and artificial bones, has been identified in human lungs and associated with pulmonary embolism. While PMMA NP toxicity has been reported in vitro, their in vivo effects, as well as the underlying mechanism, remain poorly understood. In this study, we investigated the pulmonary effects of inhaled PMMA NPs in mice. Mice received 20 or 100 μg of PMMA NPs (25 nm) via intratracheal intubation for 28 days. PMMA-NP preparation and characterization are described in the Methods section. Exposed mice exhibited body weight loss and pulmonary accumulation of PMMA NPs. Bronchoalveolar lavage fluid (BALF) analysis revealed increased cell count and elevated inflammatory cytokines in serum and BALF. Histopathology (H\u0026amp;E staining) revealed abnormalities in lung tissue and alterations in protein and RNA expression. The findings demonstrate that respiratory exposure to PMMA NPs induces lung inflammation, tissue damage, and molecular dysregulation.\u003c/p\u003e","manuscriptTitle":"Pulmonary Toxicity of Polymethyl methacrylate nanoplastics via Intratracheal Intubation in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 10:48:34","doi":"10.21203/rs.3.rs-7466865/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-12T07:06:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"244295798397280532192463360303835442366","date":"2025-11-10T08:54:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-08T16:40:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90197594264814531405585072403836197436","date":"2025-11-08T15:34:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82246576424683504169314732361795384739","date":"2025-11-06T04:00:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12929133592353266311138433173007134458","date":"2025-11-03T16:24:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-17T00:44:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119969500271371842815187265779073294708","date":"2025-09-08T04:22:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-04T20:03:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T19:59:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-04T19:41:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-31T13:07:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-31T13:03:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f18c9bc-dc81-4631-b7d7-7374163f01ad","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54513722,"name":"Biological sciences/Biotechnology"},{"id":54513723,"name":"Health sciences/Diseases"},{"id":54513724,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-01-12T16:07:19+00:00","versionOfRecord":{"articleIdentity":"rs-7466865","link":"https://doi.org/10.1038/s41598-025-31615-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-06 15:57:35","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-09-11 10:48:34","video":"","vorDoi":"10.1038/s41598-025-31615-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31615-9","workflowStages":[]},"version":"v1","identity":"rs-7466865","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7466865","identity":"rs-7466865","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}