Abstract Nanoplastics (NPs) are emerging environmental pollutants that pose growing concerns due to their potential health risks. However, the effects of inhaled NP exposure during pregnancy on fetal brain development remain poorly understood. In this study, we investigated the impact of maternal exposure to polypropylene nanoplastics (PP-NPs) on fetal brain development and neurobehavioral outcomes in a mouse model and further explored its mechanism in human cerebral organoids. Maternal exposure to PP-NPs significantly impaired neuronal differentiation and proliferation in the fetal cortex. Neurobehavioral assessments revealed significant deficits in offspring following maternal exposure, including impaired spatial memory, reduced motor coordination, and heightened anxiety-like behavior. Furthermore, human brain organoids exposed to PP-NPs exhibited reduced growth and neuronal differentiation, with significant downregulation of key neuronal markers such as TUJ1, MAP2, and PAX6. Transcriptomic analysis identified alterations in gene expression, particularly in neuroactive ligand-receptor interaction pathway. Molecular docking and fluorescence co-localization analysis further suggested CYSLTR1 and PTH1R as key molecular targets of PP-NPs. These findings provide novel insights into the toxicological effects of NPs on the developing brain and emphasize the need for preventive measures to protect fetal neurodevelopment during pregnancy. Graphical abstract [38]graphic file with name 12951_2025_3561_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03561-1. Keywords: Polypropylene nanoplastics, Cerebral organoid, Early-life exposure, Neurodevelopmental toxicity, Neuroactive ligand-receptor interaction pathway Introduction Plastics have become an integral part of modern society, extensively utilized in applications ranging from manufacturing, packaging, and medical devices. At present, the annual production of plastic is estimated to be around 450 million tons, and this number is projected to double by 2045 [[39]1, [40]2]. However, their widespread use has led to the emergence of environmental pollution problems associated with plastic waste. In the natural environment, plastic waste is degraded into microplastics (MPs; < 5 mm) and even further into nanoplastics (NPs; < 1 μm) through mechanical and photochemical processes [[41]3, [42]4], contaminating air, soil, and water ecosystems and further affecting human health. MPs/NPs can enter and accumulate in human and animal organs through oral ingestion, respiratory inhalation, and skin contact [[43]5–[44]7]. Among them, air inhalation is the most important way of MPs/NPs intake in humans [[45]7]. However, most existing studies on MPs/NPs have primarily focused on gastrointestinal exposure and the potential health risks associated with airborne MPs/NPs have received far less attention. Various types of microplastics, including polypropylene (PP), polyethylene terephthalate (PET) and polyethylene (PE), have been detected in human lung tissue, with PP being the most abundant type, accounting for 23% of the detected particles [[46]8]. Consistently, a study on atmospheric MPs reported that PP accounts for 59% of total detected MP fragments, highlighting its widespread presence in airborne particles [[47]9]. PP is one of the most widely used plastics, with applications ranging from food packaging to medical packaging [[48]10]. It is also a primary material used in personal protective equipment such as face masks. The COVID-19 pandemic has further increased PP usage. It is estimated that disposable masks made of PP are being discarded at a rate of approximately 129 billion per month worldwide during the COVID-19 pandemic, amounting to around 645,000 tons of PP waste [[49]11, [50]12], which may serve as a significant source of airborne MPs and NPs [[51]13]. Despite its prevalence in the environment, the toxicological effects of inhaled PP-MPs/NPs on human health remain poorly understood. Therefore, it is crucial to investigate the potential risks and mechanisms of PP-MPs/NPs through inhalation exposure and their impact on human health. Compared to MPs, NPs have smaller particle sizes and larger surface areas, which makes them more capable of penetrating biological barriers and exerting greater toxic effects. Notably, the accumulation of NPs is not limited to a single organism but can also be transferred from mother to offspring. Grafmueller et al. examined bidirectional transfer of polystyrene nanoplastics using an ex vivo human placental perfusion model and observed polystyrene translocation in the placenta and accumulation in the syncytiotrophoblast [[52]14]. Another study indicated that nanoplastics can be transferred to the placenta, fetal brain and other organs after exposure to the maternal mice lungs [[53]15]. Additionally, maternal exposure to polystyrene nanoplastics via gavage led to spatial memory deficits in rat offspring [[54]16]. These findings suggested that NPs have the ability to cross the placental and fetal blood-brain barriers, contributing to neurotoxicity during embryonic development. Given the challenge of preventing inhalation exposure to NPs, it is of utmost importance to evaluate the potential effects and toxic mechanisms of respiratory PP-NPs exposure on pregnant women and their offspring. 3D cerebral organoids derived from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have emerged as novel tools for neurotoxicity assessment. Unlike traditional two-dimensional (2D) cell cultures, 3D organoids can replicate the cell type diversity, tissue architecture and function of the organ they represent [[55]17, [56]18], potentially offering toxicological data that can be directly extrapolated to humans [[57]19]. Cerebral organoids partially reproduce embryonic brain development and recapitulate the complex structures and developmental trajectories found in the human brain [[58]20]. These cerebral organoids have been used to assess the impact of environmental pollutants such as cadmium [[59]21], arsenic (As) [[60]22], lead (Pb) [[61]22], and diesel particulate matter [[62]23] on the human brain. In this study, we examined the effects of PP-NPs inhalation exposure during pregnancy on mouse fetal brain development and further investigated the underlying mechanisms on human cerebral organoids. Our study aimed to provide novel insights into understanding the possible toxic effects of NPs on the developing brain and their associated mechanism. Materials and methods Animals and PP-NPs exposure Seven-week-old specific-pathogen-free (SPF) C57BL/6J female and male mice weighing 20–25 g were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China). All mice were housed in a 12/12-hour light-dark cycle for one week with free access to food and water to adapt to the environment. Eight-week-old female mice were individually caged with aged-match males for fertility. Following mating, pregnancy was confirmed by vaginal plugs, and male mice were subsequently removed. Pregnant mice were randomly divided into two groups (Control group, PP-NPs group), each containing five pregnant mice, and ensuring that there was no significant difference in mean body weight at the beginning of the experiment. The pristine and red fluorescently labeled PP-NPs with a diameter of 100 nm were purchased from Beijing Zhongke Keyou Technology Co., Ltd (Beijing, China). The morphology and the size distribution of PP-NPs were observed using transmission electron microscopy and Beckman-Coulter LS230 laser granulometer. The weekly intake of MPs in humans is estimated to be 0.1 to 5.0 g [[63]24], equivalent to approximately 0.2 to 11.0 mg/kg body weight/day (assuming 65 kg body weight in adults). For NPs, the exposure level may be higher. In this study, pregnancy was determined by the presence of a vaginal plug (day 0 of gestation, GD0), and pregnant mice were treated with PP-NPs suspension at 5 mg/kg body weight (PP-NPs group) or with normal saline (Control group) for every two days during gestation through intratracheal instillation as previously described [[64]25]. Dams were maintained on normal diet and drinking water during lactation. Pups were weaned at postnatal day 21 and received normal drinking water until the end of the experiment. In addition, according to previous studies, pregnant women could be exposed to approximately 6.67 × 10^16 nanoplastic particles per day by inhalation [[65]15, [66]26]. Considering the lung surface area between human (62.7 m^2) and the mouse (0.05 m^2) model used in our experiment [[67]27, [68]28], it was inferred that the exposure dose to pregnant mice was approximately 5.31 × 10^13 nanoplastic particles. In our study, mice in the PP-NPs group were exposed to PP-NPs suspension (approximately 2.3 × 10^11 nanoplastic particles, assuming a pregnant mice body weight of 25 g) every two days. The extrapolated real-world exposure estimates exceed the dose used in our study, thereby supporting the biological relevance of the selected exposure dose. This study received ethical approval from the animal experimental ethics committee in the department of laboratory animal science at Tongji University (TJBG09724101). Hematoxylin & Eosin (HE) staining of paraffin sections Cerebral tissues were fixed in 4% paraformaldehyde for 48 h and subsequently processed for routine paraffin embedding. Paraffin-embedded tissues were sectioned into 5 μm thickness and then stained with hematoxylin (DH0004, leagene, Beijing, China) and eosin (G1108, Solarbio, Beijing, China). Samples were finally visualized using a microscope (Nikon Corporation, Tokyo, Japan) and representative images were captured for analysis. Immunofluorescence staining of paraffin sections Cerebral cortex tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were deparaffinized using xylene and dehydrated in graded alcohol followed by high-pressure antigen retrieval in 0.02 M sodium citrate buffer for 15 min. After cooling to room temperature, the sections were washed three times with PBS, permeabilized with 0.3% Triton X-100 for 15 min at room temperature, and then blocked with 5% goat serum for 1 h. Subsequently, the sections were incubated with primary antibody anti-beta III Tubulin antibody or KI67 Polyclonal antibody overnight at 4 °C. The details of primary antibodies are summarized in supplementary Table [69]1. The next day, sections were incubated with Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (1:200, A0423, Beyotime Biotechnology, Shanghai, China) for 1 h at room temperature, and then stained with 4, 6-diamidino-2-phenylindole (DAPI, C1002, Beyotime Biotechnology, Shanghai, China) for 10 min. Finally, the sections were sealed with anti-fluorescence quenching tablets and observed by fluorescence microscope (Nikon Corporation, Tokyo, Japan), and representative images were collected for analysis. The mean fluorescence intensity of TUJ1 and the percentage of the KI67-positive cells were analyzed using Image J software. Open field test The open field test apparatus consisted of an open field response box (40 cm × 40 cm × 40 cm) and an automatic data acquisition and analysis system (Model 63041, RWD Life Science, Shenzhen, China). The open field response box was divided into 9 regions (3 rows × 3 columns). Mice were allowed to freely explore in the open field response box for at least 6 min before the start of the experiment. During testing, mice were placed on the same side of the open field response box and allowed to freely explore for 5 min. The open field response box was cleaned with 75% alcohol after removing the mice each time to avoid residual odor affecting the test results of the next mice. The total movement distance, average speed, the movement distance in the central area, central area residence time, and total number of central area entry were recorded. Rotarod test The rotarod test was used to assess the ability of motor coordination and balance of mice. Rotarod fatigue instrument was purchased from Shanghai XinRuan Information Technology Co., Ltd. (Model XR-6 C, Shanghai, China). Mice were given adaptive training one day before the experiment to adapt to the experimental environment and become familiar with equipment. For formal experiments, mice were placed on an accelerating rod, the speed of which gradually increased from 2 rpm to 50 rpm over 3 min. Each mouse were subjected to three consecutive trials separated by 30–60 min. The average time for mice to fall from the rotating rod was recorded. Morris water maze test Morris water maze test was used to assess spatial learning and memory ability of mice. The experimental apparatus consisted of water maze device, automatic image acquisition and software analysis system (Model XR-XM101, Shanghai XinRuan Information Technology Co., Ltd., Shanghai, China). Testing was performed in a round white pool of 120 cm diameter and 50 cm depth. During the training phase, the platform (8 cm in diameter) was immersed 1–2 cm below the water surface. The water temperature was kept around 23 ± 1 °C. Each mouse was trained four trials per day with a 20-minute interval between training sessions for 4 consecutive days. Each mouse had 60 s to locate the platform. If mice failed to reach the platform within 60 s, they were guided to the platform and left on the platform for 10 s before returning them to their cages. The average time taken to locate the platform (escape latency) was recorded for statistical analysis. Probe trial was performed on the fifth day, the platform was removed and mice were placed in water from the opposite side of the platform. Each mouse was allowed to explore the maze for 60 s. The swimming route of mice, the times of crossing the original platform, and the times of crossing the quadrant of the original platform were recorded. Establishment and maturation of human cerebral organoids (COs) Human COs were generated from iPSCs by using the STEMdiff™ Cerebral Organoid Kit (08570 and 08571, STEMCELL Technologies, Vancouver, Canada). Briefly, iPSC cells were removed from the plates using Gentle Cell Dissociation Reagent (100–0485, STEMCELL Technologies, Vancouver, Canada) and plated at a density of 9,000 cells per well in low-attachment, round-bottom 96-well plates (7007, Corning, New York, USA) to form Embryoid bodies. On day 5, embryoid bodies were transferred to ultra-low attachment 24-well plates (3473, Corning, New York, USA). On day 7 to day 10, COs were embedded in Matrigel ^® (354277, Corning, New York, USA) and cultured in ultra-low attachment 6-well plates (3471, Corning, New York, USA). After embedding, COs were transferred to an orbital shaker (5% CO[2]) and the medium was changed every 3 days. By day 40, COs will exhibit dense cores and optically translucent edges, and will typically be ready for analysis. The immunofluorescence staining analysis of COs was performed on day 40. Organoids exposure to PP-NPs On day 10 of human COs differentiation, the culture was replaced with medium containing 0, 10, 25, and 50 µg/mL of PP-NPs (including control group, PP-10 group, PP-25 group, PP-50 group). The medium containing NPs was changed every 3 days for 30 days. The exposure concentrations were selected based on previous study and calculations derived from animal exposure dose (5 mg/kg, assuming a pregnant mice body weight of 25 g). Specifically, considering the maternal blood volume (6 mL/100 g body weight) [[70]29] and the placental transfer rate of nanoplastics (17.5-31.5%) [[71]30], the estimated concentration of PP-NPs on the fetal side was approximately 14.6–26.25 µg/mL. Finally, considering both previous study [[72]31] and human real-world exposure level (0.1–5.0 g per week) [[73]24], this study selected three exposure concentrations (10, 25, and 50 µg/mL) to investigate the impact of PP-NPs on brain development. Immunofluorescence staining of frozen sections COs were fixed with 4% paraformaldehyde for 16–24 h at 4 °C and subsequently dehydrated overnight with 30% sucrose solution. Following embedding in gelatin solution and rapid freezing in dry ice/ethanol slurry, COs was cut into 18-µm-thick sections. The sections were washed with PBST at 37 °C to completely remove gelatin, and then incubated with 0.2% Triton X-100 for 15 min at room temperature. After blocking for 1 h, the sections were incubated with primary antibodies overnight at 4 °C. The details of primary antibodies are summarized in supplementary Table [74]1. The next day, sections were incubated with the corresponding anti-species Alexa Fluoro secondary antibodies (1:500, Thermo Fisher Scientific, Shanghai, China) for 1 h and DNA was labeled with DAPI. Cerebral cortex tissues were embedded in Tissue-Tek^® O.C.T. Compound (4583, Sakura Finetek, USA) and cryosectioned at a thickness of 10 μm. For immunofluorescence staining, sections were fixed in 4% paraformaldehyde for 30 min and then incubated with 0.2% Triton X-100 for 15 min at room temperature. After blocking for 1 h, the sections were incubated overnight at 4 °C with primary antibodies (details provided in supplementary Table [75]1). On the following day, sections were incubated with the corresponding anti-species Alexa Fluoro secondary antibodies (1:500, Thermo Fisher Scientific, Shanghai, China) for 1 h and DNA was labeled with DAPI. Transcriptome sequencing Transcriptome sequencing and analysis were conducted by Applied Protein Technology (Shanghai, China). Briefly, the total RNA was first extracted from COs using Trizol reagent, and the RNA integrity number (RIN) value was determined to assess RNA quality. Subsequently, paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, Wuhan, China) according to the manufacturer’s instructions, involving mRNA purification, mRNA fragmentation, cDNA synthesis and PCR amplification. Library quality was assessed on an Agilent Bioanalyzer 4150 system. Finally, sequencing was performed using a MGISEQ-T7 platform. The resulting sequencing data was used for bioinformatics analysis, including quality control, quantification of gene expression level, differential expression analysis and enrichment analysis. Differential expression analysis was performed using DESeq2 [[76]32] and the screening thresholds for differentially expressed genes (DEGs) were|log2FC| > 2 and Padj < 0.05. Gene Ontology (GO) [[77]33] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [[78]34] enrichment analysis were conducted to explore the biological functions and pathways associated with DEGs. Additionally, Gene Set Enrichment Analysis (GSEA) [[79]35] was performed to identify pathway-level changes in gene expression without relying on arbitrary thresholds. Finally, visualization was performed using the SRplot platform [[80]36]. Quantitative real-time PCR Total RNA was extracted from COs and cerebral tissues using TRIzol^® reagent (Invitrogen, Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s protocol. One microgram of total RNA was reversed to cDNA using ABScript Neo RT Master Mix (RK20433, ABclonal, Wuhan, China) and quantitative realtime PCR (RT-qPCR) was performed using Genious 2X SYBR Green Fast qPCR Mix (RK21206, ABclonal, Wuhan, China). The 2^−ΔΔCt method was used to evaluate the mRNA expression. Relative expression was calculated and normalized to GAPDH. The sequences of oligonucleotide primers were synthesized by General Biol(Anhui)Co.,Ltd (Anhui, China) and the forward and reverse primer sequences are shown in supplementary Table [81]2. Western blot Proteins were extracted from brain tissue and cell lysates were prepared with RIPA lysate supplemented with PMSF (Solarbio, Beijing, China), followed by protein quantification using the BCA protein assay kit (Sangon, Shanghai, China). Proteins were subsequently separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 h and then incubated with primary antibodies overnight at 4 °C. The details of primary antibodies are summarized in supplementary Table [82]1. The next day, membranes were incubated with HRP-labeled secondary antibodies (1:3000; CST, Shanghai, China) for 1 h at room temperature. Finally, immunoreactive bands were visualized using BeyoECL Moon (Beyotime Biotechnology, Shanghai, China). Molecular Docking Molecular docking is a widely used computational approach for evaluating the binding affinity and interaction modes between ligands and target proteins [[83]37]. Based on previous studies [[84]38–[85]40], we used PP monomer to represent PP-NPs for molecular docking analysis. The propylene downloaded from the PubChem database [[86]41] was imported into DS2019.3 for energy minimization. The crystal structures of CYSLTR1 (PDB ID: 6rz4) and PTH1R (PDB ID: 8flr) were obtained from the PDB database [[87]42]. Original ligands and water molecules were removed using PyMOL, and the processed files were saved as PDB files. Finally, virtual screening was performed using DS2019.3. Statistical analysis Statistical analysis was performed using GraphPad Prism 8.0.1 software (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM). For comparisons between two groups, an F test was first conducted to assess the homogeneity of variances. If the variances were equal (P > 0.05), an unpaired two-tailed t-test was used. If the variances were significantly different (P < 0.05), an unpaired two-tailed t-test with Welch’s correction was applied. Comparisons among three groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. A P value < 0.05 was considered statistically significant. Results Prenatal exposure to PP-NPs inhibited neuronal development in mouse fetal cerebrum We used the synthesized 100 nm PP-NPs to model real world pollutants, and their particle size was confirmed by the dynamic light scattering analysis and transmission electron microscopy (Fig. [88]1B and C). Then we investigated the adverse effects on offspring neurodevelopment following exposure to PP-NPs via intratracheal instillation during pregnancy using an in vivo model (Fig. [89]1A). The results of HE staining did not show significant structural differences between the two groups (Fig. [90]1D). However, further immunofluorescence analysis revealed that the expression level of neuronal marker TUJ1 was significantly lower in the PP-NPs group than that in the control group, indicating that neuronal differentiation was inhibited (Fig. [91]1E and F). Furthermore, the expression of KI67, a cell proliferation marker, was significantly reduced in the PP-NPs group (Fig. [92]1G and H). These results suggested that PP-NPs exposure during pregnancy negatively affected the fetal cerebral cortical development by inhibiting neuronal development and cell proliferation. Fig. 1. [93]Fig. 1 [94]Open in a new tab Effects of prenatal exposure to PP-NPs on fetal cerebral cortical development. (A) Schematic overview of the mouse experiment. (B) Transmission electron microscopy images of PP-NPs. Scale bars: top, 500 nm; bottom, 100 nm. (C) Particle size analysis of PP-NPs. (D) Representative HE staining of cerebral cortex sections. Scale bar, 500 μm. (E-F) Representative immunofluorescence images of TUJ1 staining in the cerebral cortex sections and quantification of the TUJ1 mean fluorescent intensity. Scale bar, 100 μm. (G-H) Representative immunofluorescence images of KI67 staining in the cerebral cortex sections and percentage of KI67-positive cells. Scale bar, 50 μm. Two-tailed t-test; *, P < 0.05; ****, P < 0.0001 Prenatal exposure to PP-NPs impaired neurobehavior in mouse offspring To further investigate the long-term effects of PP-NPs exposure on offspring neurodevelopment, we conducted neurobehavioral tests on 6-week-old mice following maternal exposure. Open field test was used to assess exploratory behavior. No significant differences between the PP-NPs exposure group and the control group were observed in terms of total movement distance and average speed (Fig. [95]2A-C). However, PP-NPs group showed a significant reduction in movement distance in the central area, number of entries, and the time spent in the central area (Fig. [96]2D-F), suggesting that they might display anxiety-like behavior. In the rotarod test, the average latency to fall was significantly shorter in the PP-NPs group, indicating decreased motor coordination ability (Fig. [97]2G). Additionally, the Morris water maze test was used to evaluate learning and spatial memory abilities in the offspring. The escape latency on day 4 was shorter than on day 1 for both groups (Fig. [98]2H). However, the escape latency on day 4 in the PP-NPs group was significantly longer than in the control group (Fig. [99]2H), suggesting impaired learning ability. In the probe trial, PP-NPs group showed significantly longer platform latency and fewer platform and target quadrant crossing times compared to the control group (Fig. [100]2I-L), further indicating that their spatial memory was significantly impaired. Fig. 2. [101]Fig. 2 [102]Open in a new tab Effects of prenatal exposure to PP-NPs on neurobehavior in mouse offspring. (A-F) Open field test (n = 6): (A) Representative trajectory map of open field test. Red dot: starting position; Blue dot: end position. (B) Total movement distance. (C) Average speed. (D) Movement distance in the central area. (E) The time spent in the central area. (F) Number of central area entries. (G) The average latency to fall in rotarod test (n = 6). (H-L) Morris water maze test (n = 6): (H) Escape latency in training phase. (I) Representative trajectory map. Red dot: starting position; Blue dot: end position. (J) Escape latency at day 5. (K) Platform crossing times. (L) Target quadrant crossing times. Two-tailed t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 Generation and characterization of human COs Next, we further explored the toxicology of PP-NPs using a human-derived in vitro organoid model. Figure [103]3A depicts the entire process of differentiation from iPSCs to COs. Morphological changes of COs were observed at different time points and the organoids presented significant structural features after day 40 (Fig. [104]3B). By day 40, the organoids exhibited dense core with regions of the organoids displaying optically translucent edges. Immunofluorescence analysis of cryosections of COs showed that cortical regions within organoids were labeled by the neural progenitor marker PAX6 (orange) and the neuronal marker TUJ1 (green). PAX6 + neural progenitor cells were localized to a ventricular zone-like region, while TUJ1 + neurons were positioned adjacent to the ventricular zone (Fig. [105]3C). Additionally, the deep layer cortical neuron marker CTIP2 was co-expressed with TUJ1 in cortical plate-like region, indicating the formation of cortical plate layer (Fig. [106]3C). These findings collectively confirm the successful generation of COs with distinct neuronal layers and regions of differentiation. Fig. 3. [107]Fig. 3 [108]Open in a new tab Characterization of the human COs. (A) Protocol diagram of COs culture. (B) Morphological changes of COs at different time points (2, 5, 7, 10, 40, 60, 80 and 100 days). Scale bar, 500 μm. (C) Immunofluorescent staining of COs on day 40. Scale bar, 100 μm PP-NPs inhibited growth and differentiation of COs To evaluate the effects of NPs on the development of COs, we exposed COs to different concentrations of PP-NPs (0, 10, 25, and 50 µg/mL) from day 10 to day 40 (Fig. [109]4A). As shown in Fig. [110]4B and C, the surface area of COs was decreased when exposed to 50 µg/mL of PP-NPs. To further assess the impact on neurodevelopmental processes, we performed immunofluorescence staining and RT-qPCR analysis targeting markers of neural stem cells, proliferating cells, neurons, and glial cells. Immunofluorescence and RT-qPCR results showed a significant decrease in the expression of TUJ1, MAP2, RBFOX3, and PAX6 in COs exposed to 50 µg/mL PP-NPs (Fig. [111]4D-F and Supplementary Fig. [112]1A-C, 1G), indicating impaired neuronal differentiation. CTIP2 also showed a decreasing trend without statistical significance (Fig. [113]4D-F). At lower dosage of exposure (25 µg/mL), neuron outgrowth and differentiation (MAP2), rather than proliferation of progenitor cells (PAX6), were affected (Supplementary Fig. [114]1A-C). Furthermore, RT-qPCR analysis showed that the expression of NES and PCNA, markers associated with neural stem cell and proliferation, was significantly decreased in COs exposed to 50 µg/mL PP-NPs (Supplementary Fig. [115]1D and 1E). In addition, the expression of OLIG2 and MOG, markers of oligodendrocytes, and S100B and SLC1A3, markers of astrocytes, was also significantly reduced (Supplementary Fig. [116]1F), indicating impaired glial differentiation. In contrast, the expression of the microglial marker AIF1 was significantly increased (Supplementary Fig. [117]1F), suggesting microglial activation and potential neuroinflammatory response. Taken together, these findings demonstrated that PP-NPs exposure disrupted multiple neurodevelopmental processes, including neural stem cell maintenance and proliferation, neuronal differentiation, and glial cell maturation, in a dose-dependent manner. Fig. 4. [118]Fig. 4 [119]Open in a new tab PP-NPs inhibited growth and neuronal differentiation of human COs. (A) Schematic diagram of PP-NPs exposure on COs. (B) Morphological changes of COs in four groups on day 40. Scale bar, 500 μm. (C) Surface area of COs on day 40. (D-F) Immunofluorescence staining and quantification of mean fluorescence intensity for TUJ1 and CTIP2 in COs from four groups on day 40. Scale bar, 100 μm. One-way ANOVA test; *, P < 0.05; **, P < 0.01; ***, P < 0.001 Molecular mechanisms of PP-NPs neurotoxicity on human COs To investigate the molecular mechanisms by which NPs affect the neurodevelopment of COs, we collected COs from control group, PP-10 (10 µg/mL) group and PP-50 (50 µg/mL) group at day 40 for transcriptomic analysis. The screening thresholds for differentially expressed genes (DEGs) were|log2FC| > 2 and Padj < 0.05. Sequencing results showed that compared with the control group, there were 342 down-regulated genes and 76 up-regulated genes in the PP-10 group, and 1023 down-regulated genes and 135 up-regulated genes in the PP-50 group (Fig. [120]5A and B). Venn diagrams showed that 310 common DEGs were observed in PP-10 and PP-50 groups (Fig. [121]5C). The up-regulated genes were significantly enriched in a variety of biological processes associated with immune response and interferon signaling pathways, including interleukin-27-mediated signaling activity pathway, negative regulation of immune response, type I interferon signaling pathway and cellular response to type I interferon (Fig. [122]5D and E). In addition, the down-regulated DEGs were significantly enriched in multiple biological processes related to development and morphogenesis, such as animal organ morphogenesis, skeletal system development, embryonic organ development and embryonic morphogenesis (Fig. [123]5D and E). Furthermore, in the PP-50 group, down-regulated genes were also significantly enriched in cell fate specification (Fig. [124]5E), indicating that high-concentration PP-NPs might cause more serious interference with the differentiation of developing cells. These results suggest that PP-NPs may interfere with the normal formation and development of fetal brain by inhibiting multiple biological processes closely related to organ morphogenesis and development. Fig. 5. [125]Fig. 5 [126]Open in a new tab Transcriptomic alteration in COs with PP-NPs exposure. (A) Volcano plot of the DEGs in PP-10 group and control group. (B) Volcano plot of the DEGs in PP-50 group and control group. (C) Venn diagram of DEGs among three groups. (D) Top 15 terms of the gene ontology biological process (GO-BP) enrichment analysis of up-/down-regulated DEGs in PP-10 and control groups. (E) Top 15 terms of GO-BP enrichment analysis of up-/down-regulated DEGs in PP-50 and control groups To further explore the mechanisms of neurotoxicity induced by PP-NPs, we performed KEGG pathway analysis on DEGs and identified the top 10 pathways with the most significant enrichment based on P-value, as shown in Fig. [127]6A and B. Notably, neuroactive ligand-receptor interaction (NLRI) pathway was the most significantly enriched pathway in both sets of DEGs. Additionally, gene set enrichment analysis (GSEA) revealed varying degrees of enrichment of this pathway in the two comparison groups (Fig. [128]6C and D). These findings suggested that the NLRI pathway may play a central role in mediating the neurodevelopmental toxicity of PP-NPs. To validate these findings, we performed heatmap analysis and RT-qPCR validation of six common DEGs in the NLRI pathway. Consistent with the RNA-seq results, cysteinyl leukotriene receptor 1 (CYSLTR1), parathyroid hormone 1 receptor (PTH1R), gastric inhibitory polypeptide receptor (GIPR), endothelin 3 (EDN3), and proenkephalin (PENK) showed significant transcriptional downregulation in the PP-NPs group, while neuropeptide VF precursor (NPVF) exhibited transcriptional upregulation (Fig. [129]6E and F). These results further support the critical role of NLRI pathway in PP-NPs-induced neurodevelopmental toxicity and confirm the reliability of the RNA-seq data. Fig. 6. [130]Fig. 6 [131]Open in a new tab Pathway enrichment analysis and expression of common DEGs in the NLRI pathway. (A) KEGG pathway enrichment analysis based on DEGs between PP-10 group and control group. (B) KEGG pathway enrichment analysis based on DEGs between PP-50 group and control group. (C-D) GSEA analysis of neuroactive ligand-receptor interaction pathway. (E) Heatmap of the common DEGs in NLRI signaling pathway. (F) RT-qPCR analysis of CYSLTR1, PTH1R, GIPR, EDN3, PENK, and NPVF mRNA (n = 3). One-way ANOVA test; compared to control, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 Experimental validation of common DEGs in vivo and molecular docking To further confirm the transcriptomic findings obtained from COs, we assessed the mRNA expression levels of six common genes identified in the NLRI pathway using fetal mice cerebral tissues. The gene expression changes observed in fetal mice cerebral tissues were consistent with the findings from COs. Compared to the control group, the mRNA levels of CYSLTR1, PTH1R, GIPR, EDN3, and PENK were significantly downregulated in the PP-NPs group, while the mRNA level of NPVF was significantly upregulated (Fig. [132]7A). Normal function of receptor proteins is essential for maintaining homeostasis in the nervous system, and their dysregulation may lead to neurodevelopmental abnormalities or dysfunction. Therefore, we next examined the expression levels of the three receptor proteins, CYSLTR1, GIPR, and PTH1R. Compared to the control group, protein levels of CYSLTR1 and PTH1R were significantly reduced, while protein level of GIPR did not change significantly (Fig. [133]7B and C). To further investigate the interaction between PP-NPs and these receptor proteins, we performed molecular docking analysis. The results demonstrated successful docking of the PP monomer with CYSLTR1 and PTH1R, with binding scores of -7.41 and − 6.91, respectively. As shown in Fig. [134]7D-F, propylene formed alkyl interactions with CYSLTR1 and PTH1R. Furthermore, to validate the predicted interactions, we performed immunofluorescence co-localization analysis [[135]43, [136]44] on frozen cerebral cortex sections of fetuses from pregnant mice exposed to fluorescently labeled PP-NPs. We observed co-localization of PP-NPs with the target receptors CYSLTR1 and PTH1R, whereas GIPR, which showed no binding affinity in our docking analysis, served as a negative control and showed no co-localization (Supplementary Fig. [137]2). These results support the specificity of the predicted interactions from our molecular docking analysis and suggest that CYSLTR1 and PTH1R may serve as key molecular targets in mediating the neurotoxic effects of PP-NPs. Fig. 7. [138]Fig. 7 [139]Open in a new tab Experimental validation of common DEGs in vivo and molecular docking. (A) RT-qPCR analysis of CYSLTR1, PTH1R, GIPR, EDN3, PENK, and NPVF mRNA (n = 4). (B-C) The expression levels of CYSLTR1, PTH1R, and GIPR were detected by Western blot and the relative intensity was quantified by Image J software (n = 3). (D) Docking pattern diagram of propylene with PTH1R. Left, 3D pattern diagram; right, 2D pattern diagram. (E) Docking pattern diagram of propylene with CYSLTR1. Left, 3D pattern diagram; right, 2D pattern diagram. (F) Binding scores of propylene with CYSLTR1 and PTH1R. Two-tailed t-test; *, P < 0.05; **, P < 0.01 Discussion Pregnancy and childhood are sensitive windows of environmental exposure during which the developing brain is particularly vulnerable to environmental toxins [[140]45]. Despite growing concern over the potential health risks of plastic pollution, the impact of NPs on fetal brain development remains poorly understood. Recent studies have highlighted the widespread presence of MPs in the water and soil environments [[141]46, [142]47]. However, human exposure to airborne microplastics may be even more significant, as their abundance and variety in the atmosphere may exceed those found in water and soil environments [[143]7, [144]26]. In addition, airborne microplastics can directly enter the human body without the need for processing or filtration, presenting a potentially greater health risk. Importantly, as MPs continue to degrade in the environment, they can generate smaller sized NPs, which may pose greater biological challenges due to their enhanced ability to cross biological barriers. In this study, we investigated the effects of PP-NPs inhalation exposure during pregnancy on fetal brain development using pregnant mice and human brain organoids as experimental models. Our findings provide novel insights into the toxicological effects of airborne NPs on the developing brain. First, we evaluated the impact of maternal PP-NPs inhalation exposure on fetal brain development. The weekly intake of MPs in humans is estimated to be 0.1 to 5.0 g [[145]24], equivalent to approximately 0.2 to 11.0 mg/kg body weight/day (assuming a 65 kg body weight in adults). For NPs, the exposure level may be higher. Therefore, the 5 mg/kg PP-NPs dose used in our study is a reasonable reflection of the current human exposure level. The results showed that exposure to PP-NPs during pregnancy led to decreased expression of neuronal differentiation marker TUJ1 and proliferation marker KI67 in the fetal cortical region. These findings highlight the vulnerability of the developing brain to environmental toxins during pregnancy. The impaired neuronal differentiation and proliferation observed in the fetal brain may contribute to long-term neurodevelopmental deficits, as early developmental processes such as neurogenesis and neurodifferentiation are crucial for establishing a functional neural network [[146]48]. Then, we performed neurobehavioral assessments of the offspring. Morris water maze results indicated that PP-NPs exposure during pregnancy resulted in impaired spatial memory ability and cognitive performance in offspring, which is consistent with a previous study [[147]16]. Moreover, motor coordination and balance, assessed by the rotarod test, were also impaired in offspring, suggesting that prenatal NPs exposure may affect the motor-related regions of the brain. In addition, open field test demonstrated that gestational exposure to PP-NPs resulted in increased anxiety-like behavior in offspring. This is in agreement with earlier study that showed maternal intake of polystyrene particles (100 nm and 1000 nm) during pregnancy led to anxiety-like behavior in offspring [[148]49]. Interestingly, another study has shown that maternal exposure to polystyrene nanoplastics (50 nm) may lead to anti-anxiety behaviors in offspring [[149]16], suggesting that the mechanism and biological effects of NPs may differ depending on the type and size of NPs. COs have emerged as an important model for studying human neurodevelopment and neurotoxicity [[150]50–[151]52]. Unlike traditional 2D cell cultures, COs better replicate the complex structure and cellular interactions found in the developing brain, offering more biologically relevant insights into how environmental toxins impact neurodevelopment [[152]51]. In the present study, we further investigated the potential effects of PP-NPs exposure on neurodevelopment using cerebral organoid model. By exposing brain organoids to PP-NPs, we found that the size of COs and the expression of neuronal markers TUJ1, MAP2, PAX6, and RBFOX3 significantly decreased with increasing exposure concentration. These changes suggested that PP-NPs exposure inhibited neuronal differentiation, which was consistent with in vivo experiments. Other studies have also reported that polystyrene microplastics or nanoplastics can inhibit neuronal differentiation and maturation in brain organoids [[153]31, [154]53]. Unlike our study, Hua et al. investigated the effects of polystyrene microplastics on human brain organoids [[155]53], while Chen et al. focused on the effects of polystyrene nanoplastics during the maturation stage of human brain organoids [[156]31]. Furthermore, the expression of NES, PCNA, OLIG2, MOG, S100B, and SLC1A3 was also significantly decreased in COs exposed to 50 µg/mL PP-NPs, while the expression of the microglial marker AIF1 was significantly increased. Thus, these findings suggested that PP-NPs exposure disrupted multiple neurodevelopmental processes, including neural stem cell maintenance and proliferation, neuronal differentiation, and glial cell maturation, highlighting its potential neurotoxic effects. Transcriptomic analysis revealed significant alterations in gene expression following PP-NPs exposure, and it was found that PP-NPs exposure effectively activated NLRI signaling pathway. The NLRI pathway has an important role in neural development, involving interactions between neuroactive ligands and their corresponding receptors [[157]54, [158]55]. These interactions are essential for regulating neuronal activity, neurotransmitter release, and signal transduction [[159]56]. Aberrations in the NLRI signaling pathway are closely associated with neurobehavioral abnormalities such as memory impairment and cognitive decline [[160]57, [161]58]. Its dysregulation may induce neurotoxicity by disrupting neurotransmitter homeostasis [[162]59]. Notably, enrichment of this pathway has also been observed in fish models following nanoplastic exposure, accompanied by behavioral abnormalities and altered neurotransmitter levels [[163]60, [164]61]. In the present study, genes related to the NLRI pathway, CYSLTR1, PTH1R, GIPR, EDN3, PENK, and NPVF were significantly regulated in both in vivo and in vitro models, indicating that PP-NPs may interfere with ligand-receptor signaling cascades, potentially leading to disrupted neurotransmission and downstream neurotoxic effects. To validate the potential interactions between PP-NPs and key neuroreceptors, we performed molecular docking analysis. Molecular docking is a widely used computational approach for assessing the binding affinity and interaction modes between plastic particles and biomacromolecules. Previous studies have applied this approach to evaluate the interaction of MPs/NPs with neurotransmitter receptors [[165]62], human serum albumin [[166]39], mitochondrial complexes [[167]40] and cytochrome P450 proteins [[168]63], thereby providing mechanistic insights into MPs/NPs-induced toxicity. In the present study, the molecular docking, immunofluorescence and Western blot results suggested that CYSLTR1 and PTH1R may be key molecular targets for the neurotoxic effects of PP-NPs. CYSLTR1 is a G protein-coupled receptor (GPCR) that is widely expressed in the cerebral cortex, hippocampus, and nigrostriatum in both rodents and humans and is closely associated with neuroinflammation [[169]64]. Studies have demonstrated that CYSLTR1 plays a role in lipopolysaccharide-induced depressive behaviors and memory deficits [[170]65, [171]66], N-methyl-D-aspartate-induced neuronal injury [[172]67] and streptozotocin-induced memory impairment [[173]68] by modulating neuroinflammatory responses. Interestingly, previous study has also reported a dual role for CYSLTR1 in inflammation. It primarily mediates pro-inflammatory effects during acute injury, whereas it exerts anti-inflammatory function under chronic injury conditions. Loss of CYSLTR1 disrupts the balance of cysteinyl leukotrienes (CysLTs), thereby exacerbating chronic inflammation [[174]69]. Thus, down-regulation of CYSLTR1 in chronic exposure to PP-NPs in our study may impair its protective effect and further exacerbate neuroinflammation and neuronal injury. PTH1R, another GPCR, is primarily involved in regulating calcium homeostasis and bone metabolism and can be expressed in different tissues of the whole body such as bone, kidney and brain [[175]70, [176]71]. This receptor can be activated by two endogenous peptide agonists, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), which regulate multiple signaling pathways and achieve different biological functions [[177]72]. Studies have shown that the PTH/PTHrP/PTH1R system protects against neuroinflammation and neurodegeneration and positively impacts memory and hyperalgesia [[178]70, [179]73]. Activation of the PTH/PTHrP/PTH1R system has been found to inhibit neuroinflammation and protect astrocytes, thereby improving learning and memory function in 5XFAD mice [[180]73]. These findings are consistent with our observation that downregulation of PTH1R following chronic PP-NPs exposure may reflect the loss of a critical neuroprotective mechanism, thereby contributing to enhanced neuroinflammation and neurotoxicity. There are still some limitations in the current study. First, the exposure dose and duration of NPs used in the experiment may differ from actual environmental exposure levels. Future research could further explore the impact of different doses and exposure durations on neurotoxic effects. Second, the specific mechanisms by which NPs affect particular cell types involved in neurodevelopment, such as neurons and glial cells, require further in-depth investigation. Third, sex-specific differences were not analyzed in the behavioral experiments. A recent study by Teng et al. [[181]74] demonstrated that polystyrene nanoplastics showed sex-specific accumulation and neurotoxicity in zebrafish. This finding suggests that biological sex may influence susceptibility to nanoplastic-induced neurotoxicity. Therefore, future research should further investigate the potential regulatory role of sex differences in nanoplastic-induced neurotoxicity. Conclusion In summary, our results indicated that maternal inhalation exposure to PP-NPs during pregnancy disrupted fetal brain development and contributed to neurobehavioral disorders in offspring. Using a mouse model and a human brain organoid model, we demonstrated that PP-NPs inhibited neuronal differentiation and neural stem cell maintenance and proliferation, impaired cognitive and motor functions, and altered key signaling pathways involved in neurodevelopment. These findings provide important evidence for assessing the health risks of nanoplastic pollution, particularly during critical developmental windows. Electronic supplementary material Below is the link to the electronic supplementary material. [182]Supplementary Material 1^ (2.4MB, docx) Acknowledgements