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. 2026 Apr 8;4(6):1061–1072. doi: 10.1021/envhealth.5c00862

Tracing the In Vivo Fate of Polystyrene Nanoplastics via a Lanthanide Copolymerization Labeling Strategy

Jiaqi Shen †,‡, Zhuda Song †,‡, Yun Wang †,‡, Yuhui Ma †, Xiao He †, Lingna Zheng †, Meng Wang †, Weiyue Feng †,‡,*, Zhiyong Zhang †,‡,*
PMCID: PMC13288247  PMID: 42344582

Abstract

The widespread detection of plastic particles in environmental and biological matrices has raised health concerns, yet quantitative evidence on the in vivo behavior of submicron and nanoplastics (S/NPs) remains limited. Here, we developed a sensitive tracking platform by stably incorporating lanthanide elements into polystyrene (PS) S/NPs via emulsion copolymerization. Using this methodology, we systematically investigated in mice the gastrointestinal transit, barrier translocation, biodistribution, and pharmacokinetics of lanthanide-doped PS particles with distinct surface properties: 80 nm (negative), 80 nm (positive), and 500 nm (positive). Following oral gavage (500 mg/kg), only 0.01–0.04% of the administered dose translocated to major organs; 500 nm particles exhibited slower gastrointestinal transit but higher barrier translocation than their 80 nm counterparts. After intravenous injection (10 mg/kg), both PS NPs accumulated predominantly in the liver and spleen, with positively charged particles showing enhanced uptake. Laser ablation-ICP-MS imaging revealed time-dependent redistribution within splenic microregions and preferential retention in iron-enriched brain areas. Complementary analyses, including zeta potential evolution in simulated gastrointestinal media, immunofluorescence staining of intestinal tight junctions, and transmission electron microscopy, provided mechanistic insights into particle behavior. By integrating a stable labeling strategy with advanced mass spectrometry-based tools, this study establishes a quantitative platform for accurately assessing S/NP in vivo fate, offering critical insights for environmental health risk assessment.

Keywords: polystyrene nanoplastics, quantitative in vivo tracking, LA-ICP-TOF-MS imaging, splenic microdistribution, brain vascular retention

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Introduction

Plastic particles have been detected across water, soil, air, and the food chain, raising growing concern about human exposure. Recent studies have reported their presence in human stool, lung tissue, blood, placenta, and even breast milk, indicating that internal exposure is already occurring and may begin as early as fetal development. These findings highlight an urgent need to understand how plastic particles enter, distribute, and persist within mammalian systems. However, their real in vivo fate, barrier-crossing capability, and tissue-level distribution remain poorly defined, particularly submicron- and nanoplastics (S/NPs). ,

Progress in this area has been limited by analytical constraints. Spectroscopic techniques such as Fourier-transform infrared (FTIR) and Raman microscopy lack sufficient spatial resolution for particles below the micrometer scale. Pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS), while not limited by particle size and offering high sensitivity for polymer identification, provides limited biological specificity. , Labeling strategies have therefore been introduced; however, fluorescence-based methods may suffer from signal instability and quantitative uncertainty in vivo, − and carbon-14 radiolabeling, although sensitive, remains constrained by accessibility and limited spatial mapping capability. , These methodological gaps underscore the need for a stable, mass-conserved, and spatially resolvable tracking strategy for quantitative in vivo analysis of S/NPs.

Metal-element labeling combined with mass spectrometric detection provides a compelling alternative. Metal tracers generate element-specific signals and permit highly sensitive quantitative analysis by inductively coupled plasma-mass spectrometry (ICP-MS). When integrated with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), this approach further enables rapid two-dimensional spatial mapping within tissues. These features position metal-based labeling as a robust framework for in vivo tracking of S/NPs.

Several metal-based labeling strategies have been developed, including surface adsorption, polymerization-based encapsulation, and swelling-diffusion approaches. Surface adsorption enables highly sensitive detection by single particle-inductively coupled plasma-mass spectrometry (sp-ICP-MS) but inevitably alters surface properties and may suffer from tag desorption under physiological conditions. Polymerization-stage embedding improves labeling stability; , however, incorporation of dense metal species can broaden particle size distributions and increase particle density, potentially confounding biodistribution and clearance behavior. Swelling-diffusion better preserve morphology, yet solvent-induced surface modification and partial tracer leakage in biological media have been reported. Therefore, it highlights a pressing need for a tracking strategy that preserves the intrinsic physicochemical characteristics of S/NPs while providing high labeling stability and quantitative robustness.

Polystyrene (PS) is among the most frequently detected microplastics in environmental and daily life settings and has been repeatedly identified in human samples. ,, Accumulating evidence suggests that PS S/NPs can induce adverse biological effects; , however, their in vivo burden, organ-specific distribution, and dose–effect relationships remain poorly characterized. Here, we establish a quantitative plastic particle tracking platform by homogeneously incorporating lanthanide complexes into PS S/NPs via emulsion copolymerization. This strategy largely preserves the intrinsic physicochemical properties of the particles while enabling ultralow-background and stable quantitative detection of lanthanide tracers by ICP-MS. Conventional oral and intravenous (IV) exposure experiments were conducted to obtain baseline biodistribution data of PS(−) NPs (80 nm), PS­(+) NPs (80 nm), and PS­(+) SPs (500 nm), and to benchmark the quantitative robustness of this strategy. Crucially, integration of this labeling approach with laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOF-MS) enables high-resolution, suborgan, multielement imaging of plastic particles in the spleen and brain, resolving particle migration across splenic microanatomical compartments and spatial localization in cerebral tissue.

Materials and Methods

Materials

Europium­(III) chloride hexahydrate (EuCl3·6H2O), terbium­(III) chloride hexahydrate (TbCl3·6H2O) and 1,10-Phenanthroline (Phen) were obtained from Aladdin (China). Methacrylic acid (MAA) and styrene were supplied by Tokyo Chemical Industry (TCI, Japan). Azobis­(isobutyronitrile) (AIBN), potassium persulfate (KPS), sodium dodecyl sulfate (SDS), and cetyltrimethylammonium bromide (CTAB) were purchased from Macklin (China). Nitric acid (trace metal grade) was obtained from Beijing Chemical Reagents Company (China). Bovine serum albumin (BSA), 1X phosphate buffered saline pepsin (PBS), pepsin and trypsin were sourced from Sigma-Aldrich (USA).

Synthesis and Characterization of Lanthanide-Doped Polystyrene Particles

Lanthanide-doped polystyrene particles were generated through a two-stage process involving the preparation of polymerizable Eu and Tb complexes followed by emulsion copolymerization, as described in our previous study with minor modifications. Lanthanide (Eu3+/Tb3+) complexes were prepared via refluxing methacrylic acid and 1,10-phenanthroline in ethanol (pH-adjusted) with EuCl3·6H2O or TbCl3·6H2O. Precipitates were rinsed and dried to obtain Eu/Tb­(MAA)3Phen dopants. For PS-Eu/Tb synthesis, dopants were mixed with styrene, emulsified via sonication in an aqueous surfactant solution, and polymerized under nitrogen at 70 °C for 24 h using thermal initiators. Adjusting surfactants/initiators enabled synthesis of negatively/positively charged nanoparticles (∼80 nm) and submicron particles (∼500 nm) (Table S1). Final products were purified by exhaustive dialysis to remove residual impurities. Particle morphology and elemental distribution were analyzed by transmission electron microscopy coupled with energy-dispersive spectroscopy (TEM-EDS; FEI Tecnai G2 F30, Thermo Fisher Scientific, USA). Hydrodynamic diameter, polydispersity index (PDI), and surface zeta potential were measured in triplicate (n = 3) using the Malvern Mastersizer 2000 (Malvern Panalytical, UK).

Lanthanide content was quantified the following literature-reported procedure. Briefly, particle suspensions were digested in 5 mL of concentrated nitric acid using a microwave-assisted digestion system (MARS6, CEM Corporation, USA). The temperature was increased to 190 °C for 25 min and held at 190 °C for 40 min. The resulting digests were subsequently analyzed by ICP-MS (X7, Thermo Fisher Scientific, USA).

Animal Experiment

Male CD-1 (ICR) mice, weighing 15–18 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The experimental protocol was reviewed and approved by the Animal Ethics Committee of the Institute of High Energy Physics (approval No. IHEPLLSC). All mice were housed in a conventional animal care facility under controlled conditions: temperature of 25 ± 2 °C, relative humidity of 60 ± 2%, and a 12-h light/dark cycle. Standard commercial pellet diets and deionized water were provided ad libitum throughout the study period. A 7-day acclimatization period was enforced prior to the commencement of experimental procedures. The experimental design is shown in Figure S1. For oral exposure, mice were randomly assigned to four groups (n = 6/group): Control, PS(−) NPs, PS­(+) NPs, and PS­(+) SPs. Animals received a single gavage dose of lanthanide-labeled PS suspension (500 mg/kg, 400 μL) or deionized water. For IV exposure, mice were assigned to three groups (n = 6/group): Control, PS(−) NPs, and PS­(+) NPs. Animals received a single tail-vein injection of lanthanide-labeled PS suspension (10 mg/kg, 250 μL) or sterile 1X PBS.

Sample Collection

For the oral exposure cohort, mice were euthanized at 1, 4, and 24 h postgavage. Major organs, including the stomach, small intestine, large intestine, brain, heart, liver, spleen, lung, and kidney, were collected for elemental analysis. Feces were collected over a 24 h period using metabolic cages. For the IV cohort, blood samples were collected at 0.5, 1, 4, 12, and 24 h postinjection under isoflurane anesthesia into EDTA-coated tubes for pharmacokinetic analysis. Mice were subsequently euthanized, and organs were harvested as described above. Feces were collected over 24 h. For brain analysis, at 24 h post-IV injection, mice were further divided into perfused and nonperfused groups. In the perfusion group, transcardial perfusion with sterile saline (30 mL) was performed prior to brain harvest to remove residual blood.

Quantitative Determination of PS M/NPs in Samples by ICP-MS

All samples were weighed, stored at −20 °C, and digested according to the procedure described above, followed by ICP-MS analysis to quantify Eu and Tb concentrations. The measured lanthanide concentration was converted to the corresponding PS mass based on the known lanthanide doping fraction in the particles.

Zeta Potential Evolution in Simulated Gastrointestinal Media

Unlabeled PS particles (10 μg/mL) were incubated in simulated gastric fluid (SGF) at 37 °C under continuous agitation (200 r/min) for 30 min, after which zeta potential was measured in triplicate (n = 3) using a Malvern Mastersizer 2000 (Malvern Panalytical, UK). Samples were then centrifuged (15,000 × g, 10 min), resuspended in simulated intestinal fluid (SIF), incubated under the same conditions for an additional 30 min, and measured again for zeta potential.

AB/PAS and Immunofluorescence Staining

Intestinal tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned (3–4 μm). Alcian blue-periodic acid-Schiff (AB-PAS) staining was performed following standard protocols. The stained area in AB-PAS sections was quantified using ImageJ (n = 3). For immunofluorescence (IF) staining, sections were blocked and incubated with primary antibodies against ZO-1, Occludin, and Claudin-1 overnight at 4 °C. After washing, sections were incubated with appropriate secondary antibodies and developed using TSA. Images were acquired using a fluorescence microscope (Nikon Eclipse C1, Japan). Detailed antibody information is provided in Table S2.

TEM Analysis

Intestinal tissues were processed for TEM according to standard procedures. Briefly, samples were fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, embedded in resin, sectioned (70–90 nm), stained, and examined using a Zeiss TEM 109 microscope (Germany).

LA-ICP-MS Imaging

Spleens were collected from one mouse per group at 1, 4, and 24 h post-IV injection. Brains were collected at 4 h post-IV injection, with one mouse undergoing transcardial perfusion (30 mL sterile saline) prior to harvest and another processed without perfusion. All tissues were stored at −20 °C until sectioning (20 μm). Both spleen and brain sections were analyzed via LA-ICP-TOF-MS line scanning to map the spatial distribution of 56Fe and 159Tb, using a laser spot size of 20 μm × 20 μm. Elemental signals were normalized to 13C as an internal standard, and images were generated using Igor Pro 6.10 software.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10. The statistical tests employed are specified in each figure caption. Statistical significance is denoted in the graphs using the following convention: *P < 0.05, **P < 0.01, ***P < 0.001; # P < 0.05, ## P < 0.01, ### P < 0.001, and NS = not significant (P > 0.05).

Results and Discussion

Synthesis and Characterization of Lanthanide-Labeled S/NPs via Copolymerization

To enable quantitative in vivo detection, we labeled PS S/NPs with lanthanide elements through an emulsion copolymerization strategy (Figure A). We first synthesized polymerizable Eu­(III)/Tb­(III) tris­(methacrylic acid)-1,10-phenanthroline lanthanide complexes via coordination chemistry, verified by organic elemental analysis, FTIR and fluorescence spectroscopy (Table S3, Figure S2). These complexes were subsequently copolymerized with styrene under controlled emulsion conditions. By adjusting surfactant type and concentration, we generated three lanthanide-doped PS particle typesPS(−) NPs, PS­(+) NPs, and PS­(+) SPs. Specifically, we added anionic surfactants SDS to produce negatively charged PS particles, cationic surfactants CTAB to produce positively charged PS particles, and tuned the ratio of surfactant to monomer to synthesize PS particles with different sizes (Table S1). The average hydrodynamic diameter and zeta potential measurements confirmed the successful synthesis of particles with the intended sizes and stable surface charges (Figure B). Scanning electron microscopy (SEM) verified monodisperse spherical morphologies (Figure S3). TEM-EDS mapping showed uniform Tb incorporation in PS(−) NPs and PS­(+) NPs and uniform Eu incorporation in PS­(+) SPs (Figure C and Figure S4). Additional FTIR and thermogravimetric analysis (TGA) data are provided in Figure S5.

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Synthesis and characterization of PS S/NPs. (A) Lanthanide-copolymerized PS S/NPs: synthesis and quantitative detection principle in mice. (B) The hydrodynamic diameter (mean size and PDI are labeled for each sample) and zeta potential measurements of the three PS S/NPs. (C) Representative TEM-EDS diagrams of Tb-doped PS(−) NPs and Eu-doped PS­(+) SPs.

Benefiting from the intrinsically low background of lanthanides in biological tissues, the labeled PS S/NPs achieve detection sensitivities in the tens-of-ppb range (Table S4). Lanthanide incorporation in the PS S/NPs was consistently maintained from 0.86 to 0.94 wt %, as detected through microwave-assisted nitric acid digestion followed by ICP-MS quantification. All three PS S/NPs exhibited excellent stability, showing minimal cumulative lanthanide release over 7 days across several simulated biological fluids (generally <0.5%, Figure S6). Slightly higher release (1–1.5%) was observed in PBS containing 10% (w/v) BSA, likely due to enhanced particle dispersion by serum proteins, which can reduce separation efficiency during centrifugation and result in an apparent increase in measured leakage.

Gastrointestinal Transit and Fecal Recovery of PS S/NPs Following Oral Exposure

Since ingestion is considered one of the most relevant human exposure routes for PS S/NPs, mice were first orally administered the three lanthanide-labeled PS S/NPs to assess how particle size and surface charge influence their gastrointestinal (GI) transit and elimination. Following gavage, all three PS particle types exhibited broadly similar distribution patterns across the gastrointestinal tract (GIT) during the early phase of transit (Figure A–C). At 1 h, the majority of PS S/NPs resided in the stomach and small intestine, followed by distal progression at 4 h and substantial fecal elimination by 24 h. These overall transit profiles closely parallel findings from previous in vivo study using fluorescently labeled polymer particles, which reported rapid gastric emptying, gradual intestinal progression, and predominant fecal excretion.

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Time-dependent distribution of PS S/NPs in the GIT and feces following oral gavage at 1, 4, and 24 h. Fractional distribution of (A) PS(−) NPs, (B) PS­(+) NPs, and (C) PS­(+) SPs. Panel D summarizes the differences in gastrointestinal–fecal transit kinetics as a function of particle charge and size.

Direct comparison between PS(−) NPs and PS­(+) NPs showed no significant differences in gastric emptying or small- and large-intestinal transit across all time points (Figure D). This indicates that surface charge exerts a minimal influence on macroscopic GI transport following oral exposure. This observation accords with prior studies, suggesting that electrostatic effects are largely masked by luminal contents, digestive fluids, and mucus interactions during intestinal transit. , In contrast, PS­(+) SPs displayed noticeably slower progression through the small–large intestinal junction, with increased small-intestinal retention compared with PS­(+) NPs (P < 0.01) at 24 h. This size-dependent retardation is consistent with studies showing that larger particles experience increased mechanical hindrance and stronger interactions with intestinal mucus and microstructures, leading to prolonged small-intestinal residence. , Notably, despite these kinetic delays, total fecal recovery at 24 h was comparable across all particle types.

Systemic Translocation and Organ Biodistribution Following Oral Exposure

Following oral administration, all three PS S/NPs exhibited low but measurable systemic translocation across the GI barrier, with cumulative bioavailability in major organs (heart, liver, spleen, lung and kidney) ranging from 0.01 to 0.04%ID (Figure A). Potential interference from free lanthanide tracer were negligible in the present study. Lanthanide leakage in simulated gastric and intestinal fluids remained below 0.05% at oral-exposure-relevant concentrations (Figure S7), and free lanthanides themselves are known to exhibit extremely low oral bioavailability (<1%). , Thus, these controls support that the measured systemic signal predominantly reflects intact PS S/NPs, rather than dissociated tracer. This magnitude is comparable to that reported in earlier radiotracer-based and isotope-labeling studies, which generally report very limited systemic uptake of polymeric or plastic particles following oral exposure. , In contrast, substantially higher apparent organ burdens have been reported in several fluorescence-based studies. − As highlighted in a recent methodological review, fluorescence-based tracking can be susceptible to signal-particle decoupling under biological conditions, including partial dye dissociation and signal persistence independent of intact particles. Such effects may introduce analytical artifacts and potentially inflate estimates of tissue-associated particle burden.

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GI translocation and interaction dynamics of PS S/NPs. (A) Time-dependent systemic bioavailability of PS S/NPs in major organs at 1, 4, and 24 h following oral exposure. (B) Schematic illustration of interaction dynamics between PS S/NPs and the GI barrier. (C) In vitro evolution of zeta potential of PS particles in SGF and SIF. (D) AB-PAS staining of intestinal mucus layer and the percentage of the mucus secretion area to total intestine area. (E) TEM visualization of tight junction morphology and IF staining of Claudin-1, ZO-1, and Occludin expression in intestinal tissue. (F) Hemodynamic pathway of absorbed particles following oral exposure. The starting point (marked by the black box) follows this sequence: mesenteric veinhepatic portal veinhepatic sinusoidcentral veinabdominal aortaperipheral organs. (G) Organ distribution of PS S/NPs at 24 h following oral exposure. Statistical significance is calculated using a one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05; ***P < 0.001; # P < 0.05; ## P < 0.01.

The temporal profile of bioavailability further provides insight into transport kinetics. Systemic bioavailability displayed a peak at 4 h rather than 24 h (Figure A), suggesting that systemic burden is governed by time-dependent intestinal transit, barrier interaction, and clearance dynamics rather than progressive accumulation. At 24 h, both PS(−) NPs and PS­(+) SPs displayed higher cumulative organ bioavailability in major organs compared to PS­(+) NPs (P < 0.05, P < 0.01, respectively). The enhanced bioavailability of PS(−) NPs aligns with the previous observations by Walczak et al., who reported greater uptake of negatively charged PS particles in both in vivo and in vitro systems. However, particle size effects appeared more nuanced. While smaller nanoparticles are often assumed to exhibit greater systemic accumulation due to facilitated epithelial translocation, , our data show that larger PS­(+) SPs can achieve greater cumulative bioavailability. Notably, our earlier transit analysis showed that PS­(+) SPs exhibited prolonged residence in the small intestine compared with PS­(+) NPs, which may increase the probability of barrier interaction and subsequent translocation. In addition, a previous study has reported that larger PS particles can more markedly disrupt mucin structure and tight-junction integrity, leading to increased intestinal permeability, and potentially facilitating enhanced entry of larger particles.

We next investigated particle interactions with the GI barrier (Figure B), focusing on dynamic surface transformations during transit, mucus interactions, and modulation of epithelial integrity (Figure B). We first examined the evolution of surface charge following sequential exposure to SGF and SIF. All particles underwent marked zeta potential shifts (Figure C). After SGF (pH = 1.2) treatment, the measured surface potentials of both positively and negatively charged particles approached near-neutral values. For PS­(+) S/NPs, the near-zero zeta potential in SGF likely reflects electrostatic compression and interfacial destabilization under highly acidic and high ionic strength conditions, rather than a true loss of intrinsic positive charge. In contrast, for PS(−) NPs, the near-neutral potential suggests acid-induced charge neutralization of surface functional groups. Upon subsequent exposure to SIF (pH = 6.8), positively charged PS particles remained close to neutral, consistent with ion-mediated charge balancing, whereas PS(−) NPs partially recovered their negative potential, indicating dynamic interfacial reorganization during GI transit. These observations collectively suggest that nanoparticle surface characteristics are continuously reshaped along the GI pathway, a process that is likely to influence their subsequent in vivo behavior and systemic fate.

We then examined intestinal tissue responses. AB-PAS staining showed a reduction in mucus area in all PS-treated groups relative to controls, with a more pronounced decrease for PS(−) NPs relative to PS­(+) NPs (P < 0.05, Figure D). This observation is consistent with previous reports, whereby transient gastric neutralization may reduce electrostatic repulsion between PS(−) NPs and anionic mucin glycoproteins, thereby facilitating deeper mucus penetration and enhancing potential adverse effects. TEM imaging of intestinal tissue showed localized epithelial alterations, including microvilli disruption and widened paracellular spaces, across all PS-exposed groups (Figure E). IF staining further indicated reduced expression of Claudin-1, with more pronounced changes in the PS­(+) SP group than in PS­(+) NPs, suggesting particle size-dependent effects on epithelial tight junction integrity. These findings provide a plausible mechanistic framework linking gastrointestinal interfacial reprogramming to differences in barrier interaction and systemic entry.

Once translocated across the intestinal barrier, particles followed the expected portal circulation pathway, entering the liver via the mesenteric and portal veins prior to systemic redistribution (Figure F). At 24 h, approximately 80% of the systemic particle burden localized to the liver for all particle types (Figure G, Figure S8), consistent with first-pass hepatic filtration reported for orally absorbed nanoparticles. , The kidney represented the second most enriched organ (40–60% of hepatic values), in agreement with its high blood perfusion fraction, whereas only minimal levels were detected in the spleen, lung, and heart. No particles were detected in the brain under the present conditions (Figure S9), indicating either inefficient blood–brain barrier (BBB) penetration or concentrations below the current limit of quantification. In agreement with the total bioavailability profile, PS­(+) NPs exhibited significantly lower deposition than PS(−) NPs and PS­(+) SPs in both the liver and lung (P < 0.01, P < 0.05).

In Vivo Pharmacokinetics, Biodistribution, and Hepatobiliary Clearance of PS NPs

Although PS plastic particles are primarily ingested, their detection in human blood and evidence of systemic entry from our oral exposure studies highlight the need to examine their behavior directly in the bloodstream. We therefore focused on intravenously injected PS­(+) NPs and PS(−) NPs, as nanoparticles are more likely than submicron particles to circulate systemically and access internal organs via blood exposure.

Following IV administration, both PS­(+) NPs and PS(−) NPs exhibited a rapid and sustained decline in blood concentrations (Figure A). Circulating levels decreased sharply within the first 4 h and approached baseline by 24 h. Importantly, a clear surface charge-dependent difference was observed, with PS(−) NPs maintaining significantly higher blood levels than PS­(+) NPs at all time points (P < 0.01 to P < 0.001), indicating prolonged circulation and slower systemic clearance of negatively charged nanoplastics.

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In vivo kinetics, tissue distribution, and excretion of PS NPs following IV injection. (A) Time-dependent biodistribution of PS NPs in blood and major organs at 0.5, 1, 4, 12, and 24 h. (B) Temporal retention profiles of PS NPs in the GIT. (C) Amount of PS NPs excreted in feces over 24 h post-IV administration. For panel A, * indicates significant differences in organ concentrations across different time points, and # denotes significant differences between exposure groups at the same time point. Statistical significance is calculated using a one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; # P < 0.05; ## P < 0.01; ### P < 0.001.

In line with established in vivo observations, , the liver and spleenthe primary organs of the reticuloendothelial system (RES)served as the major sites of PS NP uptake following entry the systemic circulation (Figure A). The liver was the dominant deposition organ, with both NP types reaching peak accumulation at 24 h. Across nearly all time points, PS­(+) NPs accumulated to significantly higher levels in RES organs than PS(−) NPs (P < 0.01 to P < 0.001). While this trend appears to differ from the report describing enhanced hepatic targeting of negatively charged PS particles, it is important to note that charge effects in vivo are highly context-dependent. After incubation in serum-mimicking conditions, all three particle types exhibited negative surface potentials (−12.6 to −15.1 mV; Figure S10). These results suggest, upon entering the bloodstream, nanoparticles rapidly acquire a biomolecular corona, which can attenuate or even override the influence of their initial ζ-potential. Because the surface potentials of the particles used here differ from those employed in previous studies, the resulting protein corona compositions are also expected to differ. Consequently, the effective or “apparent” surface charge governing in vivo interactions should be interpreted as an emergent property defined by the adsorbed protein corona rather than by the pristine particle ζ-potential alone.

The lung displayed a distinct charge-dependent pattern. From 1 h onward, PS(−) NPs accumulated significantly more than PS­(+) NPs (P < 0.05 to P < 0.01). This observation is consistent with recent reports showing that negatively charged PS nanoparticles preferentially target lung tissue via adsorption of vitronectin, which mediates interactions with pulmonary endothelium. Particle concentration in the heart and kidney remained low, confirming minimal involvement of these organs in NP clearance.

Regarding GI transit, both NP types moved progressively from the small intestine into the large intestine over 24 h (Figure B) and total fecal recovery at 24 h did not differ significantly between PS (+) and PS(−) NPs (Figure C).

Intrasplenic Distribution and Translocation of PS NPs

The spleen is a key immunological organ and a major site for filtering blood-borne particulate matter, making its microanatomical landscape critical for understanding how the immune system processes poorly degradable materials such as PS S/NPs. However, information on their spatial fate within distinct splenic compartments remains largely unexplored.

To resolve the spatial fate of PS NPs within splenic subregions, we employed LA-ICP-TOF-MS to obtain high-resolution multielement imaging (Figure A). This technique allows for the simultaneous detection of endogenous tissue markers and exogenous nanoparticle tracers. The endogenous iron signal (56Fe+) was utilized to delineate the splenic architecture, allowing clear distinction of the iron-rich red pulp (RP) from the white pulp (WP) and marginal zone (MZ). In parallel, the terbium signal (159Tb+) served as a proxy for tracking Tb-labeled PS NPs. Overlaying these elemental channels enabled precise colocalization of NP localization relative to the splenic architecture. Importantly, the combination of lanthanide labeling and LA-ICP-TOF-MS enables element-specific, background-independent detection and allows semiquantitative spatial mapping of plastic distribution. In contrast to fluorescence-based imaging, , elemental imaging provides improved specificity and more robust spatial quantification of particle localization.

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LA-ICP-MS multielement imaging of PS NP distribution in the spleen tissues. (A) Multielement imaging was performed using ICP-TOF-MS. 56Fe+ elemental signal was used to distinguish the red pulp from the white pulp and marginal zone, while 159Tb+ elemental signal enabled precise tracking of Tb-labeled PS NP distribution. Overlaying these elemental channels allowed covisualization of tissue morphology and PS NP localization. (B) Representative 56Fe+ and 159Tb+ multielement images of PS­(+) NPs and PS(−) NPs at 1, 4, and 24 h post IV-injection. (C) Co-localization analysis of 56Fe+ and 159Tb+ signals. Pearson correlation coefficients are shown (0.6–1.0: strong positive; 0.4–0.6: moderate positive; 0–0.4: weak positive). (D) PS NPs gradually translocated into the red pulp within 24 h.

Following IV injection, both PS (+) NPs and PS(−) NPs were detected across the WP, MZ, and RP as early as 1 h postexposure (Figure B and Figure S11). At early time points, PS NPs preferentially localized in the WP and MZ, likely due to slower blood flow and the presence of MZ macrophages that efficiently capture circulating nanoparticles. To quantitatively evaluate NP–tissue spatial relationships, Pearson correlation coefficients (PCCs) were calculated between the56Fe+ and 159Tb+ signals (Figure C). PCC values increased progressively from 1 to 24 h, shifting from weak to moderate positive correlations for both PS NPs, indicating a time-dependent redistribution of PS NPs toward the iron-rich RP (Figure D). This redistribution toward the RP is in agreement with established nanoparticle biodistribution paradigms, , in which the RP functions as a dominant long-term sequestration site due to its dense macrophage network and physical filtration properties. ,

Importantly, the colocalization metrics reported here reflect spatial association at the tissue-compartment level rather than direct evidence of cellular internalization. As such, elevated PCC values should be interpreted as indicative of compartmental redistribution and retention, rather than enhanced cellular uptake per se. Two-dimensional elemental colocalization captures spatial proximity but is not, on its own, sufficient to resolve intracellular localization.

Can 80 nm PS NPs Cross the Blood–Brain Barrier?

Following oral exposure, no detectable Tb signal associated with PS NPs was observed in brain tissue, although trace entry below the detection limit cannot be excluded. To directly assess whether PS NPs could access the brain via systemic circulation, we performed IV administration, thereby bypassing the intestinal barrier. After IV injection, both NP types exhibited significantly elevated brain Tb signals relative to controls (P < 0.01–0.001; Figure A), indicating that 80 nm PS NPs can access the cerebral circulation.

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(A) Lanthanide concentrations in brain tissue before and after perfusion at 24 h IV exposure. (B) Perfusion experiments illustrating the distinction between vascular retention and parenchymal penetration of brain-detected particles. (C) Representative 56Fe+ and 159Tb+ multielement images of PS(−) NPs in brain tissues at 4 h post-IV injection, without cerebral blood perfusion and rinsing. Statistical significance is calculated using a one-way ANOVA with Tukey’s multiple comparison test. **P < 0.01; ***P < 0.001; NS = not significant (P > 0.05).

However, the presence of PS S/NPs in brain tissue does not necessarily imply penetration of the BBB. To distinguish intravascular localization from parenchymal translocation, perfusion was performed prior to tissue collection. Perfusion markedly reduced brain Tb signals to near-control levels (P < 0.01–0.001; Figure A), indicating that the detected S/NPs were predominantly confined to the vascular lumen rather than the brain parenchyma (Figure B). Subregional analysis showed clear Tb signals before perfusion that were nearly abolished after perfusion (Figure C, Figure S12). Spatial colocalization revealed overlap between 159Tb+ and 56Fe+ signals, the latter largely derived from hemoglobin, supporting intravascular retention of S/NPs and potential association with blood components. These findings collectively suggest that 80 nm PS NPs do not cross the BBB under the conditions tested but are retained within cerebral vessels.

Although several studies have reported plastic-associated signals in brain tissue following microplastic exposure, most relied on fluorescence labeling approaches. , As discussed above, fluorescence-based tracking may be affected by signal–particle decoupling in biological environments. Rigorous in vitro leakage validation is essential to confirm tracer stability prior to in vivo interpretation, as noted by Catarino et al. Moreover, studies lacking perfusion validation or correlative morphological analysis cannot reliably distinguish intravascular from extravascular signals. In contrast, intravital two-photon microscopy has demonstrated thrombus formation within cerebral vessels without BBB penetration, consistent with the present findings.

It should be noted that this study focused exclusively on 80 nm PS NPs. Whether smaller nanoparticles are capable of penetrating the BBB remains to be systematically determined. Although one report suggested BBB translocation of 20 nm PS NPs following multiple exposure routes, the mechanisms underlying such transport remain unclear, particularly in light of the highly restrictive nature of the BBB. In addition, the upper size threshold for nanoparticle translocation across the BBB has yet to be systematically established.

Methodological Limitations and Future Perspectives

This study establishes a quantitative and spatially resolved platform for in vivo tracking of S/NPs by integrating stable lanthanide copolymerization labeling with bulk ICP-MS quantification and high-resolution LA-ICP-TOF-MS imaging. While this framework offers clear methodological advantages, several limitations should be acknowledged.

First, the exposure doses employed in this work exceed currently reported environmental levels of micro- and nanoplastics, which are typically estimated in the ng−μg per day intake range for humans. However, environmental concentrations may be underestimated due to analytical detection limits, particularly for nanoparticles below the size threshold of conventional infrared and Raman microscopy. Consequently, actual environmental exposure levels remain uncertain. The present work should be interpreted as a mechanistic and toxicokinetic investigation rather than a direct human health risk assessment. Future studies could further refine this approach by employing lanthanide radionuclides with enhanced analytical sensitivity, enabling investigations under more environmentally relevant exposure conditions and facilitating quantitative dose–response analyses.

Second, the in vivo experiments focused on short-term exposure and early distribution and clearance phases. While this design is appropriate for resolving initial absorption, organ distribution, and early retention, it does not capture long-term accumulation, redistribution, or chronic biological effects that may arise from prolonged or repeated exposure. Given growing evidence that S/NPs may persist in specific organs, future studies incorporating long-term, low-dose, and repeated exposure paradigms are warranted.

Third, the present work focuses on PS particles with defined sizes and surface charges. Although PS is among the most commonly detected plastics in environmental and human samples and serves as a well-established model material, extrapolation of the findings to other polymer types, shapes, or weathered plastics should be made with caution. Extending this copolymerization-based labeling strategy to additional polymer systems will further enhance its general applicability.

Conclusion

This study demonstrates that the systemic absorption of PS S/NPs following oral exposure was extremely low, and particle size (80/500 nm) markedly influenced their transport kinetics and organ distribution. Upon entry into the bloodstream, surface charge further modulated PS NP accumulation within the RES system. Within the spleen, PS NPs exhibited time-dependent accumulation within the red pulp over 24 h. PS NPs were also able to reach the brain via the circulation, but did not penetrate the BBB or enter the brain parenchyma. This work establishes a robust methodological framework for quantitative studies of the in vivo behavior of S/NPs and provides critical evidence to support more accurate health risk assessments. Future investigations could build on these findings by exploring long-term, low-dose exposure scenarios and systematically comparing different polymer types.

Supplementary Material

eh5c00862_si_001.pdf (1.6MB, pdf)

Acknowledgments

We acknowledge BioRender (https://biorender.com/) for providing the professional platform and templates used to generate the charts (Figures A, D, B, F, A, D, B, S1) in this study.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00862.

  • Figures S1–S12 and Tables S1–S4 (PDF)

J.S. and Z.S. contributed equally. J.S., W.F., and Z.Z. conceived and designed the experiments. J.S. and Z.S. synthesized and characterized polystyrene submicroplastics and nanoplastics. J.S. and Y.W. conducted the animal experiments. J.S., Z.S., and Z.Z. provided materials and analytical tools. M.W. and L.Z. assisted with LA-ICP-MS imaging. J.S. and Z.Z. analyzed the data. Z.Z., Y.M., and X.H. supervised the project. J.S., W.F., and Z.Z. drafted the manuscript. All authors contributed to the results, discussion, and manuscript writing.

The study was funded by the National Natural Science Foundation of China (12535020, 12175263, and 12075262), the National Key R&D Program of China (2021YFA1200100 and 2022YFA1207600), and the Beijing Natural Science Foundation (Z230008).

The authors declare no competing financial interest.

Published as part of Environment & Health special issue “Micro- and Nano-plastics: What’s Hidden Beneath”.

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eh5c00862_si_001.pdf (1.6MB, pdf)

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