Nanoplastics Enhance Transmembrane Transport and Uptake of Carcinogens: Transcriptional Changes and the Effects of Weathering

Abstract

Nanoplastics are generated from common consumer plastics (polyethylene terephthalate, high-density polyethylene, polystyrene, polyvinyl chloride) and exposed to simulated marine weathering for up to 10 weeks. Fourier-transform infrared spectroscopy and ζ-potential measurements reveal continuous changes in the composition of the nanoplastics, consistent with oxidation. Although the chemical composition and oxidation of the nanoplastics influence their ability to sorb polycyclic aromatic hydrocarbons (PAHs), for all investigated conditions, sorption of PAHs to nanoplastics achieves effective PAH concentrations that are orders of magnitude higher than the solubility limit in water. In an intestinal co-culture model membrane consisting of M cells and enterocytes, PAH-loaded nanoplastics enhance the overall PAH transport into and across the membrane, with HDPE achieving the highest intracellular PAH concentration. RNA sequencing of cell membranes exposed to nanoplastics reveals significant transcriptional changes, including upregulation of oxidative stress and detoxification pathways (NQO1, CYP1A1, CYP1B1), especially in response to PAH-loaded nanoplastics, while genes associated with basic cell functions, such as DNA repair (MACROD2) and division (KIF20A), are downregulated. These findings confirm the feasibility of nanoplastics to increase bioaccessibility and bioavailability of hydrophobic carcinogens and enhance cellular stress, which underscores the potential environmental and health impacts associated with nanoplastics as carriers of hydrophobic environmental toxins.

Keywords: RNA; intestinal membrane; nanoplastics; polycyclic aromatic hydrocarbons (PAHs); transmembrane transport.

Figure 1

Nanoplastics characterization. A) Number mean (nm) and polydispersity index (PDI) of the hydrodynamic diameter (nm) determined by dynamic light scattering (DLS) for native and 10 weeks weathered nanoplastics. B) ζ‐potentials (mV) for native and weathered (week 4, 10) nanoplastics. Error bars represent the mean ζ‐potential ± standard deviation (SD, n = 3 repeat measurements). C) Fourier‐transform infrared (FTIR) spectra for PET, PS, PVC, and HDPE nanoplastics with different degrees of weathering (native, week 4, and week 10).

Figure 2

Quantification of PAH sorption. PAH concentration in µg per mg of nanoplastics for PVC, PET, PS, HDPE nanoplastics weathered for 0 weeks (native), 4 weeks, or 10 weeks. Error bars represent the mean ± SD (n = 8 replicates).

Figure 3

Characterization of intestinal membranes by SEM and confocal microscopy. A) SEM images of Caco‐2 only cell membrane at magnifications of 1.5k× (left) or 4k× (right). High magnification (right) reveals the surface microvilli. Scale bar: 20 µm (left) and 10 µm (right). B) SEM images of Caco‐2/Raji B co‐culture model (M Cell model) at 1.5k× (left) or 4k× (right) magnification. The area with a loss of microvilli indicates the presence of M‐cell (M), whereas microvilli‐rich areas indicate Caco‐2 cells (C). High‐magnification (right) images show smooth cell areas for the M‐cell phenotype. Scale bar: 20 µm (left) and 10 µm (right). C) Confocal images of the M cell model and Caco‐2 cells before addition of nanoplastics (0 h) as well as 1 and 24 h after the addition of nanoplastics. The cells were stained with Hoechst (blue), WGA (red), and nanoplastics stained with rhodamine B (green). Scale bar: 100 µm.

Figure 4

Quantification of nanoplastics‐mediated PAH uptake and transmembrane transport. Dot plots depict the mass (ng) of PAHs from PAH‐loaded nanoplastics that traversed across the M cell model cell membrane (Basolateral) or were taken up into cells (Intracellular) for native, and 4 and 10 weeks weathered PVC, PET, PS, HDPE nanoplastics. Error bars represent the mean mass ± SD (n = 6 biological replicates).

Figure 5

PCA of all conditions and subclustering of differentially expressed genes (DEGs). A–C) 2D PCA plot (PC2 vs PC1) for A) all individual samples as well as Water_PAH and NoTreat controls, B) samples grouped by composition HDPE, PVC, PS, PET with and without PAH loading as well as Water_PAH and NoTreat controls, C) samples grouped by 0 weeks weathering (native), 4 weeks weathering (W4), and 10 weeks weathering (W10) with and without PAH loading as well as Water_PAH and NoTreat controls. Water_PAH and NoTreat controls are circled in red. D–F) PC2 versus PC1 Loading scores plotted of 2D PCA plots generated in (A)–(C). Gray shaded area denotes 10 SD from the origin. The top 15 genes furthest away from the origin are highlighted and numerically labeled in red. G) Subclusters of genes captured from hierarchical clustering heatmap in Figure S11 (Supporting Information). Gray lines represents gene expression relative to the corrected expression levels for the individual genes and blue line represents the mean for all genes in the cluster.

Figure 6

DEG visualizations of transcriptomic changes in treated membranes. A) Two‐way hierarchical clustering heat map of RNA‐Seq transcriptome analysis of the genes from subclusters 4, 5, 6, 8 (in Figure 5G). x‐axis represents conditions, where y‐axis represents genes. Data Log₂ normalized. Scale bar illustrates the intensity of Log₂‐transformed expression value. B) Bar chart of the DEG count for upregulated (Up), downregulated (Down), and sum (All) of all genes relative to the NoTreat control. |Log₂(Fold Change)| ≥ 1 and p _adj ≤ 0.05. C) Representative volcano plots displaying DEGs for native, week 4 (W4), week 10 (W10) HDPE with and without PAHs relative to the NoTreat control group. |Log₂(Fold Change)| ≥ 1 and p _adj ≤ 0.05. Red dots represent genes above this threshold in the positive direction (Up), blue dots above this threshold in the negative direction (Down), black dots do not meet the criteria (N.S.). Remaining plots for plastics are provided in Figure S12 (Supporting Information). D) List all DEGs that are shared across all nanoplastic samples loaded with PAHs (12), but not observed in any unloaded nanoplastic samples (12). |Log₂(Fold Change)| ≥ 1 and p _adj ≤ 0.05. Reporting average Log₂(Fold Change) and p _adj values.