A. In vitro cellular interactions with PS nanoplastics and PS/protein corona complexes

A549 cells were tested for 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to investigate the cytotoxicity of PS nanoplastics and PS/protein corona complexes. First, bare PS nanoplastics, with different sizes and charges, were introduced to confluent cell monolayers at concentrations of 50 and 10 μg ml−1 (typical concentrations tested in the literature).24,2524. A. Lesniak, F. Fenaroli, M. P. Monopoli, C. Åberg, K. A. Dawson, and A. Salvati, ACS Nano 6, 5845 (2012). https://doi.org/10.1021/nn300223w25. C. C. Fleischer and C. K. Payne, J. Phys. Chem. B 118, 14017 (2014). https://doi.org/10.1021/jp502624n At both concentrations, mild cytotoxicity was observed, although there were no clear size and/or charge related trends observed (at p Fig. 1 and Table II). When the proteins (15 μg ml−1, approximately covering the surface area of PSsmall) were introduced to the PS nanoplastics, cytotoxicity was mitigated to Fig. 1 and Table III). The significance of the different trends was confirmed, again, by two-tailed (p 

TABLE II. Summary of MTT cytotoxicity assay using A549 cells. The cell viability % is shown for the A549 cells exposed to two concentrations of uncoated PS nanoplastics were used. The results are the average of triplicate, and the error is shown inside the parentheses.

50 μg ml−110 μg ml−1PS(−)large60% (25)82% (10)PS(−)small73% (4)85% (5)PS(+)large85% (16)83% (16)PS(+)small80% (5)81% (5)

TABLE III. Summary of the MTT cytotoxicity assay using A549 cells. The cell viability % is shown for the A549 cells with different nanoplastics exposure conditions. The concentration of PS nanoplastics and proteins were fixed at 50 and 15 μg ml−1, respectively. The results are the average of triplicate, and the error is shown inside the parentheses.

HSA (−)LYS (+)PS(−)large95% (5)Soft corona89% (7)Hard coronaPS(−)small97% (2)84% (7)PS(+)large87% (8)Hard corona89% (4)Soft coronaPS(+)small81% (6)98% (6)The effect of PS nanoplastics and protein corona on A549 cells was studied using flow cytometry (FC). Scatter plots (Fig. 2) show the presence of A549 cells, indicated in the major scattering population (80%−90%). Other populations appearing in the low side scattering (SSC) and forward scattering (FSC) intensities are the debris and/or noise. The introduction of PS(−)small, with or without the presence of protein coronae, did not alter the cytogram profiles significantly. The results imply that a majority of the cells are intact, aligning with the cytotoxicity assay results. The same trend can be observed for the PS(−)large complexes with both types of protein coronae (Fig. S1 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. However, for their positively charged analogs, PS(+)small and PS(+)large, another scattering population, in the low FSC, and a broad range of SSC intensities appear (Fig. S1 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. Although strong scattering from the PS(+)small and PS(+)large nanoplastics is evident in the control FC experiment, without A549 cells (Fig. S2 in the supplementary material),5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. the additional scattering population appears to have a different SSC (Fig. S2 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. Colloidal aggregation is observed when PS(+)small and PS(+)large nanoplastics are dispersed in DMEM (evidenced in cytograms found in Fig. S1 in the supplementary material),5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. which led to the appearance of the scattering profile.The FSC and SSC histograms are shown in Fig. 3. The FSC scale increased slightly when PS(−)small (with or without the protein corona) was incubated with the cells, implying little influence on overall cell sizes according to the Mie theory. The relative SSC scale increased in comparison to the control cells, with the bare PS(−)small showing the greatest effect followed by PS(−)small/LYS hard corona complex, and PS(−)small/HSA soft corona complex. The observed trend was also found for PS(−)large complexes (Fig. S3 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. Similarly, for the positively charged particles, the effect of the bare PS nanoplastics appeared to promote the greatest change, and the PS/hard corona complexes either caused greater or a similar extent of change compared to the PS/soft corona complexes. The increase on the SSC scale is related to the cytoplasmic complexity for eukaryotic cells.32,3332. R. Foldbjerg, D. A. Dang, and H. Autrup, Arch. Toxicol. 85, 743 (2011). https://doi.org/10.1007/s00204-010-0545-533. H. Suzuki, T. Toyooka, and Y. Ibuki, Environ. Sci. Technol. 41, 3018 (2007). https://doi.org/10.1021/es0625632 We speculate that the increase in the cellular complexity is attributed to the additional cellular environment present inside the cells and/or internalised PS particles.The cell uptake process was visualized using fluorescently labeled PS(−)small and A549 cells with nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) (Fig. 4) by fluorescence microscopy. Prior to and after the staining process, the A549 cells were extensively rinsed, to remove freely floating particles. Under all conditions [except for PS(+)small, which we could not obtain the florescence particles], the cellular uptake of the PS nanoplastics was confirmed (Figs. 5 and 6). The experiment was repeated in the presence of 10% fetal bovine albumin (FBS) to simulate the high concentration of serum albumin (the main component of 10% FBS is bovine serum albumin) in the cells treated with PS(−)small/HSA soft corona (Fig. S4 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. Once more, the PS particles were observed within the cells even in the presence of the 10% FBS. Overall, the penetrated particles appear to surround the nuclei, though it is unclear if the particles are inside the nuclei or not. To verify this, three-dimensional images were created via Z-stacking different focal planes (Fig. 7 and Fig. S5 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. While the particle adhesion on the nuclear membrane is evident visually, no particles were found to be inside the nuclei.In rare cases, the chromosomes, in the absence of nuclear membrane, are surrounded by PS(−)small though no such case was found for the cells treated with the large nanoplastics. To further investigate this size-dependent effect, the cells were treated with the two size-combination of PS(−) nanoplastics tagged with two distinct fluorescent molecules (image shown in Fig. 7). Indeed, the small PS(−)small particles are shown to interact with the chromosomes more favorably than the PS(−)large. Provided that transmembrane diffusion is an implausible uptake mechanism, the chromosomal interactions likely took place during the prophase or anaphase mitotic stages, which takes place prior to the nuclear membrane formation.

B. Molecular interactions between cell membrane and PS nanoplastics and PS/protein corona complexes

EIS was used to analyze the effect of PS nanoplastics with and without the presence of soft/hard protein coronae. The EIS spectra were described by an electrical equivalent circuit (EEC) model, shown in Fig. 8(d). The fitting parameters, Rmembrane and constant phase element (CPE) (shown in Tables IV and V), are used to describe the physical properties of differently treated lipid bilayers. Rmembrane shows the resistance of the lipid bilayer—how closely packed the bilayers are—to mitigate the ion flows across the membrane. CPEs describe the capacitance and ideality of the capacitor (perfectly homogeneous bilayer when α = 1.0).29,34,3529. R. Naumann et al., Langmuir 19, 5435 (2003). https://doi.org/10.1021/la034206034. J. Andersson, M. A. Fuller, K. Wood, S. A. Holt, and I. Köper, Phys. Chem. Chem. Phys. 20, 12958 (2018). https://doi.org/10.1039/C8CP01346B35. J. Andersson and I. Köper, Membranes 6, 30 (2016). https://doi.org/10.3390/membranes6020030 Additionally, bulk solution resistance (Rsolution) and submembrane capacitance (Csub), describing the Au-tether submembrane reservoir, are used in the model.34,3634. J. Andersson, M. A. Fuller, K. Wood, S. A. Holt, and I. Köper, Phys. Chem. Chem. Phys. 20, 12958 (2018). https://doi.org/10.1039/C8CP01346B36. J. J. Knobloch, A. R. Nelson, I. Köper, M. James, and D. J. McGillivray, Langmuir 31, 12679 (2015). https://doi.org/10.1021/acs.langmuir.5b02458 Hereafter, Rmembrane and CPE values are reported.

TABLE IV. EIS fitting parameters for tethered lipid bilayers before and after the introduction of PS nanoplastics, at different concentrations. Uncertainties are reported in parentheses. The change in the fitting parameters with respect to the control data (bare lipid bilayers) is shown as a percentage.

Fitting parametersControlPS(−)small 125 μg ml−1PS(−)small 250 μg ml−1ControlPS(+)small 125 μg ml−1PS(+)small 250 μg ml−1Rmembrane/MΩ cm21.26 (0.03)1.06 (0.02)/−16%0.70 (0.03)/−44%1.30 (0.05)1.21 (0.04)/−7.1%0.87 (0.01)/−33%CPE/μF cm−2 s(1−α)1.29 (0.02)1.38 (0.01)/+7.0%1.57 (0.04)/+22%1.51 (0.07)1.52 (0.01)/+0.4%1.60 (0.04)/+6.0%α0.954 (0.006)0.950 (0.006)/−0.4%0.939 (0.01)/−1.5%0.958 (0.001)0.953 (0.01)/−0.5%0.949 (0.01)/−0.9%

TABLE V. EIS fitting parameters for tethered lipid bilayers before and after the introduction of PS(−)/protein corona complexes at different protein concentrations, with a fixed concentration of PS(−)small nanoplastics of 250 μg ml−1. Uncertainties are reported in parentheses. The change in the fitting parameters with respect to the control data (bare lipid bilayers) are shown as a percentage.

Fitting parametersControlSoft corona PS(−)small/HSA 50 μg ml−1PS(−)small/HSA 150 μg ml−1ControlHard corona PS(−)small/LYS 50 μg ml−1PS(−)small/LYS 150 μg ml−1Rmembrane/MΩ cm20.75 (0.02)0.68 (0.02)/−9.3%0.81 (0.02)/+8.0%0.91 (0.05)0.63 (0.04)/−31%0.55 (0.04)/−40%CPE/μF cm−2 s(1−α)1.51 (0.05)1.46 (0.02)/+3.3%1.44 (0.02)/−0.7%1.43 (0.03)1.56 (0.03)/+9.0%1.54 (0.05)/+7.7%α0.955 (0.02)0.947 (0.02)/−0.8%0.946 (0.01)/−1.3%0.953 (0.002)0.936 (0.001)/−1.8%0.925 (0.005)/−2.9%Representative EIS Bode plots of lipid bilayers before and after the introduction of bare PSsmall (20 nm), PSsmall with soft protein corona, and hard corona are presented in Figs. 8(a) and 9(a). Regardless of the surface charge, bare PSsmall nanoplastics caused a notable decline in Rmembrane (as high as a 44% decrease compared to the bare bilayer) was observed with an increasing concentration of the PS nanoplastics. Additionally, a systematic increase in the capacitance values and decreasing α values were observed (fitting summary is found in Tables IV and V). Larger PS nanoplastics, on the contrary, showed insignificant changes to CPE values (Fig. S6 and Table S1 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. Rmembrane was observed to increase for both negatively and positively charged particles. These increasing Rmembrane values are thought to be attributed to the large insulating PS nanoplastics adsorbed on the bilayer surface, inhibiting ion transport across the membrane, without causing significant disruption to the membrane structure.The effect of free-standing HSA and LYS was also tested prior to assessing the impact of nanoplastics with soft or hard protein corona (Fig. S7 and Table S3 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. After the introduction of both HSA and LYS, marginal changes to the Rmembrane and CPE values were observed, in some cases, a slight increase. This indicates that both proteins interact with the membrane surface without disturbance to the structural integrity.Finally, the relevance of the protein corona in nanoplastic-membrane interactions was tested by introducing the PS nanoplastics with either soft or hard protein corona to the bilayer. In these complex systems, the relevance of corona protein was explored by varying the concentration of proteins (50–150 μg ml−1) while the concentration of PS nanoplastics was fixed (250 μg ml−1). The EIS Bode plots of PS(−)small with HSA soft protein corona, at two different HSA concentrations, are presented in Figs. 8(b) and 8(c), and the fitting parameters are summarized in Table V. The disruption of the bilayer was significantly reduced when the concentration of HSA soft corona was 50 μg ml−1 [16% decline in Rmembrane vs 44% decline with bare PS(−)small]. When the HSA soft corona concentration reached 150 μg ml−1, the Rmembrane value surpassed that of the control membrane, resembling the behavior of free-standing HSA. For PS(−)small with LYS hard corona, the mitigation effect was found to be minimal, if not, a similar degree of bilayer disruption was observed (40% decline in Rmembrane). Noticeably, when the concentration of hard corona LYS increases from 50 to 150 μg ml−1, the bilayer stability deteriorated further as evidenced by the significant change in Rmembrane and CPE values while free-standing LYS cause marginal effect (Table V and Table S2 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data.Another set of nanoplastics/hard corona combination [PS(+)small/HSA] was also tested [Fig. 9(b) and Table VI]. Likewise, the membrane stability declined with an increasing concentration of hard corona participating HSA, as seen by the decreasing Rmembrane and CPE values. Intriguingly, LYS soft protein corona around PS(+)small did not mitigate the membrane disruption (Fig. 9 and Table VI), instead, it amplified the destabilization effect with increasing LYS concentration (e.g., the Rmembrane % change increased from −46% to −63% with respect to the control bilayer when the LYS concentration increased from 50 to 150 μg ml−1). This shows that the membrane stability (or reduction of disruption caused by bare PS nanoplastics) is not simply dependent on the type of corona formed but a combination of corona and protein types.

TABLE VI. EIS fitting parameters for tethered lipid bilayers before and after the introduction of PS(+)small/protein corona complexes at different protein concentrations, with a fixed concentration of PS(+)small nanoplastics of 250 μg ml−1. Uncertainties are reported in parentheses. The change in the fitting parameters with respect to the control data (bare lipid bilayers) are shown as a percentage.

Fitting parametersControlHard corona PS(+)/HSA 50 μg ml−1PS(+)/HSA 150 μg ml−1ControlSoft corona PS(+)/LYS 50 μg ml−1PS(+)/LYS 150 μg ml−1Rmembrane/MΩ cm20.87 (0.02)0.54 (0.02)/−38%0.48 (0.01)/−45%0.70 (0.03)0.38 (0.10)/−46%0.26 (0.09)/−63%CPE/μF cm−2 s(1−α)1.11 (0.02)1.04 (0.06)/+6.4%1.04 (0.07)/+6.4%1.22 (0.02)1.19 (0.02)/−2.5%1.28 (0.03)/+0.5%α0.970 (0.001)0.957 (0.001)/−1.3%0.951(0.001)/−2.0%0.954 (0.002)0.937 (0.003)/−1.8%0.940 (0.005)/−1.5%

The extent of membrane structural alteration caused by the PS nanoplastics and the nanoplastic/protein corona complexes was studied using NR. The seven slab layers were used to accommodate the NR profiles of the tethered lipid bilayer, namely, Si, SiOx, Cr(CrOx), Au, tether, hydrophobic chains, and POPC headgroup. The inorganic layers and the tether regions were fixed to the values fitted for the control samples, while the hydrophobic chains and POPC headgroups were fitted. Hydrophobic chains of DPhyTL and POPC merged in this fitting model. The lack of neutron scattering length density (nSLD) contrast between the hydrophobic moieties of these two lipid molecules did not justify the presence of two independent slabs. The nSLD of the hydrophobic chains were fixed to −0.5 × 10−6 Å−2. Herein, we report the change in the thickness, nSLD, and solvent vol. % of the hydrophobic chains and POPC headgroup before and after the introduction of PS nanoplastics in the presence or absence of the protein corona.

The PSsmall, regardless of their surface charges, increased solvent penetration on the POPC headgroup by approximately 20% and lowered the nSLD from ∼2.0 × 10−6 to ∼1.5 × 10−6 Å−2 (NR and SLD profiles are shown in Fig. 10 and Fig. S8 in the supplementary material,5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. and fitting parameters are shown in Tables VII and VIII). Furthermore, the tightly packed hydrophobic chains were found to be occupied by the solvent when bare PS(−)small nanoplastics were introduced [0%(0.1) for control vs 2%(0.1)]. The size effect was also investigated using PS(−)large (Fig. S9 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. Addition caused insignificant changes (Table S3 in the supplementary material)5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. to the structural properties of the bilayer, which agrees with the EIS measurements. The effects of proteins on the POPC tethered bilayer were independently assessed. A notable effect was seen in the thinning of the hydrophobic chains [3.0 Å (±0.1)], and the decrease in the solvent penetration in the POPC headgroup [by 29% (±2)]. For LYS (Fig. S10 in the supplementary material),5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. thinning on the hydrophobic chains was observed while the POPC headgroup appeared to experience a thickening effect [by 2.4 Å (±0.2)] (Table S4 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. The nSLD of the POPC headgroup increased by ∼0.5 × 10−6 Å−2. Finally, a weakly increasing trend in the solvent penetration in the POPC headgroup could be observed [increase in ∼6% (±2)].

TABLE VII. Summary of NR fitting parameters for POPC tethered lipid bilayer with and without PS(−)small or PS(−)small/protein corona complexes. The concentration of the PS nanoplastics was fixed at 250 μg ml−1 and at 150 μg ml−1 for the proteins.

Control bilayerPS(−)smallPS(−)small/HSA soft coronaPS(−)small/LYS hard coronaThickness/ÅHydrophobic chains34.0 (0.1)34.3 (0.3)33.1 (0.2)34.3 (0.1)POPC headgroup12.0 (0.4)11.9 (0.4)12.0 (0.3)7.5 (0.5)nSLD/10−6 Å−2Hydrophobic chains−0.5 (fixed)−0.5 (fixed)−0.5 (fixed)−0.5 (fixed)POPC headgroup1.98 (0.02)1.53 (0.04)1.61 (0.10)1.52 (0.02)Solvent vol. %Hydrophobic chains0.0 (0.1)2.0 (0.1)0.0 (0.1)2.0 (0.3)POPC headgroup50 (2)67 (2)53 (2)57 (2)

TABLE VIII. Summary of NR fitting parameters for POPC tethered lipid bilayer with and without PS(+)small or PS(+)small/protein corona complexes. The concentration of the PS nanoplastics was fixed at 250 μg ml−1 and at 0.15 μg ml−1 for the proteins.

Control bilayerPS(+)smallPS(+)small/HSA hard coronaPS(+)small/LYS soft coronaThickness/ÅHydrophobic chains30.2 (0.2)30.7 (0.2)34.4 (0.2)30.3 (0.4)POPC headgroup11.9 (0.1)11.9 (0.4)12.0 (0.6)11.9 (0.1)nSLD/10−6 Å−2Hydrophobic chains−0.5 (fixed)−0.5 (fixed)−0.5 (fixed)−0.5 (fixed)POPC headgroup1.92 (0.09)1.52 (0.02)1.53 (0.03)1.76 (0.04)Solvent vol. %Hydrophobic chains10 (1)4 (2)11 (1)11 (1)POPC headgroup59 (1)78 (2)87 (2)76 (3)When PS(−)small combined with the HSA soft corona, the change in the solvent penetration in both the hydrophobic chains [0%(±0.1) for control vs 0%(±0.1) with PS(−)small/HSA soft corona] and the POPC headgroup became insignificant [50%(±2) for control vs 53%(±3) for PS(−)small/HSA soft corona]. A decline in the nSLD of the POPC headgroup was also observed, the extent of which may be comparable to the effect of the bare PS(−)small nanoplastics [1.61 × 10−6 Å−2 (±0.10)]. Furthermore, PS(−)small/HSA soft corona appears to thin the POPC headgroup by ∼1.0 Å (±0.2)—such an effect is also seen when bilayer interacts with HSA alone (Fig. S10 and Table S5 in the supplementary material).5454. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001124 for additional flow cytometry, florescence microscopy, EIS, and NR data. PS(−)small/LYS hard corona caused a significant thinning effect on the POPC headgroup [∼4.5 Å(±0.5)], and a greater amount of the aqueous solvent penetration in the headgroup was observed [+7%(±2) compared to the control], although not as great as that caused by bare PS(−)small [+17%(±2)]. Here, a similar extent of disruption in the hydrophobic chains could be observed for bilayers with PS(−)small/LYS hard corona complexes to the bilayer with bare PS(−)small [2%(±0.1)].

The reflectivity profiles of POPC tethered bilayers with PS(+)small and protein coronae were also collected. When HSA formed a hard corona with PS(+)small, the complex caused a notable thickening effect in the hydrophobic chains [+4.2 Å (±0.2)]. A structural change in the headgroup was observed, along with greater solvent penetration [28% (±2)] and a decrease in the nSLD [from 1.92 × 10−6 (±0.09) to 1.53 × 10−6 (±0.03) Å−2]. The addition of PS(+)small/LYS soft corona complex provided a similar structural change to PS(+)small, except that the decrease in the nSLD of the headgroup was not as pronounced.