Nanoplastics penetration across the blood–brain barrier

Abstract

Microplastics and nanoplastics (MNPs), originating from plastic degradation, have arisen to be a threat to ecology and human health. Alarmingly, the penetration of MNPs across the highly selective blood–brain barrier (BBB) poses an emerging and urgent risk, yet its molecular mechanism remains unexplored. In this work, using long-time-scale (over 28 microseconds) all-atom explicit solvent steered molecular dynamics, we examine the free energy of the passive permeation of four polymer nanoparticles: polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PET). PE, PP, and PS nanoparticles exhibited a remarkable preference for entering the BBB, attributed to their high hydrophobicity, though they are kinetically trapped in the aqueous phase. In contrast, the PET nanoparticle was energetically unfavored for entering the BBB. Our study further reveals that the PE, PP, and PS polymers can enter the BBB as polymerized nanoplastics, dissolve within the BBB, and eventually exit as dispersed polymer chains, which is specifically true for the amorphous PP and PS nanoparticles. Moreover, the crystalline structure of the PE nanoparticle drives distinct orientations during the penetration process, and the insertion of the PET nanoparticle elevates the hydration of the bilayer interior. Our work advances the knowledge about the mechanism of nanoplastic penetration across the BBB, which could aid in the rational design of therapeutics for nanoplastic penetration inhibitors.

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1. Introduction

Despite their relatively short history of just over a century,¹ synthetic polymers, such as plastics, have become integral to modern life. Their popularity stems from the unique properties, including durability, lightweight design, cost-effectiveness, and customizability. However, while their durability is advantageous in practical applications, this poses a significant challenge when it comes to degradation after end-of-life use.² For example, polyethylene terephthalate (PET) plastics can take approximately 450 years to degrade under ambient conditions.³ As a result, plastic pollution has become an emerging and urgent threat to the environment and human health,⁴ given the slow degradation and the generation of microplastics and nanoplastics (MNPs), which refer to plastic particles at the micrometer and nanometer scales, respectively. Human exposure to MNPs is ubiquitous: it is estimated that humans may ingest 0.1–5 g of microplastic particles weekly,⁵ or 39 000–52 000 microplastic particles annually.⁶ Specifically, their presence has been reported in the food chain,^7–9 human organs (e.g., liver¹⁰ and kidneys¹¹), infant formula,¹² and other sources.^13–15 Recent studies have demonstrated that microplastics in the bloodstream can lead to neurobehavioral abnormalities,¹⁶ cellular toxicity in mammals,^17,18 and numerous diseases.^19,20 Compared to microplastics, the abundance of nanoplastics is not well-documented or understood due to methodological and technological limitations of nanoplastics detection.²¹

More alarmingly, MNPs have been very recently detected in the human brain,²² further highlighting the potential risks to human health. Specifically, through the autopsy of cadaver brains, livers, and kidneys, polyethylene (PE) and many other polymers were found to be abundant in these organs, with the highest concentration in the blood–brain barrier (BBB) and brains of dementia patients. The BBB protects the human brain against foreign particles by strictly regulating the exchange of substances across the capillary walls. Its dysfunction is associated with neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.²³ A study on mice found that exposure to polystyrene (PS) microplastics caused disruption in the BBB, cognitive defects, and inflammation of the hippocampus.²⁴ These observations underscore the importance of analyzing the penetration of MNPs across human tissues, for instance, the BBB. To the best of our knowledge, no molecular-level understanding has been reported on MNPs crossing the BBB. Related studies are present for nanoplastics,²⁵ polymer-based nanoparticles,^26–28 or short polymer chains²⁹ interacting with model lipid bilayers, small organic molecules crossing the BBB,^30,31 and the penetration of nanoparticles of oligomer PE chains across a variety of membranes.³²

In this study, we quantified the free energy of the passive permeation of polymer nanoparticles crossing the apical bilayer of the BBB using long-time-scale all-atom explicit solvent steered molecular dynamics (MD) simulations. Four types of small-sized polymer nanoplastics were examined: PE, polypropylene (PP), PS, and PET, which have a diameter of approximately 3.1–3.5 nm. These polymers are broadly employed in the industry and everyday applications, including food packaging, wire insulation, carpets, appliances, automotive components, and many more. The passive permeation mechanism³³ is examined here as it was found to dominate for nanoplastics with a diameter of 0.293 µm across the BBB,³⁴ while the other mechanisms (e.g., endocytosis) apply to larger, micrometer-sized particles.³⁵ Smaller-sized nanoplastics were found to dominate in personal care products³⁶ and cause more serious damage to carp myocardium.³⁷

2. Methods

All-atom explicit solvent MD simulations were carried out to examine the interactions between the polymer nanoparticles and the BBB. The open-source package GROMACS (version 2024.5) was employed.³⁸ The CHARMM 36m force field,^39,40 which is known to accurately describe lipid bilayers,⁴¹ was applied to the lipids and metal ions (Na⁺ and Cl⁻). The CGenFF (CHARMM general force field) potential (version 4.6)^42,43 was generated for the polymers using the CGenFF web server (https://cgenff.com). The recommended CHARMM TIP3P water model⁴⁴ was used with the structures constrained using the SETTLE algorithm.⁴⁵

The neighbor searching was calculated up to a cutoff distance of 12 Å via the Verlet particle-based algorithm and was updated every 20 timesteps. The short-range Coulombic interactions were truncated at the cutoff distance of 12 Å, with the long-range interactions calculated using the Smooth Particle Mesh Ewald algorithm.^46,47 The Lennard-Jones 12–6 interactions were switched off from 10 to 12 Å via the force-switch method. The temperature coupling was implemented using the V-rescale algorithm, with the temperatures of the BBB, polymers, and water/ions separately coupled at 303 K, and a characteristic time of 1 ps. The semi-isotropic pressure was managed using the C-rescale algorithm⁴⁸ at the reference pressure of 1 bar with a compressibility of 4.5 × 10⁻⁵ bar⁻¹ and a coupling constant of 5.0 ps. The integration time step of 2 fs was used with all hydrogen-involved covalent bonds constrained using the LINCS algorithm.^49,50

2.1. BBB bilayer preparation

The BBB bilayer was generated using the CHARMM-GUI web server.⁴¹ The composition of the bilayer (Table S1, SI) represents the apical bilayer of the human brain microvascular endothelial cells (Fig. 1A and B).³⁰ Given the large size of the BBB, the apical bilayer and the basolateral bilayer have to be simulated separately.^30,31 Here, a total of 192 lipids (including cholesterol) were included in the symmetric bilayer, with a water-to-lipid number ratio of 50 : 1. Control simulations of the BBB bilayer (without polymers) with 150 mM NaCl were first conducted, where we examined the cross-sectional area per lipid (APL) of the BBB at varying temperatures. The obtained APLs agree well with the literature data (Table S2, SI). The APL was obtained to be 48.0 ± 0.7 Ų at 303 K. The BBB bilayer thickness was estimated to be approximately 4.5 nm at 303 K based on the Z-dimensional distributions of the lipid phosphorus atoms (Fig. S1, SI).

Fig. 1 Schematic representation of (A) the BBB, and (B) a PE nanoparticle (colored in cyan) crossing the apical layer of the BBB. The Na⁺ and Cl⁻ ions are represented by the blue/cyan dots, respectively. (C) Four types of nanoplastics are employed, composed of PE, PP, PS, and PET. Each nanoparticle consists of 10 polymer chains and has a diameter of approximately 3.4/3.5/3.1/3.1 nm for the PE/PP/PS/PET nanoparticles, respectively. The chemical formulas are presented below. The polymers have similar molecular weights (around 1 kDa) with the degree of polymerization n = 36/24/9/6 for PE/PP/PS/PET, respectively.

To further verify the simulation protocol above, we simulated a POPC bilayer as another control system at varying temperatures. The calculated APLs agreed well with the literature values (Table S2, SI), validating the force field and the simulation configurations employed here.

2.2. Assembly of polymer nanoparticles

Four types of nanoparticles, mimicking nanoplastics, were simulated. Each nanoparticle was assembled with 10 polymer chains. The degree of polymerization is 36, 24, 9, and 6 for PE (C₇₂H₁₄₆, molecular weights (m.w.) = 1012.9 Da), PP (C₇₂H₁₄₆, m.w. = 1012.9 Da), PS (C₇₂H₇₄, m.w. = 939.4 Da), and PET (C₆₀H₅₀O₂₄, m.w. = 1155.0 Da), respectively. The molecular weights are similar for all polymer chains for the convenience of comparison. The polymer chains were generated using the CHARMM-GUI web server. The atactic configuration was employed for the PP and PS chains. First, ten extended polymer chains were randomly placed in a cubic box with an edge length of 10 nm in vacuum and were then relaxed for 200 ns via the annealing process (gradually dropping the temperature from 500 K to 200 K). All ten polymer chains were condensed into a nanoparticle (except the PE chains), which was subsequently dissolved in a water box with an edge length of 6.6 nm. It was further equilibrated for another 200 ns with the isotropic pressure coupling. The polymer chains were found to assemble into one nanoparticle and reorganize over the 200 ns simulation duration. Specifically, the PE nanoparticle reorganized itself from a random (amorphous, roughly globular) structure into a well-ordered (crystalline, anisotropic) structure. The obtained nanoparticles are presented in Fig. 1C. Their diameters are estimated to be around 3.1–3.5 nm, comparable to those in recent publications.^25,34

The PE chains form a crystalline structure (Fig. 1C), consistent with the crystalline solid phase at room temperature.⁵¹ This qualitatively supports the accuracy of this work. To quantitatively verify the models, we estimate the density of these nanoparticles, which is 0.85 g cm⁻³, 0.75 g cm⁻³, 1.00 g cm⁻³, and 1.28 g cm⁻³ for the PE, PP, PS, and PET nanoparticles, respectively. These values are in agreement with the experimental data of 0.917–0.94 g cm⁻³ for low-density PE,⁵² 0.85 g cm⁻³ for amorphous PP,⁵³ 1.04–1.05 g cm⁻³ for PS,⁵⁴ and 1.38 g cm⁻³ for PET.⁵⁵ The minor difference is ascribed to the absence of high pressure when preparing these structures⁵¹ and their lower molecular weights (1 kDa here versus tens of kDa or higher in practical applications).

2.3. Steered MD (sMD) simulations of nanoparticle insertion into the BBB

The pre-equilibrated BBB and nanoparticle systems were merged to prepare the BBB/nanoparticle complexes with a salt concentration of 150 mM. We first ran classical (unbiased) MD simulations, which supported the absence of the insertion of these nanoparticles into the BBB bilayer for a duration of up to 2 µs, necessitating the advanced sampling simulations.

Steered MD is one of the advanced sampling approaches that has been historically developed to accelerate the sampling of rare events.⁵⁶ For instance, sMD has been applied to describe the penetration of cell-penetrating peptides,⁵⁷ antimicrobial peptides,⁵⁸ and gold-based nanoparticles.⁵⁹ Here, the sMD simulations were conducted using the pulling code in GROMACS. It is a two-step protocol. Firstly, the nanoparticle was pulled downwards along the Z-dimension from 7 nm above the BBB (based on the center-of-mass, COM, of the nanoparticle and the COM of the BBB) to the BBB center. In what follows, the center refers to the COM of the symmetric lipid bilayer or the COM of the whole nanoparticle. A pulling force constant of 5000 kJ mol⁻¹ nm⁻² was employed. To better preserve the structure of the BBB bilayer and relax the nanoparticle, a very slow pulling rate of 0.1 nm ns⁻¹ was used. To prevent the flip of the lipid chains, position restraints were applied to their headgroups and tail groups in the pulling stage. The simulation trajectory was saved every 10 ps. The distance between the COM of the nanoparticle and the COM of the BBB was calculated as a function of the simulation time.

Subsequently, the simulation configurations were extracted for a 1 Å interval of the COM(polymer)–COM(BBB) distance in the range of 0–6 nm, generating 61 windows. Umbrella sampling was carried out by setting the pull rate to zero, along with the removal of the position restraints on the lipids. Each of the 61 sampling windows was equilibrated for 60 ns, with a saving frequency of 100 ps per frame. Around 3.66 µs of sMD simulation was conducted for each of the four systems. Therefore, a total of 14.64 µs was conducted for the four systems.

The GROMACS program (gmx wham) was employed to calculate the potential of mean force (PMF), using the Weighted Histogram Analysis Method (WHAM) algorithm.⁶⁰ The last 40 ns of the simulation of each sampling window were used. The convergence of the PMF calculations is presented in Fig. S2, SI. To calculate the standard deviations of the PMF, each sampling window was divided into 4 blocks, with 10 ns each.

2.4. Unbiased MD simulations on nanoparticle dissolution in the BBB interior

We carried out classical MD simulations to examine the dissolution of the nanoparticles when positioned in the interior of the BBB. The simulation protocol was the same as that in the sMD simulations above, except that the pulling code was turned off here. Each simulation run for 600 ns (totaling 2.4 µs), and the trajectory was saved every 1 ns. The dissolution of the nanoparticles was characterized using the interaction energy between the polymer chains and the BBB bilayer and the solvent-accessible surface area (SASA).

2.5. sMD simulations of single polymer chains exiting the BBB

The initial structures were prepared by deleting 9 out of 10 polymer chains from the systems where the nanoparticles were located in the BBB center. They were subsequently equilibrated for 40 ns. The polymer chain was then pulled outwards along the Z-dimension. The range of 0–5 nm was examined for the distance between the COM of the polymer chain and the COM of the BBB bilayer, with an interval of 1 Å, leading to 51 sampling windows for each system. The simulation for each sampling window lasted 50 ns for PE, PP, and PS (2.55 µs each) and 120 ns for PET (6.12 µs). A total of 13.77 µs sMD simulations were carried out for the four systems.

Similarly, the first 20 ns simulations for each sampling window were abandoned for the PMF calculation. The convergence of the calculated PMFs is presented in Fig. S3, SI. To calculate the standard deviations of the PMFs, each sampling window was divided into 3 blocks with 10 ns each for PE/PP/PS and 4 blocks with 25 ns each for PET.

3. Results and discussion

3.1. Free energy of polymer nanoparticle insertion into the BBB

We first conducted sMD simulations to quantify the free energy of the nanoparticle insertion into the BBB bilayer using the pulling module of the GROMACS package.³⁸ The calculated potential of mean force is presented in Fig. 2.

Fig. 2 Free energy profile of polymer nanoparticle insertion into the BBB. The error bars represent the standard deviations of 4 blocks with 10 ns each. ξ stands for the Z-dimensional distance between the COM of the BBB and the COM of the nanoparticle. The shadowed vertical region represents the distribution of the lipid phosphorus atoms in the control system (Fig. S1, SI), defining the BBB/water interface. Presented on the right are the simulation snapshots with the nanoparticles located in the water phase (top) and the interior of the BBB (bottom). Salt ions, water, and lipid hydrogen atoms are omitted for the display. Highlighted with the magenta circles are the polymers that are partially dissolved inside the membrane.

The free energy of the nanoparticle insertion into the BBB center from the bulk water phase can thus be obtained from the difference between the PMF in the membrane interior (i.e., 0 kJ mol⁻¹) and the values in the water phase. The latter was obtained based on the PMF value at ξ = 6 nm, where the convergence of PMF has been reached, evidenced by the plateau starting at around 5 nm. The values were obtained to be −479 ± 22 kJ mol⁻¹ for PE, −562 ± 26 kJ mol⁻¹ for PP, −237 ± 33 kJ mol⁻¹ for PS, and 108 ± 30 kJ mol⁻¹ for PET. They roughly align with the hydrophobicity of these polymers that PE ≈ PP > PS > PET. For instance, the water contact angles on solid polymers have been experimentally reported to be 86°–93° for PE, 85°–96° for PP, 72°–86° for PS, and 72°–77° for PET.⁶¹ PE and PP are highly nonpolar and have no measurable dipole moment, whereas the styrene group on PS provides an elevated polarity (the styrene monomer has a dipole moment of around 0.18 D⁶²) compared to PE and PP. It was also suggested that the interactions between styrene and cholesterol could enhance the attraction between lipid membranes and PS nanoparticles, thereby promoting PS nanoparticle insertion.³⁴ In contrast to PS, the introduction of the ester functional groups in PET further improves the polarity with a dipole moment of around 2.7 D,⁶³ which is close to the dipole moment of liquid water (2.9 D⁶⁴). PET is thus the most hydrophilic nanoparticle investigated here.

To further validate the increase in the hydrophilicity of these polymer nanoparticles, we defined a feature of the relative hydrophilicity.⁶⁵ In this regard, we first calculated the short-range Coulombic and LJ interaction energies, denoted as E(Coulombic) and E(LJ), respectively, between the nanoparticles and all neighboring water molecules. Accordingly, the relative hydrophilicity is defined to be the ratio of the Coulombic and the Lennard-Jones interactions, E(Coulombic)/E(LJ) (Table 1). Specifically, a positive value of the ratio, originating from the attractive Coulombic interaction, stands for a higher hydrophilicity; in contrast, a negative value of the ratio, originating from the repulsive Coulombic interactions, describes a higher hydrophobicity. For instance, the repulsive Coulombic interactions for the PE and PP nanoparticles support their highly nonpolar nature. In contrast, the PET nanoparticle displays attractive Coulombic interactions with water, which are much stronger than the corresponding LJ interactions with a ratio of E(Coulombic)/E(LJ) = 2.0, supporting the relatively hydrophilic feature of the PET nanoparticle.

Table 1 Interaction energy (kJ mol⁻¹) between polymers and water in control systems^a

		PE - water	PP - water	PS - water	PET - water
a In these control simulations, the BBB bilayer and 0.15 M NaCl were not included. The nanoparticle (or polymer chain) was dissolved in a cubic water box with an edge length of around 6.5 nm (or 6.0 nm for the single chains). Each simulation run for 200 ns, with the last 100 ns used for the energy calculations. b Coulombic and Lennard-Jones 12–6 (LJ) energies were calculated up to 1.2 nm, which is the cut-off distance for the short-range interactions in the atomistic simulations. Such post-simulation calculations were conducted using the gmx mdrun -rerun program, which excludes the long-range Coulombic interactions. c Ratio = E(Coulombic)/E(LJ).
Nanoparticle (10 chains)	Coulombic^b	45 ± 15	75 ± 14	−438 ± 47	−2792 ± 130
	LJ^b	−1554 ± 41	−1360 ± 62	−1443 ± 48	−1396 ± 63
	Ratio^c	−0.03	−0.06	0.30	2.0
Single Chain	Coulombic^b	10 ± 7	14 ± 6	−86 ± 21	−431 ± 41
	LJ^b	−332 ± 19	−307 ± 18	−312 ± 20	−294 ± 35
	Ratio^c	−0.03	-0.04	0.28	1.46

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By comparing the results for the nanoparticles and the single polymer chains, we further find that the assembly of the PE, PP, and PS chains into nanoparticles does not affect the polarity, as demonstrated by the comparable values of the ratio. Whereas the assembly of PET chains remarkably elevates its polarity (ratio = 1.46 for single chains vs. 2.0 for nanoparticles). That is, its assembly into nanoparticles could embed the nonpolar groups within the nanoparticle and expose the polar ester groups to the surrounding aqueous environment. Such local structural orientation was also found in nanoparticles assembled using a homopolymer poly(propylene sulfone) in our recent work.⁶⁶

We also notice that the PP nanoparticle displayed a relatively stronger preference for entering the BBB than the PE nanoparticle, though their hydrophobicity is highly similar. Such a difference is ascribed to the nanoparticle dissolution process when positioned in the BBB center, which is energetically more preferred for the amorphous PP nanoparticle. As demonstrated in the simulation snapshots in Fig. 2, the PP and PS nanoparticles displayed elevated dissolution when located inside the BBB compared to the PE and PET nanoparticles. A more detailed discussion will be presented in the following section.

Consequently, with the increase in the polarity from PE/PP, to PS, to PET, their hydration becomes more energetically favorable, and the interactions with the nonpolar interior of the BBB bilayer become less favorable, which collectively play a decisive role in the free energy of insertion in Fig. 2.

It is noteworthy that, though the insertion of the PE, PP, and PS nanoparticles from the water phase to the BBB center is energetically favored, such transports need to overcome a free energy barrier on the BBB/water interface (around 3.5–3.7 nm from the BBB center). The free energy barrier is obtained to be approximately 52, 46, and 32 kJ mol⁻¹ for the PE, PP, and PS nanoparticles, respectively. Therefore, though their insertion into the BBB bilayer from a water phase is energetically favored, these nanoparticles are kinetically trapped in the aqueous solution.

3.2. Anisotropic orientation of the PE nanoparticle

The PE chains assembled into a crystalline structure in water, and a strong orientation was observed for the PE nanoparticle during its insertion into the BBB bilayer. To quantify its anisotropic orientation, we calculated the second-order Legendre polynormal orientational function using an in-house script based on our previous work.⁶⁷where r stands for the Z-dimensional distance between the BBB center and the center of the PE nanoparticle, N(r) denotes the number of PE nanoparticle neighbors over the simulation duration at a distance r from the BBB center with a bin width of 0.1 nm, and θ(r) represents the angle between the normal of the BBB bilayer (the Z-dimension) and the principal vector of the nanoparticle. This orientation function has characteristic values: O(r) = −0.5 indicates that the nanoparticle is parallel to the bilayer plane (i.e., perpendicular to the bilayer normal), O(r) = 1 indicates that the nanoparticle is perpendicular to the bilayer plane (i.e., parallel to the bilayer normal), and O(r) = 0 denotes no preferred orientations.

The result is presented in Fig. 3. At the BBB center, the value of O(r) = 0.96 ± 0.06, suggesting a strong vertical orientation of the PE nanoparticle relative to the bilayer plane (Fig. 3A). A similar orientation was found when the PE nanoparticle crosses the BBB surface (Fig. 3B) with O(r) = 0.94 ± 0.04 at 3.0 nm above the BBB center. A similar orientation preference was also observed for the “forever chemical”, per- and polyfluoroalkyl substances (PFAS) chains, at the lipid bilayer/water interface.⁶⁸ When the nanoparticle is detached from the BBB, it prefers to lie on the BBB surface horizontally with O(r) = −0.48 ± 0.02 at 4.0 nm above the BBB center (Fig. 3C). Note that half of the bilayer thickness is around 2.75 nm based on the lipid phosphorus atoms (Fig. S1, SI). In contrast, when the PE nanoparticle is in the water phase (more than 5 nm above the BBB center), it shows no preferred orientation with O(r) ≈ 0 (Fig. 3D). The random orientations displayed much larger fluctuation (0.0 ± 0.4) owing to a greater degree of freedom of 3-dimensional rotation in the water phase compared to those inside the bilayer and on the BBB/water interface.

Fig. 3 Orientation of the PE nanoparticle as a function of the distance from the BBB center. The error bars represent the ensemble-based standard deviations. Demonstrated in the insert are the definitions of the angle and the distance in eqn (1). (A) In the BBB interior and (B) at the BBB/water interface, the vertical orientation dominates. (C) On top of the BBB/water interface, the parallel orientation dominates. (D) In the water phase, random orientations exist. Three representative orientations (tilted in gray, vertical in cyan, and parallel in orange) are overlaid on the same plot.

Therefore, the insertion of the PE nanoparticle is subject to the change in the PE nanoparticle orientation from a horizontal orientation on top of the surface (no touching) to a vertical orientation when touching the BBB.

3.3. Dissolution of polymer nanoparticles inside the BBB

The high free energies for the insertion of the PE, PP, and PS nanoparticles into the BBB (−562 to −237 kJ mol⁻¹ in Fig. 2) indicate that entering the bilayer is energetically favored, whereas the escape is highly challenging. A previous in vivo mice experiment suggested that BBB permeation is size-dependent and that green fluorescent signals were detected in mice brain tissues after 2 hours of exposure to small-sized PS nanoplastics (i.e., 0.293 µm, where passive permeation dominates).³⁴ The driving force of the passive permeation of water molecules across lipid bilayers was found to be predominantly ascribed to the fluctuations in the system's potential energy and the membrane morphology.⁶⁹ The fluctuations in the total potential energy are only around ±100 kJ mol⁻¹ in all systems investigated here. Therefore, unlike the passive permeation of water molecules, energy fluctuations are unable to play a decisive role in transporting nanoparticles across the BBB bilayer.

In the sMD simulations (Fig. 2), we observed that the nanoparticles could dissolve inside the BBB bilayer. To confirm the observation, we conducted unbiased MD simulations. Each simulation lasted 600 ns, which supported the gradual dissolution of the polymer nanoparticles when positioned in the BBB interior (Fig. 4A and B). The dissolution of nanoparticles inside the lipid membrane interior is a phenomenon that has been consistently observed in previous all-atom and coarse-grained simulations.^26,32,70,71 Specifically, the dissolution was evidenced by the increases in the BBB-nanoparticle potential energy and the solvent-accessible surface area (SASA) of the nanoparticles as a function of the simulation time. The dissolution is relatively more favored for the amorphous and nonpolar PP and PS nanoparticles compared to the PE (crystalline) and PET (relatively polar) nanoparticles.²⁵ Specifically, the gradual dissolution of the PP nanoparticle lasted over the whole course of the 600 ns simulations. Such slow kinetics make it highly computationally challenging to achieve the precise convergence of the PMF calculations. As demonstrated in Fig. S1, the PMF curves undergo minor changes throughout the sMD simulations. The complete convergence of the sMD simulations requires a much longer simulation duration, which is inaccessible to us, given the fact that each of the eight sMD simulations took around 30 days on our GPU-equipped computers.

Fig. 4 Dissolution of the nanoparticles when embedded in the BBB interior. (A) The initial and final (600 ns) simulation snapshots of the PP nanoparticle. (B) The total potential energy (Coulombic + Lennard-Jones) between the polymers and the BBB and the SASA of the nanoparticles as a function of the simulation time. (C) Number density profile of water oxygen atoms in the presence of polymer chains inside the BBB. The number density profile of the lipid phosphorus atoms (labelled as P) is included to indicate the BBB/water interface. The insert shows a close view of the central region, highlighting the elevated distribution of water in the presence of PET chains. (D) The APL with the polymer chains dissolved in the BBB interior, which are all larger than the value of 48.0 ± 0.7 Ų in the control simulation without polymers.

We further noticed that when the PET chains are located inside the BBB, the BBB interior becomes more hydrated (Fig. 4C), similar to the observation for amphiphilic random heteropolymers when embedded inside lipid bilayers.⁷² The insertion of the nonpolar PS, PP, and PS polymers showed no impact on the hydration of the BBB interior.³² Moreover, all BBB bilayers are found to expand with the nanoparticles positioned inside, which is most pronounced for the PET nanoparticle (around an 8.8% increase; Fig. 4D and Table S3, SI). Taken together, these findings suggest that, in contrast with the nonpolar PE, PP, and PS polymers, PET displayed elevated polarity due to the presence of the relatively hydrophilic ester group, which inhibited the preference of entering the BBB (Fig. 2) and deforming (i.e., expanding) the structure of the BBB bilayer (Fig. 4).

3.4. Free energy of a single polymer chain exiting the BBB

Therefore, one plausible mechanism is that polymers enter the BBB membrane in their solid form (i.e., nanoplastics), dissolve in the hydrophobic interior of the membrane, and eventually exit the membrane as single polymer chains. To validate the escape of single-chain polymers, we conducted sMD simulations to quantify the free energy profiles of a single polymer chain (Fig. 5). The free energy barrier substantially drops for the single polymer chains compared to that for the nanoparticles: from 479 ± 22 to 145 ± 11 kJ mol⁻¹ for PE, from 562 ± 26 to 153 ± 3 kJ mol⁻¹ for PP, from 237 ± 33 to 97 ± 1 kJ mol⁻¹ for PS. Therefore, escaping from the BBB is energetically much more favorable for single polymer chains for PE, PP, and PS. We also conducted sMD for a single PET chain, which led to the free energy barrier of 9 ± 8 kJ mol⁻¹ to escape from the BBB.

Fig. 5 Free energy for a single polymer chain exiting the BBB bilayer. The shadowed vertical region denotes the interface of the BBB. Demonstrated on the right are the representative structures of the polymer chains located in the BBB interior (bottom) and the water phase (top). Salt ions, water, and lipid hydrogens are omitted for the display.

Furthermore, the PMF profiles of the nanoparticles reach a plateau at a distance of around 5 nm from the BBB center (Fig. 2), supporting the convergence of the interactions between the BBB membrane and the nanoparticles. Given that the radius of the nanoparticles is around 1.55–1.75 nm and the half of the thickness of the BBB bilayer is around 2.75 nm (Fig. S1, SI), it is supported that the nanoparticles display negligible long-range interactions with the BBB slightly beyond their contact range. Similarly, the PMF profiles for the single-chain polymers are converged at a shorter distance of around 3.5 nm from the BBB center, given the smaller size of the single polymer chains (Fig. 5).

Note that the diameters of the nanoparticles investigated here (3.1–3.5 nm) are smaller than the thickness of the BBB bilayer (4.5 nm). The size impact of nanoplastics crossing the BBB needs further study.³⁴ Specifically, larger-sized nanoplastics (than the BBB bilayer thickness) might disrupt the membrane to a larger extent and display different energetic preferences crossing the bilayer.

Here, we exclusively examined the penetration of nanoparticles across the apical bilayer of the BBB. Given the similar compositions between the apical and basolateral bilayers,³⁰ the overall penetration hypothetically follows a stepwise mechanism: nanoplastics enter the apical bilayer from blood, dissolve, and exit as dispersed polymer chains; the hydrophobic polymer chains aggregate into nanoplastics in the cytosol; eventually, the new nanoplastics enter the basolateral bilayer, dissolve, and exit as dispersed polymer chains into the brain. Collectively, the dural barrier of the apical and basolateral bilayers underscores the BBB's highly selective nature.

4. Conclusions

We investigated the molecular mechanism and computed the free energy profiles for the passive permeation of PE, PP, PS, and PET nanoparticles across the BBB apical bilayer using all-atom explicit-solvent steered MD simulations. It is found that the PE, PP, and PS nanoparticles are energetically more preferred for entering the BBB bilayer, though they are kinetically trapped in the aqueous phase, whereas the PET nanoparticle is energetically unfavored for entering the BBB. The energetic preference of the nanoparticles entering the BBB follows the order PP (–562 ± 26 kJ mol⁻¹) > PE (–479 ± 22 kJ mol⁻¹) ≫ PS (–237 ± 33 kJ mol⁻¹) ≫ PET (108 ± 30 kJ mol⁻¹). The trend of the permeation free energy roughly follows the hydrophobicity of the polymers (PE ≈ PP > PS > PET): the highly hydrophobic PE and PP result in a strong preference for entering the BBB bilayer, while the elevated polarity of PET due to its ester groups leads to an unfavored preference for entering the BBB. Furthermore, the dissolution of the amorphous PP nanoparticle is more energetically spontaneous compared to that of the crystalline PE nanoparticle. Consequently, the free energy is collectively determined by the polarity of the nanoparticle and their dissolution in the bilayer interior, which play a decisive role and a minor role, respectively.

Unbiased atomistic MD simulations showed that these nanoparticles can dissolve when located in the BBB interior. This behavior is particularly pronounced for the nonpolar, amorphous PP and PS nanoparticles, as evidenced by their interactions with the BBB and their surface area. The high free energy barrier for the nanoparticles exiting the BBB, combined with their dissolution in the BBB interior, suggests that they enter the BBB as polymerized nanoplastics but exit as single polymer chains. This is supported by the substantial drop in the free energy barrier for single polymer chains exiting the BBB.

We further find that the PE nanoparticle adopts different anisotropic orientations depending on its location: parallel to the bilayer normal when located in the BBB interior and at the BBB/water interface, parallel to the bilayer plane right on top of the BBB bilayer, and no preferred orientation when positioned in the bulk water phase. In contrast to PE, PP, and PS nanoparticles, the relatively more polar PET nanoparticle increases hydration of the hydrophobic BBB interior and expands the BBB bilayer, despite showing an unfavorable preference for entering the BBB.

Our study represents a first step toward understanding the molecular mechanism by which nanoplastics passively penetrate across the apical bilayer of the BBB. For a complete picture of the MNP penetration, further studies are needed to investigate the factors, such as nanoplastic size, polymer degree of polymerization, polymer cross-linking, and other possible penetration mechanisms (e.g., endocytosis). These studies together will aid in the rational design of therapeutics to prevent MNPs from crossing the BBB. Research on the de novo design of peptides to capture plastics exists.^51,73–76 Nevertheless, the abundance of the hydrophobic (tryptophan and phenylalanine) and positively charged (arginine) amino acids⁷³ makes such peptides inappropriate as plastic-penetration inhibitors: the former will lower the solubility of peptides, and the latter could promote their cell penetration.⁷⁷ Therefore, the rational design of plastic-penetrating inhibitors is still in its infancy.

Author contributions

A. I., J. Z., and T. Q. conducted the simulation, analyzed the data, and wrote the manuscript. T. W. contributed to the discussion and wrote the manuscript. B. Q. conceived the project, conducted the simulation, analyzed the data, and wrote the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

All data have been included in the manuscript and the supplementary information (SI) document. Supplementary information: supporting tables of the composition of the BBB bilayer, and the area per lipid; supporting figures of the BBB bilayer thickness and the convergence of the PMF calculations. See DOI: https://doi.org/10.1039/d5cp04125b

Acknowledgements

This work was supported by the Eugene M. Lang Junior Faculty Fellowship to B. Q.

Notes and references

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Footnote

† A. I. and J. Z. contributed equally.

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