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Biological and Environmental Phenomena at the Interface

The Role of Lipid Coating in Transport of Nanodroplets across the Pulmonary Surfactant Layer Revealed by Molecular Dynamics Simulations Yan Xu, Shixin Li, Zhen Luo, Hao Ren, Xianren Zhang, Fang Huang, Yi Y. Zuo, and Tongtao Yue Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01547 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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The Role of Lipid Coating in Transport of Nanodroplets across the Pulmonary Surfactant Layer Revealed by Molecular Dynamics Simulations

Yan Xu,† Shixin Li,† Zhen Luo,† Hao Ren,† Xianren Zhang,‡ Fang Huang,† Yi Y. Zuo,§,|| and Tongtao Yue*,†

†

State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology,

College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, China ‡

State Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical

Technology, Beijing 100029, China §

Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, Hawaii

96822, United States ||

Department of Pediatrics, John A. Burns School of Medicine, University of Hawaii,

Honolulu, Hawaii 96826, United States

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ABSTRACT: Hydrophilic drugs can be delivered into lungs via nebulization for both local and systemic therapies. Once inhaled, ultrafine nanodroplets preferentially deposit in the alveolar region where they first interact with the pulmonary surfactant (PS) layer, with nature of the interaction determining both efficiency of the pulmonary drug delivery and extent of the PS perturbation. Here, we demonstrate by molecular dynamics simulations the transport of nanodroplets across the PS layer being improved by lipid coating. In the absence of lipids, bare nanodroplets deposit at the PS layer to release drugs that can be directly translocated across the PS layer. The translocation is quicker under higher surface tensions, but at the cost of opening pores that disrupt the ultrastructure of the PS layer. When the PS layer is compressed to lower surface tensions, the nanodroplet prompts collapse of the PS layer to induce severe PS perturbation. By coating the nanodroplet with lipids, the disturbance of the nanodroplet on the PS layer can be reduced. Moreover, the lipid-coated nanodroplet can be readily wrapped by the PS layer to form vesicular structures, which are expected to fuse with the cell membrane to release drugs into secondary organs. Properties of drug bioavailability, controlled drug release and enzymatic tolerance in real systems could be improved by the lipid coating on nanodroplets. Our results provide useful guidelines for molecular design of nanodroplets as carriers for pulmonary drug delivery.


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INTRODUCTION Inhalation therapy has emerged for decades and already been used in clinical medicine.1 It appears to be a desirable route of administration for treatment of many respiratory disorders, such as asthma, pneumonia and cystic fibrosis.2 Such a ‘topical’ or targeted treatment with inhaled drugs allows the use of smaller doses that reduce the systemic side effect.3 Compared with other drug delivery routes, the human lung provides numerous advantages, including a large absorptive surface area, thin alveolar-capillary barrier and abundant underlying vasculature.4 Therefore, developing effective strategies to promote pulmonary drug delivery has attracted remarkable scientific interests in the past two decades.5 However, such a progress has been hindered by the lack of knowledge on how the inhlaed drugs or drug carriers interact with different biological entities in the pulmonary system. Hydrophilic drugs, such as insulin, can be delivered into lungs via nebulization to achieve efficient alveoli deposition.6 Nebulizers use compressed gas or ultrasound to break up drug solutions into ultrafine aerosol nanodroplets that can be directly inhaled from the mouthpiece of the device.7 The therapeutic efficacy is largely dependent on the drug stability, bioavailability, targeting and uptake efficiency, and biological activity.8 Once inhaled, nanodroplets need to cross the pulmonary surfactent (PS) layer to achieve pulmonary entry. The PS layer is a complex material lining at the air-water interface of lung alveoli. It is composed of approximately 90% lipids (phospholipids and cholesterol) and 10% proteins (SP-A, SP-B, SP-C and SP-D), and plays dual roles in tension reduction and innate host defense.9-14 After transport across the PS layer, nanodroplets enter the alveolar fluid and get ready to act on other biological entities. Obviously, the interaction of inhaled nanodroplets with the PS represents the initial nanobio interaction occurring in the lungs and critically influences the subsequent fate of drugs. Besides, both structures and biophysical functions of the endogenous PS could be perturbed by interacting with nanodroplets. There exist numerous experimental evidences convincingly showing that interactions with inhaled nanoparticles can adversely affect biophysical functions of the natural PS.15-17 Therefore, designing better nanodroplets as carriers for promoting the drug delivery efficiency and reducing the PS perturbation is in an urgent need. In spite of the importance of interactions between inhaled nanodroplets and the PS, relevant studies at the molecular level are still lacking. Here, we employ a model nanodroplet representing nebulized hydrophilic drugs to investigate interactions with the PS layer using molecular dynamics (MD) simulations. We also designed a nanodroplet coated with lipids that


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make the nanodroplet surface more hydrophobic. Simulation results showed that bare nanodroplets rapidly adhered onto the PS layer to release drugs diffusing across the layer. The trans-layer diffusion was enhanced under higher surface tensions, but at the cost of opening pores that disrupt the ultrastrucutre of the PS layer. When the PS layer was under compression, the nanodroplet prompted collapse of the PS layer. By coating the nanodroplet with lipids, the PS perturbation by nanodroplet was reduced. Moreover, the lipid-coated nanodroplet can be readily wrapped by the PS layer to form vesicular structures, which may enhance fusion with cell membranes to improve drug release into secondary organs. Our simulations suggest that premixing drug solutions with lipids could let nebulizers generate lipid-coated nanodroplets which may improve the efficiency of pulmonary drug delivery and reduce the PS perturbation.

MODEL AND METHODOLOGY Coarse-grained Models and System Setup. The entire system was simulated using the coarse-grained (CG) models, which allow MD simulations to be conducted in adequate temporal and spatial scales. Among CG methods,18 the MARTINI force field,19 which maps four heavy atoms into one interactive bead, has been widely used to simulate biomolecules, such as lipids,20 proteins,21 and DNA,22 and especially the nano-bio interactions.23-24 The demonstrated ability to reproduce biological phenomena makes it an adequate tool to simulate the nanodroplet-PS interactions. The system setup includes a water slab in vacuum with two symmetric PS monolayers at the two air-water interfaces (Fig. 1a). Each layer contains 2,176 dipalmitoylphosphatidylcholine (DPPC), 704 palmitoyloleoylphosphatidylglycerol (POPG) and 704 cholesterol molecules, doped by 13 SP-B and 13 SP-C proteins (Fig. 1b). Both types of proteins were derived from their all-atom models in the protein data bank (PDB IDs: 2DWF (Mini-B); 1SPF (SP-C)). The aqueous phase contains 238,858 water beads and 1148 Na+ ions to neutralize the system. The bare nanodroplet was constructed by stacking 11,690 hydrophilic beads into a spherical shape of 14 nm, in which the relative position of each bead was derived from an equilibrated water box to ensure liquid phase of the droplet (Fig. 1c). To construct the lipid-coated nanodroplet, 1,150 DPPC molecules were physically adsorbed onto the nanodroplet (Fig. 1d). It accordingly has a larger diameter of 16 nm. The choice of DPPC for lipid coating is because it is the major component of the natrual PS, thus having higher biocompatibility. Note that the specific structure can be generated in reality via nebulizing a drug solution mixed with lipids.25 To simulate interactions between nanodroplets and the cell membrane, a lipid bilayer system containing 8,192 POPC molecules and 1,275,104 water beads was also prepared (Fig. 1e). This model has been widely used by other researchers to


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represent the alveolar epithelial cell membrane.26 Simulation Details. Both bare and lipid-coated nanodroplets were equilibrated in air for 10 ns before positioned above the pre-equilibrated PS layer at the air-water interface. After energy minimization using the steepest descent algorithm, the CGMD simulations with constant particle number, surface tension and temperature were then carried out. For all simulations, a cutoff of 1.2 nm was used for van der Waals interactions. The Lennard-Jones potential was smoothly shifted to zero between 0.9 nm and 1.2 nm to reduce the cutoff noise. For electrostatic interactions, the coulombic potential, with a cutoff of 1.2 nm, was smoothly shifted to zero from 0 to 1.2 nm. Temperature was kept constant at 310 K using the Berendsen weak coupling algorithm with a time constant of 1 ps. The surface tension was varied between 0 and 40 mN m-1 via the Berendsen barostat to represent different respiration conditions.27 The compressibility was set to be 5 ×10−5 bar −1 in the lateral direction and 0 bar −1 in the normal direction. Periodic boundary conditions were implemented in all three directions. The time step of simulations was 20 fs, and the neighbor list for non-bonded interactions was updated every 10 steps. All simulations were performed using the open source code Gromacs 4.6.7.28 Snapshots were rendered by VMD.29

RESULTS AND DISCUSSION Bare Nanodroplets. We began CGMD simulations of the interaction between a bare nanodroplet of 14 nm and the PS layer under a fixed surface tension of 40 mN m-1, close to the alveolar surface tension during inspiration.30 As shown in Fig. 2a (the dynamic process can be found in Video S1), upon contacting the PS, the nanodroplet immediately adhered onto the layer. Unfavorable interactions between the hydrophilic nanoroplet and the hydrophobic PS tails were generated to disturb the ultrastructure of the PS layer. Shortly, a PS layer pore was opened, from which the nanodroplet was quickly transported (Fig. 2a), being reflected by a sudden decrease of the interaction energy between nanodroplet and water (Fig. 2b) and a simultaneous increase of the PS layer area (Fig. 2c). By contrast, no obvious change of the PS layer area was observed in the absence of nanodroplet (Fig. 2c). This is reminiscent of the instability of the natrual PS under highly humid conditions, as has been previously measured by Zuo et al. using the biophysical method.31 Recently, Hu et al. found that graphene oxide nanosheets have adverse effects on the ultrastructure of the PS by generating pores in the PS layer.32 Our simulations are consistent with those findings, suggesting that the unfavorable interactions between hydrophilic drugs or drug carriers and hydrophobic PS tails may induce the PS instability and the biophysical inhibition. ACS Paragon Plus Environment

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In our simulations, once a pore was generated, it rapidly expanded and developed into a more severe PS layer rupture (Fig. 2). Despite the fact that extra PS molecules exist in PS reserviors that can be transferred back to the interface to prevent the PS layer rupture,33 our results at least suggested that high dose of inhalation of nanodroplets may aggravate the PS perturbation.32 Even in the presence of PS reserviors, the opened PS layer pore may not close before completion of the nanodroplet translocation. To elaborate the PS perturbation by inhaled nanodroplets, we extracted the structural evolution of the PS layer before 30 ns and calculated the order parameters of all PS molecules (Fig. 3a, b). The order parameter has the form S =

1 (3cos 2 θ n − 1) , where θ n is the angle between bond connecting two adjacent tail 2

beads and the PS layer normal.32,

34

Before opening the PS layer pore, deposition of the

nanodroplet was found to disturb the local PS arrangement, as reflected in the transient order parameter diagram at 24 ns (Fig. 3b). The extent of the PS perturbation was quantified by counting the defected number of PS molecules with the order parameter smaller than 0.0. It appears that opening the PS layer pore was accompanied with a sudden increase of the defected number, while subsequent rupturing reduced the number by releasing surface energy of the PS layer (Fig. 3c). The larger defected number compared with that before opening the pore was due to the rearrangement of PS molecules around the nanodroplet, as revealed by radial distributions of both the density and the order parameter of PS molecules as a function of distance with center of the nanodroplet (Fig. 3d). Using a larger reference value of 0.25, middle between -0.5 and 1 from the formular, similar results were observed expect that a much larger number of PS molecules were identified to be defective even before rupturing of the PS layer (Fig. S1). Since the average order parameter of PS molecules under surface tension of 40 mN m-1 was 0.55, the reference value of 0.0 should be more appropriate to measure the PS perturbation by nanodroplets. During respiration, surface tension of the PS layer varies from tens mN m-1 to near zero.10, 30 Above simulations suggested that nanodroplets can be transported across the PS layer via opening destructive pores under deep inspiration (40 mN m-1). We next investigated the effect of surface tension on the nanodroplet interaction. As shown in Fig. 4a-c, the nanodroplet was found to adhere onto the PS layer under surface tensions of 30 mN m-1, 20 mN m-1 and 10 mN m-1. To decrease the interaction energy, the nanodroplet was partially wrapped by the PS layer, with the wrapping extent increasing with decrease of surface tension of the PS layer. A number of drug particles were found to diffuse across the PS layer to enter the water phase (Fig. 4a-c).


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We calculated the number of drug particles diffusing across the PS layer, and found a roughly linear increase as a function of the simulation time (Fig. 4d). The transport rate being defined as r = ∂N / ∂t was found to increase with surface tension of the PS layer. In fact, the PS layer has been demonstrated to accelerate diffusion of oxygen through the interface, manifesting permeability of the PS layer for small molecules.35 At the molecular level, the

enhanced penetration of drug particles through the PS layer under higher surface tensions can be ascribed to the less compact stacking of PS molecules, similar with the enhanced permeability of the lipid bilayer membrane.36 Both experimental and simulation studies have demonstrated that nanoparticles can more easily penetrate through lipid bilayers under higher surface tensions.37-38 Once diffusing into the air or crossing the PS layer to enter the water phase, drug particles dispersed randomly and barely formed clusters. We thus used this property to count the number of drug particles in the nanodroplet. Specifically, one drug particle was regarded as part of the nanodroplet if the number of its neighbors was larger than 4. Two particles were defined as neighbors if their separation distance was smaller than 0.5 nm. As expected, we observed a corresponding decrease of the nanodroplet size (Fig. 4e), albeit with slight fluctuations due to the counting error. By summing the numbers of drug particles crossing the PS layer and staying in the nanodroplet, interestingly, they were found to keep nearly unchanged, regardless of different tensions (Fig. 4f). The fluctuations arose from both the diffusion of drug particles and the error of counting number of drug particles in the nanodroplet. These results suggested that inhaled nanodroplet deposit onto the PS layer to release drug particles that may directly diffuse across the PS layer. When the PS layer was compressed to a lower surface tension, the nanodroplet prompted collapse of the PS layer (Fig. 5 and Video S2), another indication of the PS perturbation. In the absence of nanodroplets, the PS layer was buckled under compression.39 Upon contacting the nanodroplet, the PS layer rapidly collapsed to wrap the nanodroplet to both release high surface energy of the PS layer under compression and decrease the interaction energy between nanodroplet and the PS (Fig. S2a, b). The wrapped nanodroplet was connected with the upper layer through a bilayer, which kept growing under constant compression of the layer to drive the nanodroplet continuously moving downward (Fig. S2c). Earlier simulations reported accumulation of water molecules in cave of the collapsed PS layer deposited with fullerenes,40 similar to the structure observed in our simulations. Note that rigid hydrophilic nanoparticles were found to directly penetrate through the PS layer regardless of different surface tensions.41 For hydrophilic nanoparticles entering the alveolar fluid, they were found to recruit PS molecules forming PS corona with a bilayer conformation, while a monolayer


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conformation was formed on the hydrophobic NP surface.42 Those results suggest that nature of interactions between inhaled particles and the PS is determined by physicochemical properties of the particle, but also influenced by the local biological environment.

Lipid-coated Nanodroplets. To both improve efficiency of the pulmonary drug delivery and reduce the PS perturbation, we next designed a lipid-coated nanodroplet, with the inspiration coming from the promoted membrane transport of nanoparticles coated with polymer ligands.43-47 On the other hand, it has been evidenced that the PS layer is far from being a barrier against inhaled particles, but could be used as an efficient shuttle for pulmonary drug delivery.48 Therefore, rational design of surface coating on nanodroplets should better involve the PS layer facilitating the pulmonary drug delivery. Note that lipid nanoparticles have been synthesized by Liu et al. using the reverse micelle-double emulsion method,25 and showed performance for delivering insulin into the lungs.49 However, it still remains unclear about whether and how the lipid coating on nanodroplets promotes transport across the PS layer and reduce the PS perturbation. As shown in Fig. 6a-d, lipid-coated nanodroplets were partially or fully wrapped by the PS layer, depending on surface tension of the PS layer. Nine independent simulations were performed under different surface tensions ranging from 40 mN m-1 to 0 mN m-1. We found that the lipid-coated nanodroplet with a diameter of 16 nm and coated with 1,150 DPPC molecules can be fully wrapped by the PS layer if the surface tension was below 15 mN m-1 (Fig. 6e, f). Otherwise, only partial wrapping was achieved in the limited simulation time. Under a higher surface tension of 50 mN m-1, the PS layer immediately ruptured upon contacting the lipid-coated nanodroplet (Fig. S3a), owing to the PS depletion by interacting with the nanodroplet. By furthe increasing surface tension of the PS layer to 60 mN/m, it rapidly ruptured even in the absence of nanodroplets (Fig. S3b), being consistent with previous experimental and simulation results.50-51 We performed five independent simulations under a fixed surface tension of 40 mN m-1 to estimate properties of the interaction between lipid-coated nanodroplets and the PS layer under the inspiration condition.31 Repeated simulations showed that a number of drug particles slightly leaked from the nanodroplet during interaction with the PS layer (Fig. S4). Upon contacting the PS, the lipid-coated nanodroplet rapidly adhered onto and was partially wrapped by the PS layer (Fig. 7a). To further increase contact with the PS layer, it was deformed into an oblate shape, being reflected by an increase of the aspect ratio, defined as

AS = WXY / H Z , where WXY and H Z represent width and height of the nanodroplet, respectively (Fig. 7b). Notably, spreading deformation of the nanodroplet increased its surface ACS Paragon Plus Environment

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tension,52 which induced a structural defect appearing at the wrapping front, as characterized by a sudden increase of the aspect ratio (Fig. 7b). We also observed a sudden striking decrease of drug particle number in the nanodroplet (Fig. 7b), suggesting that drug particles leaked from the nanodroplet and diffused in air or across the PS layer (Fig. 7c). Note that such behavior of slight drug leakage may not occur in real systems because extra PS molecules in reserviors can be transferred back to the layer to prevent drastic increase of surface tension of the PS layer. On the other hand, surface tension of the PS layer dynamically change during respiration. Thus the transient drug leakage may stop as further decrease of the surface tension. Nevertheless, considering that lipid-coated nanodroplets can be generated in experiments via nebulizing a drug solution admixed with lipids,25 our simulations suggest that a proper increase of the lipid concentration in drug solutions may increase density of lipids adsorbing onto nanodroplets, decreasing surface tension to prevent drug leakage during interactions with the PS layer. Similarly, it has been demonstrated that adding anionic surfactants into nebulisers decreases surface tension of the generated aerosols.53 Another feasible way to prevent the drug leakage is to decrease surface tension of the PS layer, because the lipid-coated nanodroplet can be wrapped by the PS layer more efficiently to reduce the nanodroplet deformation (Fig. 6). More importantly, when surface tension of the PS layer was decreased below 15 mN m-1, nanodroplets were fully wrapped by the PS layer (Fig. 8a, for the dynamic process, see Video S3), with nearly no nanodroplet deformation being accompanied (Fig. 8b). The more efficient wrapping compared with bare nanodroplets was attributed to the more favorable interactions between hydrophobic lipid tails coating on the nanodroplet and hydrophobic PS tails lining at the layer (Fig. S5). After completion of the wrapping, a bilayer vesicular structure was formed, in which the nanodroplet was encapsulated. The vesicle was stable and connected with the PS layer through a short neck, in which two SP-B proteins were anchored to help stablize the connection between the vesicle and the PS layer (Fig. 8c). We performed seven independent simulations, in five of which at least one SP-B protein was found to finally locate at the connection between the vesicle and the PS layer (Fig. S6). This observation suggests that SP-B proteins may preferentially locate at the connection between the PS layer and the formed vesicle, in agreement with one of the hypothesized functions of the SP-B protein in maintaining connection between the PS layer and the generated PS reserviors.54

Cell Membrane Interactions with Nanodroplets. Once transported across the PS layer, nanodroplets enter the alveolar fluid and get ready to interact with the epithelial cells or be cleared by the macrophages. Especially for the systemic therapy, they need to enter the


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systemic circulation via cell uptake and translocation through epithelial cells or paracellularly via intercellular spaces particularly if tight junctions are disrupted.55 Both transport and diffusion of nanoparticles across the cell membrane can be modulated by changing the nanoparticle property, such as size, shape, elasticity and surface charge.56-58 Here, we consider the interaction between transported nanodroplets and the model epithelial cell membrane. Previous studies have revealed that vesicles can readily fuse with the plasma membrane of cells, requiring less time and energy than endocytosis,59-60 though the endocytic pathway can be preferred by increasing elasticity of the vesicles.61 Above simulations suggested that bare nanodroplets can be dispersively translocated across or wrapped by the PS layer (Fig. 4 and 5), whereas lipid-coated nanodroplets are readily wrapped by the PS layer to form bilayered vesicular structures (Fig. 6). The two wrapped structures were equilibrated in water for 10 ns before positioned above a preequilibrated lipid bilayer. It was found that the bare nanodroplet wrapped by the PS monolayer kept suspending above the lipid bilayer with no apparent interaction occuring in the limited simulation time (Fig. S7a). For the lipid-coated nanodroplet wrapped by the PS layer, it released a number of PS molecules into the upper leaflet of the membrane (Fig. S7b), indicating a vesicle fusion pathway. To facilitate fusion, an external force (1000 kJ/mol/nm) mimicking that generated by the trans-SNARE complex formation was exerted on centers of nanodroplets toward the membrane.62 Consequently, several PS molecules were transferred from the nanodroplet to the membrane (Fig. 9a). Such behavior increased surface tension of the nanodroplet to induce rupture occurring on top of the nanodroplet, releasing drug particles into the water phase (Fig. 9a). By contrast, the bilayered vesicular structure formed by the PS layer wrapping on the lipid-coated nanodroplet was more stable during interaction with the membrane. They are expected to fuse with the membrane to release drugs inside a cell.63-64 Otherwise, they might be internalized via endocytosis, penetrate through cell junctions to enter the systemic circulation,55, 65 or be cleared by the macrophages. Our result showed that only partial fusion was achieved in the limited simulation time (Fig. 9b),66 manifesting energy barriers exisiting in the fusion process.67 Note that the vesicle was formed by the PS layer wrapping on the lipid-coated nanodroplet. Such structure exhibits a lower surface tension to further increase the barrier for fusion with the plasma membrane.

CONCLUSIONS In summary, we have investigated transport of nanodroplets across the PS layer using CGMD


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simulations. It was found that bare nanodroplets preferentially adhere onto the PS layer, releasing drugs to be directly translocated across the PS layer. The transport is quicker under higher surface tensions, but at the cost of opening pores that disrupt the ultrastructure of the PS layer. When the PS layer is compressed to lower surface tensions, the nanodroplet prompts collapse of the PS layer, another important indication of the PS perturbation. By coating the nanodroplet with lipids, it can be readily wrapped by the PS layer with slight PS perturbation. Moreover, the formed vesicular structures are expected to fuse with the cell membrane to release drugs into secondary organs. Properties of drug bioavailability, controlled drug release and enzymatic tolerance in real biological systems can be improved by the lipid coating strategy. Taken together, our simulations may assist in the design of more efficient and safe nanocarriers for pulmonary drug delivery.

ASSOCIATED CONTENT Supporting Information The Supporting information is available free of charge on the ACS Publication website at DOI: Comparison of the calcualted defected number using two different reference values; Detailed characterization of the PS layer collapse prompted by the bare nanodroplet; Interactions between the lipid-coated nanodroplet and the PS layer under higher surface tensions of 50 mN m-1 and 60 mN m-1; Repeated simulation results of the interaction between lipid-coated nanodroplets and the PS layer under a lower surface tension of 40 mN m-1; Comparison of the interaction energy between two different nanodroplets and the PS layer under different surface tensions; Repeated simulations results of preferential location of SP-B proteins in connection between the vesicle and the PS layer; Unbiased simulation results on interactions between nanodroplets and the plasma membrane (PDF). Rupturing of the PS layer induced by a bare nanodroplet (AVI) PS layer collapse prompted by a bare nanodroplet (AVI) PS layer wrapping on a lipid-coated nanodroplet (AVI)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ORCID


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Tongtao Yue: 0000-0002-8329-167X

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science foundation of China (no. 21303269), Science and Technology Major Project of Shandong Province (2016GSF117033),

the

Natural

Science

Foundation

of

Shandong

Province

(No.

ZR2018MC004), and Qingdao Science and Technology Project (no. 16-5-1-73-jch). This work was also partly supported by the Fundamental Research Funds for the Central Universities. MD simulations were performed at the National Super-computing Center in Shenzhen.

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Figure 1. Coarse-grained models and simulation system setup. (a) Schematic of the PS layer system. (b) Coarse-grained lipid and protein models used in our simulations. (c) The bare nanodroplet model. (d) The lipid-coated nanodroplet model. (e) The lipid bilayer model.


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Figure 2. The PS layer rupture and dispersive translocation of nanodroplet across the layer under tension of 40 mN m-1. (a) Time sequence of typical snapshots showing the process of PS layer rupture and nanodroplet translocation. (b) Time evolutions of interaction energies of nanodroplet with both water (black) and PS molecules (red). (c) Time evolutions of the monolayer area with (red) and without (black) deposition of nanodroplets.


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Figure 3. Characterization of the PS perturbation by deposited nanodroplets. (a) Time sequence of typical snapshots from the top view. The nanodroplet was not shown for clarity. (b) Time sequence of the PS order parameter diagram. (c) Time evolution of the defected number. (d) Radial distributions of both the PS density and PS order parameter as a function of distance from the nanodroplet at t = 30 ns. Surface tension of the PS layer is fixed at 40 mN m-1.


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Figure 4. Effect of surface tension of the PS layer on the interaction with bare nanodroplets. (a-c) Final simulated snapshots from the side view under surface tensions of 30 mN m-1, 20 mN m-1 and 10 mN m-1, respectively. (d) Time evolutions of the number of drug particles diffusing across the PS layer under different surface tensions. (e) Time evolutions of the number of drug particles in the nanodroplets. (f) Time evolutions of the total number of drug particles both diffusing across the PS layer and staying in the nanodroplets.


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Figure 5. Bare nanodroplet prompts collapse of the PS layer under zero surface tension.


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Figure 6. Effect of surface tension of the PS layer on interactions with lipid-coated nanodroplets. (a-d) Final simulated snapshots under surface tensions of 40 mN m-1, 30 mN m1

, 20 mN m-1 and 10 mN m-1, respectively. (e) Time evolutions of the wrapping percentage of

lipid-coated nanodroplets by the PS layer under tensions ranging from 40 mN m-1 to 0 mN m-1 with an interval of 5 mN m-1. (f) Time evolutions of the distance between centers of nanodroplets and the PS layer.


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Figure 7. Partial PS layer wrapping on a lipid-coated nanodroplet under a higher surface tension of 40 mN m-1. (a) Time sequence of typical snapshots showing the partial wrapping, with the rupturing location marked with blue arrows. (b) Time evolutions of the nanodroplet drug number and the aspect ratio. The time of drug leakage reflected by a sudden decrease of the nanodroplet drug number and a striking increase of the aspect ratio is marked by a dashed blue line. (c) Enlarged local structure with the drug leakage labeled by a white dashed circle.


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Figure 8. Full PS layer wrapping on a lipid-coated nanodroplet under a lower surface tension of 10 mN m-1. (a) Time sequence of typical snapshots showing the wrapping process. (b) Time evolutions of the nanodroplet drug number and the aspect ratio. (c) Final structure showing locations of both SP-B (gray) and SP-C (orange) proteins in the formed vesicular structure.


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Figure 9. Cell membrane interactions with two different nanodroplets wrapped by the PS layer. (a) The bare nanodroplet undergoes rupture by interacting with the cell membrane. (b) The lipid-coated nanodroplet partially fuses with the cell membrane in the limited simulation time.


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