Adsorption of Plasma Proteins onto PEGylated Lipid Bilayers: The

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Adsorption of Plasma Proteins onto PEGylated Lipid Bilayers: The Effect of PEG Size and Grafting Density Hwankyu Lee*,† and Ronald G. Larson‡ †

Department of Chemical Engineering, Dankook University, Yongin, 448-701, South Korea Department of Chemical Engineering, Biomedical Engineering, Mechanical Engineering, and Macromolecular Science and Engineering Program, University of Michigan, Ann Arbor, Michigan 48109, United States

‡

ABSTRACT: Lipid bilayers grafted with polyethylene glycol (PEG) of different sizes (Mw = 750, 2000, and 5000) and grafting densities (1.6−25 mol % of PEGylated lipid in dipalmitoylphosphatidylcholine (DPPC) lipid molecules) were simulated with human serum albumin (HSA) using coarsegrained force fields. At low enough grafting density, the PEG has a conformation similar to that of an isolated chain in water, and its Flory radius RF is smaller than the distance between the grafting points (d), which is the so-called “mushroom” regime. In contrast, densely grafted PEG chains (RF > d) extend like brushes, with brush layer thickness given by the Alexander-de Gennes theory. A nearly spherical HSA added to this simulation migrates to the bilayer surface because of the charge interactions between anion residues of HSA and cationic cholines of DPPC, but this HSA-bilayer binding can be sterically suppressed by the PEG chains to an extent that depends on the PEG size and grafting density. In particular, regardless of the extent of the coverage of the PEG on the bilayer, the binding between HSAs and bilayers is suppressed by the PEG layer in a brush but not in a mushroom, indicating that the attractive force between proteins and bilayers can overcome the steric effect of the PEG layer in the mushroom state or in the transition region from mushroom to brush. This helps explain and clarify experiments that show much less adsorption of plasma proteins onto the particle or membrane surface when PEGs are in the brush rather than in the mushroom state.

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INTRODUCTION Genes or drug molecules can be encapsulated into transporters such as liposomes, biopolymers, and nanoparticles and delivered to specific cancer cells.1−8 To achieve this, drug carriers need to be less toxic and more soluble,9−11 and thus, their surfaces have been often modified by attaching polyethylene glycol (PEG), a process called PEGylation.12,13 PEGylation not only reduces the toxicity of drug molecules but also sterically shields them, leading to longer circulation lifetime.14−16 In particular, when drug-encapsulating liposomes flow through the bloodstream, PEG chains can inhibit the adsorption of plasma proteins onto the liposome membrane, which protects the encapsulated drug and reduces interparticle aggregation, leading to prolonged circulation.17−20 This delivery efficiency can be modulated by the size and grafting density of PEGs, which has motivated many experimental and theoretical studies on the interactions between plasma proteins and PEGylated membranes.21−24 Needham et al. pioneered experimental studies of the conformation of PEG chains grafted to the liposome. They found that PEG becomes extended into a brush at high grafting density, which induces interbilayer repulsion and inhibits liposome aggregation, leading to increased circulation lifetime.18,25 Kuhl et al. and Kenworthy et al. determined the dependence of PEG conformation on its size and grafting density,26,27 obtaining results that agreed with the Alexander-de © 2016 American Chemical Society

Gennes theory that describes the transition of polymers between hemisphere (mushroom) and brushlike conformations.28 They also determined the maximum amount of PEGylated lipids that can be incorporated into the liposome while still maintaining liposome stability.29,30 Du et al., using a supported lipid monolayer, showed that the presence of PEG (PEG Mw = 5000; PEG5000) inhibits protein adsorption if the lipid surface is nearly completely covered by PEG5000, and so is in the brush state.31 More specifically, Efremova et al. and Bartucci et al., respectively, performed surface plasmon resonance (SPR) and electron spin resonance (ESR) experiments with PEGylated liposomes and found that plasma proteins do not adsorb onto lipid bilayers that contain more than 3−4.7 mol % PEGylated lipids (PEG2000), indicating the dependence of protein adsorption on the PEG size and grafting density.32,33 In particular, protein adsorption was observed only when the PEG in the bilayer was in the “mushroom” regime, indicating that PEG conformation modulates the extent of protein adsorption.34,35 However, Dos Santos et al. performed protein binding assays and showed that the inclusion of 5 mol % PEG2000-lipids reduces liposome aggregation, but does not inhibit the binding of plasma proteins to liposomes.36 The Received: January 28, 2016 Revised: March 29, 2016 Published: April 5, 2016 1757

DOI: 10.1021/acs.biomac.6b00146 Biomacromolecules 2016, 17, 1757−1765

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without HSA. The root mean squared end-to-end distances of PEGs and the thickness of the PEG layer are computed and compared with the predictions for the mushroom and brush regimes of the Alexander-de Gennes model. We then simulate an HSA protein interacting with PEGylated lipid bilayers and analyze the extent of protein adsorption and binding to the bilayer surface, which are correlated with the PEG length and grafting density. We will show that these results help explain some experimental observations regarding the dependence of the protein adsorption on the mushroom-brush transition of the grafted polymer.

concentration at which PEG5000 significantly reduces protein adsorption was determined by Gref et al. and Perry et al. to be at 2−5 and 3.1−6.3 mol %, respectively.37,38 Recently, Schneck et al. performed neutron reflectometry measurements with antiPEG antibody proteins, which more strongly interact with PEG chains than do other typical plasma proteins, showing the deep insertion of anti-PEG antibodies into the PEG layer, even when they do not bind to the membrane surface.39 Besides the PEGylated membrane, the protein adsorption into the solid surface grafted with PEG or block copolymers has been also studied.40−43 These experimental observations from different methodologies, conditions, and plasma proteins are not completely consistent, although they generally suggest that protein adsorption decreases strongly upon transition of the grafted PEG from the mushroom to the brush state. Also, note that because of inability to directly quantify PEG surface concentration, experimental results were interpreted under the assumption of complete binding of all PEGs onto the surface, an assumption that might not always hold. Hence, in addition to experiments such as the above, theory and molecular simulations are needed to clarify the conditions needed for inhibiting protein adsorption by PEG layers grafted onto lipid bilayers. To understand and complement these experimental results, many theoretical studies have in fact been performed. Jeon et al. calculated the steric repulsion, van der Waals and hydrophobic attraction free energies for the interactions between spherical model proteins and poly(ethylene oxide) (PEO) layers on a hydrophobic surface in the brush state only, qualitatively showing that the least protein adsorption occurs onto surfaces with the longest PEO and at the highest grafting density.44,45 In particular, they found that the protein−surface interaction is mainly modulated by steric repulsion and hydrophobic interaction. Szleifer showed a significant dependence of those interactions on the protein structure and PEO chain length, again in the brush state.46 Using the self-consistent field (SCF) theory, Halperin and co-workers showed that the penetration of small proteins into the brush layer can be inhibited by increasing the grafting density, while the adsorption of large proteins can be inhibited by increasing the brush thickness,47 which was verified by calculating free energies from their Monte Carlo simulations.48 They also found the difference in the strengths of specific and nonspecific binding between various plasma proteins and PEG brushes.49 Recently, Lehtinen et al. performed molecular dynamics (MD) simulations of small peptides interacting with PEGylated lipid bilayers, showing that the adsorption extents of hydrophobic and hydrophilic peptides differ.50 Taylor and Jones observed an exponential decrease in protein adsorption with increasing PEG-brush coverage.51 These theoretical and computational studies have revealed that the conformations of proteins and polymers influence the adsorption and binding of proteins onto the surface, but the dependence of protein adsorption on the mushroom-brush transition of polymers has not yet been systematically quantified through computation. As a further step toward understanding the relationship between the polymer conformation and the protein adsorption onto membrane, here we report coarse-grained (CG) MD simulations of a human serum albumin (HSA), which has been widely used for experiments on the adsorption of plasma proteins,33,38 on lipid bilayers grafted with PEGs of different sizes and grafting densities. First, PEGylated lipid bilayers at different PEG lengths and grafting densities are simulated

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METHODS

All simulations and analyses were performed with the GROMACS4.6.7 simulation package.52−54 Potential parameters for the HSA protein and dipalmitoylphosphatidylcholine (DPPC) lipid were taken directly from the “MARTINI” CG force field (FF), which lumps three or four heavy atoms into each CG bead.55−57 The structure and coordinates of the protein HSA were downloaded from the Protein Data Bank (PDB code: 1AO6), which shows that the HSA protein is folded with a heart-like shape.58 Note that previous simulations with the MARTINI amino acid FF have successfully captured the protein− membrane interaction,57,59 although the secondary structure of the protein needs to be assigned and held fixed, so that changes in secondary structure cannot be modeled. Thus, the heart-like folded structure of HSA was retained for whole simulation time by fixing secondary structures in the CG model, although it cannot be ruled out that structural changes might influence the interaction between HSA and PEGylated bilayer. To model the PEGylated lipid, we previously developed the CG FF for PEGylated dipalmitoylglycerophosphoethanolamine (DPPE) within the framework of the MARTINI FF.60,61 Briefly, the terminal CG bead of PEGs (Mw of 750, 2000, and 5000, which, respectively, correspond to 17-, 45-, and 113-mers of the CG PEG bead) was attached to the headgroup (choline) of a DPPC, and the CG bead for the DPPC choline was converted to represent the amide group of the linkage between PEG and lipid, leading to a net charge of −1 for the PEGylated DPPE. The CG amide and PEG beads were linked with the bond and angle potentials, as described in our previous work.60 These CG PEG and PEGylated-lipid models have successfully captured the self-assembly of liposomes, bicelles, and micelles at the expected ratios of lipids and PEGylated lipids,61 and the mushroom-brush transition of PEG chains grafted onto peptides,62 dendrimers,63 and carbon nanotubes,64 giving results in agreement with experiments and polymer theories. PEGylated lipids were evenly distributed within the DPPC bilayer at different concentrations of 1.6−25 mol % (Table 1 and Figure 1). The final simulation system consists of 1152 DPPC or PEGylated lipids (576 lipids/leaflet) and 40000−45000 CG water beads (representing 160000−180000 real waters) in a periodic box of size 16.4 × 16.4 × 23−33 nm3. Since each PEGylated lipid has a net charge of −1, counterions of 33, 51, 87, 159, and 303 Na+ were added to neutralize the systems at PEG concentrations of 1.6, 3.1, 6.3, 12.5, and 25.0 mol %, respectively. For the systems with HSA, a single HSA protein was initially positioned above the bilayer with a distance of 10−16 nm between protein and bilayer centers, so that HSA is not touching the PEG layer. A cutoff of 12 Å was used for the Lennard-Jones potential with a smooth shift to 0 between 9 and 12 Å. For the Coulomb potential, a short-range interaction with a cutoff of 12 Å and a longrange interaction with particle mesh Ewald summation (PME) were used.65 A pressure of 1 bar and a temperature of 283 K, which reproduce the experimentally observed gel phase of DPPC liposome at 293 K (area per lipid of 48 Å2),66 were maintained by applying a velocity-rescale thermostat67 and Parrinello−Rahman barostat68 in the NPxyPzT ensemble with semi-isotropic pressure coupling. The LINCS algorithm was used to constrain the bond lengths.69,70 Simulations were carried out for 0.5−2 μs with a time step of 8 fs on computer facilities supported by the National Institute of Supercomputing and Networking/Korea Institute of Science and Technology Information 1758


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Biomacromolecules Table 1. List of Simulations No. of molecules

pure bilayer (no protein)

Mw of PEG

mol % of PEG

PEGylated lipid

DPPC

750

1.6 3.1 6.3 12.5 25.0 1.6 3.1 6.3 12.5 25.0 1.6 3.1 6.3 12.5 25.0 1.6 3.1 6.3 12.5 25.0 1.6 3.1 6.3 12.5 25.0 1.6 3.1 6.3 12.5 25.0

18 36 72 144 288 18 36 72 144 288 18 36 72 144 288 18 36 72 144 288 18 36 72 144 288 18 36 72 144 288

1134 1116 1080 1008 864 1134 1116 1080 1008 864 1134 1116 1080 1008 864 1134 1116 1080 1008 864 1134 1116 1080 1008 864 1134 1116 1080 1008 864

2000

5000

bilayer with a protein

750

2000

5000

HSA

simulation time (μs)

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.5 0.5 0.5 0.5 0.5 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

with supercomputing resources including technical support (KSC2015-C3−057). The last 200−500 ns trajectories were used for analyses.

Figure 1. Density probabilities of PEG chains as a function of distance from the PEG−bilayer interface. In the snapshot, red, blue, and lightblue colors indicate PEGs, lipid head groups, and tails, respectively. The images were created using Visual Molecular Dynamics.81

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RESULTS AND DISCUSSION Conformation of PEGs Grafted on the Lipid Bilayer. Lipid bilayers grafted with PEG of different sizes (Mw = 750, 2000, and 5000) and grafting densities (1.6−25 mol %) were simulated for 0.5−0.8 μs (Table 1). Figure 1 shows a schematic illustration of the PEGylated bilayer and density probabilities of PEGs as a function of distance from the average position of the choline and phosphate beads (normal to the bilayer surface). Longer chains at higher grafting densities form thicker PEG layers, as expected, since they are more crowded and, thus, more extended toward water. The Alexander-de Gennes theory describes the conformational transition of the polymer chain grafted onto a nonadsorbing surface.28 At very low grafting density, the grafted chain behaves like an isolated chain in solution, leading to a hemisphere (mushroom) conformation with a size given by the Flory radius, RF = aN3/5, where N is the degree of polymerization and a is the monomer size (3.3 Å for the bond length in the CG model). At high grafting density (d < RF), polymer chains become crowded and repel each other, and thus, they extend like a brush with a thickness given by L = Na(a/d)2/3, where d is the distance between the grafting points of polymers, leading to a relatively thick layer of uniform

concentration. Here, d equals a root of the average squarelattice area per each grafted PEGylated lipid (d = (area of the bilayer surface/number of PEGylated lipids)1/2). Alexander-de Gennes theory only defines the brush thickness precisely in the limit a = d from L = Na(a/d)2/3; the intermediate values are left unspecified. Here, density profiles in Figure 1 were used to reasonably define the thickness of the PEG layer. The root mean squared end-to-end distance (⟨h2⟩1/2) from one end of the PEG graft to the other should be comparable to the thickness of the PEG layer above the bilayer surface. This is confirmed in Table 2, which shows that at a distance ⟨h2⟩1/2 above the head groups, the PEG density is in the range of 80− 90% of its peak value at the top of the lipid layer. Thus, hereafter, the “thickness” of the PEG layer is defined as the position at which the PEG density reaches 80−90% of its maximum, which is then compared with the size of mushroom and brush in the Alexander-de Gennes theory. Table 2 compares the simulation results with the Alexanderde Gennes theory. For PEG750, where the d values are either close to RF or larger, the thickness of the PEG layer is close to 1759


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Table 2. Root Mean Squared End-to-End Distance (⟨h2⟩1/2), Thickness of PEG Layer Calculated from Simulations, and Size of Mushroom and Thickness of Brush Calculated from the Alexander-de Gennes Theorya simulation Mw of PEG PEG750

PEG750 in waterb PEG2000

PEG2000 in waterb PEG5000

PEG5000 in waterb a

theory

thickness of PEG layer on the bilayer (80−90 % density)

size of mushroom (RF)

thickness of brush (L)

1 1 1 1 1 1

18−21 19−22 19−22 19−22 19−22

18 18 18 18 18

9 11 13 17 21

± ± ± ± ± ±

1 1 1 1 1 1

31−36 31−37 32−39 35−42 41−49

32 32 32 32 32

23 29 36 45 57

± ± ± ± ± ±

1 1 2 2 2 1

51−61 57−67 64−76 65−76 74−89

56 56 56 56 56

57 72 90 112 142

mol % of PEG

N

d

d2/RF2

1.6 3.1 6.3 12.5 25.0

17 17 17 17 17

55 39 28 20 14

9.34 4.69 2.42 1.23 0.60

23 23 23 23 23 21

± ± ± ± ± ±

1.6 3.1 6.3 12.5 25.0

45 45 45 45 45

55 39 28 20 14

2.95 1.49 0.77 0.39 0.19

37 37 38 40 43 36

1.6 3.1 6.3 12.5 25.0

113 113 113 113 113

55 39 28 20 14

0.96 0.49 0.25 0.13 0.06

57 61 67 68 80 56

⟨h2⟩1/2

See ref 28. bThe ⟨h2⟩1/2 values for a single PEG chain in water are from ref 60. All lengths are in Angstroms.

RF and ⟨h2⟩1/2 at all grafting densities in the range 1.6−25.0 mol % are similar to the value of ⟨h2⟩1/2 for a single chain in water, indicating a mushroom conformation. For PEG2000, the PEGlayer thickness is close to both RF and ⟨h2⟩1/2 over the graftingdensity range 1.6−6.3 mol %, while at higher grafting densities, the PEG-layer thickness exceeds RF and becomes close to the theoretical brush thickness L, indicating a conformational transition from mushroom to extended brush. For PEG5000, the mushroom-brush transition occurs between 3.1 and 6.3 mol %, where d ≈ RF. These results are plotted as a function of the grafting distance (d) in Figure 2. For PEGs of all sizes, the thickness of the PEG layer corresponds to the size of mushroom at low grafting density (d ≥ RF) and increases beyond the mushroom regime at high grafting density (d < RF). Note that, at high grafting density of PEG5000, PEG layers are thinner than expected. This discrepancy between simulation and brush theory is presumably because the bilayer surface is not a nonadsorbing surface and, hence, may somewhat attract PEG beads, as previously observed in all-atom MD simulations by Stepniewski et al.71 However, our simulations clearly show that for PEGs of all sizes the transition between mushroom and brush states occurs at d ≈ RF, indicating that this CG model can accurately reproduce the mushroom-brush transition in Alexander-de Gennes theory. Diffusivity and Hydrodynamics of HSA in Water. To understand the conformation of the protein HSA, a single HSA was simulated with water at 298 K, and its diffusivity and hydrodynamic radius were calculated. To obtain the diffusion coefficient of HSA, the slope of the mean-square displacement of the center of mass (COM) of HSA was calculated and then corrected for finite size effects using the formula D = DPBC + kBTξ/6πηL,72 where kB is Boltzmann’s constant, L is the equilibrated cubic-box length, ξ = 2.837297, and η is the solution viscosity. Since the viscosity increases with higher solute concentration, the solution viscosity was corrected using

Figure 2. Thickness of the PEG layer, calculated from the height above the headgroup beads at which the PEG density falls to 80−90% of the maximum density, as a function of the distance between the average grafting points of PEG (d). Red dotted and black dashed lines respectively represent the size of the mushroom (RF) and the thickness of the brush (L) calculated from the Alexander-de Gennes theory.28

the Einstein formula η = ηw(1 + 2.5⌀),73 where ⌀ is the volume fraction of the solutes, and ηw is the viscosity of pure water (taken to be 0.75 cP at 298 K for CG water).74 Thus, the final diffusion constant is given by D = [DPBC + kbTξ/(6π × 0.0075(1 + 2.5⌀)L)]. Using this diffusion coefficient, we calculated the hydrodynamic radius (Rh) from the Stokes− 1760


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Biomacromolecules Einstein equation for a sphere with no-slip boundary conditions, Rh = kBT/6πηD.75 Note that viscosities of water for the CG model and experiment do not significantly differ. Also, we previously simulated isolated PEO chains in water using the MARTINI FF, and the Rh values calculated from the diffusion coefficients for PEOs agreed well with experimental values,60 without the use of a dynamical scaling factor of around 4 often used to correct for the faster dynamics often observed with the MARTINI CG model.55 The absence of the scaling factor for relatively large objects such as proteins is reasonable, because once the diffusing object becomes much larger than the solvent beads with the diameter of 4.7 Å, the artificial smoothness of the CG beads ceases to be relevant, and the object diffuses according to the Stokes−Einstein theory.60 The diffusion coefficient for a given particle thus depends primarily on solvent viscosity, and this is within 25% of the experimental value. Thus, we do not use this scaling factor here. Table 3 Table 3. Diffusion Coefficient (D), Hydrodynamic Radius (Rh), and Radius of Gyration (Rg) of a Single HSA Molecule in Water diffusivity (10

−7

2

−1

cm s )

Figure 3. Snapshots at the end (2 μs) of simulations of bilayers with HSA at PEGylated-lipid concentrations of 1.6, 12.5, and 25.0 mol %. Black and red colors, respectively, represent HSA and PEG, while blue and light-blue colors indicate lipid head groups and tails, respectively. For clarity, water and ions are omitted.

radius (nm)

DPBC

D

Rh

Rg

2.83 ± 0.42

9.07

2.90 ± 0.13

2.46 ± 0.02

shows that D of HSA is 9.07 × 10−7 cm2 s−1, yielding the Rh value of 2.9 (±0.13) nm, roughly half of the HSA dimension in the crystal structure (a heart-shaped molecule with an apparent size of 8 × 8 × 3 nm3).58 Note that radius of gyration (Rg) of HSA is 2.46 nm, leading to the ratio of Rg/Rh = 0.848, which is slightly higher than experimental and theoretical values of Rg/ Rh = ∼0.775 for globular (sphere-like) proteins.76,77 This indicates that HSA is nearly but not completely spherical, as expected, since the heart-like shape and folded structure of HSA are fixed in the CG model. These results confirm that this simulated conformation of HSA matches experimental observations and thus can be considered for the HSA−bilayer interaction. Effects of the PEG Size and Grafting Density on the Adsorption of HSA onto the Bilayer. With HSA and PEGylated bilayer equilibrated as described above, we simulated the adsorption of a single HSA protein onto the PEGylated bilayer for 2 μs (Table 1). Figure 3 shows final snapshots of simulations at PEG concentrations of 1.6, 12.5, and 25 mol %. HSA molecules, which were initially positioned above the PEG layer, migrate toward the bilayer surface, presumably because of electrostatic interactions between HSAs and lipid head groups. HSAs insert into the PEG layer and bind to the bilayer with small PEGs at low grafting density, while HSAs are not able to bind to the bilayers grafted with larger PEGs at higher grafting density, implying that thick PEG layers block the binding between HSA and the bilayer surface. These configurations are also confirmed by calculating minimum distances between HSAs and bilayers. Figure 4 shows that the minimum distances between HSA and bilayer become less than 0.5 nm within 1.5−1.7 μs for the bilayers with PEG750 at 1.6− 12.5 mol %, those with PEG2000 at 1.6−6.3 mol %, and those with PEG5000 at 1.6−3.1 mol %, indicating that adsorption and binding of HSA onto the bilayer surface occur when the PEG is in the mushroom regime. Note that although HSAs do not insert to the PEG layer in the brush regime, they still bind to the outer edge of the PEG brush, which is considered to be

Figure 4. Minimum distances between any atoms of HSA and the bilayer surface as a function of time.

“non-adsorption” in this work. In experiments, nonadsorption indicates no adsorption to the membrane surface as well as to the PEG layer,39 in contrast to our simulations. This discrepancy might be an artifact of the CG FF, since strong attractive interactions between amino acids and PEGs have been also reported in our previous work.62 Figure 5 compares the PEG-layer thickness with the PEG’s COM and the location of the bottom of the HSA. These results show that HSAs bind to the bilayer surface at 1.6−12.5 mol % PEG750, 1.6−6.3 mol % PEG2000, and 1.6−3.1 mol % PEG5000, while they do not bind to the bilayer at higher PEG 1761


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interaction strength of anionic and cationic residues, radial distribution functions (RDFs) between lipid head groups and charged residues of HSA were calculated. Figure 6 shows that

Figure 6. Radial distribution functions (RDFs) between anionic residues (Asp and Glu) of HSA and cationic cholines of lipids, and between cationic residues (Lys and Arg) of HSA and anionic phosphates of lipids, for the systems with PEG2000.

for the system with 1.6 mol % PEG2000, the RDF peaks between anionic residues and lipid cholines are much higher at ∼0.5 nm than those between cationic ones and lipid phosphates, indicating a stronger interaction of the bilayer with anionic residues than with cationic residues. This is presumably because HSAs do not insert into the bilayer and thus cannot strongly interact with anionic phosphates of lipids that are located deeper in the bilayer than are the cationic cholines, which are at the surface. Note that this result seems to conflict with experiments, which have shown that cationic colloids more deeply penetrate into the lipid bilayer than do anionic ones because of their interactions with anionic lipid phosphates.78,79 However, experiments have also shown that both anionic and cationic colloids bind to the bilayer surface, indicating that the charge type influences the extent of particle insertion into the bilayer rather than the binding to the bilayer surface.80 In our simulations, HSAs bind to the bilayer surface but do not insert into the bilayer, and thus their strong interactions with cationic lipid cholines can be reasonably considered especially for the gel-phased bilayer. For the systems with 12.5 and 25 mol % PEG2000, there are no sharp RDF peaks at ∼0.5 nm, again confirming that HSA do not interact with the bilayer surface at those high grafting densities. To compare the strength of electrostatic interactions at different grafting densities, RDFs between anionic residues of HSA and lipid cholines were calculated. Figure 7 shows the first peaks at ∼0.5 nm only for PEG750 of 12.5 mol % or less, PEG2000 of 6.3 mol % or less, and PEG5000 of 3.1 mol % or less, indicating strong electrostatic interactions at relatively lower grafting densities, where HSAs bind to the bilayer, consistent with Figures 3-5. These indicate that the HSA adsorption onto the bilayer is induced by electrostatic interactions between anionic residues of HSA and choline lipid head groups, which can be modulated by PEG size and density.

Figure 5. Distance from the average position of lipid head groups to the center of mass (COM) of the protein (squares) and to the lowest residue of the protein (circles), and the thickness of PEG layer (dashed line).

concentrations, consistent with Figure 4. These results, combined with the mushroom-brush transition of PEG in Table 2 and Figure 2, indicate that HSAs do not bind to the bilayer surface with PEGs in the brush regime, implying that the HSA adsorption can be effectively inhibited by PEG chains in the extended-brush state rather than in the mushroom regime, which agrees with experiments showing that PEG2000 at a concentration of 2−5 mol %, which is still in the mushroom state, does not reduce the extent of protein adsorption onto the liposome,36 and showing that there is much less adsorption of plasma proteins through PEG5000 chains that are in the brush regime than that are in the mushroom regime.38 Interactions between HSA and the Bilayer Surface. The HSA protein is highly charged with anionic (aspartic acid and glutamic acid) and cationic (lysine and arginine) amino acids, leading to a net charge of −15. To compare the 1762


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Figure 7. RDFs between anionic residues of HSA and lipid cholines.

Figure 8. Snapshots of the top and side view of simulations with 3.1 and 25 mol % PEG2000 (top) and percentages of the exposed portions (uncovered by PEGs) of the bilayer headgroup (cholines).

Since PEG does not have a charge in the CG model, PEG chains may sterically suppress the binding between HSA and lipid head groups. To check this, the extent of the coverage of PEG on the bilayer was quantified by calculating solvent accessible surface areas (SASA) of the bilayer surface, which measures the surface area uncovered by PEGs. Here, the radius of the solvent probe set to 1.5 nm, since the length along the shortest axis of HSA is approximately 3 nm. With this solvent criteria, we calculated the SASA of the lipid choline for each PEGylated bilayer, where grafted PEG chains are considered to be impenetrable, and then divided those by the SASA of the lipid choline for pure bilayer without PEG, which we interpret as the percentage of the exposed portions of the bilayer surface in Figure 8. Figure 8 shows that the bilayers with smaller and fewer PEGs are more exposed than those with larger and more PEGs, as expected. In particular, bilayer surfaces are almost covered by PEG750 at grafting densities of 12.5 mol % or more, and PEG2000 and PEG5000 at grafting densities of 6.3 mol % or more, where HSAs barely bind to the bilayer as observed in Figures 4−7, indicating the steric effect of PEG. For instance, PEG5000 chains become extended like brushes at such a high grafting density and thus cannot be easily sterically penetrated by HSA. However, note that at PEG concentrations of 6.3 mol % or more, HSAs still bind to those bilayers with PEG750 (6.3 and 12.5 mol %) and PEG2000 (6.3 mol %), where PEG chains have the mushroom conformation (Table 2 and Figure 2). This indicates that, although bilayers are still mostly covered by PEGs, those PEGs in the mushroom state can be penetrated by HSA. Figure 9 summarizes simulation results regarding the HSA-bilayer binding in mushroom and brush regimes, clearly showing that the binding of HSA to the bilayer is suppressed only in the brush regime, regardless of PEG size, again confirming that the mushroom-brush transition of the grafted polymer modulates the adsorption of plasma protein onto the membrane surface. Experimentally, Bartucci et al.33 and Efremova et al.32 observed much less adsorption of HSA onto the bilayer at a PEG2000 concentration of more than around 3 to 4.5 mol %, while Dos Santos et al.36 found no decrease in the adsorption of

Figure 9. Boundary between regions of binding and no-binding between HSA and the bilayer surface, as functions of PEG size and grafting density (top) and of d2/RF2 (bottom). A thick red line represents the boundary between mushroom and brush states.

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Biomacromolecules

binds to the bilayer when the PEG is in a mushroom but not in a brush state. In particular, we find that the PEG layer sterically blocks the binding between HSA and lipid bilayer, but when PEG has a mushroom conformation, HSA can penetrate the PEG layer and bind to the bilayer, indicating that this steric effect depends on the PEG conformation. These simulation findings help explain experimental results showing much less absorption of plasma protein to the surface with PEGs with the brush state than with the mushroom state.

serum albumins at PEG2000 concentration up to 5 mol %, which favorably compares with our results showing no effect of PEG2000 at grafting densities of 6.3 mol % or less. Also, Gref et al. observed significant reduction of the plasma-protein adsorption at a PEG5000 concentration of around 2 to 5 mol %,37 similar to our simulation results showing a significant difference at a concentration of 3.1 to 6.3 mol %. Perry et al. showed a significant reduction of the adsorption of bovine serum albumin (BSA) at d = 3.9 nm (corresponding to a PEG5000 concentration of ∼3.1 mol %, which is in the brush regime),38 but much less reduction at d = 6.7 nm (∼1.1 mol % PEG5000 in the mushroom regime), which also agrees with our simulations. Simulation findings favorably compare with some experiments, and indicate that HSAs bind electrostatically to the bilayer surface, but this HSA-bilayer binding can be sterically suppressed by PEG, to an extent that depends on the PEG size and grafting density. Note that the size and electrostatic property of plasma proteins can influence the extent of the protein adsorption,31,37 and thus, our results should be applied to some plasma proteins with caution. Also, it cannot be ruled out that the protein adsorption onto the bilayer might be not only induced by the steric effect, but also influenced by electrostatics, since PEGylated lipids are negatively charged and thus make the HSA-bilayer interaction weaker at higher grafting density. In fact, the analytical SCF calculation yields a parabolic density profile of grafted polymers,43 which can be more directly compared with simulation densities than does a step-like profile from the Alexander-de Gennes theory. It would obviously be interesting to investigate the adsorption dependence on protein types and bilayer charges using the SCF theory, which we hope to report on elsewhere. However, based on our detailed investigation of the interactions, it is the anionic protein residues that lead to electrostatic binding of the protein onto the choline groups of DPPC lipids, and this information should be of relevance in determining binding, and its suppression by PEG, of other proteins. Most importantly, our systematic simulations with HSA clearly show that when PEG is in the mushroom regime, the PEG layer can be penetrated by the protein presumably because of bare lipid patches between mushrooms. This indicates that the ability of the HSA to overcome the steric effect of the PEG layer and bind to the bilayer depends on the conformational transition of PEG from the mushroom to the brush state. This helps explain recent experiments that show that protein adsorption is much more inhibited when the PEG layer is in a brush than when it is in a mushroom.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2014R1A1A2054016).

(1) Popielarski, S. R.; Pun, S. H.; Davis, M. E. Bioconjugate Chem. 2005, 16, 1063−1070. (2) Segura, T.; Shea, L. D. Annu. Rev. Mater. Res. 2001, 31, 25−46. (3) Ferrari, M. Nat. Rev. Cancer 2005, 5, 161−171. (4) Rolland, A. P. Crit. Rev. Ther. Drug Carrier Syst. 1998, 15, 143− 198. (5) Davis, M. E. Curr. Opin. Biotechnol. 2002, 13, 128−131. (6) Luo, D.; Haverstick, K.; Belcheva, N.; Han, E.; Saltzman, W. M. Macromolecules 2002, 35, 3456−3462. (7) Liu, Z.; Robinson, J. T.; Sun, X.; Dai, H. J. Am. Chem. Soc. 2008, 130, 10876−10877. (8) Torchilin, V. P. Pharm. Res. 2006, 24, 1−16. (9) Nel, A.; Xia, T.; Madler, L.; Li, N. Science 2006, 311, 622−627. (10) Leroueil, P. R.; Hong, S. Y.; Mecke, A.; Baker, J. R.; Orr, B. G.; Holl, M. M. B. Acc. Chem. Res. 2007, 40, 335−342. (11) Leroueil, P. R.; Berry, S. A.; Duthie, K.; Han, G.; Rotello, V. M.; McNerny, D. Q.; Baker, J. R.; Orr, B. G.; Holl, M. M. B. Nano Lett. 2008, 8, 420−424. (12) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40, 539−551. (13) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discovery 2003, 2, 214−221. (14) Caliceti, P.; Veronese, F. M. Adv. Drug Delivery Rev. 2003, 55, 1261−1277. (15) Lee, H.; Larson, R. G. J. Phys. Chem. B 2009, 113, 13202− 13207. (16) Steinmetz, N. F.; Manchester, M. Biomacromolecules 2009, 10, 784−792. (17) Maruyama, K.; Yuda, T.; Okamoto, A.; Kojima, S.; Suginaka, A.; Iwatsuru, M. Biochim. Biophys. Acta, Lipids Lipid Metab. 1992, 1128, 44−49. (18) Needham, D.; Hristova, K.; McIntosh, T. J.; Dewhirst, M.; Wu, N.; Lasic, D. D.; Alving, C. R.; Wassef, N. M.; Senior, J. H.; Ghosh, P. C.; Bachhawat, B. K.; Allen, T. M. J. Liposome Res. 1992, 2, 411−430. (19) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett. 1990, 268, 235−237. (20) Gabizon, A.; Barenholz, Y. Liposomes: Rational Design; Marcel Dekker: New York, 1999; pp 343−362. (21) Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yauyoung, A. Biochim. Biophys. Acta, Biomembr. 1991, 1066, 29−36. (22) Garbuzenko, O.; Barenholz, Y.; Priev, A. Chem. Phys. Lipids 2005, 135, 117−129.

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CONCLUSIONS We performed coarse-grained MD simulations of lipid bilayers grafted with polyethylene glycols (PEGs) of different sizes and grafting densities. Root mean squared end-to-end distances of PEG and thickness of the PEG layer show that at low grafting density (with graft spacing d greater than the coil radius RF) the PEG chain has a hemispherical (mushroom) conformation, while at high grafting density (d < RF) the PEG layer becomes an extended brush, as expected from both theory and previous work on chains grafted to surfaces. In particular, for PEGs of all sizes, the transition between mushroom and brush states occurs at d ≈ RF, in quantitative agreement with the Alexander-de Gennes theory. A single HSA simulated near the equilibrated PEGylated bilayer migrates toward the bilayer surface because of the electrostatic interaction between anionic residues of the HSA and the cationic choline lipid head groups. The HSA 1764


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Biomacromolecules (23) Marsh, D.; Bartucci, R.; Sportelli, L. Biochim. Biophys. Acta, Biomembr. 2003, 1615, 33−59. (24) Park, Y.; Franses, E. I. Langmuir 2010, 26, 6932−6942. (25) Needham, D.; McIntosh, T. J.; Lasic, D. D. Biochim. Biophys. Acta, Biomembr. 1992, 1108, 40−48. (26) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479−1488. (27) Kenworthy, A. K.; Hristova, K.; Needham, D.; McIntosh, T. J. Biophys. J. 1995, 68, 1921−1936. (28) de Gennes, P. G. Adv. Colloid Interface Sci. 1987, 27, 189−209. (29) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903−1920. (30) Hristova, K.; Kenworthy, A.; McIntosh, T. J. Macromolecules 1995, 28, 7693−7699. (31) Du, H.; Chandaroy, P.; Hui, S. W. Biochim. Biophys. Acta, Biomembr. 1997, 1326, 236−248. (32) Efremova, N. V.; Bondurant, B.; O’Brie, D. F.; Leckband, D. E. Biochemistry 2000, 39, 3441−3451. (33) Bartucci, R.; Pantusa, M.; Marsh, D.; Sportelli, L. Biochim. Biophys. Acta, Biomembr. 2002, 1564, 237−242. (34) Chiu, G. N. C.; Bally, M. B.; Mayer, L. D. Biochim. Biophys. Acta, Biomembr. 2001, 1510, 56−69. (35) Li, W. M.; Mayer, L. D.; Bally, M. B. J. Pharmacol. Exp. Ther. 2002, 300, 976−983. (36) Dos Santos, N.; Allen, C.; Doppen, A. M.; Anantha, M.; Cox, K. A. K.; Gallagher, R. C.; Karlsson, G.; Edwards, K.; Kenner, G.; Samuels, L.; Webb, M. S.; Bally, M. B. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 1367−1377. (37) Gref, R.; Lück, M.; Quellec, P.; Marchand, M.; Dellacherie, E.; Harnisch, S.; Blunk, T.; Müller, R. H. Colloids Surf., B 2000, 18, 301− 313. (38) Perry, J. L.; Reuter, K. G.; Kai, M. P.; Herlihy, K. P.; Jones, S. W.; Luft, J. C.; Napier, M.; Bear, J. E.; Desimone, J. M. Nano Lett. 2012, 12, 5304−5310. (39) Schneck, E.; Berts, I.; Halperin, A.; Daillant, J.; Fragneto, G. Biomaterials 2015, 46, 95−104. (40) Currie, E. P. K.; Van Der Gucht, J.; Borisov, O. V.; Cohen Stuart, M. A. Pure Appl. Chem. 1999, 71, 1227−1241. (41) Bosker, W. T. E.; Iakovlev, P. A.; Norde, W.; Cohen Stuart, M. A. J. Colloid Interface Sci. 2005, 286, 496−503. (42) Norde, W.; Gage, D. Langmuir 2004, 20, 4162−4167. (43) Currie, E. P. K.; Norde, W.; Cohen Stuart, M. A. C. Adv. Colloid Interface Sci. 2003, 100−102, 205−265. (44) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149−158. (45) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159− 166. (46) Szleifer, I. Biophys. J. 1997, 72, 595−612. (47) Halperin, A. Langmuir 1999, 15, 2525−2533. (48) Ermilov, V.; Lazutin, A.; Halperin, A. Macromolecules 2010, 43, 3511−3520. (49) Halperin, A.; Kröger, M. Langmuir 2009, 25, 11621−11634. (50) Lehtinen, J.; Magarkar, A.; Stepniewski, M.; Hakola, S.; Bergman, M.; Róg, T.; Yliperttula, M.; Urtti, A.; Bunker, A. Eur. J. Pharm. Sci. 2012, 46, 121−130. (51) Taylor, W.; Jones, R. A. L. Langmuir 2013, 29, 6116−6122. (52) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem. Theory Comput. 2008, 4, 435−447. (53) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7, 306−317. (54) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701−1718. (55) Marrink, S. J.; de Vries, A. H.; Mark, A. E. J. Phys. Chem. B 2004, 108, 750−760. (56) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. J. Phys. Chem. B 2007, 111, 7812−7824. (57) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S. J. J. Chem. Theory Comput. 2008, 4, 819− 834.

(58) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. Protein Eng., Des. Sel. 1999, 12, 439−446. (59) Yelimov, S.; van der Giessen, E.; Onck, P. R.; Marrink, S. J. Biophys. J. 2008, 94, 2994−3002. (60) Lee, H.; de Vries, A. H.; Marrink, S. J.; Pastor, R. W. J. Phys. Chem. B 2009, 113, 13186−13194. (61) Lee, H.; Pastor, R. W. J. Phys. Chem. B 2011, 115, 7830−7837. (62) Woo, S. Y.; Lee, H. Langmuir 2014, 30, 8848−8855. (63) Lee, H.; Larson, R. G. Macromolecules 2011, 44, 2291−2298. (64) Lee, H. J. Phys. Chem. C 2013, 117, 26334−26341. (65) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577−8593. (66) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta, Rev. Biomembr. 2000, 1469, 159−195. (67) Bussi, G.; Donadio, D.; Parrinello, M. J. Chem. Phys. 2007, 126, 014101−014101. (68) Parrinello, M.; Rahman, A. J. Appl. Phys. 1981, 52, 7182−7190. (69) Hess, B. J. Chem. Theory Comput. 2008, 4, 116−122. (70) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1997, 18, 1463−1472. (71) Stepniewski, M.; Pasenkiewicz-Gierula, M.; Rog, T.; Danne, R.; Orlowski, A.; Karttunen, M.; Urtti, A.; Yliperttula, M.; Vuorimaa, E.; Bunker, A. Langmuir 2011, 27, 7788−7798. (72) Yeh, I. C.; Hummer, G. J. Phys. Chem. B 2004, 108, 15873− 15879. (73) Tanford, C. Physical Chemistry of Macromolecules. John Wiley & Sons: New York, 1961. (74) Fuhrmans, M.; Sanders, B. P.; Marrink, S. J.; de Vries, A. H. Theor. Chem. Acc. 2010, 125, 335−344. (75) Doi, M.; Edwards, S. F. The Theory of Polymer Dynamics; Clarendon Press: Oxford, 1986. (76) van Holde, K. E. Physical Biochemistry; Prentice-Hall: New Jersey, U.S.A., 1971; pp 180−202. (77) Kunz, D.; Thurn, A.; Burchard, W. Colloid Polym. Sci. 1983, 261, 635−644. (78) Letellier, D.; Sandre, O.; Ménager, C.; Cabuil, V.; Lavergne, M. Mater. Sci. Eng., C 1997, 5, 153−162. (79) Mishra, A.; Lai, G. H.; Schmidt, N. W.; Sun, V. Z.; Rodriguez, A. R.; Tong, R.; Tang, L.; Cheng, J.; Deming, T. J.; Kamei, D. T.; Wong, G. C. L. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16883−16888. (80) Verma, A.; Stellacci, F. Small 2010, 6, 12−21. (81) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33−38.

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Adsorption of Plasma Proteins onto PEGylated Lipid Bilayers: The

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