Research Note pubs.acs.org/IECR
Assessment of the Antifouling Properties of Polyzwitterions from Free Energy Calculations by Molecular Dynamics Simulations Ryo Nagumo,† Kazuki Akamatsu,‡ Ryuji Miura,§ Ai Suzuki,† Hideyuki Tsuboi,† Nozomu Hatakeyama,§ Hiromitsu Takaba,§ and Akira Miyamoto*,†,§ †
New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan Department of Environmental and Energy Chemistry, Faculty of Engineering, Kogakuin University, 2665-1 Nakano-machi, Hachioji-shi, Tokyo 192-0015, Japan. § Department of Chemical Engineering, Graduate School of Engineering, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan ‡
ABSTRACT: Polyzwitterions (PZs), such as carboxybetaine and phosphobetaine polymers, show remarkable suppression of protein adsorption and have potentially widespread application as bioengineering materials. We show that free energy profiles, from molecular dynamics simulations in explicit water, for hydrophilic and hydrophobic amino acids approaching a PZ monomer, provide thermodynamic insights into protein adsorption. The predicted profiles for PZ have almost no energetically stable points, regardless of the type of residue. In contrast, the profiles for conventional polyester show some energetically remarkable minima, particularly for the hydrophobic residue. These results agree with recent experimental reports of differences in the amounts of protein adsorbed on these polymers, suggesting that free energy calculations for hydrophobic residue can play a significant role in assessing antifouling properties. Our simple strategy, which investigates the affinities between residues and monomers, can become a convenient approach to predicting protein antifouling properties of polymeric materials.
1. INTRODUCTION Suppression of protein adsorption on the surface of a biomaterial is one of the most crucial factors affecting its biocompatibility, because foreign body response is largely influenced by this adsorption which occurs within seconds of implantation.1,2 Polyzwitterions (PZs), such as carboxybetaine, phosphobetaine, and sulfobetaine polymers, are among the most widely studied types of biomaterial because of their excellent biocompatibility.3−6 To understand how the body responds to implanted biomaterials such as PZs, it is essential to gain an insight into the behavior of proteins in the vicinity of the biomaterial surface. Ishihara et al.3 suggested that the drastically reduced protein adsorption of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer is closely related to the water structure in the hydrated polymers, which possess a high free water fraction. Feng et al.4 also suggested that the water layer near the MPC surface does not change the native conformation of adsorbed proteins, in contrast to the surfaces of conventional copolymers. Kitano et al.5 concluded that a 1carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl)methanaminium inner salt (CMB) monomer does not disturb the hydrated structure of water molecules. These experimental studies have speculated that the structure of water in the vicinity of PZs influences prevention of protein adsorption. Molecular dynamics (MD) simulations have also provided detailed information in various fields,7−9 including protein adsorption phenomena.10−15 Chen et al.10 showed the strong antifouling properties of zwitterionic phosphorylcholine (PC) self-assembled monolayers (SAMs), suggesting that their strong resistance to fouling is because of their capacity for hydration via electrostatic interactions. He et al.11 further supported these ideas using MD, estimating the interactions between a protein © 2012 American Chemical Society
and PC SAMs in explicit water molecules. They indicated that water molecules in the vicinity of the SAM surface act to produce a strong repulsive force on the approaching protein, because of a tightly bound water layer on the surface. It seems that their speculations do not corroborate the experimental consensus,3−6 although the length scales which can be investigated in MD are certainly of higher resolution than those achievable with experimental methods. They focus on the influence of tightly bound water molecules, whereas the significance of free water molecules has been emphasized in the experimental reports. Consequently, protein adsorption behavior and dynamics of water molecules near PZ surfaces remain poorly understood at the microscopic level. To elucidate the correlation between antifouling properties and dynamics of the solvent, more detailed physicochemical investigations are essential. In particular, adsorption free energy profiles for proteins approaching the biomaterial surface are thermodynamically decisive factors for assessing the adsorption properties.16 Free energy profiles have been calculated using all−atom MD for the adsorption of linear peptides onto several types of functionalized SAM surfaces.14,15 In these studies, the calculated free energies were compared with experimental data from surface plasmon resonance. The studies are significant, particularly because various combinations of residues and functionalized surfaces were investigated to evaluate the transferability of empirical force fields. As for the PZ surfaces, we have not found previous studies on evaluation of free energy Received: Revised: Accepted: Published: 4458
December 14, 2011 February 3, 2012 February 16, 2012 February 16, 2012 dx.doi.org/10.1021/ie2029305 | Ind. Eng. Chem. Res. 2012, 51, 4458−4462
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Research Note
profiles for the adsorption of residues or peptides. We aim to conduct free energy calculations for PZ surfaces, although it is at present not possible to model realistic amorphous PZ materials within finite−size simulation cells, because the simulations would take too long to complete on current computers. We hypothesize that free energy calculations of protein adsorption phenomena on PZ surfaces can be feasible, by using much simpler molecular structures, consisting of only monomers, as illustrated in Figure 1. If such a simplified
amounts of protein that would be expected to adsorb on the surfaces. On the basis of this hypothesis, we have evaluated the affinities using free energy profiles. For reference, the profiles for the residues approaching a conventional polyethylene terephthalate (PET) monomer, whose chemical structural formula is also described in Figure 2, were evaluated. Here we have studied the affinities of the following pairs of residues and monomers in explicit water molecules: Gly−PET, Gly− CMB, Phe−PET, Phe−CMB, Asp−CMB, and Lys−CMB. Potential of mean force (PMF)17−21 was calculated to evaluate free energy profiles for the amino acid residues approaching the monomers. We calculated PMF from the summation of the difference in the Helmholtz free energy, ΔA, given by
ΔA =
∫a
b
⟨F(z)⟩ dz
where F(z) is the force acting on the molecules at the distance z between the centers of mass of a residue and a material monomer; a and b denote the distances at the initial and terminal points, respectively, and ⟨...⟩ is the ensemble average obtained from the MD. In our simulations, ⟨F(z)⟩ was computed by fixing the distance over a range of 0.1−1.2 nm. In this study, PMF was calculated based on simulations conducted for 1−10 ns in real time. The residues, monomers, and explicit water molecules were modeled with the all−atom CVFF force field.22 Nonbonded interactions were described with a 12-6 Lennard-Jones potential, which was truncated at 10.0 Å. Long-range electrostatics were treated with the particle mesh Ewald method.23 Each pair of residues and monomers was simulated in ca. 25 × 25 × 25 Å boxes, whose densities was set at 1.0 g/cm3, where a total of 512 water molecules were solvated. A periodic boundary condition was applied in all three directions. In our MD simulations, an NVT ensemble (number of atoms, volume, and total energy in the simulation box are kept constant) was applied. The temperature was set at 293 K by using a scaling method, and the initial velocity of each molecule was set corresponding to the Boltzmann distribution. The time steps for each MD step were 1.0 fs for PET and 0.5 fs for CMB, respectively. It was necessary to set a smaller value for CMB, because the time scales of conformational changes for a CMB monomer are smaller than those for a hydrated PET monomer. A schematic of a phenylalanine residue approaching a PET monomer in aqueous solution is illustrated in Figure 3.
Figure 1. Schematic of our simplified approach to assessing antifouling properties of polymeric biomaterials. For clarity, explicit water molecules are not shown.
approach is effective, a more combinatorial and efficient biomaterial design would be achievable, and new detailed insights into protein adsorption could be provided, leading to the significant contribution to biomedical engineering.
2. SIMULATION DETAILS In this study, free energy profiles were calculated by MD, particularly for four amino acid residues of different types approaching a carboxybetaine CMB monomer in aqueous solution,5 whose chemical structural formula is given in Figure 2: glycine (Gly), phenylalanine (Phe), asparaginic acid (Asp),
Figure 2. Chemical structural formulas. (Top) 1-Carboxy-N,Ndimethyl-N-(2′-methacryloyloxyethyl)methanaminium inner salt (CMB) monomer; (bottom) polyethylene terephthalate (PET) monomer.
and lysine (Lys). The 20 main amino−acid residues making up proteins can be generally categorized into four groups: hydrophilic (subdivided into acidic, basic, and neutral residues) and hydrophobic residues. It is noted that the charge states of amino acid residues should be considered, but here we deal with un-ionized probe residues as a first step. Our assumption is that the inhibition of protein adsorption onto PZs is achieved, when the approach of all four groups of amino acids to the surfaces is prohibited. The weaker the affinities between PZ monomers and the four types of residue are, the smaller are the
Figure 3. Schematic of a phenylalanine residue approaching a PET monomer. For clarity, explicit water molecules are not shown. 4459
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3. RESULTS AND DISCUSSION The calculated profiles for the Gly−PET and Gly−CMB pairs are illustrated in Figure 4 panels a and b, respectively. The
Figure 5. Free energy profiles for a phenylalanine residue approaching a monomer, (a) PET and (b) CMB, in explicit water molecules at 293 K. The individual profiles from three equal time intervals are also shown. Figure 4. Free energy profiles for a glycine residue approaching a monomer, (a) PET and (b) CMB, in explicit water molecules at 293 K. The individual profiles from three equal time intervals are also shown.
with solvent molecules, suggesting that they cannot easily separate from each other over a wider range of intermolecular distances. On the other hand, for Phe−CMB, the profile also becomes nearly flat in Figure 5b, as is the case with Gly−CMB, regardless of the simulation time evolution. This indicates that even hydrophobic residues do not readily remain near the CMB surface. We also calculated the PMFs for Asp and Lys. Figure 6 panels a and b show the free energy profiles for Asp−CMB and
horizontal axes represent the distance between the centers of mass of the two approaching molecules. To see if the fluctuations of the profiles varied with time, we have also plotted the individual profiles obtained by dividing the overall simulation into three equal time intervals. Figure 4 demonstrates a remarkable difference between the profiles obtained for PET and CMB. Figure 4a exhibits three usual features of PMF curves: a contact minimum, a desolvation barrier, and a solvent-separated minimum.21 They appear at a intermolecular distances of ca. 0.3, 0.5, and 0.8 nm, respectively. This tendency does not change with time over our simulations, although the profiles plotted every 2 ns fluctuate within a range of ca. 1 kJ/ mol. The characteristic barrier is certainly due to the contribution from the void between the residue and the monomer, where water molecules rarely exist, resulting in the double minimum shape of the profiles, as has been pointed out previously.24 Figure 4a suggests that Gly can approach PET without any anomalously high barriers and that it can remain in the vicinity of a contact minimum, meaning that Gly can adsorb onto PET. On the other hand, Figure 4b shows a nearly flat profile, indicating that although Gly can approach the surface of CMB, it can also readily separate from the CMB surface. Moreover, the profiles barely fluctuate over the simulation time. To evaluate the affinity between hydrophobic residues and surfaces, the profiles for Phe−PET and Phe−CMB in explicit water are shown in Figure 5. As a result, fluctuations on simulation times are insignificant, as compared with Figure 4a. In addition, the profile for Phe−PET is notably different from that for Gly−PET. As for Phe−PET, an energetic minimum is deeper and wider than that for Gly−PET. Moreover, no solvation barriers are observed, probably because the affinities between Phe and PET are so strong, as compared with those
Figure 6. Free energy profiles for (a) an asparaginic acid and (b) lysine residue approaching a CMB monomer, in explicit water molecules at 293 K. The individual profiles from three equal time intervals are also shown. 4460
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that this approach can be a convenient theoretical method for the initial screening of optimal fouling-free materials. As a next target, substitution of head and/or tail groups in a repeat unit is intriguing, because it enables us to correlate antifouling properties with others, such as wettability. Application of our approach to nonionic polymers, such as poly(ethylene oxide) and poly(ethylene glycol), is also significant to compare the thermodynamic differences between zwitterionic and nonionic materials. Various applications of polyzwitterions have been already conducted,31,32 and now we are also starting a collaborative research for the development of antifouling membranes, using the CMB copolymer as a facile surface modifier.33
Lys−CMB, respectively. As a result, almost no fluctuations are observed in both figures. Moreover, it should be noted that the both profiles for Asp−CMB and Lys−CMB show almost completely flat curves. For the profiles for Gly−PET, as can be seen in Figure 4a, sufficient sampling cannot be taken from our conventional MD simulations, and fluctuations are observed over the elapsed time, whereas more data sampling for Phe−PET in Figure 5a can be taken. This is probably because time scales of conformational changes of the hydrated PET monomers in the vicinity of more hydrophilic glycine residues are larger than those which can be computed by conventional MD. In addition, more detailed investigations are needed to discuss the accuracy of the absolute values of energetic minima obtained from our simulations. To confirm them, as a next step, it is necessary to take sufficient sampling, by considering several approaches, such as high-temperature configuration-space exploration and replica exchange method.15,25−29 On the other hand, all the four types of residue approaching a CMB monomer show remarkably almost flat PMF profiles, regardless of their hydrophobicities or acidities. It confirms that amino acid residues in the vicinity of CMB can readily separate from the surface without particularly high energetic barriers. Moreover, we suppose that sufficient samplings are achieved because time scales of conformational changes of CMB monomers are smaller enough to be simulated by MD. It may be pointed out that the profiles can depend on the initial conformations of residues, PZs, and water molecules. However, it should be noted that these nearly flat profiles are achieved for all the four types of residue. Considering that these residues are naturally different in molecular and steric structures, these four flat profiles certainly offer indirect evidence that the shape of profiles is independent of the initial conditions of the systems. We consider that the agreement with the experimental data,30 which compares the amounts of adsorbed proteins onto CMB and PET, reinforces the suitability of CVFF, although it is not always appropriate to adopt a specific force field without allowing for its transferability for a given type of molecular system.15 Therefore, we can conclude that all the four types of residue do not adhere tenaciously on the CMB surface. Furthermore, considering that the profiles for a hydrophobic phenylalanine residue show the most marked difference between PET and CMB, as shown in Figure 5, prevention of the adsorption of hydrophobic residues can be an indispensable prerequisite for the development of antifouling biomaterials, which can inhibit protein adsorption. It turns out that a hydrophobic residue can be used as a convenient probe molecule to predict the antifouling properties of polymeric biomaterial surfaces, by conducting free energy calculations. It should be noted that the comprehensive phenomena of protein adsorption on the polymeric surface cannot be simulated by our simplified approach. Our aim is focused on the achievement of efficient and theoretical screening of fouling-free bioengineering materials. In considering the overall interactions between a protein molecule and a polymeric surface, the affinities between a residue and a repeat unit must be summed. When each free energy profile for a residue approaching a repeat unit is nearly flat, the total sum of these affinities certainly remains almost unchanged. Therefore, we consider that the antifouling properties can be efficiently screened by seeing whether the profiles for amino acid residues approaching a repeat unit are nearly flat. Our results suggest
4. CONCLUSION To provide physicochemical insights into protein adsorption phenomena on biocompatible interfaces, we show that free energy profiles, from MD simulations in explicit water, for hydrophilic and hydrophobic amino acids approaching a carboxybetaine CMB monomer. The predicted profiles for CMB have almost no energetically stable points, regardless of the type of amino-acid residue. On the other hand, the profiles for PET show some energetically remarkable minima, particularly for the hydrophobic residue. These results agree with recent experimental reports of differences in the amounts of protein adsorbed on these polymers. Our simplified approach suggests that hydrophobic residues can play a convenient role as a probe molecule for assessing protein antifouling properties of polymeric biomaterials. Furthermore, our strategy certainly contributes to the development of antifouling polymers in industrial situations, such as membrane separation processes.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.: +81-22-7957233. Fax: +81-22-795-7235. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work has been supported by the research project, “Application of Integrated Intelligent Satellite System (IISS) to Construct Regional Water Resources Utilization System,” sponsored by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST).
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REFERENCES
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