Effect of End-Grafted Polymer Conformation on Protein Resistance

Subscriber access provided by READING UNIV

Article

Effect of end-grafted polymers conformation on protein resistance Yuanyuan Han, Jiani Ma, Yu Hu, Jing Jin, and Wei Jiang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03930 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Effect of end-grafted polymers conformation on protein resistance Yuanyuan Han, Jiani Ma, Yu Hu, Jing Jin* and Wei Jiang* State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China Abstract: Monte Carlo simulation combined with experimental method was used to investigate the effect of conformational structure of polymer brushes on their protein resistance. The end-grafted polymers with two conformational structures, i.e., linear and looped, were considered. Protein adsorption behaviors on the surfaces grafted with either linear or looped polymers were investigated. Different chain lengths and grafting numbers of end-grafted polymers were employed in this simulation. The simulation results indicated that for long polymer brushes, the conformational change from linear to looped generally improved their protein resistant property for all the grafting numbers investigated here, and a remarkable improvement of protein resistance can be achieved at a certain grafting number. Moreover, the simulations revealed that the smoothness of surface and the formation of a dense impenetrable layer are the two significant characteristics of the looped polymer brush in resisting protein adsorption. Meanwhile, experiment results also showed that for a given chain length and grafting number, the protein resistant property of the looped polymer brush was superior to that of the surface grafted with linear polymers, which is quite consistent with the simulation results. These results further elucidated the difference in protein resistant property between the linear and looped polymer brushes, which provided useful information for preparing excellent antifouling materials in future experiments.

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Key words: looped polymer, protein adsorption, antifouling, Monte Carlo simulation

AUTHOR INFORMATION Corresponding authors *Wei Jiang. E-mail: [email protected] *Jing Jin. E-mail: [email protected]


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1. INTRODUCTION Due to the devastating effects of protein adsorption on surfaces that include surrounding tissue irritation, bacterial adhesion, hull surface roughness increase, and coating deterioration acceleration,1–5 nonspecific protein adsorption is unwanted in many applications, such as medical implants, marine and industrial equipment. Various methods for preparing antifouling coatings have been developed to prevent protein adsorption.6–8 The most common strategy is to immobilize well-solvated polymers, such as poly(ethylene glycol) (PEG), on substrates by covalent coupling or chemical deposition. The immobilized hydrophilic polymer chains can shield the hydrophobic substrate and generate a kinetic barrier to prevent protein adsorption.9 Previous investigations suggested that only surfaces grafted with long PEG chains can resist protein adsorption.10,11 However, Prime and Whitesides12 prepared protein-resistant surfaces based on self-assembled monolayers of alkanethiolates with oligo(ethylene oxide) groups and suggested that short chains can resist protein adsorption as well. Protein adsorption is mainly determined by surface coverage, and not solely by chain length.4,13 Polymers with complex architectures,14–17 such as hyperbranched, dendritic, and comb-like, have been synthesized to prepare protein-resistant surfaces because they can provide larger surface coverage than linear analogs.18,19 However, the synthesis of polymers with well-defined complex architectures is relatively difficult and costly, thereby limiting its practical applications. Polymer loop (i.e., polymer with both ends attached to the substrate to form a loop conformation) is an alternative to linear polymers or polymers with complex architectures. The substrate covered with looped polymer brushes (such as lubricin loops) are


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 29

generally considered to have good lubricating abilities.20–22 Recently, Kang et al.23 synthesized a triblock copolymer, which has two catechol-functionalized end blocks and a neutral poly(ethylene oxide) midblock. The triblock copolymer could adsorb onto silicon-oxide-based surface and adopt loop conformation. Their experimental results demonstrated that such polymer loop surface combines excellent lubricating and antifouling properties. Most recently, Morgese et al.24 prepared a cyclic polymer brush based on poly-2-ethyl-2-oxazoline on titanium oxide substrates. The cyclic polymer was synthesized and formed highly dense brushes due to the unique topology and had improved antifouling properties with respect to linear analogs. A series of molecular dynamics simulations on the surfaces grafted with polymer loops were performed by Pei et al.25 They found that the physical properties of polymer loops, such as rigidity and polydispersity, affected the geometries of loop grafted surfaces, which were closely related to the surface wettability. The antifouling property of the surfaces grafted with polymer chains are closely related to their surface geometries, which can be tuned by changing the conformation and topology of grafted chains.25-27 Looped polymer brushes are promising candidates for preparing high-performance antifouling coatings.23 Simply

changing the

conformational structure of polymer brush from linear to looped can improve its protein resistant property. However, the protein resistant mechanism of looped polymer brush is still unrevealed. Meanwhile, it is unclear yet whether the protein resistant property of looped polymer brushes is always better than that of linear polymer brushes under all conditions. To address these issues, Monte Carlo


Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

simulations were employed in this study. On the other hand, fabricating looped polymer brush reported in the literature was also still involved a tedious processing. A relatively simple and more feasible method to prepare looped polymer brush based on chemical reactions in situ was presented in this study.

2. COMPUTATIONAL DETAILS 2.1. Lattice Model in Monte Carlo Simulation. The Lattice Monte Carlo (MC) method was used in this study. The volume of simulation box is V = LX × LY × LZ , where

LX = LY = 50 and LZ = 100 (the unit of the simulation box is one lattice spacing). Periodic boundary conditions were used in the X- and Y-directions, and an impenetrable substrate is introduced at Z = 0 . Each monomer (or solvent molecule) occupied one lattice site, and the excluded volume effect was employed for avoiding two or more monomers occupying one lattice site at the same time. The single-site bond fluctuation model28,29 was employed, and the permitted bond length is 1 and

2 . The conformation evolutions of grafted polymers and

proteins were all achieved through the exchange movement which was defined as follows: A monomer is randomly selected to exchange with one of its 18 nearest neighbors. If the neighbor is a solvent and the exchange does not violate the bond length restriction, and no bond crossing occurs, then exchange with the solvent is attempted. The acceptance or rejection of the attempted move is further governed by the Metropolis rule30: if the energy change ∆E is negative, the exchange is accepted. Otherwise, the exchange is accepted with a probability of p = exp[−∆E / (kBT )] , in which ∆E = ∑ ∆Nijε ij is the energy change caused


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

by the attempted move; ∆Nij is the number difference of the nearest neighbor pairs between different components before and after the movement, and ε ij is the interaction energy between different components and is in units of kBT ; kB is the Boltzmann constant, T is the temperature. 2.2. Construction of protein solution in MC Simulation. The hydrophobic (H)-polar (P) model31 was adopted to obtain the native structure of protein. The amino acids were classified into H and P beads neglecting their other chemical and structural details. The HP sequence of 67-mer protein designed by Yue et al.32 was adopted. Protein folding was mainly governed by the relative hydrophobic character of the amino acids33, therefore, an attractive interaction between two non-bonded nearest neighbor H monomers ( ε HH = −5 ) was introduced to induce protein folding and then obtained the native structure of protein. For evaluating the compact degree of protein, the H-H contact number ( NHH ) between non-bonded nearest neighbor H monomers was calculated. A compactly folded protein with NHH = 75 was selected as the starting state. The selected protein was rotated 90, 180, and 270 degrees around the X-, Y-, and Z-axis, respectively, to obtain proteins with different orientations. A total of 60 proteins with different orientations were randomly placed into the simulation box in which the bottom was grafted with polymers, and the unoccupied lattice sites were set as solvents. The substrate ( Z = 0 plane) is hydrophobic, which attracts H monomers ( ε H-Substrate = −1). Except for the substrate-H interaction ( ε H-Substrate ), the H-H interaction ( ε HH ), and the polymer-solvent interaction ( ε PS ), all other interactions were set to zero. Proteins can adsorb onto the substrate


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

and denatured. A value of NHH ( NHH = 68 ) was selected as the criterion for evaluating whether the protein was denatured, i.e., the protein with NHH < 68 was considered denatured. To prevent protein aggregation in solution, the H-H interactions between different proteins possessing native structures were neglected. The movement of protein as a whole was introduced to improve the computational efficiency, and detailed information of the mode of this movement can be found in our previous work34. 2.3. Construction of polymer brushes with different conformations in MC Simulation. Polymer chains (the chain length is leP = 50 if not specified) were randomly placed into the simulation box and the unoccupied lattice sites were assumed to be solvents, thereby forming a homogenous polymer solution (Figure 1a). The polymer chain concentration in the solution is C P = 10% , and the interaction energy between polymers and solvents is ε PS = −0.1 if not specified for simulating a good solvent environment. The ends of the polymers can be attached onto the substrate via covalent bonding. The linear polymer brushes can be formed in the case that only one end of the polymer chains can be covalently bonded with the substrate, while the looped polymer brushes can be formed in the case that both ends of the polymer chains can be covalently bonded with the substrate. As simulation time ( t ) increased, the polymer chains were randomly attached onto the substrate. The adsorption process was terminated when a certain number ( nP ) of polymer loops or linear polymers were grafted on the substrate. After the unbonded polymer chains were removed, the formation of looped polymer brushes (or linear polymer brushes) was done. The looped polymer brush constructed


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

by 100 polymer loops was shown in Figure 1b as an example. Note that the grafting amount of this looped polymer brush can be calculated as N P = nP × leP = 100 × 50 = 5000 . For each polymer loop in Figure 1b, the distance between the two grafting points was calculated. Figure 1c shows the distribution of the distances. The distribution of the distances is wide, thereby indicating that the immobilization process in MC simulation is a random process, which is quite consistent with the real experimental process.

Figure 1. (a) Snapshots of the morphologies of the homogenous polymer solution in the simulation. The polymer chain length is leP = 50 , and the hydrophilicity of polymers is ε PS = − 0.1 ; (b) The looped polymer brush constructed by 100 polymer loops obtained from

the polymer solution shown in (a); (c) distribution of the distances between the two grafting points of the polymer loops in the brush shown in (b). For the purpose of providing more useful information about the preparation of looped polymer brush, the factors affecting the adsorption rate of polymer chains onto substrate, such as the initial concentration ( C P ) and the hydrophilicity ( ε PS ) of polymers in solution, were also investigated. Figure 2 shows the variations of the number of polymer loops formed in


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

each system with simulation time ( t ) under the conditions with different C P and ε PS . The simulation results indicate that the adsorption rate is closely depended on the initial concentration and the hydrophilicity of polymers in solution. The adsorption rate of polymers onto the substrate increases as C P increases (Figure 2a), whereas it decreases as the hydrophilicity increases (Figure 2b). These simulation results suggest that if we want to prepare a highly dense looped polymer brush within a short time in experiment, a high initial concentration and weak polymer hydrophilicity in the solution are required.

Figure 2. (a) Variations of the number of polymer loops with simulation time ( t ), when the initial polymer concentrations in the solution ( C P ) are different, the hydrophilicity of polymers in the solution is ε PS = − 0.1 . (b) Variations of the number of polymer loops with t when the hydrophilicity of polymers in the solution ( ε PS ) are different, the initial polymer concentrations in the solution is C P = 10% .

3. EXPERIMENTAL SECTION In this study, the PEG with dual thiol (SH) groups (SH-PEG-SH) was used to prepare looped polymer brushes. The SH-PEG-SH molecules were deposited on Au substrates via grafting to


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

technique. A simple thiol-Au reaction caused SH-PEG-SH to be immobilized on the substrate. Due to the high affinity of thiols for various metal surfaces,35-39 the SH-PEG-SH molecules technically can adopt loop conformations on multiple metal substrates. Furthermore, a linear polymer brush with similar grafting amount (constructed by SH-PEG) was prepared for comparison. The adsorption behaviors of Bovine serum albumin (BSA) on the Au-substrates grafted by either SH-PEG-SH or SH-PEG were investigated. The immobilization of SH-PEG or SH-PEG-SH onto Au-substrates and the subsequent BSA adsorption were performed on a quartz crystal microbalance with dissipation (QCM-D) E4 instrument (Q-Sense, Sweden) with four parallel chambers in real time. The changes in frequency ( ∆f ) and dissipation ( ∆D ) of the sensor were measured simultaneously. The third overtone ( n = 3 ) on 5 MHz gold crystal of ∆f and ∆D were presented. The PEG density and BSA mass were modeled using the Voigt model. The temperature was controlled by the instrument at 20.0 ± 0.1 °C. The clean protocol of the gold sensor was performed as previously reported.40 The SH-PEG (PLS-604, M W = 5000 , Creative PEGWorks, USA) or SH-PEG-SH (PLS-614, M W = 5000 , Creative PEGWorks, USA) solution with 1 mg/mL in water was injected into the chamber for 30 min at 30 µL/min and was rinsed with water. Thereafter, phosphate-buffered saline (PBS, 0.01 M phosphate buffer, pH 7.4) was added until the baseline was stable. BSA (20 mg/mL) in PBS solution was injected into the chamber for 30min at 30µL/min and rinsed with PBS. Figure S1 in the Surpporting Information (SI) shows the frequency change of SH-PEG or SH-PEG-SH onto gold sensors. The calculated mass values of SH-PEG and SH-PEG-SH on the surface are 1.86 ± 0.01 and 2.10 ± 0.02 ng/mm2,


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

respectively. The immobilized density values of SH-PEG and SH-PEG-SH on the surface are

0.22 ± 0.01 and 0.24 ± 0.01 chains/nm2, respectively. Hence, SH-PEG and SH-PEG-SH have similar grafting amount values on the gold surfaces.

4. RESULTS AND DISCUSSION 4.1 Protein adsorption behaviors on looped and linear polymer brushes. In this subsection, comparisons between the protein adsorption behaviors on the looped and linear polymer brushes with equal chain length ( leP ) and equal number of grafted chains ( nP ) were performed for the purpose of illustrating the effect of the conformations of grafted chains on the protein resistant properties of polymer brushes. A series values of leP and nP were considered. The hydrophilic interaction between solvents and the grafted polymers was set as ε PS = −0.1 in the following simulations. A total of 60 proteins with their native structures were randomly placed into the simulation boxes, in which the bottom surfaces were grafted with either linear or looped polymer brushes as shown in Figure S2 in the SI. The protein-polymer brush systems were run for a sufficiently long simulation time ( t = 1.0 × 108 Monte Carlo steps (MCS)). Figure 3a shows the variations of protein adsorption amounts on looped and linear polymer brushes with nP in the case of short chain length, i.e., leP = 10 . As shown in Figure 3a, for both polymer brushes, the protein adsorbed amounts always decrease with an increase in nP . Noteworthy that for all the nP investigated here, the protein


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

adsorbed amounts on linear polymer brushes are quite similar to that on looped polymer brushes with equal nP . This simulation result indicates that when the grafting length is short, the protein resistant properties of polymer brushes do not closely depend on their conformational structures. However, when the chain length of grafted polymers is increased to leP = 30 , distinct gaps between protein adsorption amounts on these two brushes can be observed (Figure 3b), i.e., the protein adsorption amounts for linear polymer brushes are always larger than that for looped polymer brushes when their nP values are equal. It should be noticed that the gap between protein adsorption amounts for these two brushes first increases, then decreases as nP increases, and the largest gap is at nP = 120 . When the chain length is further increased to leP = 50 , similar phenomenon is observed (Figure 3c). It is found that the largest gap between protein adsorption amounts on these two brushes is observed at nP = 100 , which means that the largest gap shifts to smaller nP . The simulation results obtained in the case of longer polymer chains (Figure 3b-3c) indicate that changing the conformational structure of grafted polymers from linear to looped can obviously improve the protein resistant property of polymer brushes, and the remarkable improvement of protein resistance can be achieved at a certain value of nP . Take the case of leP = 30 as an example, the optimal grafting number is nP = 120 . At nP = 120 , the number of adsorbed proteins decreases from 48 to 4 via simply changing the conformational structure of polymer brush from linear to looped. And this optimal grafting number of grafted polymers decreases as the chain length of grafted polymer increases.


Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3. Simulation results of the variations of protein adsorption amounts with polymer grafting number ( nP ) for linear and looped polymer brushes. The chain lengths of the grafted


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

polymers are (a) leP = 10 , (b) leP = 30 , and (c) leP = 50 , respectively. Data are mean ± SD,

n=6.

4.2 Protein resistant mechanism of looped polymer brushes. In this subsection, the looped and linear polymer brushes with leP = 50 and nP = 100 were selected as model systems due to the large gap between protein adsorption amounts on these two brushes as shown in Figure 3c. Detailed comparisons between the protein adsorption processes on these two brushes, as well as the structural properties of these two brushes, were done for elucidating the protein resistant mechanism of looped polymer brush. Figure 4a shows the changes in protein adsorption amounts with simulation time. As shown in Figure 4a, the protein adsorption amount on the linear polymer brush increases rapidly as simulation time increases. However, the protein adsorption amount on the looped polymer brush almost remains at zero consistently. The final protein adsorption amounts at t = 1.0 × 108 MCS shown in Figure 4b clearly indicate the difference in protein resistant

property between the two brushes. Furthermore, the morphological snapshots at t = 1.0 × 108 MCS were also given. As shown in Figure 4b1-4b2, most of the proteins passed through the linear polymer brush and were adsorbed on the hydrophobic substrate (Figure 4b1), whereas none of the proteins were trapped in the looped polymer brush or adsorbed on the substrate (Figure 4b2). These simulation results illustrate that at this chain length and grafting number, the protein resistant property of the looped polymer brush is indeed superior to that of linear polymer brush.


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 4. (a) Simulation results of the changes of protein adsorption amounts on linear and looped polymer brushes with simulation time. (b) The final protein adsorption amounts, and (b1)-(b2) the final morphological snapshots at t = 1.0 × 108 MCS. The chain length and grafting number are leP = 50 and nP = 100 . The H, P monomers of proteins, the grafted polymers and their head groups are marked with dark blue, light blue, grey and black, respectively. Data are mean ± SD, n = 6 . For exploring the protein resistant mechanism of looped polymer brushes, the structural characteristics of the looped polymer brush shown in Figure 4b2 were investigated. Meanwhile, the structural characteristics of the linear polymer brush shown in Figure 4b1 were also investigated for the purpose of comparison. Figure 5a-b shows the morphologies of the looped and linear polymer brushes. It is found that the surface of the looped polymer brush is much smoother than that of the linear polymer brush. The height distributions of the polymer chains in each brush shown in Figure 5c-d further prove this result. As shown in Figure 5c-d, the narrow peak in the height distribution (Figure 5c) indicates that the looped polymer brush is flat and uniform. However, the wide peak in the height distribution (Figure 5d) indicates that the surface


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of linear polymer brush is rugged. Furthermore, the polymer density profiles ( ρ P ) along the perpendicular direction to the substrate (Z-direction) were calculated and shown in Figure 5e-f. A peak appears close to the substrate in both brushes. However, the peak value of the looped polymer brush ( ρ P = 0.196 ) is larger than that of the linear polymer brush ( ρ P = 0.125 ), and the width of the peak is much narrower in the looped polymer brush than that in the linear polymer brush. A narrow peak with a large peak value (Figure 5e) demonstrates a dense layer formation near the substrate in the looped polymer brush. Szleifer and co-workers41 have theorized that the polymer molecule density in the region close to the substrate is an important parameter in determining the protein resistance of PEG coating layers. Moreover, our previous simulations also indicated the importance of the formation of a dense layer34. Therefore, we suggest that the formation of a dense layer near the substrate in the looped polymer brush, as well as the smooth surface of the looped polymer brush, are two crucial structural characteristics for looped polymer brushes to resist proteins.


Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. Snapshots of the morphologies of the looped (a) and linear (b) polymer brushes with leP = 50 and nP = 100 ; the height distributions of polymer chains in the looped (c) and linear (d) polymer brushes; the polymer density profiles ( ρ P ) along the Z-direction of the looped (e) and linear (f) polymer brushes, respectively. For further elucidating the protein resistant mechanism of the looped polymer brush, the dynamic processes of protein adsorption on these two polymer brushes were examined carefully. Figure 6 shows the morphological snapshots of the protein adsorption processes at different simulation time for each brush. For the looped polymer brush, the morphological snapshots (Figure 6a1-a5) clearly show that no proteins were adsorbed and trapped in the looped polymer brush throughout the simulation. Obviously, this was attributed to the smooth surface of the looped polymer brush and the dense impenetrable layer formed in the region close to the substrate in the looped polymer brush. However, for the linear polymer brush, it is seen from Figure 6b1-b5 that if proteins were trapped into the brush, then these proteins could not get out of the constraint of the surrounding chains. As a result, these trapped proteins were adsorbed on the substrate. Furthermore, we also tracked the adsorption process for a single protein in this brush (Figure 7). As shown in Figure 7, once the protein moved into the polymer brush (Figure 7b), its movement was consistently confined within the brush (Figure 7c-7e), which highly increased the contact probability between the protein and substrate. Moreover, the top views of the system (Figure


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7a′-7f′) show many defects on the linear polymer brush surface and their sizes are comparable with the protein sizes. Hence, proteins can pass through those defects and get adsorbed on the substrate. All these results reveal that different from the looped polymer brush, the rough and defective surface of the linear polymer brush increases the contact probability between protein and substrate, rather than resists the proteins.

Figure 6. Morphological snapshots at different simulation time during protein adsorption process for each brush with leP = 50 and nP = 100 . (a1)-(a5) the looped polymer brush; (b1)-(b5) the linear polymer brush. For the purpose of clarity, only 10 proteins are drawn in each figure. The colour coding of the images is the same as that in Figure 4b.


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 7. Tracking the adsorption process of a single protein in the linear polymer brush with leP = 50

and nP = 100 . Simulation time: (a) 1.490 × 10 4 MCS, (b)

1.491 × 10 4 MCS, (c) 1.492 × 10 4 MCS, (d) 1.498 × 10 4 MCS, (e) 1.499 × 10 4 MCS, (f) 1.500 × 10 4 MCS. (a’)-(f’) are the top views of (a)-(f). Note that the white parts in

(a’)-(f’) are the exposed hydrophobic substrates. The colour coding of the images is the same as that in Figure 4b.

4.3 Experimental results of protein adsorption behaviors on looped and linear polymer brushes. In this subsection, the adsorption of BSA on the looped and linear PEG brushes with equal chain length ( M W = 5000 ) and similar grafting amount ( 1.86 ± 0.01 ng/mm2 in the case of SH-PEG and 2.10 ± 0.02 ng/mm2 in the case of SH-PEG-SH) were investigated experimentally. The BSA adsorptions were monitored through QCM-D. The BSA concentration is 20 mg/ml, which is high enough to magnify the difference of BSA-adsorbed masses on the two PEG brush surfaces. Figure 8a shows the BSA-adsorbed masses on the two surfaces before and after PBS rinsing. No distinguishing difference exists on the SH-PEG


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

surface before and after PBS rinsing; moreover, the final adsorption mass is 3.40 ± 0.04 ng/mm2 (Figure 8b). However, the adsorption mass on the SH-PEG-SH surface is almost zero after PBS rinsing, which is rather low under the high protein concentration (Figure 8b). This result is quite consistent with the simulation result shown in Figure 4, indicating that the SH-PEG-SH brush possess superior protein resistance compared to SH-PEG brush with similar grafting amount and chain length. Meanwhile, the immobilized process by thiol-Au chemistry to prepare looped polymer is simple and feasible.

Figure 8. (a) QCM-D traces showing the changes of BSA-adsorbed masses on the Au-substrates grafted by SH-PEG-SH ( M W = 5000 ) and SH-PEG ( M W = 5000 ), respectively; (b) the final BSA-adsorbed mass on these two substrates after PBS rinse. Data are mean ± SD, n = 3 .

According to the discussions in the above sections, the conformational change of grafted chains from linear to looped can increase the polymer density near the substrate and improve the surface smoothness of polymer brush, and therefore improve the protein resistant property of polymer brush. Considering the polymer density is closely


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

related to the number of grafting points, another comparison between the looped and linear polymer brush with equal grafting amount ( N P ), but different chain length ( leP ) was done as well. The looped polymers are twice as long as the linear polymers, while the number of looped grafted chains is a half of that of linear grafted chains. Under this condition, the two kinds of brushes have equal number of grafting points, and then almost have the same polymer density along the direction perpendicular to the substrate (Figure S3 in the SI). Both the simulation (Figure S4 in the SI) and experimental results (Figure S5 in the SI) indicate that the looped polymer brush still has better protein resistant property than the linear ones have. Detailed information and discussions were given in the SI.

5. Conclusions Monte Carlo simulation combined with experimental method was used to investigate the effect of conformational structure of end-grafted polymers on protein resistance. The polymers with linear and looped conformations were employed. Protein adsorption behaviors on the surfaces grafted with either linear or looped polymers were investigated. Different chain lengths and grafting numbers were employed in the simulation. For long polymer chains, the conformational change from linear to looped generally improved the protein resistant property of polymer brush for all the grafting numbers investigated here, and a remarkable improvement of protein resistance can be achieved at a certain grafting amount. Both the simulation and the experiment results showed that the protein resistant property of the looped


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

polymer surface was superior to that of linear polymer surface, for a given chain length and grafting amount. The preparation of looped polymer brushes experimentally was based on the chemical reactions in situ between thiol and metal substrate by self-assembled monolayer. This technique is quite simple and feasible to be used in practical applications. In addition, the smoothness of looped polymer surface and the formation of impenetrable layer are two crucial characteristics to proteins resistance. These results further elucidated the different protein resistance between the surfaces with linear and looped polymers, which can provide useful information for preparing excellent antifouling materials in future experiments.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China for General Program (21674115, 51673196), major Program (21434007), the Natural Science Foundation of Jilin Province (20160520139JH), and the National Key Research and Development Program of China (2017YFC1104800). The resource provided by Computing Center of Jilin Province is gratefully acknowledged.


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

References (1) Anderson, J. M. Biological Responses to Materials. Ann. Rev. Mater. Res. 2001, 31, 81-110. (2) Deutsch, S.; Tarbell, J. M.; Manning, K. B.; Rosenberg, G.; Fontaine, A. A. Experimental Fluid Mechanics of Pulsatile Artificial Blood Pumps. Annu. Rev. Fluid Mech. 2006, 38, 65-86. (3) Xu, K.; Ouberai, M. M.; Welland, M. E. A Comprehensive Study of Lysozyme Adsorption Using Dual Polarization Interferometry and Quartz Crystal Microbalance with Dissipation. Biomaterials 2013, 34, 1461-1470. (4) Banerjee, I.; Pangule, R. C.; Kane, R. S. Antifouling Coatings: Recent Developments in the Design of Surfaces that Prevent Fouling by Proteins, Bacteria, and Marine Organisms. Adv. Mater. 2011, 23, 690-718. (5) Yang, W. J.; Neoh, K. G.; Kang, E. T.; Teo, S. L. M.; Rittschof, D. Polymer Brush Coatings for Combating Marine Biofouling. Prog. Polym. Sci. 2014, 39, 1017-1042. (6) Riepl, M.; Enander, K.; Liedberg, B.; Schaferling, M.; Kruschina, M.; Ortigao, F. Functionalized Surfaces of Mixed Alkanethiols on Gold as A Platform for Oligonucleotide Microarrays. Langmuir 2002, 18, 7016-7023. (7) Lasseter, T. L.; Clare, B. H.; Abbott, N. L.; Hamers, R. J. Covalently Modified Silicon and Diamond Surfaces: Resistance to Nonspecific Protein Adsorption and Optimization for Biosensing. J. Am. Chem. Soc. 2004, 126, 10220-10221.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8) Kingshott, P.; Thissen, H.; Griesser, H. J. Effects of Cloud-Point Grafting, Chain Length, and Density of PEG Layers on Competitive Adsorption of Ocular Proteins. Biomaterials 2002, 23, 2043-2056. (9) Leckband, D.; Sheth, S.; Halperin, A. Grafted Poly(Ethylene Oxide) Brushes as Nonfouling Surface Coatings. J. Biomater. Sci., Polym. Ed. 1999, 10, 1125-1147. (10)Desai, N. P.; Hubbell, J. A. Biological Responses to Polyethylene Oxide Modified Polyethylene Terephthalate Surfaces. J. Biomed. Mater. Res. 1991, 25, 829-843. (11)Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, A. S. Protein Adsorption to Poly(Ethylene Oxide) Surfaces. J. Biomed. Mater. Res. 1991, 25, 1547-1562. (12)Prime, K. L.; Whitesides, G. M. Self-Assembled Organic Monolayers - Model Systems for Studying Adsorption of Proteins at Surfaces. Science 1991, 252, 1164-1167. (13)Jin, J.; Jiang, W.; Yin, J. H.; Ji, X. L.; Stagnaro, P. Plasma Proteins Adsorption Mechanism on Polyethylene-Grafted Poly(Ethylene Glycol) Surface by Quartz Crystal Microbalance with Dissipation. Langmuir 2013, 29, 6624-6633. (14)Zhou, Y. F.; Huang, W.; Liu, J. Y.; Zhu, X. Y.; Yan, D. Y. Self-Assembly of Hyperbranched Polymers and Its Biomedical Applications. Adv. Mater. 2010, 22, 4567-4590. (15)Wyszogrodzka, M.; Haag, R. Synthesis and Characterization of Glycerol Dendrons, Self-Assembled Monolayers on Gold: A Detailed Study of Their Protein Resistance. Biomacromolecules 2009, 10, 1043-1054.


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(16)Wyszogrodzka, M.; Haag, R. Study of Single Protein Adsorption onto Monoamino Oligoglycerol Derivatives: A Structure-Activity Relationship. Langmuir 2009, 25, 5703-5712. (17)Feng, Y.; Zhao, H.; Behl, M.; Lendlein, A.; Guo, J.; Yang, D. Grafting of Poly(Ethylene Glycol) Monoacrylates on Polycarbonateurethane by UV Initiated Polymerization for Improving Hemocompatibility. J. Mater. Sci.-Mater. Med. 2012, 24, 61-70. (18)Wei, Q.; Becherer, T.; Angioletti-Uberti, S.; Dzubiella, J.; Wischke, C.; Neffe, A. T.; Lendlein, A.; Ballauff, M.; Haag, R. Protein Interactions with Polymer Coatings and Biomaterials. Angew. Chem. Int. Ed. 2014, 53, 8004-8031. (19)Xu, X. F.; Cao, D. P. Density Functional Theory for Adsorption of Colloids on the Polymer-Tethered Surfaces: Effect of Polymer Chain Architecture. J. Chem. Phys. 2009, 130, 365. (20)Zappone, B.; Ruths, M.; Greene, G. W.; Jay, G. D.; Israelachvili, J. N. Adsorption, Lubrication, and Wear of Lubricin on Model Surfaces: Polymer Brush-Like Behavior of A Glycoprotein. Biophys. J. 2007, 92, 1693-1708. (21)Greene, G. W.; Martin, L. L.; Tabor, R. F.; Michalczyk, A.; Ackland, L. M.; Horn, R. Lubricin: A Versatile, Biological Anti-Adhesive with Properties Comparable to Polyethylene Glycol. Biomaterials 2015, 53, 127-136.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(22)Zappone, B.; Greene, G. W.; Oroudjev, E.; Jay, G. D.; Israelachvili, J. N. Molecular Aspects of Boundary Lubrication by Human Lubricin: Effect of Disulfide Bonds and Enzymatic Digestion. Langmuir 2008, 24, 1495-1508. (23)Kang, T.; Banquy, X.; Heo, J.; Lim, C.; Lynd, N. A.; Lundberg, P.; Oh, D. X.; Lee, H.-K.; Hong, Y.-K.; Hwang, D. S.; Waite, J. H.; Israelachvili, J. N.; Hawker, C. J. Mussel-Inspired Anchoring of Polymer Loops that Provide Superior Surface Lubrication and Antifouling Properties. Acs Nano 2016, 10, 930-937. (24)Morgese, G.; Trachsel, L.; Romio, M.; Divandari, M.; Ramakrishna, S. N.; Benetti, E. M. Topological Polymer Chemistry Enters Surface Science: Linear versus Cyclic Polymer Brushes. Angew. Chem. Int. Ed. 2016, 55, 15583-15588. (25)Pei, H.-W.; Liu X.-L.; Liu H.; Zhu, Y.-L.; Lu Z.-Y. Enhancement of Surface Nonwettability by Grafting Loops. Phys. Chem. Chem. Phys. 2017, 19, 4710-4718. (26)Pei, H.-W.; Liu H.; Zhu, Y.-L.; Lu Z.-Y. Understanding the Wettability of a Hairy Surface: Effect of Hair Rigidity and Topology. Phys. Chem. Chem. Phys. 2016, 18, 18767-18775. (27)Xue, Y.-H.; Liu, H.; Lu, Z.-Y.; Liang, X.-Z. Layered Structure in Compatible Binary Polymer Brushes with High Graft Density: A Computer Simulation Study. J. Chem. Phys. 2010, 132, 044903. (28)Larson, R. G. Self-Assembly of Surfactant Liquid-Crystalline Phases by Monte-Carlo Simulation. J. Chem. Phys. 1989, 91, 2479-2488.


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(29)Larson, R. G. Monte Carlo Simulation of Microstructural Transitions in Surfactant Systems. J. Chem. Phys. 1992, 96, 7904-7918. (30)Metropolis, N.; Rosenbluth, A. W.; Rosenbluth, M. N.; Teller, A. H.; Teller, E. Equation of State Calculations by Fast Computing Machines. J. Chem. Phys. 1953, 21, 1087-1092. (31)Lau, K. F.; Dill, K. A. A Lattice Statistical Mechanics Model of the Conformational and Sequence Spaces of Proteins. Macromolecules, 1989, 22, 3986-3997. (32)Yue, K.; Dill, K. A. Forces of Tertiary Structural Organization in Globular Proteins. Proc. Nati. Acad. Sci. USA, 1995, 92, 146-150. (33)Anand, G.; Sharma, S.; Dutta, A. K.; Kumar, S. K.; Belfort, G. Conformational Transitions of Adsorbed Proteins on Surfaces of Varying Polarity Langmuir, 2010, 26, 10803-10811. (34)Han, Y. Y.; Jin, J.; Cui, J.; Jiang, W. Effect of Hydrophilicity of End-Grafted Polymers on Protein Adsorption Behavior: A Monte Carlo Study. Colloids Surf., B 2016, 142, 38-45. (35)Poirier, G. E.; Pylant, E. D. The Self-Assembly Mechanism of Alkanethiols on Au(111). Science 1996, 272, 1145-1148. (36)Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y. T.; Parikh, A. N.; Nuzzo, R. G. Comparison of the Structures and Wetting Properties of Self-Assembled Monolayers of Normal-Alkanethiols on the Coinage Metal-Surfaces, Cu, Ag, Au. J. Am. Chem. Soc. 1991, 113, 7152-7167.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(37)Bain, C. D.; Evall, J.; Whitesides, G. M. Formation of Monolayers by the Coadsorption of Thiols on Gold: Variation in the Head Group, Tail Group, and Solvent. J. Am. Chem. Soc. 1989, 111, 7155-7164. (38)Laibinis, P. E.; Whitesides, G. M. .omega.-Terminated Alkanethiolate Monolayers on Surfaces of Copper, Silver, and Gold have Similar Wettabilities. J. Am. Chem. Soc. 1992, 114, 1990-1995. (39)Love, J. C.; Wolfe, D. B.; Haasch, R.; Chabinyc, M. L.; Paul, K. E.; Whitesides, G. M.; Nuzzo, R. G. Formation and Structure of Self-Assembled Monolayers of Alkanethiolates on Palladium. J. Am. Chem. Soc. 2003, 125, 2597-2609. (40)Hu, Y.; Jin, J.; Liang, H.; Ji, X.; Yin, J.; Jiang, W. pH Dependence of Adsorbed Fibrinogen Conformation and Its Effect on Platelet Adhesion. Langmuir 2016, 32, 4086-4094. (41)Szleifer, I. Protein Adsorption on Surfaces with Grafted Polymers: A Theoretical Approach. Biophys. J. 1997, 72, 595-612.


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table of Content Graphic


Effect of End-Grafted Polymer Conformation on Protein Resistance

Recommend Documents