Surface Interactions by Surface Chemical

Downloaded by NORTH CAROLINA STATE UNIV on December 14, 2012 | http://pubs.acs.org Publication Date (Web): December 12, 2012 | doi: 10.1021/bk-2012-1120.ch013

Chapter 13

Regulation of Protein/Surface Interactions by Surface Chemical Modification and Topographic Design Dan Li and Hong Chen* Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China *E-mail: [email protected]

Protein/surface interaction has long been an important topic in life sciences and materials engineering. Regulating these interactions is not only of great theoretical interest but is also key in meeting the requirements of many biomedical applications. The surface properties, including chemistry and topography, of a solid material are dominant factors influencing protein adsorption. In this chapter, we summarize our contributions to understanding protein/surface interactions and regulating these interactions by surface chemical modification and topographic design. It is hoped that this report may be of help for readers who wish to improve the biological performance of their materials by surface engineering.

Protein/surface interactions have been recognized as critical in a number of fields including blood-contact materials, biological separations and biosensors (1–4). Different applications require different interactions. For example, if used as the platform for a protein sensor (5, 6), the surface should have high binding capacity for the target protein while resisting all nonspecific protein adsorption; if used as the substrate for protein purification or protein delivery (7), the surface should have the ability to regulate adsorption and desorption. Therefore, it is

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


crucial to be able to control protein/surface interactions to meet the requirements of a specific application. In addition, protein adsorption is believed to be the first significant event when a material comes into contact with a biological environment, and the events which follow, including cell/surface interactions, are largely dependent on the properties of the adsorbed layer (8). If the initial protein adsorption can be controlled, then one may be able to prevent undesirable events and promote favorable ones. Protein adsorption behavior may be affected by both surface chemistry and topography (9–12). The effect of surface chemistry is more obvious since proteins interact with materials through the chemical groups exposed on their respective surfaces. With the development of chemical modification techniques, surface chemical composition can be varied over a wide range, and the spatial distribution of the modifiers on the surface can be controlled. This allows extensive investigation of the correlations between surface chemistry and protein adsorption and the formulation of approaches for regulating surface/protein interactions by chemical modification. The effects of surface topography have recently received keen attention. Topographical properties such as roughness, curvature and geometry have been correlated to protein behavior. More importantly, the synergy of topography and chemical composition may endow a surface with unexpected properties which have not been considered in the context of regulating protein/surface interactions. Our research is mainly focused on understanding and regulating protein/surface interactions. Chemical modification strategies such as incorporating bioinert polymers to resist nonspecific protein adsorption (bioinert surface) and conjugating special ligands for specific protein recognition (bioactive surface) have been successfully applied to prepare biofunctional surfaces including antifouling surfaces, antithrombogenic surfaces and fibrinolytic surfaces. Recent work also includes stimuli-responsive surfaces, chiral surfaces and amphiphilic diblock copolymer modified surfaces. With respect to topographical surfaces, we have paid particular attention to the synergy of topography and surface chemistry, especially for stimuli-responsive polymers. It is our hope that by providing details of these examples from the work of our lab, further progress in the regulation of protein/surface interactions may be realized.

Regulation of Protein/Surface Interactions by Chemical Modification Proteins contain various chemical functions and may interact with surfaces by hydrogen bonding, hydrophobic interactions and electrostatic interactions. Therefore, surface chemistry is the main property influencing protein adsorption. Over the past several years we have made great efforts to develop methods for the regulation of protein/surface interactions by incorporating various molecules, including bioinert polymers, high affinity ligands, stimuli-responsive polymers, stereoisomeric molecules and amphiphilic diblock copolymers. 302 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Bioinert Surfaces Protein resistance or bioinertness is required in most biomedical applications of materials. Considerable effort has been made to find/develop bioinert surface modifiers. A number of hydrophilic polymers have been shown to be protein resistant such as poly(ethylene glycol) (PEG) (13), poly(N-vinylpyrrolidone) (PVP) (14), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) (15), etc. The key consideration is how to incorporate these bioinert polymers into material surfaces. For commercialization purposes, simple methods such as blending the modifier with the bulk material are preferred. For example, we prepared a protein resistant poly(dimethylsiloxane) (PDMS) surface by incorporating mono- or bis-triethoxysilyl PEG (TES-PEG-Me or TES-PEG-TES) into PDMS during curing. As shown in Figure 1A, PEG was covalently bound to the PDMS backbone through TES groups and the PEG chains migrated to the surface when exposed to water (16, 17). PEG incorporated by this method, namely chemical blending, is superior in durability to physically blended PEG, which is prone to leaching out in an aqueous environment.

Figure 1. Surface modification of PDMS with PEG: (A) blending, (B) surface grafting. Compared to bulk modification, covalent grafting of bioinert polymers to a substrate is an effective way to form a stable protein-resistant surface with minimal or no effect on the bulk mechanical properties. We have described an effective method to graft PEG chemically on PDMS surfaces in which abundant Si-H groups were created by acid-catalyzed equilibration in the presence of polymethylhydrosiloxane (MeHSiO)n. An allyl-terminated PEG was then grafted by platinum-catalyzed hydrosilylation (Figure 1B). Measurements using radiolabeling indicated that fibrinogen adsorption from buffer to the PEG-modified PDMS was reduced by more than 90% compared with controls (18). In follow on work, a chemically heterogeneous patterned surface was fabricated from the PEGylated PDMS surface by vacuum ultraviolet (VUV) 303 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


lithography. On exposure to a solution of fluorescein labeled fibrinogen, the surface exhibited distinct localization of protein on the UV exposed areas (19). Surface grafting of bioinert polymers, typically PEO, by the “grafting to” method may limit the grafting density due to steric restrictions and thus limit the protein resistance of the surface. Since surface initiated atom transfer radical polymerization (SI-ATRP) was first reported in 1997 (20), many researchers have turned to “grafting from” strategies, which can readily generate non-fouling surfaces by forming dense layers of bioinert polymer (21–23). Very recently, we prepared, for the first time, well controlled PVP-grafted silicon surfaces using SI-ATRP (Figure 2). Polymerization of the nonconjugated monomer NVP was achieved using CuCl/5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (Me6TATD) as catalyst. The surface with a PVP thickness of 15 nm reduced the level of adsorption of fibrinogen, human serum albumin (HSA), and lysozyme by 75, 93, and 81%, respectively (24). A similar ATRP system was also successfully used to prepare PVP-grafted PDMS surfaces (Figure 2). It was shown that the modified surfaces were strongly hydrophilic, and fibrinogen adsorption was reduced by 96% compared to unmodified PDMS (25). However, in spite of the extensive application of SI-ATRP, it is not suitable for the surface modification of some polymeric materials since the metal ions may penetrate into the bulk. Conventional radical polymerization therefore remains important. Recently, we introduced a facile method for radical polymerization on polyurethane (PU) surfaces. In this method, vinyl groups are incorporated into the PU surface; these groups are subsequently “copolymerized” with double-bond containing monomers. We showed that fibrinogen adsorption was reduced by 94% and 87% respectively on poly(N-isopropyl acrylamide) (PNIPAAm) and poly(2-hydroxyethyl methacrylate) (PHEMA) grafted PU surfaces prepared in this way (26). In addition, the molecular weight of the surface-grafted PNIPAAm could be controlled by adjusting the monomer concentration. Protein adsorption was found to decrease gradually with increasing molecular weight of PNIPAAm up to 7.9×104 where it reached a plateau (27).

Figure 2. SI-ATRP of N-vinylpyrrolidone on silicon and PDMS surfaces. To date, many surfaces have been reported to be protein resistant after chemical modification with different chemical compounds. Great efforts have been made to correlate the properties of single molecules or macromolecules with 304 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


protein-resistance. However, in recent years, increasing attention is being paid to the distribution of the functional groups or polymer chains. For example, Jiang and coworkers demonstrated, using mixed-charge SAMs and polymer coatings, that when the positive charges and negative charges are uniformly distributed at the molecular level, the surface has ultra-low fouling properties due to strong hydration (28, 29). In the case of polymer brush modified surfaces, structural parameters such as the length and surface density of the polymer chains should always be taken into account. For instance, on the PVP-grafted silicon surface mentioned above, fibrinogen adsorption decreased gradually with increasing PVP thickness and the critical thickness for maximum protein resistance was found to be ~13 nm (24). This behavior has been observed on most bioinert polymer grafted surfaces (15, 30). With respect to graft density, many studies have shown that protein adsorption decreases with increasing graft density of bioinert polymer (30–33), while other studies are more ambiguous. Brash et al. investigated protein adsorption on PEO grafted model surfaces with varying chain length and chain density. They found that protein resistance increased with chain density to a maximum at a critical density beyond which adsorption increased. It was suggested that, at high chain density, the surface-grafted PEO is dehydrated giving a surface that is no longer protein resistant (34). We also investigated the effect of PEG graft density and conformation on protein adsorption to a PEG-grafted surface. PU surfaces were modified using monobenzyloxy polyethylene glycol (BPEG), then PEG or monomethoxy poly(ethylene glycol) (MPEG) with various chain lengths were grafted as fillers. An increase of graft density after backfilling was demonstrated by chemical titration. However, fibrinogen and albumin adsorption increased on all surfaces after PEG or MPEG backfilling. It was suggested that BPEG was changed from a arched comformation to a extended one as a result of backfilling. This caused the hydrophobic benzyloxy end groups to be exposed to the exterior, which played a key role in the increase of protein adsorption (35). It should be noted in any case that protein-surface interactions are by no means determined by any single parameter; chemical composition, chain arrangement and nature of the adsorbed protein should be considered together. The emergence of new bioinert polymers and the development of surface modification techniques has given more choices for preparing protein-resistant surfaces. Several surfaces have exhibited protein resistance superior to that of PEG modified surfaces; the terms “superlow protein adsorption” and “zero protein adsorption” have been used to describe such surfaces. However, caution should be used in describing quantitative data on protein adsorption especially when measured using only one method. The most widely used methods for the quantification of protein adsorption, such as surface plasmon resonance (SPR), quartz crystal microbalance (QCM) and radiolabeling, are all based on different principles. To obtain adsorbed amounts, the raw data from these methods (the physical signal) must be transformed using calibration or modeling, leading in some cases to unreliable results. For labeling-based methods, the free “labels” cannot be completely cleared from the labeled protein solution and may result in an erroneous estimate of adsorbed amount. In this regard, one should first test the possibility of interactions between the free labels and the surface (36). 305 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Changes in the conformation of a protein on adsorption to a material surface, which may influence its biological activity, are always of concern, perhaps more so than the adsorbed amount. For example, Latour et al. concluded that platelet adhesion was strongly correlated with the degree of adsorption-induced unfolding of fibrinogen and not at all with the adsorbed amount (37). However, probing the conformation of an adsorbed protein remains a challenge due to the very low protein amounts that are below the detection limits of typical techniques, such as infrared spectroscopy, Raman spectroscopy and circular dichroism. Recently, conformational changes of lysozyme adsorbed on flat gold surfaces with different chemistries were measured in our laboratory using surface-enhanced Raman scattering (SERS). Instead of fabricating a metal substrate with special topographical features, colloidal silver nanoparticles were simply mixed with the protein adsorbed on gold surfaces to enhance the Raman signals. We found that the small amount of silver nanoparticles used (low laser power, short contact time 2-3 min) generated a measurable Raman signal for lysozyme while having negligible influence on the lysozyme structure. Data obtained by this technique revealed that the conformational change of lysozyme adsorbed on a hydrophilic PEG-modified surface was much smaller than that on a hydrophobic octadecane-modified surface, consistent with the high specific activity of lysozyme on the PEG surface (38). Bioactive Surfaces Promoting the binding of specific proteins on a surface is also important in many biomaterial applications such as protein purification, biosensing, anticoagulation etc. This requires the introduction of bioactive ligands with affinity for specific proteins to give so-called “bioactive” surfaces. An optimized bioactive surface should be able to bind the target protein selectively while preventing nonspecific protein adsorption. Although this may be achieved in some cases by surface grafting of bioactive ligands with high graft density, elimination of all nonspecific protein adsorption is not an easy task given that typical environments such as blood always contain surface-active components. The most commonly employed strategy is to use a bioinert polymer as a spacer to immobilize the bioactive ligand (2, 39). The inert spacer can resist non-specific protein adsorption and thus minimize unwanted biological responses. In addition, it provides a favorable microenvironment that is useful in maintaining the activity of the ligand(s) on the surface (40). Since PEG-grafted surfaces have been shown to be particularly effective for protein resistance, PEG has been extensively used as a spacer for preparing bioactive surfaces (41–44). We synthesized an asymmetric PEG with an allyl group and an N-succinimidyl carbonate (NSC) group at the respective chain termini and grafted it onto a Si-H functionalized PDMS surface by hydrosilylation. The NHS groups distal to the surface are available for covalent immobilization of amine-containing bioactive molecules (45). In the case of heparinized PDMS, high specificity for antithrombin with minimal fibrinogen adsorption was noted in plasma studies (46). Recently, PDMS and PU surfaces with fibrinolytic activity were prepared by immobilizing ε-lysine (ε-amino groups 306 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


free) on the surface through a PEG spacer. It was shown that these lysinated surfaces reduce nonspecific protein adsorption while binding plasminogen from plasma with a high degree of selectivity (Figure 3). When activated by t-PA the plasmin generated at the surface was shown to dissolve fibrin clots in vitro (47, 48). However, the PEG spacer, while effectively resisting nonspecific protein adsorption, is a deterrent to the specific binding of plasminogen due to the protein resistant properties of the PEG and the enhanced mobility of the chain-end conjugated lysines. Since the repellent effect of PEG is known to be dependent on chain length, we investigated the effect of PEG chain length on plasminogen binding to lysine at the PEG distal terminus. PEG-lysine surfaces were prepared using PEGs of different molecular weight (PEG300 and PEG1000) and their effects on the “balance” between nonspecific and specific protein binding were investigated (49). It was concluded that lysinated surfaces with PEG spacers of the relatively shorter length adsorbed plasminogen more rapidly than those with longer PEG, although the ultimate adsorbed quantities were the same. Correspondingly, the surface with the greater plasminogen binding capacity lysed fibrin clot more rapidly.

Figure 3. Fibrinogen adsorption from buffer and plasminogen adsorption from plasma on PU, PU-PEG and PU-PEG-Lys surfaces. The surface density of spacer achievable by the “grafting to” strategy is limited due to steric hindrance, and the density of chain-end conjugated bioactive ligands is correspondingly limited. Therefore a “grafting from” strategy may be more effective. Moreover, if the surface-grafted polymers have abundant side chains with active chain ends, this permits the generation of a high concentration of chemically active sites on the surface for binding bioactive molecules. Of the various polymers available to form such grafts, PHEMA and POEGMA have found the most widespread use (50–54). The pendant hydroxyl groups of PHEMA and POEGMA brushes can be activated by thionyl chloride, p-nitrophenyl chloroformate (NPC), disuccinimidyl carbonate (DSC), 1,1′-carbonyldiimidazole (CDI), succinic anhydride (SA) etc. Amine-containing bioactive molecules can 307 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


then be immobilized. Trmcic-Cvitas et al. recently compared the activation efficiency of 10 coupling agents when functionalizing a POEGMA surface with streptavidin and found that DSC activated surface afforded the highest streptavidin content (55). We have optimized our fibrinolytic PU surface using PHEMA as a spacer and DSC as a coupling agent for ε-lysine. The lysine density reached a value of 2.81 nmol cm-2 compared to 0.76 nmol cm-2 on a comparable PU-PEG-lysine surface. With increased plasminogen binding capacity, this surface showed more rapid clot lysis (20 min) in a standard in vitro assay than the corresponding PEG-lysine system (40 min) (56). To further improve the protein resistance of the spacer, a random copolymer of OEGMA and HEMA [poly(OEGMA-co-HEMA)] was grafted from PU surface as a spacer for ε-lysine immobilization. The quantity of fibrinogen adsorbed from buffer to the PU-poly(OEGMA-co-HEMA)-Lys surface was ~0.14 μg cm-2, much lower than to the PU-PHEMA-Lys surface (~0.28 μg cm-2) (57). Surface-grown spacers could also be random or diblock copolymer brushes containing both a bioinert segment and a chemically active segment for the immobilization of bioactive molecules (58–60). The main advantage of this strategy lies in the simplicity of the process since the coupling agent is introduced simultaneously with the grafting of the spacer. In addition, the contributions of the bioinert and bioactive moieties can be well defined by varying the conditions or feed composition during the copolymerization. Liu et al. developed a highly sensitive microarray immunoassay device by grafting a random copolymer of glycidyl methacrylate (GMA) and PEGMA on a glass chip via SI-ATRP and printing the probe proteins on the copolymer brush (61). Our lab developed a novel bioactive surface grafted with a diblock copolymer of POEGMA and poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA) by SI-ATRP: POEGMA was used as the protein-resistant component and PNHSMA for binding high densities of various bioactive molecules. This surface exhibited high binding capacity for three typical bioactive molecules, biotin, heparin and collagen, and high selectivity for their specific targets. For example, the biotinylated surface showed strong fluorescence intensity upon incubation with a solution of fluorescein labeled avidin, while there was essentially no detectable fluorescence signal to indicate the adsorption of fluorescein labeled HSA (Figure 4) (62).

Figure 4. Schematic illustration of POEGMA-b-PNHSMA modified silicon surface and its performance in avidin binding and HSA repelling after biotinated. (Reproduced with permission from ref. (62). Copyright 2010 Royal Society of Chemistry) 308 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Other Surfaces Smart surfaces which can undergo dramatic changes in physicochemical properties in response to specific environmental stimuli have attracted increasing attention in applications that require the regulation of protein adsorption and desorption, such as drug delivery and protein purification (63–66). PNIPAAm modified surfaces, exhibiting lower critical solution temperature (LCST) behavior, have been well studied for the regulation of surface wettability (67) and cell attachment/detachment (68, 69). It is expected that protein adsorption and desorption could also be regulated by temperature on PNIPAAm modified surfaces. In this regard, we investigated protein adsorption on PNIPAAm brushes of varying thickness prepared by SI-ATRP on silicon. As shown in Figure 5A, in the low thickness range (

Surface Interactions by Surface Chemical

Recommend Documents