- Email: [email protected]
Polymers and proteins: interactions at interfaces
Polymers and proteins: interactions at interfaces lgal Szleifer The ability of polymer molecules attached at one end to a surface to prevent or enhance protein adsorption has been studied experimentally
and theoretically.
Recent systematic
studies show that surface density seems to be the most important property of the tethered
layer that determines
ability to prevent protein adsorption.
Theoretical
its
studies
predict that the interactions
of the polymer layer with the
proteins and the adsorption
behavior do not depend
in the
same way on polymer molecular weight and on the type of interaction
delivery systems [6’]. Furthermore, tethered polymers can be used for the specific targeting of liposomes to cells by chemically inserting a specific binding reagent to the free ends of the tethered polymers [7,8*].
of the polymer with the surface.
Addresses Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393, USA; e-mail: [email protected] Current Opinion in Solid State & Materials Science 1997, 2~337-344 Electronic identifier: 1359-0286-002-00337 0 Current Chemistry Ltd ISSN 1359-0286 Abbreviations EO ethylene oxide PEG polyethylene glycol PEO polyethylene oxide PS polystyrene SCMF single-chain mean-field
Introduction Many biological processes depend upon the adsorption of proteins on a variety of surfaces. The understanding and control of the adsorption of protein molecules and their interactions with surfaces is of primary importance in the design of biomaterials. For example, the design of biocompatible materials requires (among other things) the reduction of adsorption of blood proteins to the surface of the material. It is known that the adsorption of blood proteins followed by platelet adhesion can result in surface induced thrombosis. The elimination or reduction of protein adsorption results in very low adhesion of platelets into the surface [l’]. Another example involves the elimination of lysozyme build-up on the surface of contact lenses [2,3]. In other cases, one is interested in increasing the adsorption of some types of proteins while reducing the adsorption of others [4,5]. During the last few years the use of polymer molecules as surface modifiers has been shown to be a very promising way of manipulating the interactions of surfaces with proteins [lo]. The proper tuning of these interactions will hopefully lead us to the rational design of a modified surface that will improve biocompatibility. For example, the inclusion of tethered polymers on the surfaces of liposomes results in their increased longevity in the blood stream, hence, these aggregates can be used as drug
In this review I will discuss the recent advances in our understanding of the ability of tethered polymer chains to prevent protein adsorption. The present experimental status of these systems will be discussed together with theoretical studies that enable us to calculate the interactions of proteins with tethered polymer layers as well as protein adsorption on modified surfaces. First, I will discuss some of the general properties of tethered polymer layers. The ability of tethered polymer layers to prevent protein adsorption is believed to be due to the steric repulsion associated with the perturbation that the protein molecules cause on the polymer layer [9] (see Fig. 1). In order to understand this repulsion it is appropriate to understand the structure of the polymer layers. The general picture in the good solvent regime (i.e. when the polymer is soluble in the solvent) is that [lo’], for very low surface coverages of polymers the tethered chains are isolated and their structure is not very different from that of the polymers in bulk. This is called the ‘mushroom’ regime. As the surface coverage increases the chains start to repel each other and they have to stretch out of the surface in order to avoid each other and thus reduce the polymer-polymer repulsions. The regime of highly stretched chains is generally called the ‘brush’ regime (11,121. Detailed studies performed by computer simulations [13] and molecular theories [10’,14], in particular the single-chain mean-field (SCMF) theory, show that the treatment of tethered polymers in terms of only the two above defined regimes is not appropriate for short to intermediate chain length polymers, that is from a few segments to a few hundred segments. Furthermore, the intermediate regime, that is, the ‘mushroom’ to ‘brush’ transition region, is the one that corresponds to most experimental studies. This has been shown to be the case in experimental observations of tethered polyethylene oxide (PEO) (also called polyethylene glycol [PEG]), which is the most widely used polymer in biological related applications of tethered polymer layers. This conclusion was reached by comparisons of experimental observations with the predictions of the SCMF theory on structural and thermodynamic properties [10*,14]. Figure 2 shows experimental and theoretical pressure-area isotherms of polystyrene (PS)-PEO diblock copolymers at the water-air interface. The PS block is at the air side of the interface, while the PEO chain is on the
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Figure 1
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Current Opinion
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Schematic representation of protein molecules in contact with a tethered polymer layer.
water side. The figure shows experimental observations as well as SCMF calculations. The agreement between the predicted and observed isotherms is very good. The picture that emerges from the calculations is that in the ‘mushroom’ regime the PEO chains are actually adsorbed co the interface. Thereby, forming a quasi two-dimensional adsorbed layer instead of a swollen ‘mushroom’. The attraction of the polymer monomers with the surface is due to the amphiphilic nature of PEO and this is a very important consideration in understanding and designing layers to protect hydrophobic surfaces (see below). The offset of the plateau in terms of the pressure corresponds to the area at which the adsorbed PEO chains start to strongly repel each other. Thus, the chains can release some of the pressure by stretching some of the ethylene oxide (EO) segments out of the interface into the water phase. The sharp increase in pressure at small area per molecule is due to the strong repulsions between the chains that result in a high degree of stretching of both the PEO and the PS blocks. Similar observations for pressure-area isotherms have been reported by Bijsterbosch et al. ]15]. The structural changes with surface coverage as well as the resulting (nontrivial) lateral interactions (lateral pressures) indicate that the interactions of PEO tethered layers with protein molecules are rather complex. Furthermore, in the cases of PEO on the surface of liposomes it has recently been shown that the EO monomers are not attracted to the liposome interface (161. Therefore, the structural properties and the interactions with the surrounding solution, will be different than in the case of hydrophobic surfaces. A thorough description of the properties of the tethered layers in the regime of molecuiar weights and surface coverages used experimentally requires a detailed molecular description [ 10*,14]. This is necessary to understand the interactions of the polymer layer with the protein molecules as well as to establish the amount
2000 0 1997
4000
6000
8000
a @‘J Current
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The lateral pressure (II), in mN/m, as a function of the area (a) per molecule, in As,for PS-PEO diblock copolymers at the water-air interface. The PS block has 30 styrene units and the PEO has 179 ethylene oxide units. The line with circles represents the experimental observations (P Sassereau, MC Fat&, personal ~mrnuni~t~n} while the other fine is derived from predictions of the SCMF theory (b&I Carignano, personal communication). The calculations assume that there is an attractive interaction of -k$ when an ethylene oxide unit is in contact with the interface.
of protein adsorbed on the surface and its variation with polymer surface coverage, molecular weight and type of surface. In the next. sections I will review the experimental observations of protein adsorption on a variety of surfaces with tethered polymer layers as well as recent theoretical predictions on protein adsorption and the interactions of the proteins with polymer modified surfaces.
Experimental
observations
Most of the experimental observations of protein adsorption on tethered polymer layers have been performed with PEO. However, several other polymers have also been studied. One of the main problems in measurements of the ability of polymer layers to prevent protein adsorption is that the concentration of the polymer at the surface as well as the amount of protein adsorbed need to be determined. In this way one can systematically study how different polymer surface coverages result in different amounts of adsorbed proteins. Furthermore, a complete unders~nding of the interactions between the polymer layer and the proteins also requires a study of how other parameters, such as polymer chain length, solution composition and the nature of the surface influence protein adsorption. Elbert and Hubbell [lo] describe the experimental work carried out on a variety of surfaces with tethered polymers.
Polymers
Their article reviews the different techniques used to modify surfaces and the benefits and drawbacks of each technique. The authors discuss the different strategies necessary for reduction or increase of protein adsorption. Gref et al. [ 17.1 discuss the stability of sterically stabilized nanospheres as possible drug delivery agents. The use of multiblock copolymers in which one of the blocks is hydrophilic, in particular PEG, and the others are hydrophobic to form the nanosphere core is demonstrated. The experimental work clearly shows the ability of PEG, covering the surfaces of the spheres to drastically reduce protein adsorption and the coated materials showed increased circulation times in the blood stream. A book edited applications of presents studies to increase the includes studies layers chat help as a function of
by Lasic and Martin [18’] compiles the PEO on the surface of liposomes and of the ability of the surface modifier longevity of liposomes. Furthermore, it on the basic interactions of the PEO elucidate the variation of the steric barrier polymer chain length and surface density.
A systematic study of the ability of grafted PEO layers to reduce protein adsorption has been undertaken by McPherson and coworkers [19’,20]. It was found that grafting of the surface at high polymer surface coverage was difficult to achieve. The amount of protein adsorbed was not zero even at the highest accessible surface coverages. In the case of fibrinogen, it was found that the amount of protein adsorbed was too small for the platelet to adhere to the modified surface. The small amount of protein adsorbed at the highest polymer surface coverages raises the question of whether these proteins are sitting on the top of the polymer layers due to small attractions with the polymer segments, or whether they reach the surface due to defects in the grafted layer. Atomic force microscopy and scanning tunneling microscopy studies of the structure of the grafted layers with and without proteins adsorbed on the surface may shed light as to the structure of the adsorbed layer of proteins. Gsterberg and coworkers [Zl*] have studied the ability of dextran attached to surfaces and of PEO attached to surfaces to prevent protein adsorption. They looked at different configurations of the attached dextran on the surface, in one case the polymers were attached end-on and in another it was side-on. The conclusion from their study is that end-on PEO and side-on dextran are much more effective in preventing protein adsorption than end-on dextran. Furthermore, they found that the thickness of the polymer layer did not seem to be a relevant parameter in determining the amount of protein adsorbed while the surface density is very important. Kato et a/. [S] have studied the ability of charged and neutral polymers to prevent or enhance the adsorption of charged proteins onto surfaces. They found that
and proteins: lnteradlons at interfaces Sdeifer
339
neutral surfaces reject proteins (and thus, prevent protein adsorption) regardless of the isoelectric point of the protein. However, charged surfaces increased the adsorption of oppositely charged proteins while decreasing the adsorption of equally charged proteins. They explained their results in terms of electrostatic interactions for the charged polymers and steric repulsions for the neutral chain molecules. Piehler and coworkers [4] have studied a variety of different polymer modified surfaces which reduce nonspecific protein adsorption and increase specific binding of antibodies by functionalizing the polymer with a ligand. They found that all the different polymers they studied were able to reduce nonspecific protein adsorption as compared with the surface without polymer. Furthermore, when a ligand was attached to the polymer, specific binding of antibodies increased significantly as compared to the ligand directly attached to the bare surface. The comparisons between the different polymers to prevent protein adsorption show that dextran is more effective than the other polymers, including PEO. However, the surface coverages and chain lengths of the polymers used in the comparisons were not the same. Thus, the comparison is only important for the specific preparation of the surfaces presented in that work, and the conclusions cannot be generalized for other surface conditions. Ishihara and collaborators [22-251 studied a different approach of attaching the polymer to surface by use of phospholipid polymer surfaces. They found that the ability of a modified surfaces to prevent protein adsorption increased as the PEO concentration on the surface increased.
Theoretical approaches The first theoretical description of the effect of tethered polymers on the interaction between a protein and a polymer modified surface was carried out by Jeon and coworkers [26,27]. The steric repulsion arising from the tethered polymers was described by use of the Alexander-deGennes theory for a tethered polymer in the ‘brush’ regime. Their approach is applicable only in the limit of very long polymer chains (with a thousand segments or more [10*,14,28] and only for proteins whose size is much larger than the distance between the grafting points of the polymers. It turns out that many of the experimental tethered layers do not fulfill both conditions because it is extremely hard to prepare surfaces at very high surface coverages. Furthermore, the polymers used experimentally are in the intermediate regime of molecular weights. The Jeon et al. [26,27] approach is based on a correct qualitative picture, namely that the main effect of the grafted chains is to provide a steric barrier. However, it cannot be used for estimates in many cases because its range of applicability does not correspond to the
340
Biomaterials
experimental situations. For example, the shape of the steric repulsion between a lysozyme and a PEO polymer layer is found to have a maximum [29”], however, Jeon and Andrade [27] predict a monotonically increasing repulsion as the protein approaches the surface. Again, the reason is that the lysozyme interacting with PEO is beyond the range of applicability of their approach. In more recent work Williams and MacKintosh [30] and Subramanian et ai. [31] have studied the interactions of very large particles (which could be large proteins) with grafted polymers in the ‘mushroom’ regime. Their work concentrated on studying the ability of the tethered polymer chains to escape from under the particle. They found a rich phase diagram for chains grafted to the surface and for chains that can translate on the tethering surface as is the case for liposomes formed by lipids at temperatures above the gel-liquid crystal transition temperature of the lipid chains. Subramanian et a/. [32] extended these studies to include polymer layers in the ‘brush’ regime. They used a self-consistent field approach to treat the tethered polymers and looked at the interactions of a large particle with the layer using a perturbative calculation. Their studies are relevant to the interactions,between very large proteins and tethered polymer layers in the regime of very long polymer chains. They found the existence of different force laws between the polymer layer and the large particles, depending upon the surface coverage regime (‘mushroom’ or ‘brush’) and whether the polymers were mobile or grafted onto the surface. It will be interesting to check these predictions experimentally using atomic force microscopy or direct force measurements 1331. For intermediate to short chain molecules the SCMF theory was recently generalized to the study of the adsorption of proteins on tethered polymer layers [29”]. This theory enables the incorporation of detailed molecular structure of both the polymer molecules and the proteins. The studies presented in [29**] were carried out for model PEO chains, which is the same model that was used for the calculations of Figure 2. The theory was applied for model lysozyme for which the bare (in the absence of polymer) surface-protein interaction was calculated by Lee and Park [34]. The predictions of the theory for the adsorption isotherm, that is the amount of protein adsorbed as a function of the surface coverage of polymer, were shown to be in good agreement with the experimental observations of lysozyme adsorption on surfaces with grafted PEO-PPO-PEO triblock copolymers [lo*]. Thus, the SCMF theory seems to be appropriate in describing the properties of the polymer layers (see Fig. 2 and [10*,14]) and the interactions and resulting thermodynamic properties of protein solutions in contact with the grafted layer [10*,29”]. Protein adsorption isotherms show that as the surface coverage of polymer increases the amount of adsorbed protein
decreases. The amount of protein adsorbed is determined by the interplay between the bare surface-protein attraction, the polymer-protein repulsion, the protein-protein repulsions and the loss of conformational entropy of the tethered chains due to the presence of the proteins at the surface. In the cases of surfaces that attract segments of the polymer, the amount of protein adsorbed also depends on the energetic cost associated with the desorption of polymer segments in order to have enough space on the surface to accommodate the protein molecules. The structure of the resulting mixed adsorbed protein-tethered polymer is rather different than that. of the polymer layer without the proteins. Furthermore, looking at the structure of the polymer layer in the absence of the protein does not provide enough information to predict whether the proteins will adsorb or not. A complete treatment of the mixture is necessary in order to draw adequate conclusions [29”]. Systematic studies of the adsorption behavior of model lysozyme on model PEO layers using SCMF theory have shown that in the case were the surface does not attract the ethylene oxide (EO) segments (as in the case of polymer decorated liposomes), the adsorption isotherms are independent of the molecular weight of the polymer [29”]. Actually, there is a molecular weight threshold above which the isotherms become chain length independent. This critical molecular weight depends upon the size of the adsorbing protein. For lysozyme it is around 35 EO units [29”]. The conclusion that the adsorption is independent of molecular weight is in agreement with experimental observations [21*]. The reason for this behavior is that above the molecular weight threshold the number of segments of the tethered chains that need to be stretched toward the solvent is the same independent of the chain length of the polymer. The potential of interaction between the proteins and the grafted polymer layer exhibits a very strong dependence on polymer molecular weight. Therefore, while the equilibrium adsorption remains the same the kinetic behavior is dramatically different and adsorption becomes much slower as the chain length of the polymer increases [29”]. These findings suggest that one can control the amount of protein adsorbed thermodynamically (by the proper choice of polymer surface coverage) or kinetically (by choosing the right molecular weight). The adsorption of proteins is rather different for surfaces that attract the segments of the tethered polymers [29’*]. Namely, the adsorption isotherms strongly depend on the molecular weight. For a fixed surface coverage of grafted polymer the number of polymer segments adsorbed to the surface is larger for longer chains. Thus, the adsorbing proteins need to desorb a larger number of polymer segments to be accommodated on the surface. Hydrophobic surfaces seem to fall in this category in which the EO segments attract to the surface and thus, there is
Polymers and proteins: interactions at interfaces Szleifer
a very strong competition between the polymer segments and the proteins for contact with the surface. This is to be compared to the pure stretching effect that adsorbing proteins cause to the tethered chains when the polymer segments are not attracted to the surface [29”]. Figure 3 shows the adsorption isotherm of model lysozyme onto a surface which attracts the EO segments and a surface that does not attract them. The calculations shown in Figure 3 assume that the bare protein-surface interaction is the same so that the effect of the different types of surfaces with respect to the polymer segments can be isolated. Clearly, the attracting surface is much more effective in preventing the adsorption of proteins than the nonattractive one. This is in qualitative agreement with the experimental observations on side-on as compared to end-on dextran [21*].
Figure3
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The adsorption isotherms, that is the number of proteins per unktarea, (p ,,) as a function of the number of tethered polymers per unit area (or(the area is measured in units of bond length of EO square), for model lysozyme on tethered PEO layers as predicted by the SCMF theory [29*1. The full line corresponds to a surface that does not attract the polymer segments, while the dashed line corresponds to polymer chains that have a gain in energy of k$ when they are in contact with the surface. In both cases the tethered polymers have one hundred segments.
It has been.predicted that the time scale for the protein to reach the surface is faster for the attractive surface than for the nonattractive one [29”]. Again, showing that thermodynamic and kinetic control do not necessarily behave in the same way. Protein adsorption isotherms have been studied for different types of polymer chains architectures and for mixtures of tethered chains [35*]. An interesting prediction is that chains tethered at both ends to the surface are more effective in preventing protein adsorption than the same
341
polymer with only one end grafted to the surface. This is in apparent contradiction with the argument of Gref and coworkers [ 17.1. Their conclusion was based upon the assumption that the ability of the polymer layer to prevent protein adsorption was a function of the thickness of the layer and the surface coverage. However, as discussed above, the main parameter in determining the ability of the layer to prevent protein adsorption is the density of polymer segments in that region close to the surface. That is the free energy barrier that determines the equilibrium adsorption. Certainly the thickness of the polymer layer will very strongly affect the repulsive interactions that the proteins will experience before they reach the surface and therefore the thickness of the film will change the kinetic properties of the adsorption [29”]. Proper manipulation of the polymer chain architecture may be used to prevent protein adsorption and at the same time leave the free end of the chains highly accessible to bind to receptors [4,8*]. For example, calculations show that a diblock copolymer formed by a flexible block and a rod-like block, with the flexible block grafted to the surface can fulfill both conditions [35*]. A further example that the thickness of the polymer layer is not a determining factor in the ability of the chain molecules to prevent protein adsorption is based upon the experimental work of Prime an
Conclusions The interactions of tethered polymer layers with proteins can be tuned to prevent or enhance the amount of protein that adsorbs to a surface. One of the most important applications of tethered polymer layers is biocompatibility. For this purpose the tethered polymer layer must act as a steric barrier to approaching proteins. There is
342
Btomaterials
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The amount of model lysozyme adsorbed (p r& as a function of the surface coverage of chains (o) (upper scale P and composition of PEO chains (xp& (lower scale) assuming that the surface is a mixed self assemt&d monolayer. The isotherms were calculated using the SCMF theory [29**]. The full line corresponds to model chains with four EO units, the dashed line to chains with six EO units and the dotted line to chains with seventeen EO units. The isotherms include only the proteins that are in contact with the surface in order to mimic the effect of rinsing and drying as performed in the experimental observations. Compare the surface coverage of polymer necessary to completely prevent protein adsorption for these short chains with the case of Figure 3.
clear experimental and theoretical evidence that the most important factor in determining the ability of the polymer layer to prevent protein adsorption is the surface coverage of the grafted polymer. However, it is experimentally difficult to obtain a high surface coverage of polymer of intermediate chain length due to the steric barrier that the layer also presents to other polymer molecules trying to reach the surface. The most widely used polymer for prevention of protein adsorption is PEO. This is a flexible water soluble polymer that has been used on many types of surfaces including polymeric and glass surfaces, liposomes and polymeric beads. Other polymer molecules, for example dextran, that are water soluble but not as flexible as PEO have also been shown to be effective in reducing protein adsorption. Protein adsorption isotherms in the presence of a tethered polymer layer have been calculated using a generalized SCMF theory. The predictions of the theory are in good agreement with experimental observations for short and intermediate (from a few segments to two hundred segments) chain length PEO and model lysozyme. The theory predicts qualitative different behaviors for surfaces
in which the polymer segments are attracted to the surface to those which are not. Furthermore, the kinetic and thermodynamic behavior of the adsorbing proteins exhibit different dependences on the grafted polymer molecular weight. In order to obtain a good understanding of the ability of polymer layers to prevent protein adsorption under several conditions we need to focus our attention on several aspects of the problem. On the experimental side more systematic studies of the adsorption isotherms are needed. The types of question that need to be addressed in a systematic way are; what is the influence of the type of surface on the ability of the polymer layer to prevent protein adsorption! How can one tune the interaction of the polymer layer with the surface? Can we measure the interaction of the proteins with the tethered polymers? Can we follow the kinetics of the adsorption process and study in which cases the adsorption is kinetically determined, rather than ~ermodynam~cally, in the presence of the polymer, layer? Can we study the conformational changes of the adsorbed proteins and how the preferred adsorbed conformation depends upon the surface coverage of polymer? On the theoretical side very little has been done in order to understand the competition between different conformers of the protein adsorbing on the surface and how the competition between them changes due to the presence of a tethered polymer layer fZP*]. The bare interactions between proteins and surfaces need to be studied. Furthermore, none of the theoretical approaches described above, even the molecular ones, have considered, so far, the effect that the inhomogeneities on the protein structure has on the protein adsorption behavior. For example, some regions of the proteins may be attracted to the tethered polymer segments while others are not. The agreement between the SCMF theoretical predictions and the experimental observations suggests that, at least for lysozyme, the main factor determining the adsorption behavior is dominated by the competition between the strong bare attraction between the protein and the surface and the strong repuIsive interaction resulting from the presence of the tethered polymer layer. However, in the case of much larger proteins, such as fibrinogen, the fact that the proteins are inhomogeneous may play a central role in their ability to be adsorbed on the surface or on the polymer layer. The systematic combination of experimental and theoretical studies will enable an understanding of the basic processes underlying the kinetic and equilibrium adsorption of proteins on modified surfaces and the effective interactions between them. This unders~nding will be used in the rational design of biomaterials with the desired ability to prevent (or increase) the adsorption of proteins to their surfaces.
Polymers
Acknowledgements This work is supported by the National Science Foundation, grant CTS9624268. The author is a Camille Dreyfus Teacher-Scholar.
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Lee SJ, Park K: Protein intemction with ourfeces: separation distence-dependent interaction energies. I Vat Sci Techno/A 1994, 12:1-7.
35. .
Szleifer I: Protein adsorption on tethered polymer layers: effect of polymer chain architecture and composition. PI?ysicaA 1997, in press.
Theoretical studies of the adsorption isothermsand effective interactionsof proteins with tethered polymer layers for a variety of polymer chain architectures are presented. The ability of layers formed by mixturesof polymer chains in preventingprotein adsorption was also studied. 36.
Prime K, Whitesides G: Adsorption of proteins onto surface containing end-etteched oligo(ethylene oxide): a model system using self-assembled monolayer% I Am Chem Sot 1993, 115:10714-10721.
and theoretically.
Recent systematic
studies show that surface density seems to be the most important property of the tethered
layer that determines
ability to prevent protein adsorption.
Theoretical
its
studies
predict that the interactions
of the polymer layer with the
proteins and the adsorption
behavior do not depend
in the
same way on polymer molecular weight and on the type of interaction
delivery systems [6’]. Furthermore, tethered polymers can be used for the specific targeting of liposomes to cells by chemically inserting a specific binding reagent to the free ends of the tethered polymers [7,8*].
of the polymer with the surface.
Addresses Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393, USA; e-mail: [email protected] Current Opinion in Solid State & Materials Science 1997, 2~337-344 Electronic identifier: 1359-0286-002-00337 0 Current Chemistry Ltd ISSN 1359-0286 Abbreviations EO ethylene oxide PEG polyethylene glycol PEO polyethylene oxide PS polystyrene SCMF single-chain mean-field
Introduction Many biological processes depend upon the adsorption of proteins on a variety of surfaces. The understanding and control of the adsorption of protein molecules and their interactions with surfaces is of primary importance in the design of biomaterials. For example, the design of biocompatible materials requires (among other things) the reduction of adsorption of blood proteins to the surface of the material. It is known that the adsorption of blood proteins followed by platelet adhesion can result in surface induced thrombosis. The elimination or reduction of protein adsorption results in very low adhesion of platelets into the surface [l’]. Another example involves the elimination of lysozyme build-up on the surface of contact lenses [2,3]. In other cases, one is interested in increasing the adsorption of some types of proteins while reducing the adsorption of others [4,5]. During the last few years the use of polymer molecules as surface modifiers has been shown to be a very promising way of manipulating the interactions of surfaces with proteins [lo]. The proper tuning of these interactions will hopefully lead us to the rational design of a modified surface that will improve biocompatibility. For example, the inclusion of tethered polymers on the surfaces of liposomes results in their increased longevity in the blood stream, hence, these aggregates can be used as drug
In this review I will discuss the recent advances in our understanding of the ability of tethered polymer chains to prevent protein adsorption. The present experimental status of these systems will be discussed together with theoretical studies that enable us to calculate the interactions of proteins with tethered polymer layers as well as protein adsorption on modified surfaces. First, I will discuss some of the general properties of tethered polymer layers. The ability of tethered polymer layers to prevent protein adsorption is believed to be due to the steric repulsion associated with the perturbation that the protein molecules cause on the polymer layer [9] (see Fig. 1). In order to understand this repulsion it is appropriate to understand the structure of the polymer layers. The general picture in the good solvent regime (i.e. when the polymer is soluble in the solvent) is that [lo’], for very low surface coverages of polymers the tethered chains are isolated and their structure is not very different from that of the polymers in bulk. This is called the ‘mushroom’ regime. As the surface coverage increases the chains start to repel each other and they have to stretch out of the surface in order to avoid each other and thus reduce the polymer-polymer repulsions. The regime of highly stretched chains is generally called the ‘brush’ regime (11,121. Detailed studies performed by computer simulations [13] and molecular theories [10’,14], in particular the single-chain mean-field (SCMF) theory, show that the treatment of tethered polymers in terms of only the two above defined regimes is not appropriate for short to intermediate chain length polymers, that is from a few segments to a few hundred segments. Furthermore, the intermediate regime, that is, the ‘mushroom’ to ‘brush’ transition region, is the one that corresponds to most experimental studies. This has been shown to be the case in experimental observations of tethered polyethylene oxide (PEO) (also called polyethylene glycol [PEG]), which is the most widely used polymer in biological related applications of tethered polymer layers. This conclusion was reached by comparisons of experimental observations with the predictions of the SCMF theory on structural and thermodynamic properties [10*,14]. Figure 2 shows experimental and theoretical pressure-area isotherms of polystyrene (PS)-PEO diblock copolymers at the water-air interface. The PS block is at the air side of the interface, while the PEO chain is on the
338
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Figure 1
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Current Opinion
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Schematic representation of protein molecules in contact with a tethered polymer layer.
water side. The figure shows experimental observations as well as SCMF calculations. The agreement between the predicted and observed isotherms is very good. The picture that emerges from the calculations is that in the ‘mushroom’ regime the PEO chains are actually adsorbed co the interface. Thereby, forming a quasi two-dimensional adsorbed layer instead of a swollen ‘mushroom’. The attraction of the polymer monomers with the surface is due to the amphiphilic nature of PEO and this is a very important consideration in understanding and designing layers to protect hydrophobic surfaces (see below). The offset of the plateau in terms of the pressure corresponds to the area at which the adsorbed PEO chains start to strongly repel each other. Thus, the chains can release some of the pressure by stretching some of the ethylene oxide (EO) segments out of the interface into the water phase. The sharp increase in pressure at small area per molecule is due to the strong repulsions between the chains that result in a high degree of stretching of both the PEO and the PS blocks. Similar observations for pressure-area isotherms have been reported by Bijsterbosch et al. ]15]. The structural changes with surface coverage as well as the resulting (nontrivial) lateral interactions (lateral pressures) indicate that the interactions of PEO tethered layers with protein molecules are rather complex. Furthermore, in the cases of PEO on the surface of liposomes it has recently been shown that the EO monomers are not attracted to the liposome interface (161. Therefore, the structural properties and the interactions with the surrounding solution, will be different than in the case of hydrophobic surfaces. A thorough description of the properties of the tethered layers in the regime of molecuiar weights and surface coverages used experimentally requires a detailed molecular description [ 10*,14]. This is necessary to understand the interactions of the polymer layer with the protein molecules as well as to establish the amount
2000 0 1997
4000
6000
8000
a @‘J Current
O&on
in &iii
State 6 Material soimx
The lateral pressure (II), in mN/m, as a function of the area (a) per molecule, in As,for PS-PEO diblock copolymers at the water-air interface. The PS block has 30 styrene units and the PEO has 179 ethylene oxide units. The line with circles represents the experimental observations (P Sassereau, MC Fat&, personal ~mrnuni~t~n} while the other fine is derived from predictions of the SCMF theory (b&I Carignano, personal communication). The calculations assume that there is an attractive interaction of -k$ when an ethylene oxide unit is in contact with the interface.
of protein adsorbed on the surface and its variation with polymer surface coverage, molecular weight and type of surface. In the next. sections I will review the experimental observations of protein adsorption on a variety of surfaces with tethered polymer layers as well as recent theoretical predictions on protein adsorption and the interactions of the proteins with polymer modified surfaces.
Experimental
observations
Most of the experimental observations of protein adsorption on tethered polymer layers have been performed with PEO. However, several other polymers have also been studied. One of the main problems in measurements of the ability of polymer layers to prevent protein adsorption is that the concentration of the polymer at the surface as well as the amount of protein adsorbed need to be determined. In this way one can systematically study how different polymer surface coverages result in different amounts of adsorbed proteins. Furthermore, a complete unders~nding of the interactions between the polymer layer and the proteins also requires a study of how other parameters, such as polymer chain length, solution composition and the nature of the surface influence protein adsorption. Elbert and Hubbell [lo] describe the experimental work carried out on a variety of surfaces with tethered polymers.
Polymers
Their article reviews the different techniques used to modify surfaces and the benefits and drawbacks of each technique. The authors discuss the different strategies necessary for reduction or increase of protein adsorption. Gref et al. [ 17.1 discuss the stability of sterically stabilized nanospheres as possible drug delivery agents. The use of multiblock copolymers in which one of the blocks is hydrophilic, in particular PEG, and the others are hydrophobic to form the nanosphere core is demonstrated. The experimental work clearly shows the ability of PEG, covering the surfaces of the spheres to drastically reduce protein adsorption and the coated materials showed increased circulation times in the blood stream. A book edited applications of presents studies to increase the includes studies layers chat help as a function of
by Lasic and Martin [18’] compiles the PEO on the surface of liposomes and of the ability of the surface modifier longevity of liposomes. Furthermore, it on the basic interactions of the PEO elucidate the variation of the steric barrier polymer chain length and surface density.
A systematic study of the ability of grafted PEO layers to reduce protein adsorption has been undertaken by McPherson and coworkers [19’,20]. It was found that grafting of the surface at high polymer surface coverage was difficult to achieve. The amount of protein adsorbed was not zero even at the highest accessible surface coverages. In the case of fibrinogen, it was found that the amount of protein adsorbed was too small for the platelet to adhere to the modified surface. The small amount of protein adsorbed at the highest polymer surface coverages raises the question of whether these proteins are sitting on the top of the polymer layers due to small attractions with the polymer segments, or whether they reach the surface due to defects in the grafted layer. Atomic force microscopy and scanning tunneling microscopy studies of the structure of the grafted layers with and without proteins adsorbed on the surface may shed light as to the structure of the adsorbed layer of proteins. Gsterberg and coworkers [Zl*] have studied the ability of dextran attached to surfaces and of PEO attached to surfaces to prevent protein adsorption. They looked at different configurations of the attached dextran on the surface, in one case the polymers were attached end-on and in another it was side-on. The conclusion from their study is that end-on PEO and side-on dextran are much more effective in preventing protein adsorption than end-on dextran. Furthermore, they found that the thickness of the polymer layer did not seem to be a relevant parameter in determining the amount of protein adsorbed while the surface density is very important. Kato et a/. [S] have studied the ability of charged and neutral polymers to prevent or enhance the adsorption of charged proteins onto surfaces. They found that
and proteins: lnteradlons at interfaces Sdeifer
339
neutral surfaces reject proteins (and thus, prevent protein adsorption) regardless of the isoelectric point of the protein. However, charged surfaces increased the adsorption of oppositely charged proteins while decreasing the adsorption of equally charged proteins. They explained their results in terms of electrostatic interactions for the charged polymers and steric repulsions for the neutral chain molecules. Piehler and coworkers [4] have studied a variety of different polymer modified surfaces which reduce nonspecific protein adsorption and increase specific binding of antibodies by functionalizing the polymer with a ligand. They found that all the different polymers they studied were able to reduce nonspecific protein adsorption as compared with the surface without polymer. Furthermore, when a ligand was attached to the polymer, specific binding of antibodies increased significantly as compared to the ligand directly attached to the bare surface. The comparisons between the different polymers to prevent protein adsorption show that dextran is more effective than the other polymers, including PEO. However, the surface coverages and chain lengths of the polymers used in the comparisons were not the same. Thus, the comparison is only important for the specific preparation of the surfaces presented in that work, and the conclusions cannot be generalized for other surface conditions. Ishihara and collaborators [22-251 studied a different approach of attaching the polymer to surface by use of phospholipid polymer surfaces. They found that the ability of a modified surfaces to prevent protein adsorption increased as the PEO concentration on the surface increased.
Theoretical approaches The first theoretical description of the effect of tethered polymers on the interaction between a protein and a polymer modified surface was carried out by Jeon and coworkers [26,27]. The steric repulsion arising from the tethered polymers was described by use of the Alexander-deGennes theory for a tethered polymer in the ‘brush’ regime. Their approach is applicable only in the limit of very long polymer chains (with a thousand segments or more [10*,14,28] and only for proteins whose size is much larger than the distance between the grafting points of the polymers. It turns out that many of the experimental tethered layers do not fulfill both conditions because it is extremely hard to prepare surfaces at very high surface coverages. Furthermore, the polymers used experimentally are in the intermediate regime of molecular weights. The Jeon et al. [26,27] approach is based on a correct qualitative picture, namely that the main effect of the grafted chains is to provide a steric barrier. However, it cannot be used for estimates in many cases because its range of applicability does not correspond to the
340
Biomaterials
experimental situations. For example, the shape of the steric repulsion between a lysozyme and a PEO polymer layer is found to have a maximum [29”], however, Jeon and Andrade [27] predict a monotonically increasing repulsion as the protein approaches the surface. Again, the reason is that the lysozyme interacting with PEO is beyond the range of applicability of their approach. In more recent work Williams and MacKintosh [30] and Subramanian et ai. [31] have studied the interactions of very large particles (which could be large proteins) with grafted polymers in the ‘mushroom’ regime. Their work concentrated on studying the ability of the tethered polymer chains to escape from under the particle. They found a rich phase diagram for chains grafted to the surface and for chains that can translate on the tethering surface as is the case for liposomes formed by lipids at temperatures above the gel-liquid crystal transition temperature of the lipid chains. Subramanian et a/. [32] extended these studies to include polymer layers in the ‘brush’ regime. They used a self-consistent field approach to treat the tethered polymers and looked at the interactions of a large particle with the layer using a perturbative calculation. Their studies are relevant to the interactions,between very large proteins and tethered polymer layers in the regime of very long polymer chains. They found the existence of different force laws between the polymer layer and the large particles, depending upon the surface coverage regime (‘mushroom’ or ‘brush’) and whether the polymers were mobile or grafted onto the surface. It will be interesting to check these predictions experimentally using atomic force microscopy or direct force measurements 1331. For intermediate to short chain molecules the SCMF theory was recently generalized to the study of the adsorption of proteins on tethered polymer layers [29”]. This theory enables the incorporation of detailed molecular structure of both the polymer molecules and the proteins. The studies presented in [29**] were carried out for model PEO chains, which is the same model that was used for the calculations of Figure 2. The theory was applied for model lysozyme for which the bare (in the absence of polymer) surface-protein interaction was calculated by Lee and Park [34]. The predictions of the theory for the adsorption isotherm, that is the amount of protein adsorbed as a function of the surface coverage of polymer, were shown to be in good agreement with the experimental observations of lysozyme adsorption on surfaces with grafted PEO-PPO-PEO triblock copolymers [lo*]. Thus, the SCMF theory seems to be appropriate in describing the properties of the polymer layers (see Fig. 2 and [10*,14]) and the interactions and resulting thermodynamic properties of protein solutions in contact with the grafted layer [10*,29”]. Protein adsorption isotherms show that as the surface coverage of polymer increases the amount of adsorbed protein
decreases. The amount of protein adsorbed is determined by the interplay between the bare surface-protein attraction, the polymer-protein repulsion, the protein-protein repulsions and the loss of conformational entropy of the tethered chains due to the presence of the proteins at the surface. In the cases of surfaces that attract segments of the polymer, the amount of protein adsorbed also depends on the energetic cost associated with the desorption of polymer segments in order to have enough space on the surface to accommodate the protein molecules. The structure of the resulting mixed adsorbed protein-tethered polymer is rather different than that. of the polymer layer without the proteins. Furthermore, looking at the structure of the polymer layer in the absence of the protein does not provide enough information to predict whether the proteins will adsorb or not. A complete treatment of the mixture is necessary in order to draw adequate conclusions [29”]. Systematic studies of the adsorption behavior of model lysozyme on model PEO layers using SCMF theory have shown that in the case were the surface does not attract the ethylene oxide (EO) segments (as in the case of polymer decorated liposomes), the adsorption isotherms are independent of the molecular weight of the polymer [29”]. Actually, there is a molecular weight threshold above which the isotherms become chain length independent. This critical molecular weight depends upon the size of the adsorbing protein. For lysozyme it is around 35 EO units [29”]. The conclusion that the adsorption is independent of molecular weight is in agreement with experimental observations [21*]. The reason for this behavior is that above the molecular weight threshold the number of segments of the tethered chains that need to be stretched toward the solvent is the same independent of the chain length of the polymer. The potential of interaction between the proteins and the grafted polymer layer exhibits a very strong dependence on polymer molecular weight. Therefore, while the equilibrium adsorption remains the same the kinetic behavior is dramatically different and adsorption becomes much slower as the chain length of the polymer increases [29”]. These findings suggest that one can control the amount of protein adsorbed thermodynamically (by the proper choice of polymer surface coverage) or kinetically (by choosing the right molecular weight). The adsorption of proteins is rather different for surfaces that attract the segments of the tethered polymers [29’*]. Namely, the adsorption isotherms strongly depend on the molecular weight. For a fixed surface coverage of grafted polymer the number of polymer segments adsorbed to the surface is larger for longer chains. Thus, the adsorbing proteins need to desorb a larger number of polymer segments to be accommodated on the surface. Hydrophobic surfaces seem to fall in this category in which the EO segments attract to the surface and thus, there is
Polymers and proteins: interactions at interfaces Szleifer
a very strong competition between the polymer segments and the proteins for contact with the surface. This is to be compared to the pure stretching effect that adsorbing proteins cause to the tethered chains when the polymer segments are not attracted to the surface [29”]. Figure 3 shows the adsorption isotherm of model lysozyme onto a surface which attracts the EO segments and a surface that does not attract them. The calculations shown in Figure 3 assume that the bare protein-surface interaction is the same so that the effect of the different types of surfaces with respect to the polymer segments can be isolated. Clearly, the attracting surface is much more effective in preventing the adsorption of proteins than the nonattractive one. This is in qualitative agreement with the experimental observations on side-on as compared to end-on dextran [21*].
Figure3
0.00
0.01 Q 1997
0.03 0.04 0.02 d Current Opinion in Sdid State 6. Materials Sdena
The adsorption isotherms, that is the number of proteins per unktarea, (p ,,) as a function of the number of tethered polymers per unit area (or(the area is measured in units of bond length of EO square), for model lysozyme on tethered PEO layers as predicted by the SCMF theory [29*1. The full line corresponds to a surface that does not attract the polymer segments, while the dashed line corresponds to polymer chains that have a gain in energy of k$ when they are in contact with the surface. In both cases the tethered polymers have one hundred segments.
It has been.predicted that the time scale for the protein to reach the surface is faster for the attractive surface than for the nonattractive one [29”]. Again, showing that thermodynamic and kinetic control do not necessarily behave in the same way. Protein adsorption isotherms have been studied for different types of polymer chains architectures and for mixtures of tethered chains [35*]. An interesting prediction is that chains tethered at both ends to the surface are more effective in preventing protein adsorption than the same
341
polymer with only one end grafted to the surface. This is in apparent contradiction with the argument of Gref and coworkers [ 17.1. Their conclusion was based upon the assumption that the ability of the polymer layer to prevent protein adsorption was a function of the thickness of the layer and the surface coverage. However, as discussed above, the main parameter in determining the ability of the layer to prevent protein adsorption is the density of polymer segments in that region close to the surface. That is the free energy barrier that determines the equilibrium adsorption. Certainly the thickness of the polymer layer will very strongly affect the repulsive interactions that the proteins will experience before they reach the surface and therefore the thickness of the film will change the kinetic properties of the adsorption [29”]. Proper manipulation of the polymer chain architecture may be used to prevent protein adsorption and at the same time leave the free end of the chains highly accessible to bind to receptors [4,8*]. For example, calculations show that a diblock copolymer formed by a flexible block and a rod-like block, with the flexible block grafted to the surface can fulfill both conditions [35*]. A further example that the thickness of the polymer layer is not a determining factor in the ability of the chain molecules to prevent protein adsorption is based upon the experimental work of Prime an
Conclusions The interactions of tethered polymer layers with proteins can be tuned to prevent or enhance the amount of protein that adsorbs to a surface. One of the most important applications of tethered polymer layers is biocompatibility. For this purpose the tethered polymer layer must act as a steric barrier to approaching proteins. There is
342
Btomaterials
Figure 4
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. 0,3
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04.
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0.0
0.2
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The amount of model lysozyme adsorbed (p r& as a function of the surface coverage of chains (o) (upper scale P and composition of PEO chains (xp& (lower scale) assuming that the surface is a mixed self assemt&d monolayer. The isotherms were calculated using the SCMF theory [29**]. The full line corresponds to model chains with four EO units, the dashed line to chains with six EO units and the dotted line to chains with seventeen EO units. The isotherms include only the proteins that are in contact with the surface in order to mimic the effect of rinsing and drying as performed in the experimental observations. Compare the surface coverage of polymer necessary to completely prevent protein adsorption for these short chains with the case of Figure 3.
clear experimental and theoretical evidence that the most important factor in determining the ability of the polymer layer to prevent protein adsorption is the surface coverage of the grafted polymer. However, it is experimentally difficult to obtain a high surface coverage of polymer of intermediate chain length due to the steric barrier that the layer also presents to other polymer molecules trying to reach the surface. The most widely used polymer for prevention of protein adsorption is PEO. This is a flexible water soluble polymer that has been used on many types of surfaces including polymeric and glass surfaces, liposomes and polymeric beads. Other polymer molecules, for example dextran, that are water soluble but not as flexible as PEO have also been shown to be effective in reducing protein adsorption. Protein adsorption isotherms in the presence of a tethered polymer layer have been calculated using a generalized SCMF theory. The predictions of the theory are in good agreement with experimental observations for short and intermediate (from a few segments to two hundred segments) chain length PEO and model lysozyme. The theory predicts qualitative different behaviors for surfaces
in which the polymer segments are attracted to the surface to those which are not. Furthermore, the kinetic and thermodynamic behavior of the adsorbing proteins exhibit different dependences on the grafted polymer molecular weight. In order to obtain a good understanding of the ability of polymer layers to prevent protein adsorption under several conditions we need to focus our attention on several aspects of the problem. On the experimental side more systematic studies of the adsorption isotherms are needed. The types of question that need to be addressed in a systematic way are; what is the influence of the type of surface on the ability of the polymer layer to prevent protein adsorption! How can one tune the interaction of the polymer layer with the surface? Can we measure the interaction of the proteins with the tethered polymers? Can we follow the kinetics of the adsorption process and study in which cases the adsorption is kinetically determined, rather than ~ermodynam~cally, in the presence of the polymer, layer? Can we study the conformational changes of the adsorbed proteins and how the preferred adsorbed conformation depends upon the surface coverage of polymer? On the theoretical side very little has been done in order to understand the competition between different conformers of the protein adsorbing on the surface and how the competition between them changes due to the presence of a tethered polymer layer fZP*]. The bare interactions between proteins and surfaces need to be studied. Furthermore, none of the theoretical approaches described above, even the molecular ones, have considered, so far, the effect that the inhomogeneities on the protein structure has on the protein adsorption behavior. For example, some regions of the proteins may be attracted to the tethered polymer segments while others are not. The agreement between the SCMF theoretical predictions and the experimental observations suggests that, at least for lysozyme, the main factor determining the adsorption behavior is dominated by the competition between the strong bare attraction between the protein and the surface and the strong repuIsive interaction resulting from the presence of the tethered polymer layer. However, in the case of much larger proteins, such as fibrinogen, the fact that the proteins are inhomogeneous may play a central role in their ability to be adsorbed on the surface or on the polymer layer. The systematic combination of experimental and theoretical studies will enable an understanding of the basic processes underlying the kinetic and equilibrium adsorption of proteins on modified surfaces and the effective interactions between them. This unders~nding will be used in the rational design of biomaterials with the desired ability to prevent (or increase) the adsorption of proteins to their surfaces.
Polymers
Acknowledgements This work is supported by the National Science Foundation, grant CTS9624268. The author is a Camille Dreyfus Teacher-Scholar.
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and recommended
reading
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