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Effect of polysaccharide structure on protein adsorption
Colloids and Surfaces B: Biointerfaces 17 (2000) 37 – 48 www.elsevier.nl/locate/colsurfb
Effect of polysaccharide structure on protein adsorption Sally L. McArthur a,b, Keith M. McLean a,b, Peter Kingshott a,b, Heather A.W. St John a,b, Ronald C. Chatelier a,b, Hans J. Griesser a,b,* b
a CSIRO Molecular Science, Pri6ate Bag 10, Clayton South MDC, Clayton 3169, Australia Cooperati6e Research Centre for Eye Research and Technology, Uni6ersity of New South Wales, Sydney 2052, Australia
Received 2 November 1998; accepted 7 June 1999
Abstract Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and g-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Protein adsorption; Polysaccharide; Carboxymethyldextran; Surface modification; Plasma polymer; Lysozyme; Albumin; Lactoferrin; Immunoglobulin
1. Introduction
* Corresponding author. Tel.: +61-3-95452611; fax: + 613-95452446. E-mail address: [email protected] (H.J. Griesser)
The uncontrolled adsorption of proteins from biological fluids, such as blood or tears, onto synthetic biomaterials triggers biological events including the host defense mechanisms, for instance fibrous encapsulation, complement activation and the coagulation cascade. These processes
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lead to adverse consequences that impact the long-term viability of the biomedical device. The adverse responses can span the range from being merely inconvenient, such as the fouling of contact lenses which entails the need for frequent cleaning or disposal of the lens, to the life-threatening, such as the clotting of small diameter artificial blood vessels. Thus, protein adsorption has been the subject of much research in the biomaterials field, and many reports have focused on unraveling its principles [1 – 4] in order to progress towards rational design of improved biomedical materials and coatings. For a number of applications, surfaces are desired that are highly hydrophilic and are able to prevent protein adsorption. Among the ‘non-fouling’ surface technology [5], coatings from polyethylene oxide (PEO) have attracted particularly strong attention [6,7]. Another class of molecules able to produce highly wettable, lowfouling coatings are polysaccharides, which like PEOs possess a heavily hydrated, hydrogel-like structure and mobile molecular chains that provide a steric – entropic contribution towards repulsion of protein adsorption. Surface-immobilised polysaccharides have been investigated for a variety of biomaterials and biosensor applications [8–13]. At the macroscopic level, polysaccharide coatings have been found to markedly reduce the adherence of cells [14] and bacteria [15] to synthetic biomaterials. At the molecular level, however, the terminology ‘non-fouling’ or ‘anti-fouling’ may not be applicable to many candidate coatings. Absence of cell attachment may be a consequence of the preferential competitive adsorption of ‘wrong’ proteins that are incapable of supporting cell attachment, or adsorbed celladhesive glycoproteins denaturing so extensively that cells are no longer able to bind to them [16], or the test used. As an example of the latter, whereas in in-vitro cell assays polysaccharide coatings can achieve very low levels of attachment even of relatively robust cells such as bovine corneal epithelials [17], there is significant colonization in vivo onto such coatings [18]. Moreover, protein adsorption onto biomaterials has at times been studied using only one or
very few proteins and with assays that lack monolayer sensitivity. Complex biological solutions comprise proteins, lipids, and mucins with widely different properties such as overall charge, hydrophobicity/hydrophilicity, and conformational flexibility, and repellency of all these species is a considerable challenge. Some conventional biochemical assays can also be affected by incomplete desorption of analytes and transfer losses. Physico-chemical surface analysis methods such as X-ray photoelectron spectroscopy (XPS) [19], time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) [20], Surface-MALDI mass spectrometry [21–23], and surface plasmon resonance [24] offer advantages in terms of detection levels much below monolayer coverage. These methods have been applied to various ‘nonfouling’ surface coatings [5] and have enabled documentation of protein adsorption at very low but perhaps biologically sensitive levels. Using such methods we found both in clinical trials of polysaccharide-coated contact lenses [25] and on immersing polysaccharide coatings into an artificial tear fluid formulation comprising 14 constituents representing proteins, lipids, and mucin [22], that they are not entirely resistant to protein adsorption, although it occurred at relatively low rates and to submonolayer amounts onto some of them [22,23,25,26]. The questions therefore arose, which proteins and lipids were capable of adsorbing onto these coatings, what the mechanisms of affinity interaction are between the coatings and these biomolecules, and which properties and structural features of polysaccharide coatings can be tailored to minimise protein adsorption or affinity-select particular proteins. The present report describes model studies investigating the adsorption behaviour of four representative proteins of different charge, molecular size, and structural stability onto a series of coatings produced with systematic variation in properties. The coatings consist of covalently immobilised carboxymethyldextrans (CMDs) varying in the density of carboxyl substitution on the molecular chain. We aimed to vary the density of surface ‘pinning’ (attachment points per chain) and the residual charge density, thus manipulating protein adsorption via a combination of electrostatic and
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steric–entropic interactions. The pinning density affects the mobility of chain segments and thus the steric–entropic contribution against protein adsorption, which was quantified by XPS.
2. Materials and methods
2.1. Materials Chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin and g-globulin (from Cohn fraction II and III), were purchased from Sigma-Aldrich (St. Louis, MO). Protein solutions were freshly prepared by dissolving proteins in phosphate-buffered saline (PBS) at pH 7.4, to give a final concentration of 10 mM for g-globulin and 20 mM for the others. Carboxymethyldextrans (CMDs) with different ratios of carboxyl groups to anhydroglycopyranoside ring units were prepared in a method adapted from Lo¨fas and Johnsson [8]. Dextran (10 g) (MW 70 000, Sigma-Aldrich, St Louis) was dissolved in 50 ml 2 M NaOH containing either 0.125, 0.25 or 1 M bromoacetic acid. The solution was stirred overnight, dialysed against water for 24 h, against 0.1 M HCl for 24 h and finally for 24 h against water. The solution was then lyophilised and stored at 4°C until required. The degree of carboxylation was assessed using back titration and NMR. The experimentally determined ratios were 1:2. 1:14 and 1:30, carboxyl groups per sugar unit. The resultant substitution is illustrated in Fig. 1.
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2.2. Surface modification CMD was grafted, using water soluble carbodiimide chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccin imide), to fluorinated ethylene propylene (Teflon FEP, 100 Type A; Du Pont) tape following surface amine functionalisation by radio frequency glow discharge plasma polymerisation. FEP is a convenient substrate as it does not contain low molecular weight constituents (plasticisers, other additives, oligomers) that might outdiffuse. In addition, the fluorine atom possesses a high XPS intensity and thereby facilitates detection of any coating non-uniformities and of any partial delamination that might occur on immersion in saline media. It is essential to be alert to such artefacts which might affect reliability and interpretation of protein adsorption experiments. The methods used are described elsewhere for the application of the plasma-polymerised, aminegroup-containing interlayer coating [27] and the subsequent immobilisation of CMDs [28]. Briefly, plasma polymerisation of n-heptylamine was used to deposit a coating with surface amine functional groups on the FEP substrate in a custom-built plasma apparatus [29], employing an ENI HPG-2 high frequency plasma generator. After incubation all samples were washed thoroughly in MilliQ water to remove any excess reagents and then stored in water until required.
2.3. Protein adsorption Samples were rinsed in MilliQ water and placed in clean 55 mm diameter polystyrene petri dishes to which 5 ml of the desired protein solution was added. Adsorption was allowed to proceed at room temperature for 1 h after which samples were washed three times in MilliQ water to remove loosely adsorbed proteins and salts. Samples were dried prior to analysis by XPS.
2.4. X-ray photoelectron spectroscopy
Fig. 1. Carboxymethyl substitution of dextran.
X-ray photoelectron spectroscopy (XPS) analysis was performed using a AXIS HSi spectrometer (Kratos Analytical Ltd) equipped with a
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Table 1 Elemental composition determined by XPS of the coatings Surface
FEP FEP+Happ FEP+Happ+CMD 1:2 FEP+Happ+CMD 1:14 FEP+Happ+CMD 1:30
Atomic concentration (%)
Atomic ratios
C1s
O1s
N1s
F1s
Si 2p
N:C
O:C
33.0 86.2 75.0 75.9 76.1
0.0 6.8 19.4 17.9 17.6
0.0 7.0 5.5 5.6 5.7
67.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
n/a 0.08 0.07 0.07 0.07
n/a 0.08 0.26 0.24 0.23
monochromatic Al Ka source at a power of 300 W. The total pressure in the main vacuum chamber during analysis was typically 2 ×10 − 8 mbar. Elements present were identified from survey spectra. For further analysis, high resolution spectra were recorded from individual peaks at 40 eV pass energy. Atomic concentrations of each element were calculated by determining the relevant integral peak intensities (using a linear type background) and applying the sensitivity factors supplied by the instrument manufacturer. The random error associated with elemental quantification has been determined for this instrument to be 1–2% of the absolute values for atomic percentages in the range encountered in this study (\ 5%). The systematic error is much more difficult to determine and is generally assumed to be of the order of 5 – 10% [30]. It is the random error that is more relevant when comparing samples of similar composition. A value of 285.0 eV for the binding energy of the main C1s component (CHx ) was used to correct for charging of specimens under irradiation [31]. Assuming a value of 3 nm for the electron attenuation length of a C1s photoelectron in a polymeric matrix [30], this translates into an approximate value for the XPS analysis depth (from which 95% of the detected signal originates) of 10 nm when recording XPS data at an emission angle normal to the surface, and 2 nm when recording at an emission angle of 75° relative to the surface normal (‘grazing angle’). A minimum of three samples for each experimental configuration were analysed.
2.5. Contact angle measurements A modified Kernco model G-II goniometer was used for the determination of air/water contact angles. The goniometer was equipped with a syringe comprising a micrometer-driven plunger whose direction of travel could be reversed in order to enable determination of sessile, advancing and receding contact angles [32].
3. Results
3.1. Coatings As a reference, ‘control’ surface for comparison with CMD coatings in protein adsorption experiments we used FEP coated with a thin layer of n-heptylamine plasma polymer (HApp). This coating is solid and dense, and therefore contributes minimal interfacial steric–entropic repulsion effects. It also possesses a very low density of positive surface charge in pH 7.4 buffered saline [32,33], since its low dielectric constant reduces the thermodynamic stability of charged amine groups and thus allows substantial protonation of amine groups only at pH valuesB 7. Hence, interfacial electrostatic forces are small, and protein adsorption onto the HApp layer may be presumed to occur predominantly by dispersion and hydrophobic forces. Comparison with CMD coatings thus allows assessment of additional forces, particularly of electrostatic and steric–entropic origins. XPS analysis indicated that the HApp layer was thicker than the analysis depth of XPS
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(\ 10 nm) and continuous as indicated by both the elimination of fluorine signal from the underlying FEP substrate (Table 1) and the absence of a component at 292.2 eV characteristic of CF2 structures in the XPS C 1 s high resolution spectrum (spectrum not shown). Following attachment of the CMDs onto the HApp coatings, significant increases in the XPS O:C ratios (Table 1) and the introduction of significant CO and CO components in the C1s high resolution spectra were observed [28]. No F signal was observed (Table 1), showing that the HApp layer did not partially delaminate in the aqueous carbodiimide/NHS/CMD reaction solutions. The three CMD coatings produced similar XPS results, indicating that all three CMDs produced similar amounts of surface coverage. The fact that a N signal was still observed in XPS indicates incomplete attenuation of the N 1s photoelectron emission originating from the HApp layer as this emission passes through the CMD overlayer. Therefore, the CMD coatings in the ‘dry’ state, as applies for XPS analysis, are thinner than 10 nm. XPS mapping and the uniform wettability of the samples indicated that the coatings were continuous and uniform over the sample surfaces. The sessile contact angles were 20° for all the CMD coatings and 70° for the HApp surface.
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3.2. Protein adsorption to n-heptylamine plasma polymer XPS was used to monitor the adsorption of lysozyme, human serum albumin, lactoferrin and g-globulins to HApp coated FEP samples. The presence of adsorbed proteins was clearly evident in XPS C 1s high resolution spectra; an example is shown in Fig. 2. For quantitative analysis of protein adsorption, changes in atomic concentrations measured by XPS were used, similar to reported procedures [19,34]. Fig. 3 shows the increases in the XPS N 1s signals on adsorption of proteins to the HApp surface. The increases in the N content over that of the non-immersed HApp sample are a measure of the amount of immobilised protein. They are, however, not a linear measure, and multilayer XPS algorithms [35] must be used to calculate the absolute amounts of surfacebound protein. In these cases, a three-layer model (HAppB CMDB protein) applies. While this can be done, it is not necessary for the purposes of the following discussion, for which the ‘raw’ data of XPS elemental compositions are sufficient and enable comparisons between proteins and coatings. The N contents shown in Fig. 3 after protein adsorption correspond to approximately monolayer coverage, with the size and relative stability
Fig. 2. High resolution XPS C1s spectra recorded on a HApp coated FEP sample before (solid line) and after (dashed line) immersion in an IgG solution.
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3.3. Protein adsorption to CMD coatings
Fig. 3. Atomic% nitrogen, recorded by XPS, before and after adsorption of proteins to HApp coated FEP.
of the proteins influencing the surface coverage and thus the nitrogen signal recorded using XPS. As expected on the basis of molecular size (Table 2), IgG gives the largest increase in the N content. The effect of protein stability is apparent in the HSA datum. This protein is present at lower coverage than the similarly sized lactoferrin; this may be due to ‘flattening’ of HSA as it denatures after adsorption. Each denatured HSA molecule then covers a larger surface area than expected on the basis of its solution equilibrium conformation. In addition, comparing the nitrogen signal from the lysozyme-adsorbed sample to that of the HSA-adsorbed sample shows that coverage is not solely dependent on molecular weight. We would expect that due to its approximately five-fold increase in molecular weight, HSA adsorption would result in a significant increase in the N signal when compared to the lysozyme sample, yet there is little difference between the two. This may be accounted for by the highly stable nature of lysozyme that prevents the protein from denaturing on the plasma polymer surface and produces a densely packed protein coverage. The ‘softer’ albumin molecules have greater conformational freedom; they spread and denature over the surface as they adsorb, producing a protein layer thinner than expected on the basis of molecular dimensions in solution.
All of the CMD coatings examined were found to adsorb proteins, but the amounts varied considerably with the surface and the particularprotein, and the presence of the CMDs significantly influenced the protein adsorption behaviour. For the CMD 1:2 coating, high resolution XPS C 1s spectra revealed the presence of lysozyme (Fig. 4), lactoferrin and IgG (spectra not shown) via significant increases in the spectral region where amine/hydroxyl and amide components are expected to contribute (286.5 and 288.5 eV respectively). For HSA adsorption, on the other hand, the spectra are less conclusive of adsorption (Fig. 4). There is only a very small increase in intensity in the amide spectral region. Grazing angle XPS spectra, by virtue of their higher sensitivity to the outermost 2 nm of the analyte surface, provided a clearer indication of HSA adsorption (Fig. 5) although again the small amide intensity (288.5 eV) suggested that the adsorbed amount was minimal. As above, the increases in the XPS N percentages upon protein adsorption can be used for quantitative comparisons. Fig. 6 shows data for the adsorption of the four proteins onto the CMD 1:2 coating. In agreement with the C 1s spectra, the elemental ratios showed that lysozyme, lactoferrin and IgG adsorbed to substantial amounts onto the CMD 1:2 surface (D%N =5, 8, and 3, respectively, compared with the non-exposed CMD coating), whereas there appeared to be no HSA adsorption within experimental uncertainty. There are marked differences in the protein adsorption characteristics due to the CMD 1:2 layer: this polysaccharide coating is certainly not non-fouling towards lysozyme, lactoferrin and IgG, and adsorbs even larger amounts of the first two proteins than the solid plasma polymer coating which does not possess steric–entropic interfacial effects. In turn, the CMD 1:2 surface almost entirely prevents adsorption of HSA, which adsorbs efficiently onto typical solid coatings of moderate hydrophobicities in the range similar to HApp (sessile contact angle of 70°). Thus, we
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Table 2 Isoelectric point and molecular weight of the proteins used Protein
pI
MW (Da)
Lysozyme (Lyz) Human serum albumin (HSA) Lactoferrin (Lf) Immunoglobulin G (IgG)
11.1 4.7 8 6.0–8.0
14 500 66 250 84 000 150 000
would expect that in competitive adsorption experiments HSA should compete very inefficiently with the other proteins for surface adsorption sites on the CMD 1:2 coating. As such, the CMD coating is not non-fouling but it clearly can be expected to alter the balance in competitive adsorption from multicomponent solutions. These data illustrate the dangers inherent in labelling a coating non-fouling on the basis of exposure to one protein only. The CMD coating with an intermediate carboxyl density (1:14) performed in qualitatively the same way as the CMD 1:2 coating except that the adsorbed amounts of lysozyme, lactoferrin and IgG were slightly less than on CMD 1:2 (data not shown). In contrast, the CMD 1:30 coating showed different patterns of behaviour (Fig. 7). Compared with CMD 1:2 (Fig. 6), the data manifested a significant increase in HSA adsorption
(D%N =1.4), but a decrease in adsorption of lysozyme and lactoferrin (D%N = 1.7 and 4.6, respectively). There was an increase in adsorbed g-globulin (D%N =4.7) when compared to CMD 1:2. Figs. 8 and 9 show XPS data recorded at grazing angle, which enables probing of the outermost 2 nm and hence, by comparison with data recorded at normal emission, assessment of depth distributions. Again we plot the data in terms of
Fig. 4. XPS high resolution C1s spectra of FEP+ HApp + CMD 1:2 coated samples before (solid thick line) and after adsorption of lysozyme (solid thin line) and HSA (dashed line).
Fig. 6. Percent atomic nitrogen recorded by XPS following adsorption of proteins to CMD 1:2 coatings.
Fig. 5. XPS high resolution C1s spectra recorded at grazing angle (75°) of FEP +HApp +CMD 1:2 coated samples before (solid line) and after (dashed line) HSA adsorption.
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Fig. 7. Percent atomic nitrogen recorded by XPS following adsorption of proteins to CMD 1:30 coatings.
XPS ‘raw’ data since conversion to depth distributions by algorithms such as those used for XPS depth profiling [38] relies on several assumptions whose potential error contributions are difficult to assess for the present case of CMD coatings. However, comparison of data collected at normal emission with data collected at grazing angle (75°) enables qualitative assessment of in-diffusion. If all protein molecules were located at the aqueous
Fig. 9. Percent atomic nitrogen recorded by XPS at normal and grazing angle (75°) following adsorption of proteins to CMD 1:30 coatings.
interface, ‘on top of’ the CMD coatings, then a substantially higher nitrogen content should be evident in grazing angle XPS data. However Figs. 8 and 9 do not show pronounced differences in most cases, suggesting significant diffusion of proteins into the coatings.
4. Discussion
4.1. Protein adsorption to n-heptylamine plasma polymer
Fig. 8. Percent atomic nitrogen recorded by XPS at normal and grazing angle emission (75°) following adsorption of proteins to CMD 1:2 coatings.
There are a number of interfacial interactions between surfaces and proteins that can lead to spontaneous irreversible protein adsorption. Most proteins carry a net charge in physiological media, with the sign and magnitude of the net charge depending on the isoelectric point of the protein (Table 2). Electrostatic interactions between proteins and charged surfaces therefore often play a major role in the adsorption behaviour of the proteins. The HApp coating carries little charge; its surface contains mainly amine and amide groups [27,36], with protonation of the former inefficient at pH 7.4 [32,33]. Together with charge screening in the PBS medium, this results in an low level of surface charge under our protein adsorption conditions. The putative lack of elec-
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trostatic interactions is reflected in the protein adsorption results with both overall positive (lysozyme and lactoferrin) and negative (HSA) proteins adsorbing virtually equally well to the HApp surface. The main driving forces for protein adsorption would in this case appear to be dispersive and hydrophobic interactions. This interpretation extends earlier observations of adsorption of lysozyme onto HApp coatings [21] where it was observed that this overall positively charged protein readily and rapidly adsorbed onto HApp, consistent with the absence of strong electrostatic repulsion that would occur if a substantial number of surface amine groups were protonated.
4.2. Protein adsorption to CMD coatings Application of the CMD coatings on top of the HApp layer markedly changes protein adsorption behaviours, in ways that indicate that dispersive and hydrophobic interactions no longer dominate the driving forces for adsorption. Instead, the observed differences in the adsorption of the four test proteins to CMD coatings relative to the HApp coating can tentatively be interpreted in terms of electrostatic and steric – entropic interfacial forces emanating from these hydrogel-like coatings, and, when comparing the CMD coatings, in terms of differences in these forces. As the net charge of the proteins differs, differences in their adsorption behaviour should reflect electrostatic interactions with charged surfaces. Electrostatic interactions are clearly apparent in the results obtained with the CMD 1:2 coating, with the positively charged proteins lysozyme and lactoferrin strongly adsorbing and a very low level of adsorption of the overall negatively charged HSA. The importance of electrostatic forces in adsorption onto CMD 1:2 and CMD 1:14 is evident in the data of Fig. 6; the two proteins with a net positive charge adsorb substantially more. On CMD 1:30 the effect is less pronounced. The different densities of carboxyl groups on the CMD moieties will lead to different magnitudes of negative surface charge after attachment. However, the magnitudes of the surface charge obtained after covalent immobilisation of the three
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different CMDs onto amine surfaces are currently undetermined since the techniques that have been applicable to well-defined, solid coatings [32,33] do not work with the hydrogel-like, expanded CMD coatings; we are currently attempting to use zeta potential measurements. In the meantime, one can reasonably assume that because of the relatively low surface amine density on the HApp layer [27], many of the carboxyl groups are not converted to amides during the attachment process, particularly for CMD 1:2. For all the CMDs we expect that the low ionic strength of the CMD reaction media together with the branched structure of the dextran molecules would, by electrostatic repulsion between carboxyl groups, lead to an expanded molecular structure which, on approach to the surface, would place a substantial fraction of the carboxyl groups beyond the reach of interfacial reaction with surface amine groups. The high carboxyl density particularly of CMD 1:2 will enable it, however, to react efficiently with many of the available amine groups, and hence we expect this coating to have the highest density of surface pinning points and thus the least segmental mobility, which means less steric–entropic repulsion towards proteins. We picture the CMD 1:2 coating as possessing a high density of remaining carboxyl groups and being well pinned to the HApp layer, producing a relatively dense polysaccharide structure. At the other end of the spectrum is the CMD 1:30 coating; the low density of carboxyl species in CMD 1:30 results in a lower level of pinning, producing a more open, ‘floppy’ coating, with much less residual charge but much higher interfacial entropy than the other coatings. On the CMD 1:30 coating, the relative levels of protein adsorption do not correlate with expectations based on dominant electrostatic interactions; HSA adsorbs onto the like-charged surface, although to a lesser amount than the positively charged proteins. One possible interpretation for the observed HSA adsorption on CMD 1:30 is that repulsive electrostatic interactions and steric– entropic effects (polysaccharide chain mobility) are not sufficient to counteract attractive forces, which could arise from the HApp substrate ‘shining through’ the thin CMD coating and from entropic gains when water molecules are liberated
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from within the CMD coating as protein molecules take their place. In the case of the CMD 1:2 coating, in contrast, the coating structure and/or the surface carboxyl charge density may be sufficiently dense to prevent significant in-diffusion of HSA and the concomitant energy gains. While it appears reasonable to expect that the chain segments of the CMD 1:30 coating, being pinned at large distances, have considerable mobility, the resultant interfacial entropy evidently is not sufficient to overcome the attractive forces driving towards protein adsorption. Another factor may also be relevant for HSA and IgG adsorption. Whilst the adsorption characteristics of smaller molecular weight proteins such as insulin, lysozyme and myoglobin can be interpreted in terms of single-domain, relatively homogeneous structures, larger, more complex proteins such as HSA and IgG cannot be as readily defined. It has been suggested by Andrade et al. [37] that these proteins should be considered as multidomain structures, with each domain having differing properties in terms of charge, stability and thus propensity to adsorb and denature on specific surface. Whilst proteins such as fibrinogen and fibronectin are pictured with a large number of domains (12 and 20 respectively), for albumin a simpler ‘tennis ball’ model of three 20 kDa domains has been proposed. In the model domains I and II are strongly negatively charged and contain fewer di-sulfide bonds than the weakly positively charged domain III [37]. As a result, on a negatively charged surface such as CMD 1:2, HSA would tend to adsorb weakly via domain III, but due to the domain stability would tend not to denature. The highly hydrated nature of the CMD surface would also reduce forces towards structural rearrangements of the HSA and hence, the hydrophobic regions of the protein would remain internalised and the hydrophilic (charged) residues would interact specifically with the CMD surface. The multidomain structure concept also applies to IgG. While some domains of this protein may be electrostatically attracted to the highly charged CMD 1:2 surface, others are attracted to the less charged CMD 1:30 surface and may interact with the surface via other interactions. This is reflected
in the data showing that all of the CMD surfaces attracted significant amounts of IgG. It appears to be much more challenging to reduce/prevent the adsorption of large, multidomain proteins, an issue which some studies of ‘non-fouling’ coatings have overlooked. Interpretation in terms of interfacial forces assumes protein adsorption as a discrete layer on top of the CMDs. To what extent can the protein molecules diffuse into the thin coatings? The grazing angle XPS data enable probing of the outermost region of the sample surface and hence, by comparison with data recorded at normal emission, assessment of discrete versus diffuse layer structures. For the CMD 1:2 coated samples, grazing angle XPS data compared with normal emission data (Fig. 8) suggest, by the increases in the nitrogen signals, that the proteins are somewhat enriched towards the outermost surface. It appears, therefore, that whilst there is some indiffusion, the proteins are not uniformly distributed throughout the thickness of the polysaccharide coating. For lactoferrin, assessment of in-diffusion is more difficult as the observed compositions approach the bulk composition of proteins and therefore variations with emission angle analysis are expected to be less pronounced. In the case of CMD 1:30 (Fig. 9) there are no pronounced differences when comparing normal emission and grazing angle data, suggesting a substantial extent of diffusion of the proteins into this coating. Thus, it appears that the ‘biofouling’ behaviour of these coatings must be interpreted not just in terms of interfacial forces; in-diffusion of proteins is substantial in some cases and gives rise to additional effects such as the entropic gain when water molecules are released from within the coating. A more densely attached coating such as CMD 1:2 can reduce such in-diffusion but is expected to possess less mobility of the attached chains, which reduces the effectiveness of the steric barrier.
5. Conclusions On all the hydrophilic, negatively charged carboxymethyl-dextran coatings of varying carboxyl
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group density investigated, all four test proteins can adsorb to some extent, even though for HSA, which carries a net negative charge, this would seem electrostatically unfavourable. The data suggest that elimination of adsorption of a broad spectrum of proteins is not a simple matter with negatively charged polysaccharide coatings (the majority of natural and available polysaccharides being negatively charged). While increasing the steric–entropic forces by decreasing the pinning density can lead to a reduction in adsorption of some proteins, the adsorption of others (HSA in this instance) can actually increase, and yet others (IgG) remain unaffected, adsorbing to approximately monolayer coverage regardless of structural variations in the carboxymethyl dextran coatings. It appears from our data that increasing the entropic forces by generating more flexible surface-attached macromolecular structures, also unfortunately leads to more open coatings that then allow in-diffusion of proteins. At this point in time we conjecture that ‘non-fouling’ in terms of elimination, or much reduced levels, of protein accumulation onto/into such coatings from complex mixtures containing proteins of various sizes and net charges, may not be achievable solely by an optimal balance of electrostatic and steric–entropic interfacial forces; other avenues must be invoked. Acknowledgements This work was partially supported by the Commonwealth Government under the Co-operative Research Centres Scheme (Co-operative Research Centre for Eye Research and Technology), by Australian Postgraduate Research Awards for S. McA. and P.K., and by the Ciba Vision Corporation (Atlanta, GA, USA). We thank Prof. A.S. Hoffman (University of Washington) for stimulating discussions. References [1] J.D. Andrade (Ed.), Surface and Interfacial Aspects of Biomedical Polymers, 2: Protein Adsorption, Plenum Press, New York, 1985.
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[2] J.D. Andrade, V. Hlady, Adv. Polym. Sci. 79 (1986) 1. [3] J.L. Brash, T.A. Horbett (Eds.), Proteins at Interfaces: Physicochemical and Biochemical Studies, ACS Symp. Ser. Vol. 343, American Chemical Society, Washington DC, 1987. [4] T.A. Horbett, J.L. Brash (Eds.), Proteins at Interfaces II: Fundamentals and Applications, ACS Symp. Ser. Vol. 602, American Chemical Society, Washington DC, 1995. [5] A.S. Hoffman, B.D. Ratner (Eds.), Abstracts, Symposium on Non-Fouling Surface Technologies, University of Washington, Seattle WA, July 30 – 31, 1998. [6] W.R. Gombotz, W. Guanghui, T.A. Horbett, A.S. Hoffman, J. Biomed. Mater. Res. 25 (1991) 1547. [7] M. Zhang, T. Desai, M. Ferrari, Biomaterials 19 (1998) 953. [8] S. Lo¨fa˚s, B. Johnsson, J. Chem. Soc: Chem. Comm. (1990) 1526. [9] B. Crepon, F. Maillet, M.D. Kazatchkine, J. Jozefonvicz, Biomaterials 8 (1990) 248. [10] R.C. Chatelier, L. Dai, H.J. Griesser, S. Li, P. Zientek, D. Lohmann, P. Chabrecek, PCT Application EP 93/ 02420/ WO 94/06485. [11] E. O8 sterberg, K. Bergstro¨m, K. Holmberg, J.A. Riggs, J.M. Van Alstine, T.P. Schuman, N.L. Burns, J.M. Harris, Colloids Surfaces A: Physicochem. Eng. Aspects 77 (1993) 159. [12] R.E. Marchant, S. Yuan, G. Szakalas-Gratzl, J. Biomater. Sci. Polym. Edn. 6 (1994) 549. [13] J. Toufik, M.P. Carreno, M. Jozefowicz, D. Labarre, Biomaterials 16 (1995) 993. [14] A.M. Rich, E. Pearlstein, G. Weissmann, S.T. Hoffstein, Nature 293 (1981) 224. [15] M. Morra, C. Cassinelli, L. Benedetti, L. Callegaro, Patent WO 96/24392. [16] J.G. Steele, B.A. Dalton, G. Johnson, P.A. Underwood, J. Biomed. Mater. Res. 27 (1993) 927. [17] K.M. McLean, R.C. Chatelier, G.J. Beumer, N. Brack, G. Johnson, M. Jenkins, J.G. Steele, H.J. Griesser, Trans. Am. Soc. Biomater. 22 (1999) 329. [18] R.Z. Xie, D.F. Sweeney, G.J. Beumer, G. Johnson, H.J. Griesser, J.G. Steele, Austral. N.Z. J. Ophthalmol. 25 (1997) S46. [19] R. Paynter, B.D Ratner, in: J.D. Andrade (Ed.), Surface and Interfacial Aspects of Biomedical Polymers. Vol. 2. Protein Adsorption. Plenum Press, New York, 1985, p. 189. [20] D.S. Mantus, B.D. Ratner, B.A. Carlson, J.F. Moulder, Anal. Chem. 65 (1993) 1431. [21] P. Kingshott, H.A.W. St John, R.C. Chatelier, F. Caruso, H.J. Griesser, Am. Chem. Soc. Polym. Prep. 38 (1997) 1008. [22] P. Kingshott, H.A.W. St John, R.C. Chatelier, H.J. Griesser, Polym. Mater. Sci. Eng. 76 (1997) 81. [23] P. Kingshott, H.A.W. St John, T.C. Vaithianathan,
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Effect of polysaccharide structure on protein adsorption Sally L. McArthur a,b, Keith M. McLean a,b, Peter Kingshott a,b, Heather A.W. St John a,b, Ronald C. Chatelier a,b, Hans J. Griesser a,b,* b
a CSIRO Molecular Science, Pri6ate Bag 10, Clayton South MDC, Clayton 3169, Australia Cooperati6e Research Centre for Eye Research and Technology, Uni6ersity of New South Wales, Sydney 2052, Australia
Received 2 November 1998; accepted 7 June 1999
Abstract Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and g-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Protein adsorption; Polysaccharide; Carboxymethyldextran; Surface modification; Plasma polymer; Lysozyme; Albumin; Lactoferrin; Immunoglobulin
1. Introduction
* Corresponding author. Tel.: +61-3-95452611; fax: + 613-95452446. E-mail address: [email protected] (H.J. Griesser)
The uncontrolled adsorption of proteins from biological fluids, such as blood or tears, onto synthetic biomaterials triggers biological events including the host defense mechanisms, for instance fibrous encapsulation, complement activation and the coagulation cascade. These processes
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lead to adverse consequences that impact the long-term viability of the biomedical device. The adverse responses can span the range from being merely inconvenient, such as the fouling of contact lenses which entails the need for frequent cleaning or disposal of the lens, to the life-threatening, such as the clotting of small diameter artificial blood vessels. Thus, protein adsorption has been the subject of much research in the biomaterials field, and many reports have focused on unraveling its principles [1 – 4] in order to progress towards rational design of improved biomedical materials and coatings. For a number of applications, surfaces are desired that are highly hydrophilic and are able to prevent protein adsorption. Among the ‘non-fouling’ surface technology [5], coatings from polyethylene oxide (PEO) have attracted particularly strong attention [6,7]. Another class of molecules able to produce highly wettable, lowfouling coatings are polysaccharides, which like PEOs possess a heavily hydrated, hydrogel-like structure and mobile molecular chains that provide a steric – entropic contribution towards repulsion of protein adsorption. Surface-immobilised polysaccharides have been investigated for a variety of biomaterials and biosensor applications [8–13]. At the macroscopic level, polysaccharide coatings have been found to markedly reduce the adherence of cells [14] and bacteria [15] to synthetic biomaterials. At the molecular level, however, the terminology ‘non-fouling’ or ‘anti-fouling’ may not be applicable to many candidate coatings. Absence of cell attachment may be a consequence of the preferential competitive adsorption of ‘wrong’ proteins that are incapable of supporting cell attachment, or adsorbed celladhesive glycoproteins denaturing so extensively that cells are no longer able to bind to them [16], or the test used. As an example of the latter, whereas in in-vitro cell assays polysaccharide coatings can achieve very low levels of attachment even of relatively robust cells such as bovine corneal epithelials [17], there is significant colonization in vivo onto such coatings [18]. Moreover, protein adsorption onto biomaterials has at times been studied using only one or
very few proteins and with assays that lack monolayer sensitivity. Complex biological solutions comprise proteins, lipids, and mucins with widely different properties such as overall charge, hydrophobicity/hydrophilicity, and conformational flexibility, and repellency of all these species is a considerable challenge. Some conventional biochemical assays can also be affected by incomplete desorption of analytes and transfer losses. Physico-chemical surface analysis methods such as X-ray photoelectron spectroscopy (XPS) [19], time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) [20], Surface-MALDI mass spectrometry [21–23], and surface plasmon resonance [24] offer advantages in terms of detection levels much below monolayer coverage. These methods have been applied to various ‘nonfouling’ surface coatings [5] and have enabled documentation of protein adsorption at very low but perhaps biologically sensitive levels. Using such methods we found both in clinical trials of polysaccharide-coated contact lenses [25] and on immersing polysaccharide coatings into an artificial tear fluid formulation comprising 14 constituents representing proteins, lipids, and mucin [22], that they are not entirely resistant to protein adsorption, although it occurred at relatively low rates and to submonolayer amounts onto some of them [22,23,25,26]. The questions therefore arose, which proteins and lipids were capable of adsorbing onto these coatings, what the mechanisms of affinity interaction are between the coatings and these biomolecules, and which properties and structural features of polysaccharide coatings can be tailored to minimise protein adsorption or affinity-select particular proteins. The present report describes model studies investigating the adsorption behaviour of four representative proteins of different charge, molecular size, and structural stability onto a series of coatings produced with systematic variation in properties. The coatings consist of covalently immobilised carboxymethyldextrans (CMDs) varying in the density of carboxyl substitution on the molecular chain. We aimed to vary the density of surface ‘pinning’ (attachment points per chain) and the residual charge density, thus manipulating protein adsorption via a combination of electrostatic and
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steric–entropic interactions. The pinning density affects the mobility of chain segments and thus the steric–entropic contribution against protein adsorption, which was quantified by XPS.
2. Materials and methods
2.1. Materials Chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin and g-globulin (from Cohn fraction II and III), were purchased from Sigma-Aldrich (St. Louis, MO). Protein solutions were freshly prepared by dissolving proteins in phosphate-buffered saline (PBS) at pH 7.4, to give a final concentration of 10 mM for g-globulin and 20 mM for the others. Carboxymethyldextrans (CMDs) with different ratios of carboxyl groups to anhydroglycopyranoside ring units were prepared in a method adapted from Lo¨fas and Johnsson [8]. Dextran (10 g) (MW 70 000, Sigma-Aldrich, St Louis) was dissolved in 50 ml 2 M NaOH containing either 0.125, 0.25 or 1 M bromoacetic acid. The solution was stirred overnight, dialysed against water for 24 h, against 0.1 M HCl for 24 h and finally for 24 h against water. The solution was then lyophilised and stored at 4°C until required. The degree of carboxylation was assessed using back titration and NMR. The experimentally determined ratios were 1:2. 1:14 and 1:30, carboxyl groups per sugar unit. The resultant substitution is illustrated in Fig. 1.
39
2.2. Surface modification CMD was grafted, using water soluble carbodiimide chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccin imide), to fluorinated ethylene propylene (Teflon FEP, 100 Type A; Du Pont) tape following surface amine functionalisation by radio frequency glow discharge plasma polymerisation. FEP is a convenient substrate as it does not contain low molecular weight constituents (plasticisers, other additives, oligomers) that might outdiffuse. In addition, the fluorine atom possesses a high XPS intensity and thereby facilitates detection of any coating non-uniformities and of any partial delamination that might occur on immersion in saline media. It is essential to be alert to such artefacts which might affect reliability and interpretation of protein adsorption experiments. The methods used are described elsewhere for the application of the plasma-polymerised, aminegroup-containing interlayer coating [27] and the subsequent immobilisation of CMDs [28]. Briefly, plasma polymerisation of n-heptylamine was used to deposit a coating with surface amine functional groups on the FEP substrate in a custom-built plasma apparatus [29], employing an ENI HPG-2 high frequency plasma generator. After incubation all samples were washed thoroughly in MilliQ water to remove any excess reagents and then stored in water until required.
2.3. Protein adsorption Samples were rinsed in MilliQ water and placed in clean 55 mm diameter polystyrene petri dishes to which 5 ml of the desired protein solution was added. Adsorption was allowed to proceed at room temperature for 1 h after which samples were washed three times in MilliQ water to remove loosely adsorbed proteins and salts. Samples were dried prior to analysis by XPS.
2.4. X-ray photoelectron spectroscopy
Fig. 1. Carboxymethyl substitution of dextran.
X-ray photoelectron spectroscopy (XPS) analysis was performed using a AXIS HSi spectrometer (Kratos Analytical Ltd) equipped with a
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Table 1 Elemental composition determined by XPS of the coatings Surface
FEP FEP+Happ FEP+Happ+CMD 1:2 FEP+Happ+CMD 1:14 FEP+Happ+CMD 1:30
Atomic concentration (%)
Atomic ratios
C1s
O1s
N1s
F1s
Si 2p
N:C
O:C
33.0 86.2 75.0 75.9 76.1
0.0 6.8 19.4 17.9 17.6
0.0 7.0 5.5 5.6 5.7
67.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
n/a 0.08 0.07 0.07 0.07
n/a 0.08 0.26 0.24 0.23
monochromatic Al Ka source at a power of 300 W. The total pressure in the main vacuum chamber during analysis was typically 2 ×10 − 8 mbar. Elements present were identified from survey spectra. For further analysis, high resolution spectra were recorded from individual peaks at 40 eV pass energy. Atomic concentrations of each element were calculated by determining the relevant integral peak intensities (using a linear type background) and applying the sensitivity factors supplied by the instrument manufacturer. The random error associated with elemental quantification has been determined for this instrument to be 1–2% of the absolute values for atomic percentages in the range encountered in this study (\ 5%). The systematic error is much more difficult to determine and is generally assumed to be of the order of 5 – 10% [30]. It is the random error that is more relevant when comparing samples of similar composition. A value of 285.0 eV for the binding energy of the main C1s component (CHx ) was used to correct for charging of specimens under irradiation [31]. Assuming a value of 3 nm for the electron attenuation length of a C1s photoelectron in a polymeric matrix [30], this translates into an approximate value for the XPS analysis depth (from which 95% of the detected signal originates) of 10 nm when recording XPS data at an emission angle normal to the surface, and 2 nm when recording at an emission angle of 75° relative to the surface normal (‘grazing angle’). A minimum of three samples for each experimental configuration were analysed.
2.5. Contact angle measurements A modified Kernco model G-II goniometer was used for the determination of air/water contact angles. The goniometer was equipped with a syringe comprising a micrometer-driven plunger whose direction of travel could be reversed in order to enable determination of sessile, advancing and receding contact angles [32].
3. Results
3.1. Coatings As a reference, ‘control’ surface for comparison with CMD coatings in protein adsorption experiments we used FEP coated with a thin layer of n-heptylamine plasma polymer (HApp). This coating is solid and dense, and therefore contributes minimal interfacial steric–entropic repulsion effects. It also possesses a very low density of positive surface charge in pH 7.4 buffered saline [32,33], since its low dielectric constant reduces the thermodynamic stability of charged amine groups and thus allows substantial protonation of amine groups only at pH valuesB 7. Hence, interfacial electrostatic forces are small, and protein adsorption onto the HApp layer may be presumed to occur predominantly by dispersion and hydrophobic forces. Comparison with CMD coatings thus allows assessment of additional forces, particularly of electrostatic and steric–entropic origins. XPS analysis indicated that the HApp layer was thicker than the analysis depth of XPS
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(\ 10 nm) and continuous as indicated by both the elimination of fluorine signal from the underlying FEP substrate (Table 1) and the absence of a component at 292.2 eV characteristic of CF2 structures in the XPS C 1 s high resolution spectrum (spectrum not shown). Following attachment of the CMDs onto the HApp coatings, significant increases in the XPS O:C ratios (Table 1) and the introduction of significant CO and CO components in the C1s high resolution spectra were observed [28]. No F signal was observed (Table 1), showing that the HApp layer did not partially delaminate in the aqueous carbodiimide/NHS/CMD reaction solutions. The three CMD coatings produced similar XPS results, indicating that all three CMDs produced similar amounts of surface coverage. The fact that a N signal was still observed in XPS indicates incomplete attenuation of the N 1s photoelectron emission originating from the HApp layer as this emission passes through the CMD overlayer. Therefore, the CMD coatings in the ‘dry’ state, as applies for XPS analysis, are thinner than 10 nm. XPS mapping and the uniform wettability of the samples indicated that the coatings were continuous and uniform over the sample surfaces. The sessile contact angles were 20° for all the CMD coatings and 70° for the HApp surface.
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3.2. Protein adsorption to n-heptylamine plasma polymer XPS was used to monitor the adsorption of lysozyme, human serum albumin, lactoferrin and g-globulins to HApp coated FEP samples. The presence of adsorbed proteins was clearly evident in XPS C 1s high resolution spectra; an example is shown in Fig. 2. For quantitative analysis of protein adsorption, changes in atomic concentrations measured by XPS were used, similar to reported procedures [19,34]. Fig. 3 shows the increases in the XPS N 1s signals on adsorption of proteins to the HApp surface. The increases in the N content over that of the non-immersed HApp sample are a measure of the amount of immobilised protein. They are, however, not a linear measure, and multilayer XPS algorithms [35] must be used to calculate the absolute amounts of surfacebound protein. In these cases, a three-layer model (HAppB CMDB protein) applies. While this can be done, it is not necessary for the purposes of the following discussion, for which the ‘raw’ data of XPS elemental compositions are sufficient and enable comparisons between proteins and coatings. The N contents shown in Fig. 3 after protein adsorption correspond to approximately monolayer coverage, with the size and relative stability
Fig. 2. High resolution XPS C1s spectra recorded on a HApp coated FEP sample before (solid line) and after (dashed line) immersion in an IgG solution.
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3.3. Protein adsorption to CMD coatings
Fig. 3. Atomic% nitrogen, recorded by XPS, before and after adsorption of proteins to HApp coated FEP.
of the proteins influencing the surface coverage and thus the nitrogen signal recorded using XPS. As expected on the basis of molecular size (Table 2), IgG gives the largest increase in the N content. The effect of protein stability is apparent in the HSA datum. This protein is present at lower coverage than the similarly sized lactoferrin; this may be due to ‘flattening’ of HSA as it denatures after adsorption. Each denatured HSA molecule then covers a larger surface area than expected on the basis of its solution equilibrium conformation. In addition, comparing the nitrogen signal from the lysozyme-adsorbed sample to that of the HSA-adsorbed sample shows that coverage is not solely dependent on molecular weight. We would expect that due to its approximately five-fold increase in molecular weight, HSA adsorption would result in a significant increase in the N signal when compared to the lysozyme sample, yet there is little difference between the two. This may be accounted for by the highly stable nature of lysozyme that prevents the protein from denaturing on the plasma polymer surface and produces a densely packed protein coverage. The ‘softer’ albumin molecules have greater conformational freedom; they spread and denature over the surface as they adsorb, producing a protein layer thinner than expected on the basis of molecular dimensions in solution.
All of the CMD coatings examined were found to adsorb proteins, but the amounts varied considerably with the surface and the particularprotein, and the presence of the CMDs significantly influenced the protein adsorption behaviour. For the CMD 1:2 coating, high resolution XPS C 1s spectra revealed the presence of lysozyme (Fig. 4), lactoferrin and IgG (spectra not shown) via significant increases in the spectral region where amine/hydroxyl and amide components are expected to contribute (286.5 and 288.5 eV respectively). For HSA adsorption, on the other hand, the spectra are less conclusive of adsorption (Fig. 4). There is only a very small increase in intensity in the amide spectral region. Grazing angle XPS spectra, by virtue of their higher sensitivity to the outermost 2 nm of the analyte surface, provided a clearer indication of HSA adsorption (Fig. 5) although again the small amide intensity (288.5 eV) suggested that the adsorbed amount was minimal. As above, the increases in the XPS N percentages upon protein adsorption can be used for quantitative comparisons. Fig. 6 shows data for the adsorption of the four proteins onto the CMD 1:2 coating. In agreement with the C 1s spectra, the elemental ratios showed that lysozyme, lactoferrin and IgG adsorbed to substantial amounts onto the CMD 1:2 surface (D%N =5, 8, and 3, respectively, compared with the non-exposed CMD coating), whereas there appeared to be no HSA adsorption within experimental uncertainty. There are marked differences in the protein adsorption characteristics due to the CMD 1:2 layer: this polysaccharide coating is certainly not non-fouling towards lysozyme, lactoferrin and IgG, and adsorbs even larger amounts of the first two proteins than the solid plasma polymer coating which does not possess steric–entropic interfacial effects. In turn, the CMD 1:2 surface almost entirely prevents adsorption of HSA, which adsorbs efficiently onto typical solid coatings of moderate hydrophobicities in the range similar to HApp (sessile contact angle of 70°). Thus, we
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43
Table 2 Isoelectric point and molecular weight of the proteins used Protein
pI
MW (Da)
Lysozyme (Lyz) Human serum albumin (HSA) Lactoferrin (Lf) Immunoglobulin G (IgG)
11.1 4.7 8 6.0–8.0
14 500 66 250 84 000 150 000
would expect that in competitive adsorption experiments HSA should compete very inefficiently with the other proteins for surface adsorption sites on the CMD 1:2 coating. As such, the CMD coating is not non-fouling but it clearly can be expected to alter the balance in competitive adsorption from multicomponent solutions. These data illustrate the dangers inherent in labelling a coating non-fouling on the basis of exposure to one protein only. The CMD coating with an intermediate carboxyl density (1:14) performed in qualitatively the same way as the CMD 1:2 coating except that the adsorbed amounts of lysozyme, lactoferrin and IgG were slightly less than on CMD 1:2 (data not shown). In contrast, the CMD 1:30 coating showed different patterns of behaviour (Fig. 7). Compared with CMD 1:2 (Fig. 6), the data manifested a significant increase in HSA adsorption
(D%N =1.4), but a decrease in adsorption of lysozyme and lactoferrin (D%N = 1.7 and 4.6, respectively). There was an increase in adsorbed g-globulin (D%N =4.7) when compared to CMD 1:2. Figs. 8 and 9 show XPS data recorded at grazing angle, which enables probing of the outermost 2 nm and hence, by comparison with data recorded at normal emission, assessment of depth distributions. Again we plot the data in terms of
Fig. 4. XPS high resolution C1s spectra of FEP+ HApp + CMD 1:2 coated samples before (solid thick line) and after adsorption of lysozyme (solid thin line) and HSA (dashed line).
Fig. 6. Percent atomic nitrogen recorded by XPS following adsorption of proteins to CMD 1:2 coatings.
Fig. 5. XPS high resolution C1s spectra recorded at grazing angle (75°) of FEP +HApp +CMD 1:2 coated samples before (solid line) and after (dashed line) HSA adsorption.
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Fig. 7. Percent atomic nitrogen recorded by XPS following adsorption of proteins to CMD 1:30 coatings.
XPS ‘raw’ data since conversion to depth distributions by algorithms such as those used for XPS depth profiling [38] relies on several assumptions whose potential error contributions are difficult to assess for the present case of CMD coatings. However, comparison of data collected at normal emission with data collected at grazing angle (75°) enables qualitative assessment of in-diffusion. If all protein molecules were located at the aqueous
Fig. 9. Percent atomic nitrogen recorded by XPS at normal and grazing angle (75°) following adsorption of proteins to CMD 1:30 coatings.
interface, ‘on top of’ the CMD coatings, then a substantially higher nitrogen content should be evident in grazing angle XPS data. However Figs. 8 and 9 do not show pronounced differences in most cases, suggesting significant diffusion of proteins into the coatings.
4. Discussion
4.1. Protein adsorption to n-heptylamine plasma polymer
Fig. 8. Percent atomic nitrogen recorded by XPS at normal and grazing angle emission (75°) following adsorption of proteins to CMD 1:2 coatings.
There are a number of interfacial interactions between surfaces and proteins that can lead to spontaneous irreversible protein adsorption. Most proteins carry a net charge in physiological media, with the sign and magnitude of the net charge depending on the isoelectric point of the protein (Table 2). Electrostatic interactions between proteins and charged surfaces therefore often play a major role in the adsorption behaviour of the proteins. The HApp coating carries little charge; its surface contains mainly amine and amide groups [27,36], with protonation of the former inefficient at pH 7.4 [32,33]. Together with charge screening in the PBS medium, this results in an low level of surface charge under our protein adsorption conditions. The putative lack of elec-
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trostatic interactions is reflected in the protein adsorption results with both overall positive (lysozyme and lactoferrin) and negative (HSA) proteins adsorbing virtually equally well to the HApp surface. The main driving forces for protein adsorption would in this case appear to be dispersive and hydrophobic interactions. This interpretation extends earlier observations of adsorption of lysozyme onto HApp coatings [21] where it was observed that this overall positively charged protein readily and rapidly adsorbed onto HApp, consistent with the absence of strong electrostatic repulsion that would occur if a substantial number of surface amine groups were protonated.
4.2. Protein adsorption to CMD coatings Application of the CMD coatings on top of the HApp layer markedly changes protein adsorption behaviours, in ways that indicate that dispersive and hydrophobic interactions no longer dominate the driving forces for adsorption. Instead, the observed differences in the adsorption of the four test proteins to CMD coatings relative to the HApp coating can tentatively be interpreted in terms of electrostatic and steric – entropic interfacial forces emanating from these hydrogel-like coatings, and, when comparing the CMD coatings, in terms of differences in these forces. As the net charge of the proteins differs, differences in their adsorption behaviour should reflect electrostatic interactions with charged surfaces. Electrostatic interactions are clearly apparent in the results obtained with the CMD 1:2 coating, with the positively charged proteins lysozyme and lactoferrin strongly adsorbing and a very low level of adsorption of the overall negatively charged HSA. The importance of electrostatic forces in adsorption onto CMD 1:2 and CMD 1:14 is evident in the data of Fig. 6; the two proteins with a net positive charge adsorb substantially more. On CMD 1:30 the effect is less pronounced. The different densities of carboxyl groups on the CMD moieties will lead to different magnitudes of negative surface charge after attachment. However, the magnitudes of the surface charge obtained after covalent immobilisation of the three
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different CMDs onto amine surfaces are currently undetermined since the techniques that have been applicable to well-defined, solid coatings [32,33] do not work with the hydrogel-like, expanded CMD coatings; we are currently attempting to use zeta potential measurements. In the meantime, one can reasonably assume that because of the relatively low surface amine density on the HApp layer [27], many of the carboxyl groups are not converted to amides during the attachment process, particularly for CMD 1:2. For all the CMDs we expect that the low ionic strength of the CMD reaction media together with the branched structure of the dextran molecules would, by electrostatic repulsion between carboxyl groups, lead to an expanded molecular structure which, on approach to the surface, would place a substantial fraction of the carboxyl groups beyond the reach of interfacial reaction with surface amine groups. The high carboxyl density particularly of CMD 1:2 will enable it, however, to react efficiently with many of the available amine groups, and hence we expect this coating to have the highest density of surface pinning points and thus the least segmental mobility, which means less steric–entropic repulsion towards proteins. We picture the CMD 1:2 coating as possessing a high density of remaining carboxyl groups and being well pinned to the HApp layer, producing a relatively dense polysaccharide structure. At the other end of the spectrum is the CMD 1:30 coating; the low density of carboxyl species in CMD 1:30 results in a lower level of pinning, producing a more open, ‘floppy’ coating, with much less residual charge but much higher interfacial entropy than the other coatings. On the CMD 1:30 coating, the relative levels of protein adsorption do not correlate with expectations based on dominant electrostatic interactions; HSA adsorbs onto the like-charged surface, although to a lesser amount than the positively charged proteins. One possible interpretation for the observed HSA adsorption on CMD 1:30 is that repulsive electrostatic interactions and steric– entropic effects (polysaccharide chain mobility) are not sufficient to counteract attractive forces, which could arise from the HApp substrate ‘shining through’ the thin CMD coating and from entropic gains when water molecules are liberated
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from within the CMD coating as protein molecules take their place. In the case of the CMD 1:2 coating, in contrast, the coating structure and/or the surface carboxyl charge density may be sufficiently dense to prevent significant in-diffusion of HSA and the concomitant energy gains. While it appears reasonable to expect that the chain segments of the CMD 1:30 coating, being pinned at large distances, have considerable mobility, the resultant interfacial entropy evidently is not sufficient to overcome the attractive forces driving towards protein adsorption. Another factor may also be relevant for HSA and IgG adsorption. Whilst the adsorption characteristics of smaller molecular weight proteins such as insulin, lysozyme and myoglobin can be interpreted in terms of single-domain, relatively homogeneous structures, larger, more complex proteins such as HSA and IgG cannot be as readily defined. It has been suggested by Andrade et al. [37] that these proteins should be considered as multidomain structures, with each domain having differing properties in terms of charge, stability and thus propensity to adsorb and denature on specific surface. Whilst proteins such as fibrinogen and fibronectin are pictured with a large number of domains (12 and 20 respectively), for albumin a simpler ‘tennis ball’ model of three 20 kDa domains has been proposed. In the model domains I and II are strongly negatively charged and contain fewer di-sulfide bonds than the weakly positively charged domain III [37]. As a result, on a negatively charged surface such as CMD 1:2, HSA would tend to adsorb weakly via domain III, but due to the domain stability would tend not to denature. The highly hydrated nature of the CMD surface would also reduce forces towards structural rearrangements of the HSA and hence, the hydrophobic regions of the protein would remain internalised and the hydrophilic (charged) residues would interact specifically with the CMD surface. The multidomain structure concept also applies to IgG. While some domains of this protein may be electrostatically attracted to the highly charged CMD 1:2 surface, others are attracted to the less charged CMD 1:30 surface and may interact with the surface via other interactions. This is reflected
in the data showing that all of the CMD surfaces attracted significant amounts of IgG. It appears to be much more challenging to reduce/prevent the adsorption of large, multidomain proteins, an issue which some studies of ‘non-fouling’ coatings have overlooked. Interpretation in terms of interfacial forces assumes protein adsorption as a discrete layer on top of the CMDs. To what extent can the protein molecules diffuse into the thin coatings? The grazing angle XPS data enable probing of the outermost region of the sample surface and hence, by comparison with data recorded at normal emission, assessment of discrete versus diffuse layer structures. For the CMD 1:2 coated samples, grazing angle XPS data compared with normal emission data (Fig. 8) suggest, by the increases in the nitrogen signals, that the proteins are somewhat enriched towards the outermost surface. It appears, therefore, that whilst there is some indiffusion, the proteins are not uniformly distributed throughout the thickness of the polysaccharide coating. For lactoferrin, assessment of in-diffusion is more difficult as the observed compositions approach the bulk composition of proteins and therefore variations with emission angle analysis are expected to be less pronounced. In the case of CMD 1:30 (Fig. 9) there are no pronounced differences when comparing normal emission and grazing angle data, suggesting a substantial extent of diffusion of the proteins into this coating. Thus, it appears that the ‘biofouling’ behaviour of these coatings must be interpreted not just in terms of interfacial forces; in-diffusion of proteins is substantial in some cases and gives rise to additional effects such as the entropic gain when water molecules are released from within the coating. A more densely attached coating such as CMD 1:2 can reduce such in-diffusion but is expected to possess less mobility of the attached chains, which reduces the effectiveness of the steric barrier.
5. Conclusions On all the hydrophilic, negatively charged carboxymethyl-dextran coatings of varying carboxyl
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group density investigated, all four test proteins can adsorb to some extent, even though for HSA, which carries a net negative charge, this would seem electrostatically unfavourable. The data suggest that elimination of adsorption of a broad spectrum of proteins is not a simple matter with negatively charged polysaccharide coatings (the majority of natural and available polysaccharides being negatively charged). While increasing the steric–entropic forces by decreasing the pinning density can lead to a reduction in adsorption of some proteins, the adsorption of others (HSA in this instance) can actually increase, and yet others (IgG) remain unaffected, adsorbing to approximately monolayer coverage regardless of structural variations in the carboxymethyl dextran coatings. It appears from our data that increasing the entropic forces by generating more flexible surface-attached macromolecular structures, also unfortunately leads to more open coatings that then allow in-diffusion of proteins. At this point in time we conjecture that ‘non-fouling’ in terms of elimination, or much reduced levels, of protein accumulation onto/into such coatings from complex mixtures containing proteins of various sizes and net charges, may not be achievable solely by an optimal balance of electrostatic and steric–entropic interfacial forces; other avenues must be invoked. Acknowledgements This work was partially supported by the Commonwealth Government under the Co-operative Research Centres Scheme (Co-operative Research Centre for Eye Research and Technology), by Australian Postgraduate Research Awards for S. McA. and P.K., and by the Ciba Vision Corporation (Atlanta, GA, USA). We thank Prof. A.S. Hoffman (University of Washington) for stimulating discussions. References [1] J.D. Andrade (Ed.), Surface and Interfacial Aspects of Biomedical Polymers, 2: Protein Adsorption, Plenum Press, New York, 1985.
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