Chitosan-N-poly(ethylene oxide) brush polymers for reduced nonspecific protein adsorption

Journal of Colloid and Interface Science 305 (2007) 62–71 www.elsevier.com/locate/jcis

Chitosan-N -poly(ethylene oxide) brush polymers for reduced nonspecific protein adsorption Ye Zhou a,∗ , Bo Liedberg a , Natalija Gorochovceva b , Ricardas Makuska b , Andra Dedinaite c,d , Per M. Claesson c,d a Division of Molecular Physics, Department of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden b Department of Polymer Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania c Department of Chemistry, Surface Chemistry, Royal Institute of Technology, SE-100 44 Stockholm, Sweden d Institute for Surface Chemistry, Box 5607, SE-114 86 Stockholm, Sweden

Received 24 July 2006; accepted 19 September 2006 Available online 26 September 2006

Abstract The possibility of using a novel comb polymer consisting of a chitosan backbone with grafted 44 units long poly(ethylene oxide) side chains for reducing nonspecific protein adsorption to gold surfaces functionalized by COOH-terminated thiols has been explored. The comb polymer was attached to the surface in three different ways: by solution adsorption, covalent coupling, and microcontact printing. The protein repellant properties were tested by monitoring the adsorption of bovine serum albumin and fibrinogen employing surface plasmon resonance and imaging null ellipsometry. It was found that a significant reduction in protein adsorption is achieved as the comb polymer layer is sufficiently dense. For solution adsorption this was achieved by adsorption from high pH solutions. On the other hand, the best performance of the microcontact printed surfaces was obtained when the stamp was inked either at low or at high pH. For a given comb polymer layer thickness/poly(ethylene oxide) density, significant differences in protein repellant properties were observed between the different preparation methods, and it is suggested that a reduction in the mobility of the comb polymer layer generated by covalent attachment favors a reduced protein adsorption. © 2006 Elsevier Inc. All rights reserved. Keywords: Comb polymer; Comb polyelectrolyte; Chitosan; Poly(ethylene oxides); Nonspecific binding; Adsorption; Microcontact printing; Surface plasmon resonance; Imaging null ellipsometry

1. Introduction Block copolymers in selective solvents are known to be efficient steric stabilizers and provided one block, the anchor block, adsorbs strongly to the adsorbent whereas the other block, the buoy block, experiences good solvency conditions and thus extends into solution forming a steric barrier against flocculation [1]. A related but less investigated class of polymer is the comb copolymers, with the general formula A(B)n , where a number of n buoy blocks are grafted to one anchoring chain. Surprisingly little is known about the interfacial properties of comb copolymers, and nearly all of the experimental and theoretical work reported in the literature is concerned with comb * Corresponding author. Fax: +46 1328 89 69.

E-mail address: [email protected] (Y. Zhou). 0021-9797/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2006.09.058

copolymers of rather low graft density (see, e.g., [2–4]), even though some recent publications have considered comb polymers with high density of side chains [5–8]. Comb polymers with an adsorbing backbone and nonadsorbing side chains will present a large number of tails toward solution, the density of which locally is determined by the graft density of the side chains to the polymer backbone. In cases where the backbone of a comb polymer with a high density of side chains can be assembled to fully cover the surface, the nonadsorbing side chains will be highly extended and form a brush layer that is expected to act as an efficient steric barrier, as also observed experimentally [6–8]. Such a brush layer is expected to have useful properties in many applications, for instance as steric stabilizers for colloidal dispersions [9] and as lubricants [10]. Another application area is in the biomedical and diagnostics field where surface coatings with low nonspecific protein bind-

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Fig. 1. The structural elements in the PEO-derivatized chitosan Chi-N -PEO. The polymer consists of a chitosan backbone, Chi, with grafted 44 units long poly(ethylene oxide) side chains grafted to the nitrogen atom in chitosan, EO44 N. 72% of the sugar units are deacetylated, and 56% of all sugar units carry a grafted EO44 chain. Note that the polymer is not a block copolymer but the different types of segments are randomly distributed along the backbone.

ing are needed. To this end, excellent protein repellent properties were reported for a comb copolymer consisting of a polylysine backbone and poly(ethylene oxide), PEO, grafts [11]. Similar results have previously been obtained by using PEO grafted to poly(ethylene imine), PEI [12]. PEO side-chains, and other nonionic hydrophilic side chains, are particularly interesting in this respect since protein adsorption to such surfaces is generally low, see for instance [13,14]. The forces between PEO coated surfaces and protein layers have been directly measured and found to be dominated by steric repulsion [15], and this repulsive interaction is important for the protein repellency. It has been suggested that efficient steric stabilization is achieved if the surface spacing of bound PEO units is less than the radius of gyration of PEO in solution [16]. Another related factor of importance is the significant hydration of the ethylene oxide chain, around which clathrate-like dynamic water structures are formed [17]. Surface layers containing PEO can be prepared in a large number of ways, for instance through adsorption from solution, by covalent binding or by microcontact printing, µCP. In array applications, the microcontact printing technique offers an appealingly straightforward route to achieve patterns in the µm size range [18,19]. In this work we utilized a recently described class of comb polyelectrolytes consisting of a chitosan backbone and a high density of grafted PEO side chains [20,21] to coat gold surfaces functionalized with 16mercaptohexadecanoic acid. The comb polymer surface layers were prepared by adsorption, covalent attachment and microcontact printing, and their protein repellent properties were evaluated employing surface plasmon resonance (SPR) and imaging null ellipsometry (INE). 2. Materials and methods 2.1. Materials The derivatized chitosan polymer, Chi-N -PEO, consists of a chitosan backbone where 56% of the segments carry a 44 unit

long PEO side chain, which gives a graft ratio of 1.8, i.e., 5 out of every 9 chitosan units carry a grafted PEO chain. The polymer has been synthesized and characterized as described previously [22]. The structure of Chi-N -PEO is depicted in Fig. 1. A 0.1 wt% Chi-N -PEO solution was prepared by dissolving the polymer in Milli-Q water without any added salt. The solution was ultrasonicated for 20 min to speed up the dissolution of the polymer. N -ethyl-N  -(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC, and N -hydroxysuccinimide, NHS, and ethanolamine, gifts from Biacore AB, were used as received. Bovine serum albumin (BSA) and fibrinogen was obtained from Sigma. 2.2. Surface preparation Self-assembled monolayer (SAM) coated gold surfaces were prepared in the following manner: The gold surfaces were first cleaned by rinsing in a 5:1:1 mixture of Milli-Q water, 25% hydrogen peroxide, and 30% ammonia for 5 min at 85 ◦ C, followed by rinsing in Milli-Q water and drying in a stream of N2 . Subsequently the cleaned gold surfaces were incubated in a 1 mM 16-mercaptohexadecanoic acid (HS(CH2 )15 COOH (COOH hereafter)) ethanol solution for at least 24 h, and then the surfaces were ultra-sonicated in ethanol for 3 min to remove any loosely bound thiols. Immobilization of Chi-N -PEO onto COOH modified gold surfaces was performed by utilizing electrostatic interactions as well as by covalent coupling between the primary amine groups in chitosan and carboxylic acids in SAM. The electrostatic surface attachment of Chi-N -PEO was facilitated either by adsorption directly from aqueous solution or by µCP. The µCP surfaces were prepared using a patterned PDMS rubber stamp prepared as described previously [23]. The stamp was exposed to oxygen plasma for 20 s to increase the wettability of the PDMS surface. The stamp was subsequently inked with a 0.1 wt% Chi-N -PEO for 5 min and then dried with a N2 gas stream. The stamp was brought into contact with the COOH

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modified gold surface for 30 min. The printed surface was subsequently ultrasonicated in ethanol for 2 min to remove PDMS residues that may have been transferred from PDMS stamp [24] as well as loosely attached Chi-N -PEO molecules. Finally, the µCP surfaces with Chi-N -PEO were rinsed with Milli-Q water. 2.3. Methods 2.3.1. Surface plasmon resonance SPR measurements were performed on a commercial Biacore 2000 instrument, which consists of four flow channels. In two of these flow channels the COOH-modified gold surface was activated by injecting a mixture of 1:1 EDC (0.4 M)/NHS (0.1 M) for 7 min at a flow rate of 5 µl/min. This chemical treatment creates reactive N -hydroxysuccinimide esters on the surfaces that are able to form covalent bonds with primary amine groups [25,26]; meanwhile in the other two channels the COOH-modified gold surface was left intact. Finally, a 0.1 wt% Chi-N -PEO solution was flowed over the surface through all four flow channels for 15 min at a flow rate of 5 µl/min. Several different pH-values of the Chi-N -PEO solution were used in separate experiments. The surface was subsequently deactivated with 35 µl ethanolamine solution for 7 min. This procedure allows for covalent binding between Chi-N -PEO and activated COOH surface in two flow channels, whereas physical adsorption primarily driven by electrostatic interactions occurs within the other two channels. Nonspecific protein binding (NSB) onto these modified surfaces was evaluated by using two model proteins, BSA, pI = 4.7, and fibrinogen, pI = 5.1–6.3. It was evaluated by flowing the protein solution of BSA (2 mg/ml), fibrinogen (1 mg/ml), through the four channels, respectively, for 10 min at a flow rate of 5 µl/min. The proteins were in all cases dissolved in HBS-EP buffer at pH 7.4 ((Biacore AB) containing 10 mM 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.0005% surfactant P20). The effective thickness of the adsorbed proteins were calculated from the SPR measurements using the following formula [27]: d =A

∂n/∂c , (na − n0 )

(1)

where na (1.45 for adsorbed layer) and n0 (1.33 for Milli-Q water and buffer solutions) are the refractive indices of the layer and the bulk solution, respectively and A is the surface concentration of adsorbed proteins given by SPR measurement, in which the SPR signal 1000 RU corresponds to 1 ng/mm2 divided by 1.5 taking into account the fact that binding occurs on a planar surface, rather than at an extended dextran hydrogel that is the standard surface used in the Biacore instrument [28,29]. ∂n/∂c is the refractive index increment and the same value, 0.18 cm3 /g, was used for both model proteins [30]. However, for quantification of the adsorbed chitosan-PEO comb polymer from SPR measurements, the relation 1000 RU = 1 ng/mm2 is not valid, but the effective layer thickness and the surface density of adsorbed polymer can be calculated from

the SPR measurements by using the following equation [31]: d=

R Id , 2 m(na − n0 )

(2)

where R is the measured SPR response, Id is a characteristic evanescent electromagnetic field decay length and approximately estimated as 0.37 of the light wavelength, m is the refractive index unit (RIU), which is a measure of the sensitivity factor for the sensor. This quantity is obtained from the calibration of the SPR instrument, 111◦ /RIU was used in our case [28], na (1.45) and n0 (1.33) are the refractive indices for the adsorbed layer and medium, respectively. 2.3.2. Imaging null ellipsometry The patterned surfaces were studied by INE, from which both the thickness and morphology of the patterned surface could be explored. These measurements were carried out by employing an I-Elli2000 microscope from Nanofilm, Germany, having a lateral resolution of about 1 µm. The ellipsometer is equipped with a CCD camera and works at a single wavelength of 5320 Å and an incident angle of 61◦ relative to the surface normal of the substrate. 2.3.3. Dynamic light scattering Dynamic light scattering measurements were carried out with a BI-200SM goniometer system connected to a BI-9000AT digital correlator from Brookhaven Instruments and a watercooled Lexel 95-2 laser with maximum power of 2 W. The scattered light was measured at an angle of 90◦ relative to the incident beam (λ = 5140 Å). The samples were investigated at room temperature (20–23 ◦ C). 2.3.4. Small-angle neutron scattering Small-angle neutron scattering experiments were performed at the SANS1 instrument at the FRG1 research reactor at GKSS Research Centre, Geesthacht, Germany. The neutron wavelength was 8.1 Å and the wavelength resolution was 10% (fullwidth-at-half-maximum value). The range of scattering vectors (0.005 < q < 0.25 Å−1 ) was obtained using four sampleto-detector distances (0.7–9.7 m). The samples were kept at 25.0 ± 0.5 ◦ C in quartz cuvettes with a path length of 1 or 2 mm. The raw small-angle neutron scattering (SANS) spectra were corrected for backgrounds from the solvent, sample cell, and other sources by conventional procedures [32]. The two-dimensional isotropic scattering spectra were azimuthally averaged, and corrected for detector efficiency by dividing by the incoherent scattering spectra of pure water, which was measured with a 1 mm path length quartz cell. The smearing induced by the different instrumental settings is included in the data analysis. For each instrumental setting the ideal model cross section was smeared by the appropriate resolution function when the model scattering intensity was compared to the measured one by means of least-square methods [33]. The parameters in the models were optimized by conventional least-square analysis and the errors of the parameters were calculated by conventional methods [34].

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3.2. Polymer adsorption and patterning of COOH surfaces

(a)

(b) Fig. 2. (a) The cumulative distribution of the hydrodynamic diameter of ChiN -PEO measured by using dynamic light scattering method. (b) Small-angle neutron scattering curves for Chi-N -PEO in D2 O without any added salt. The Chi-N -PEO concentration was 2.5 wt% (filled squares), 1 wt% (unfilled squares), 0.5 wt% (filled circles), and 0.2 wt% (unfilled circles).

3. Results and discussion 3.1. Solution properties of Chi-N -PEO The intrinsic viscosity of Chi-N -PEO in 0.5 M CH3 COOH/ 0.5 M CH3 COONa at 25 ◦ C is [η] = 0.98. The distribution of the hydrodynamic diameter obtained by dynamic light scattering is shown in Fig. 2a. It falls in the span between 400 and 600 Å. The solution conformation, as probed by small-angle neutron scattering, indicates an extended and stiff conformation, with a slope of the ln I vs ln q curve very close to −1 at large scattering vectors, see Fig. 2b. This should be compared with a value of −1 that is characteristic of a stiff rod whereas a Gaussian chain in a good solvent would give a slope of −1.66, decreasing to −2 in a theta solvent. The change in slope at smaller q-values is due to electrostatic interactions between the polymer chains.

The polymer Chi-N -PEO is a weak polyelectrolyte where the intrinsic pKa of the chitosan backbone is 6.5, independent of the degree of deacetylation [35]. Hence, the charge of both the polymer and the surface depends on pH, and the interaction between Chi-N -PEO and the COOH SAM will also be pH-dependent. For this reason Chi-N -PEO was attached to the COOH surface, either through solution adsorption or µCP, employing several different pH-values: 2.0, 5.7, 8.9, and 11.1. We note that with increasing pH the charge density of the polymer decreases, whereas the surface charge density of the SAM increases. The amount of Chi-N -PEO attached to the COOH coated surface through adsorption from a 0.1 wt% aqueous solution is found to increase significantly with increasing solution pH, see Table 1 and Fig. 3a. It should also be noted that the layer obtained by electrostatically driven adsorption from solution contains significantly more polymer than the layer obtained through covalent grafting (Fig. 3b), and the thicknesses of the Chi-N -PEO layers obtained by µCP is also found to be smaller than those obtained by adsorption from solution (Fig. 4, Table 1). Let us first consider the results obtained by adsorption from solution. Fig. 3a shows SPR sensorgrams obtained for solution deposition of Chi-N -PEO at different pH-values. The effective polymer layer thicknesses (using Eq. (2)) were calculated and shown in Table 1 and a plot of adsorbed layer thickness as a function of pH in solution is shown in Fig. 4. In absence of nonelectrostatic driving forces for adsorption it is expected that polyelectrolyte adsorption to an oppositely charged surface proceeds until the charges in the adsorbed layer balance the surface charge density [36,37]. Thus, for a surface with a constant surface charge density one expects that the adsorbed amount increases with decreasing polyelectrolyte charge density, as also observed experimentally [38,39]. In our case the surface charge density increases while the polyelectrolyte linear charge density decreases with pH, and from this consideration one would expect an increased adsorption with increasing pH, at least as long as a sufficient electrostatic attraction between the polymer and the surface exists. This is also the trend observed in our data (Fig. 4). However, the increase in adsorption with pH is less significant than expected by this consideration alone. The fact that both the polymer and the surface can regulate its charge density during adsorption is expected to diminish the effect of the solution pH [37]. More important is that steric repulsion within the adsorption layer limits the adsorption of a low charge density polyelectrolyte on a highly charged surface [39]. This effect is particularly important for Chi-N -PEO where the space occupied by the EO44 chains limits the adsorption at high pH. Thus, the trend observed with changes in pH-value can be rationalized by considering the variation in charge density of the polyelectrolyte and the surface, and the steric hindrance imposed by the grafted hydrophilic side chains. It is furthermore suggested that the grafting density and the length of PEO side chains could be tuned to achieve surfaces displaying favorable coverage and steric hindrance.

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(a)

(b)

(c)

(d)

Fig. 3. SPR sensorgrams of (a) electrostatic adsorption and (b) covalent binding of Chi-N -PEO from solution at different pHs onto COOH coated surfaces; ellipsometric images of microcontact printed Chi-N -PEO at pH 2.0 onto COOH surface via (c) electrostatic adsorption and (d) covalent binding, respectively.

Table 1 Layer thickness of Chi-N -PEO on COOH surfaces prepared under different conditions pH 2.0 Preparation from solution

µCP (INE)

pH 5.7

pH 8.9

pH 11.1

5.5 1.1 10.7

10.6 0.7 0.6

12.4 0.4 ∼0

20.6 0 ∼0

Layer thickness (Å) Adsorbed

Chi-N-PEOa

Layer thickness (Å) Covalent attachment

Chi-N-PEOa BSAb Fib.b

0.9 1.2 9.0

2.2 0.8 6.3

1.7 1.3 7.8

8.0 ∼0 ∼0

Layer thickness (Å) Noncovalent

Chi-N -PEOc BSAc Fib.c

4.5 ∼0 6.3

2.8 2.5 11.0

2.4 3.8 21.2

7.1 ∼0 6.3

BSAb Fib.b

a The layer thickness was calculated from SPR measurements by using Eq. (2). b The protein layer thickness was calculated from SPR measurements by using Eq. (1). c The layer thickness was measured directly from imaging null ellipsometry by averaging 10 spots at different positions on at least two patterned samples.

The covalent attachment procedure resulted in considerably lower surface coverage than adsorption from solution (Fig. 3b, Table 1). Clearly, the presence of the grafted side chains counteracts the covalent attachment due to steric hindrance. The observation that the attachment process is more efficient at pH 11.1 compared to at lower pH-values remains unexplained (Fig. 4).

The µCP process is significantly different compared to the adsorption from solution procedure. Here, the patterned stamp is inked with 0.1 wt% Chi-N -PEO solution at different pHvalues, then dried and put into contact with the COOH surface. Thus, the pH used during the inking process does not directly influence the charge state of the COOH surface. Nevertheless, we do observe a distinct pH-dependence in the amount of

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ward the stamp surface exposing the PEO chains toward the SAM surface during µCP. Clearly, this will increase the steric resistance toward forming the short-range contact between the amines of the polymer and the activated ester of the SAM that is needed in order to achieve covalent attachment. 3.3. Protein adsorption to Chi-N -PEO coated surfaces

Fig. 4. Effective layer thickness of Chi-N -PEO as a function of pH for samples prepared by solution adsorption (filled squares), µCP (filled circles), and covalent attachment (unfilled squares).

microcontact printed Chi-N -PEO, with higher amounts transferred at high (in particular) and low pH (Fig. 4). It suggests that the inking conditions influence the amount of Chi-N -PEO taken up by the stamp and/or the binding of Chi-N -PEO to the stamp, which in turn affects the ease with which the polymer is transferred to the COOH surface. Furthermore, we expect that both the grafting density and the length of the PEO side chains on the chitosan backbone will influence the transferred amounts of polymer since the charge density of the backbone (the number of underivatized NH+ 3 groups available for anchoring) is determined by the graft density of PEO, and the steric shielding of the underlying chitosan is increased with the length of the PEO side chains. The ellipsometric images (see supporting material) of the patterned COOH surfaces when using Chi-N -PEO as ink molecule at different pHs via electrostatic interaction showed that the best quality of the patterned structures was obtained at pH 2.0 (also illustrated in Fig. 3c) and pH 11.1, which is in line with the thickness measurements (Fig. 4). When the COOH modified gold surface was activated with EDC/NHS for 20 min and then printed with a 0.1 wt% Chi-N -PEO solution for 1 h to facilitate covalent attachment a blurred patterned surface was obtained both when the stamp was inked at pH 2 (see Fig. 3d) and pH 11.1 (image not shown). After the surface was ultrasonicated in ethanol for 2 min, the patterns disappeared, suggesting that the great majority of the Chi-N -PEO layer seen in Fig. 3d was physisorbed onto the activated COOH surface. The observation that covalent binding of Chi-N -PEO cannot be achieved by µCP contrasts to the case of solution adsorption. The reason is, most likely, that Chi-N -PEO in solution can orientate to achieve a conformation that is suitable to bind to the activated COOH, even though steric hindrance due to the presence of the PEO side chains makes the process less favorable. In contrast, Chi-N -PEO molecules are more constrained at the PDMS stamp surface, because it has been oxidized with oxygen plasma to become hydrophilic and negatively charged. As a result the amines in chitosan are preferentially oriented to-

The proteins used to evaluate the NSB were BSA and fibrinogen. The NSB of these two proteins from HBS-EP buffer at pH 7.4 onto plain COOH coated gold surfaces was first studied by SPR measurements. The SPR responses were calculated into an effective thickness of 5.6 and 32.6 Å (using Eq. (1)) for BSA and fibrinogen, respectively. By coating the COOH surface with Chi-N -PEO the level of NSB, is considerably reduced (Table 1). It is, however, worthwhile emphasizing that the degree of reduction depends on the preparation conditions and the method used for attaching the polymer layer (Table 1). In general, the samples prepared from solution adsorption display a different pH dependence in terms of the degree of NSB than those prepared by covalent attachment and by µCP through noncovalent interaction, see Fig. 5. In fact, the only clear trend that can be seen in Fig. 5 is for the samples prepared by solution adsorption, where the NSB decreases with increasing pH during the Chi-N -PEO adsorption process. The significant difference observed between the three attachment processes; solution adsorption, covalent attachment, and µCP, can be due to variations in surface coverage and/or orientation of the polymer. To gain further insight, the NSB of BSA and fibrinogen is plotted as a function of the effective Chi-N -PEO layer thickness in Fig. 6. In this representation clear trends are observed. First, for all preparation methods the NSB of both BSA and fibrinogen is reduced with increasing adsorption of Chi-N -PEO. This result is as expected. However, an interesting result becomes evident when comparing the three methods for a given effective layer thickness of the Chi-N -PEO layer. In such a comparison the covalently attached layer is most efficient in suppressing the NSB of both BSA and fibrinogen. It is also clear that the layer prepared by µCP is least efficient in preventing NSB of fibrinogen. This is a very strong indication that the structure of the layers formed differs between the methods even when the coverage is the same. From Fig. 6b it is seen that covalently attached polymer provides the lowest “threshold” layer thickness to resist the NSB of fibrinogen. This is most likely due to the following reason: the Chi-N -PEO in the covalently attached layer has by necessity some primary amines in the polymer locked at a given position on the surface. This reduces the mobility of the polymer chain on the surface and adsorbing proteins will not be able to force the Chi-N -PEO polymer to change conformation on the surface to any large extent. Thus, the covalently attached Chi-N -PEO will efficiently block the surface area to which it is attached. The other two preparation methods rely largely on the electrostatic affinity between the polymer and the surface. In this case it is conceivable that adsorbing proteins can compete with surface attached polymer segments and replace some of these segments to facilitate their binding to the surface, as observed in other studies [15].

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(a)

(a)

(b)

(b)

Fig. 5. Nonspecific binding of BSA (a) and fibrinogen (b) in terms of effective protein layer thickness as a function of pH during preparation of the Chi-N PEO coated COOH surfaces with different methods.

Fig. 6. Nonspecific binding of BSA (a) and fibrinogen (b) in terms of effective protein layer thickness as a function of the effective thickness of the Chi-N PEO layer on COOH surfaces using different preparation methods and pHs. Note the difference in thickness scale.

At higher adsorption densities of the polymer, the density of the PEO chains anchored to the surface will increase and provide a steric barrier for the proteins to reach the surface that counteracts NSB also in case of electrostatically adsorbed Chi-N -PEO layers. Thus, to reach a low NSB the protective layer should be dense and immobile on the surface, and the less mobile the layer is the lower density is needed to prevent NSB of proteins. It is also interesting to discuss the protein repellency in relation to the spacing of PEO units on the surface. As mentioned before, the efficient steric stabilization could be achieved if the surface spacing of bound PEO units (L) is less than the radius of gyration of PEO (Rg ) in solution. According to the work performed by Selser et al. [40], the radius of gyration of the PEG side chains was calculated using a direct power law fit Rg = 0.215Mw0.583 (Å),

(3)

where Mw is the molecular weight of PEO. From this equation, the radius of gyration of the PEO side chain in Chi-N -PEO, can be calculated to equal 18 Å, which is close to 17 Å ob-

tained from the work of Textor et al. [16] as using an empirical formula based on static light-scattering measurements [41]. The average spacing of bound PEO units on the surface can be further calculated using a formula presented by Textor et al. [42]  0.5 2 L= √ (4) , 3nPEO where nPEO is the PEO chain surface density (number of molecules per unit area), is determined from the adsorbed mass of polymer and its grafting ratio [42]. nPEO can be calculated from nPEO =

mpol , Mchitosan g + MPEO

(5)

where mpol represents the mass of polymer adsorbed per unit area, g is the grating ratio, Mchitosan and MPEO are the molecular weights of chitosan monomer and PEO chain, respectively. In our case, the values of mpol for layers prepared by electrostatic adsorption, covalent attachment, and µCP were obtained

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Table 2 Calculated values for the different characteristic parameters, nPEO , L, L/2Rg of adsorbed PEO chains with respect to different preparation methods and pHs pH 2.0

pH 5.7

pH 8.9

pH 11.1

Electrostatic binding

nPEO (No./nm2 ) L (Å) L/2Rg

0.14 29 0.8

0.26 21 0.6

0.31 19 0.5

0.51 15 0.4

Covalent binding

nPEO (No./nm2 ) L (Å) L/2Rg

0.022 72 2.0

0.054 46 1.3

0.042 52 1.4

0.20 24 0.7

µCP

nPEO (No./nm2 ) L (Å) L/2Rg

0.10 34 0.9

0.060 44 1.2

0.051 48 1.3

0.152 28 0.8

from SPR measurements and INE, respectively (Table 1). The values of nPEO , L, and L/2Rg for the different pHs are listed in Table 2. The ratio of L/2Rg can be used to evaluate the extent of packing density of adsorbed PEO chains adsorbed on surface. It suggests that if L/2Rg < 1, PEO side chains overlap to form stretched brush-like structures, then the NSB of proteins are expected to be low [42]. The graphical representations of BSA and fibrinogen adsorption as a function of this ratio is illustrated in Fig. 7. It could be seen from Fig. 7 that the required packing density, i.e., the lowest value of L/2Rg to suppress NSB is different for the Chi-N -PEO layers prepared by the different methods. For layers prepared by electrostatic adsorption, a L/2Rg value of ∼0.4 is shown to completely eliminate the NSB of BSA and fibrinogen, which agrees well with recent data from the work of Textor et al. [16,42]. For a µCP layer, the L/2Rg ratio is required to be ∼0.8 to give low BSA adsorption (Fig. 7a), but for low NSB of fibrinogen, the value obviously need to be much lower than 0.8 (Fig. 7b). In contrast, for covalently attached Chi-N -PEO layers, a L/2Rg value of 0.7 appears to be sufficient repel both BSA and fibrinogen adsorption. It means that a lower packing density for covalent attachment is required to provide protein suppression compared to the layers prepared by electrostatic adsorption as well as µCP. This observation is in agreement with aforementioned result shown in Fig. 6. Thus, our results clearly point out that the methods used to prepare the surfaces have a significant influence on their protein repellent properties. Patterned surfaces could, however, be created by using µCP relying on physical transfer of Chi-N -PEO to the COOH surfaces. The images of the Chi-N -PEO patterned surfaces before and after exposure to a 2 mg/ml BSA solution and 1 mg/ml fibrinogen solution, respectively, are illustrated in the supporting material section. The patterned surfaces exhibited higher pattern resolution when the stamp was inked at pH 2.0 and 11.1, as compared to at pH 5.7 and 8.9. Although all patterned samples showed some irregularities, samples prepared at pH 2.0 using µCP provided the best uniformity in the printed region, Fig. 8a, indicating that a higher charge density of the polymer is preferable. When the surfaces were incubated in BSA and fibrinogen solutions, respectively, it was observed that BSA (Fig. 8b) and fibrinogen (Fig. 8c) preferentially adsorbed to the

(a)

(b) Fig. 7. Nonspecific binding of BSA (a) and fibrinogen (b) in terms of the ratio between the average spacing of bound PEO units on surface and the diameter of gyration of the PEO side chains in solution using different preparation methods and pHs. Note the difference in thickness scale.

COOH square areas. This is particularly evident in the case of fibrinogen where the patterns on the surface become reversed, Fig. 8c. Although there are somewhat NSB of fibrinogen on the printed Chi-N -PEO regions, the level of NSB is relatively low. We note that in a previous study [11], in which a copolymer poly(L-lysine)-g-poly(ethylene glycol) layers was coupled to metal oxide surfaces, the adsorption of fibrinogen from a 2.5 mg/ml solution at pH 7.4 was reduced to 22 ng/cm2 , corresponding to an effective thickness of 3.3 Å. In our case we achieve a comparable reduced fibrinogen layer thickness of 6.3 Å (42 ng/cm2 ) via µCP, and ∼0 Å via electrostatic and/or covalent binding at pH 11.1 as the surface was treated with fibrinogen at a concentration of 1 mg/ml. It is worth to note that an adsorbed fibrinogen mass less than 5 ng/cm2 , corresponding to an effective thickness of about 0.6 Å, does not activate the pathways leading to blood coagulation and thrombosis [43]. As mentioned before, the relatively high NSB of fibrinogen on the µCP printed polymer surface is due to the steric hindrance

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(a)

(b)

(c) Fig. 8. Ellipsometric images of Chi-N -PEO patterned surfaces prepared by µCP at pH 2.0 (a), after incubation of 2 mg/ml BSA (b), and 1 mg/ml fibrinogen (c), respectively. The surface consists of 100 × 100 µm spots that are interspaced by 50 µm wide barriers of Chi-N -PEO.

during transfer of the bulky polymer to the surface. Thus, different architectures of such polymers need to be tested in order to find the optimal graft density and PEO side chain length on the chitosan backbone to achieve both efficient transfer during printing and low NSB of proteins. 4. Conclusions The NSB of proteins is significantly reduced by coating COOH surfaces with the comb polymer Chi-N -PEO. The reduction in protein binding decreases with increasing effective layer thickness of the polymer for all preparation methods used: solution adsorption, covalent coupling and µCP. However, it also depends on the preparation method since for a given effective layer thickness covalently attached Chi-N -PEO films show a better performance than electrostatically adsorbed layers. This provides a strong indication that a reduced mobility of

the Chi-N -PEO molecule favors protein repellency. The same observation was also obtained by studying the packing density of the adsorbed PEO chains in relation to the NSB of proteins. It revealed that the packing density of PEO chains, characterized as L/2Rg , on surfaces prepared by electrostatic adsorption appears to be higher than that obtained via covalent attachment in order to reach the same level of protein suppression. It was also observed that the acquired PEO-density for µCP samples was too low to completely prevent adsorption of fibrinogen. The optimal pH condition for preparing the most proteinrepelling Chi-N -PEO layer from solution adsorption through physical adsorption is at high pH where the COOH surface is strongly negatively charged and the polymer only weakly charged in solution. The polymer may, however, due to its charge regulating capacity acquire some positive charge in contact with the surface. In contrast, using µCP the most homogeneous and uniform printed layer is obtained when the oxidized PDMS stamp is inked at pH 2.0 and 11.1, respectively. Covalent interaction between amine groups in Chi-N -PEO and activated esters of COOH could only take place to a significant degree at pH 11.1 in solution and failed when utilizing µCP. This result reflects that steric hindrance imposed by the PEO side chains counteract formation of the intimate contacts between the primary amine and the activated esters that are needed to achieve covalent coupling, and this hindrance is enhanced during µCP when the polymer is adsorbed to the oxidized, negatively charged PDMS stamp. Comb polymers of chitosan with grafted side chains of PEO show significant promise as efficient vehicles for reducing nonspecific protein adsorption. It is suggested that even better performance than reported here can be achieved by optimizing the graft density and the length of the PEO side chains. The aim is to reduce the steric hindrance for covalent attachment and thus achieve high packing density on the surface, while maintaining the steric hindrance for proteins to reach the surface through the comb polymer layer. Acknowledgments This work was supported by the Swedish Foundation for Strategic Research (Y.Z., B.L.); P.C., A.D., R.M., and N.G. acknowledge support by the Marie Curie network “Self-organization under confinement, SOCON”; P.C. and A.D. acknowledge financial support from the Swedish Research Council, VR. Supplementary material The online version of this article contains additional supplementary material. Please visit DOI: 10.1016/j.jcis.2006.09.058. References [1] G.J. Fleer, M.A. Cohen Stuart, J.M.H.M. Scheutjens, T. Cosgrove, B. Vincent, Polymers at Interfaces, Chapman & Hall, London, 1993. [2] H.D. Bijsterbosch, M.A. Cohen Stuart, G.J. Fleer, Macromolecules 31 (1998) 8981.

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