Stability and Nonfouling Properties of Poly(poly(ethylene glycol

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Biomacromolecules 2008, 9, 906–912

Stability and Nonfouling Properties of Poly(poly(ethylene glycol) methacrylate) Brushes under Cell Culture Conditions Stefano Tugulu and Harm-Anton Klok* École Polytechnique Fédérale de Lausanne (EPFL), Institut des Matériaux, Laboratoire des Polymères, Bâtiment MXD, Station 12, CH-1015 Lausanne, Switzerland Received November 24, 2007; Revised Manuscript Received January 4, 2008

This paper investigates the stability and nonfouling properties of poly(poly(ethylene glycol) methacrylate) (PPEGMA) brushes prepared by surface-initiated atom transfer radical polymerization from SiOx substrates modified with a trimethoxysilane-based ATRP initiator. At high chain densities, PPEGMA brushes were found to detach rapidly from glass or silicon substrates. Detachment of the PPEGMA brushes could be monitored with contact angle measurements, which indicated a decrease in the receding water contact angle upon detachment. Detachment of the PPEGMA brushes also resulted in an increase in nonspecific protein adsorption. The stability, and as a consequence the long-term nonfouling properties, of the PPEGMA brushes could be improved by tailoring the brush density and, to a lesser extent, the molecular weight of the polymer chains. By appropriate decrease of the grafting density, the stability of the brushes in cell culture medium could be improved from less than 1 to more than 7 days, without compromising the nonfouling properties.

Introduction The surface chemistry, physics, and topography of a biomaterial are important parameters that influence the materials properties.1–3 Polymer brushes are an attractive means to control surface properties.4,5 The structure and properties of polymer brushes can be controlled at the molecular level and polymer brushes are relatively easily accessible via a number of simple and reproducible techniques.6 Of particular interest is the use of surface-initiated controlled/“living” polymerization methods as these offer precise control over many important parameters, including brush thickness, composition, and grafting density.7,8 Among the different methods that are available, surface-initiated atom transfer radical polymerization (SI-ATRP) has attracted increasing interest due to its robustness and synthetic flexibility.9,10 SI-ATRP has been used to fabricate a variety of polymer brushes that are of interest for a range of biomedical and bioanalytical applications. Polymer brushes based on poly(2-hydroxyethyl methacrylate) (PHEMA),11 poly(poly(ethylene glycol) methacrylate) (PPEGMA),12–16 and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC),18 for example, can effectively suppress nonspecific protein adsorption and cell adhesion. The use of temperature-sensitive polymers, such as poly(N-isopropyl acrylamide) (PNIPAm) has allowed the preparation of polymer brushes that can be thermally switched from a hydrophilic, biologically inert state to a hydrophobic protein and cell adhesive state.14,19 Polymer brushes based on PPEGMA have also been successfully used as platforms for the development of protein function microarrays.20,21 The protein-resistant, i.e., nonfouling, polymer brushes mentioned above have been grafted from a range of different materials using various approaches for the surface immobilization of the ATRP initiator. Surface-initiated polymerization of poly(ethylene glycol) methacrylate (PEGMA) from gold surfaces has been carried out from ATRP-initiator modified alkyl thiol self-assembled monolayers (SAMs).15 Stainless steel and Ti substrates have been coated with PPEGMA brushes using * Corresponding author: E-mail: [email protected].

catechol-modified ATRP initiators.12,13 SI-ATRP of 2-hydroxyethyl methacrylate (HEMA) and PEGMA from Si and SiOx surfaces has been achieved using a variety of silane, chlorosilane, and alkoxysilane-modified ATRP initiators.11,14,16,17 An important issue with respect to possible biomedical applications is the long-term stability of the brushes. PPEGMA brushes grafted from gold substrates have been demonstrated to prevent nonspecific cell adhesion for up to 30 days.15 Ti substrates coated with catechol-anchored PPEGMA brushes were shown to possess excellent cell fouling resistance for up to 3 weeks.13 This contribution discusses the stability and nonfouling properties of PPEGMA brushes grown from SiOx substrates functionalized with trimethoxysilane based ATRP initiators. The results presented in this paper provide some first guidelines for the design of trialkoxysilane-anchored PPEGMA brushes for longterm biomedical applications.

Experimental Section Materials. Poly(ethylene glycol) methacrylate (PEGMA) (∼526 g · mol-1) and poly(ethylene glycol) dimethacrylate (PEGDMA) (∼330 g · mol-1) were obtained from Aldrich and freed from the inhibitor as previously described.20 2,2′-Bipyridine (bipy) was obtained from Fluka and recrystallized twice from cyclohexane. Gibco D-MEM Cell Culture Medium (DMEM 2188-025, pH 7.1) and fibrinogen-Alexa Fluor 647 conjugate were obtained from Invitrogen. Cu(II) bromide (99.999%) and Cu(I) chloride (purum, g97%) were purchased from Sigma-Aldrich and used as received. The ATRP initiator, 3-(2-bromoisobutyramido)propyl(trimethoxy)silane (1), and the ATRP inactive 3-(pivaloylamido)propyl(trimethoxy)silane (2) were synthesized as previously described20 and purified by distillation in oil-pump vacuum. Anhydrous toluene was freshly distilled over sodium before use. Deionized water was obtained from a Millipore Direct-Q 5 Ultrapure Water System. Quartz slides were obtained from Plano GmbH. Standard glass slides were obtained from Marienfeld GmbH. Analytical Methods. For the determination of brush thicknesses, samples supported on silicon wafers were analyzed with a computercontrolled Phillips Plasmon SD 2300 null-ellipsometer operating with a He-Ne laser at λ ) 632.8 nm and an angle of incidence of 70°. Film thicknesses were calculated using a three-layer silicon/polymer brush/

10.1021/bm701293g CCC: $40.75  2008 American Chemical Society Published on Web 02/09/2008

Properties of Poly(poly(ethylene glycol) methacrylate) Brushes

Biomacromolecules, Vol. 9, No. 3, 2008 907

Scheme 1

ambient air model, assuming the polymer to be isotropic and homogeneous. Refractive indices and thicknesses were calculated simultaneously from the experimental Ψ and ∆ values. Optical microscopy was carried out on a Zeiss Axiovert 200 microscope. Contact angle measurements were carried out using a DataPhysics OCA 35 contact angle measuring instrument. Relative fluorescence intensity values of adsorbed fibrinogen Alexa Fluor 647 conjugate were measured by using a GenePix 4000B microarray scanner in combination with the Genepix Pro software from Axon Instruments. Scanning electron microscopic images of gold-sputtered samples were recorded on a Phillips XLS 30 microscope. Preparation of the Polymer Brushes. First glass, quartz, or silicon substrates were sonicated 5 min in ethanol and water, activated for 30 min at 150 °C with piranha solution (H2O2 (30 wt % in H2O)/H2SO4 (98 wt %) 7:3 (v:v)), thoroughly rinsed with water and ethanol, and dried in a stream of nitrogen. Next, the substrates were modified with 1 or with mixtures of 1 and the inert pivaloyl-terminated trimethoxysilane 2 by placing the substrates in a 10 mM solution of 1, or a mixture of 1 and 2, in anhydrous toluene for 30 min at room temperature. After that the substrates were rinsed with anhydrous toluene, sonicated in acetone and in a water/tert-butanol mixture (1:1/v:v) for 1 min, rinsed with water, dried in a stream of nitrogen, and subsequently transferred to the appropriate reactors for polymerization. Surface-initiated atom transfer radical polymerization of PEGMA was carried out using a reaction system consisting of PEGMA, CuCl, CuBr2, and bipy in the following molar ratios: 1000:10:2:25. The polymerizations were performed in a water/methanol mixture (8:2/v: v). In a typical experiment 14.06 mg (0.063 mmol) of CuBr2 and 122.98 mg (0.787 mmol) of bipy were dissolved in a mixture of 15 mL of PEGMA (31 mmol), 12 mL of water, and 3 mL of methanol. After the mixture was degassed by two freeze–pump–thaw cycles, 31.18 mg (0.315 mmol) of CuCl was added and degassing was continued for two cycles. The resulting solution was subsequently transferred with a cannula to the nitrogen-purged reaction vessel containing the initiator functionalized substrates, and the reaction was allowed to proceed at 60 °C. After a certain time, the reaction mixture was removed and the substrates were rinsed with water, extracted overnight with DMSO, rinsed thoroughly with water, and finally dried in a stream of nitrogen. PHEMA functionalized substrates were prepared as described previously.20 PEGDMA functionalized glass substrates were obtained following a similar procedure using 26 mL (28.08 g, 85 mmol) of PEGDMA, 540 mg (3.76 mmol) of CuBr, 42 mg (188 µmol) of CuBr2, and 1.47 g (9.4 mmol) bipy in a mixture of 13 mL of water, 14 mL of methanol, and 2 mL of ethanol. The polymerization of PEGDMA was carried out at 60 °C for 1 h. Stability Tests. Polymer brush modified substrates were sterilized by immersion in a mixture of 70% ethanol in water and subsequently incubated under sterile conditions in D-MEM cell culture medium for a defined time at 37 °C. After being taken out from the medium, the substrates were washed three times for 10 min with distilled water and dried in a stream of nitrogen. Nonspecific Adsorption of Fibrinogen Alexa Fluor 647 Conjugate. Polymer brush functionalized glass slides were incubated for 30 min in a freshly prepared 1 µM solution of fibrinogen-Alexa Fluor 647 conjugate in 0.1 M NaHCO3 buffer (pH ) 8.3) at room temperature.

The slides were subsequently taken from the protein solution, rinsed with buffer, washed two times for 30 min in buffer by gently shaking the sample, rinsed with deionized water, and finally dried in a stream of nitrogen. All incubation and washing steps were carried out under exclusion of direct light. The amount of residual adsorbed protein on the surfaces was estimated by measuring the relative fluorescence intensities at 635 nm excitation wavelength.

Results and Discussion Brush Synthesis and Characterization. Surface-initiated atom transfer radical polymerization (SI-ATRP) allows precise tailoring of the grafting density and thickness of polymer brushes. While the film thickness can be controlled via the polymerization time, the grafting density can be adjusted by varying the surface density of ATRP initiator groups on the surface. This has been achieved by immobilizing mixtures of ATRP initiator molecules and ATRP inactive molecules using both gold-thiol as well as SiOx-alkoxysilane chemistry.15,22,23 Our approach, which is outlined in Scheme 1, is based on the deposition of mixtures of the “ATRP active” 2-bromoisobutyryl functionalized trimethoxysilane 1 with the structurally related, but “ATRP inactive”, pivaloyl-terminated trimethoxysilane 2. PPEGMA brushes were prepared by SI-ATRP on substrates modified with 1 or mixtures of 1 and 2 using a catalyst system consisting of CuCl, CuBr2, and bipy in a water–methanol mixture at 60 °C. Figure 1 shows a typical example of the evolution of brush thickness with polymerization time. Under the chosen reaction conditions, the film thickness increased linearly during the first 2 h up to a thickness of 60 nm. At longer polymerization times, the film thickness leveled off, which is indicative for the loss of the “living” character of the polymerization. Figure 2 shows the thickness of PPEGMA brushes prepared from silicon substrates modified with mixtures of 1 and 2 of different composition. The cross-linked nature of the PPEGMA brushes (vide infra) prevented gel permeation chromatography analysis of the cleaved polymer, which would have provided

Figure 1. Representative plot of the ellipsometric PPEGMA film thickness vs the polymerization time. For this example, the polymerization was initiated from a silicon surface modified with a solution containing 100 mol % 1.

908 Biomacromolecules, Vol. 9, No. 3, 2008

Tugulu and Klok

Figure 2. Ellipsometric thickness of PPEGMA brushes as a function of the mol % ATRP initiator 1 that was used to modify the silicon substrate. SI-ATRP of PEGMA was carried out on silicon substrates for 3 h at 60 °C. The solid line is drawn to guide the eye.

Figure 3. Nonspecific adsorption of fibrinogen Alexa Fluor 647 conjugate on PPEGMA brushes grafted from surfaces modified with different amounts of ATRP initiator 1. The relative fluorescence intensity, which is taken as a measure for the amount of nonspecifically adsorbed protein, is plotted against the mol % of 1 in the trimethoxysilane mixture that was used to modify the substrates. The solid line is drawn to guide the eye.

the molecular weight of the polymer chains and allowed an estimate of the brush density (chains/unit area). Consequently, Figure 2 plots ellipsometric film thicknesses versus the mol % of 1 that was used to modify the silicon substrate. Figure 2 shows that starting from a surface covered with only 2, the ellipsometric film thickness first increased slowly up to ∼26 nm at 40 mol % 1. Any further increase in the relative amount of 1 resulted in an almost linear increase in layer thickness. For a surface covered with only 1, thicknesses of about 90 nm were obtained. Figure 2 indicates a nonlinear dependence of the ellipsometric film thickness on the mol % 1 used to modify the silicon substrate. This finding is in agreement with the results of a previous study in which similar observations were made for poly(methacrylic acid) brushes grafted from silicon substrates modified with mixtures of 1 and 2.24 The nonlinear dependence of the ellipsometric film thickness on the composition of the ATRP initiator modified substrate was attributed to the nonideality of the surface modification with mixtures of the trimethoxysilanes 1 and 2. XPS experiments indicated that although it is possible to adjust the surface mole fraction of 1 by varying the relative amounts of 1 and 2 in the surface modification step, the surface mole fraction of 1 was always lower than expected based on the mole fraction of 1 in the reaction mixture that was used to modify the silicon substrate.24 The reasons for this anomalous behavior are not fully clear at the moment, but we hypothesize that they may be related to the fact that trialkoxysilanes such as 1 and 2 very often do not form well-defined monolayers, but rather thicker layers of a few nanometers on silicon substrates (cf. the ellipsometric film thickness at 0 mol % 1 in Figure 2). Nonspecific Protein Adsorption. Nonspecific protein adsorption on the PPEGMA brushes was studied by incubating samples that were polymerized for 3 h from surfaces modified with different mixtures of 1 and 2 in a 1 µM solution of a fibrinogen-Alexa Fluor 647 conjugate. After 30 min, nonphysisorbed protein was removed by washing and the relative fluorescence intensity, which was taken as a measure for the amount of adsorbed protein, was determined using a microarray scanner. Figure 3 shows the measured relative fluorescence intensities as a function of the mol % 1 in the trimethoxysilane mixture that was used to prepare the ATRP-initiator-modified substrates. For comparison, the results of a control experiment in which an unmodified glass slide was used are also included. Figure 3 shows that in comparison with the unmodified glass substrate, the PPEGMA brushes reduce nonspecific protein adsorption. The data also indicate that PPEGMA brushes become more effective at suppressing nonspecific protein

adsorption with increasing mol % of 1 that was used to modify the underlying SiOx substrate. Brushes grown from surfaces that were modified with trimethoxysilane mixtures containing less than 20 mol % 1 showed relatively high fluorescence intensities. For 20–50 mol % 1, a plateau regime was observed, characterized by relatively low fluorescence intensities. Nonspecific protein adsorption was most effectively suppressed when PPEGMA brushes were prepared from surfaces modified with more than 60 mol % 1. The results shown in Figure 3 are in agreement with observations by Genzer et al., who utilized the nonspecific adsorption of fibronectin on poly(2-hydroxyethyl methacrylate) gradient brushes to tailor cell adhesion.11 Similar effects of the brush density on nonspecific protein adsorption are also known from PEG brushes prepared by tethering endreactive PEG chains to surfaces.25–27 Stability of PPEGMA Brushes. To evaluate their long-term stability, PPEGMA brushes grafted from substrates modified with solutions containing different mol % 1 were incubated in cell culture medium at 37 °C and analyzed by contact angle measurements and phase contrast microscopy at regular time intervals. Figure 4 shows micrographs of PPEGMA brushes, which were grown from glass substrates modified with different amounts of ATRP initiator 1, after 7 days of incubation in cell culture medium. The images in panels a and b of Figure 4, which were recorded from brushes produced from surfaces modified with 100 and 70 mol % 1, respectively, show detachment of the polymer brush layer. In contrast, the PPEGMA brush prepared from a glass substrate modified with a 1/1 mixture of 1 and 2 did not reveal any signs of detachment. Interestingly, brushes produced from surfaces modified with solutions containing 70 mol % or more 1 seem to detach in the form of continuous films. This is illustrated by the scanning electron micrographs shown in Figure 5 and suggests that the PPEGMA brushes are cross-linked. Cross-linking of the PPEGMA brushes may be due to transesterification reactions28 that occur during the SI-ATRP of PEGMA and/or to autoxidation processes.29 The structures in the micrographs in Figures 4 and 5 bear some resemblance to the buckling patterns that have been observed upon pulse electrolysis induced delamination of cross-linked poly(glycidyl methacrylate) brushes grown from ATRP initiator modified thiol self-assembled monolayers on gold substrates.30 The detachment of the PPEGMA brushes was accompanied by a strong decrease in the receding water contact angle. This is illustrated in Figure 4d and Figure 4e, which plot the advancing and receding water contact angles as a function of the mol % 1 in the solution that was used to modify the substrate



Figure 4. Stability of PPEGMA brushes grafted from glass substrates modified with different mol % ATRP initiator 1 upon incubation in cell culture medium. Top: phase contrast microscopy images of PPEGMA brushes produced with 100 mol % (a), 70 mol % (b), and 50 mol % (c) ATRP initiator 1 after 7 days of incubation in cell culture medium at 37 °C. Bottom: Advancing (d) and receding (e) water contact angles of PPEGMA brush functionalized glass slides as a function of the mole fraction of ATRP initiator 1 and incubation time in cell culture medium at 37 °C.

Figure 5. Representative scanning electron microscopy images of PPEGMA brushes grown from a substrate modified with 100 mol % of ATRP initiator 1 after 7 days of incubation in cell culture medium at 37 °C.

and incubation time. The data in Figure 4e show that PPEGMA brushes prepared from surfaces with g70 mol % 1 rapidly deteriorate and lose their stability within 1 to 2 days. For PPEGMA brushes grown from surfaces modified with 100 mol % 1, this process was particularly fast, and detachment occurred already after 10-12 h incubation in cell culture medium. In contrast, PPEGMA brushes fabricated using less than 70 mol % 1 were stable for at least a week in cell culture medium, as demonstrated by the absence of any significant changes in the receding water contact angle (Figure 4e) and the optical micrograph shown in Figure 4c. In addition to the effect of the mol % of ATRP initiator moieties 1 on the substrate, also the effect of brush thickness was studied. To this end, a series of PPEGMA brushes with thicknesses from 23 to 106 nm was grown from substrates modified with 100 mol % 1 by variation of the polymerization time. These brushes were incubated in cell culture medium and subsequently studied as described above. The corresponding

advancing and receding water contact angles as a function of brush thickness and incubation time are summarized in Figure 6. Figure 6 shows that decreasing brush thickness retards the drop in the receding contact angle. For all except the two thinnest brushes, the receding contact angles approached zero within 4 days, indicating detachment of the polymer brush. Even though the receding water contact angle for the two thinnest brushes does not reach zero, it shows a significant decrease with time. The contact angle measurements are supported by the phase contrast microscopy images in Figure 6c and Figure 6d, which, in particular for the 106 nm thick brush, show the typical textures of surface detached brushes. Varying the mol % of ATRP initiator moieties 1 clearly seems to be a more powerful parameter to control brush stability as compared to brush thickness. To understand the origin of the detachment of the PPEGMA brushes, a number of additional experiments were carried out. In a first series of experiments, the ATRP initiator immobiliza-


Tugulu and Klok

Figure 6. Evaluation of the stability of PPEGMA functionalized glass substrates as function of brush thickness upon incubation in cell culture medium at 37 °C. SI-ATRP was carried out on glass slides modified with 100 mol % ATRP initiator 1. Top: Time-dependent advancing (a) and receding (b) water contact angles as a function of brush thickness. Bottom: phase contrast microscopy images of 32 nm thick (c) and 106 nm thick (d) PPEGMA brushes after 7 days of incubation in cell culture medium at 37 °C. Scale bar 100 µm.

tion procedure and the type of ATRP initiator were varied. However, neither vapor phase deposition or postbaking procedures nor the use of 4-(chloromethyl)phenyltrichlorosilane or 4-(chloromethyl)phenyltrimethoxysilane resulted in improved stabilities. Huang, Baker, and Bruening have proposed that cross-linked as opposed to linear polymer brushes should provide improved mechanical and chemical stability.31 SI-ATRP of PEGDMA, however, was not found to prevent detachment of the polymer brushes. Interestingly, however PHEMA brushes in contrast to PPEGMA brushes did not reveal any signs of detachment upon incubation in cell culture medium. Furthermore, detachment of the PPEGMA brushes was also not observed when the cell culture medium was replaced by pure water. For all the PPEGMA brush thicknesses and densities that were studied, no loss of stability could be observed upon incubation in pure water over a period of at least 7 days. The scanning electron micrographs in Figure 5 suggest that detachment of the PPEGMA brushes occurs at the interface with the glass or silicon substrate. We hypothesize that detachment of the brushes involves cleavage of Si-O bonds that are located at the interface between the brush and the substrate.32,33 Possible explanations for the detachment of the PPEGMA brushes may be osmotic stresses that act on the brushes in the cell culture medium as well as steric crowding. Both of these factors could induce additional tension along the already stretched polymer brush backbones, which could promote hydrolysis of the Si-O bond and detachment of the brush. In two recent reports, it has been demonstrated that polymer/surface interactions can generate

tensions along polymer backbones that are sufficient to mechanically break covalent bonds.34,35 Compared to PPEGMA brushes grown from alkyl SAM modified gold substrates15 or catechol-anchored PPEGMA brushes on Ti,13 the trialkoxysilane immobilized PPEGMA brushes studied in this contribution only show limited stability. In contrast to the alkyl thiol SAM and catechol-modified ATRP initiators, the trimethoxysilane initiators used here form a relatively ill-defined initiator layer, which may also facilitate cleavage of the PPEGMA brushes from the substrate. Stretching, steric crowding, and swelling of the PPEGMA brush layers is due to the water solubility of this polymer. In addition, the poly(ethylene glycol) side chains can bind alkali and earth alkali metal ions,36 which could lead to increased salt concentrations in the PPEGMA brushes as compared to the cell culture medium. At high brush densities, the resulting osmotic stresses would add to the entropically unfavorable stretched chain conformation and promote detachment of the brush. This proposal is in line with the observations that PPEGMA brushes do not detach in pure water and that PHEMA brushes are stable in cell culture medium. In pure water, PPEGMA brushes merely swell, but do not take up any salt, which could result in additional osmotic stress. In contrast to PPEGMA, PHEMA is generally considered as being only water swellable rather than water soluble.37 Furthermore PHEMA brushes do not contain the poly(ethylene glycol) chains that are present in the PPEGMA brushes and, as a result, are less effective in complexing alkali and earth alkali metal ions. As a result, the PHEMA brushes


will not be exposed to the osmotic stress that acts on the PPEGMA counterparts. It is also interesting to compare our observations with the results from related studies on the stability of poly(ethylene glycol) monolayers grafted onto SiOx surfaces using silane chemistry.38,39 Incubation of these films in phosphate buffer at pH 7.4 was found to lead to a gradual decrease in film thickness, which was ascribed to detachment of the PEG chains from the surface, most likely also via cleavage of the Si-O bonds. Detachment of the PEG chains, however, was not found to affect the nonfouling properties of the films for a period of 4 weeks. There are two important differences between these surfacegrafted PEG monolayers and the PPEGMA brushes reported in this contribution. First of all, aging of surface-grafted PEG monolayers involves detachment of individual grafted PEG chains, whereas PPEGMA brushes are released in the form of a continuous film. The free surface area that becomes exposed upon detachment of a single polymer chain in a tethered PEG monolayer may be accommodated for by neighboring chains, which will adopt a less stretched conformation. As a result, the nonfouling properties of these PEG brushes are not necessarily affected by cleavage of polymer chains. Due to their cross-linked character, such a “repair mechanism” at the molecular level is not possible for the PPEGMA brushes. In this case, after cleavage of a sufficient number of Si-O bonds, a macroscopic film is released, which will influence the nonfouling properties (vide infra). The second important difference between the surface-grafted PEG monolayers and the PPEGMA brushes is related to the molecular weight and density of the polymer chains. In most cases, both of these parameters are larger for the PPEGMA brushes as compared to the PEG monolayers. As a result, the polymer chains in the PPEGMA brushes are packed in a more extended, entropically less favorable conformation. This may explain the large difference between the stability of PPEGMA brushes grown from SiOx substrates modified with 100 mol % 1 and their surface-grafted PEG monolayer analogues. Correlating the Stability and Nonfouling Properties of PPEGMA Brushes. In the next series of experiments, the effect of detachment of the PPEGMA brushes on their ability to suppress nonspecific protein adsorption was investigated. These studies were performed with a series of 80 nm thick PPEGMA brushes, which were grown from glass slides modified with 100 mol % 1. The substrates were incubated in cell culture medium at 37 °C. The incubation time of the samples was varied from 0 to 24 h. After the brushes were taken out of the cell culture medium, the advancing and receding water contact angles were determined and the samples were immersed in a 1 µM fibrinogen-Alexa Fluor 647 conjugate solution. After 30 min, the samples were removed from the protein solution, washed, and analyzed with a microarray scanner. Figure 7 compares the water contact angles and relative fluorescence intensities measured on these brushes as a function of their incubation time in cell culture medium. The water contact angles rapidly drop if PPEGMA brushes are incubated in cell culture medium for a period longer than 6 h. Earlier in this paper, it was demonstrated that this rapid drop in water contact angle marks the onset of brush detachment. The relative fluorescence intensities that were measured after exposing the brushes to a solution containing fibrinogen-Alex Fluor 647 conjugate clearly indicate that detachment of the PPEGMA brushes is accompanied by an increase in the nonspecific protein adsorption, i.e., a loss of the nonfouling character of the PPEGMA brushes. This process can also be directly observed by analyzing the


Figure 7. Top: Water contact angles of a series of PPEGMA brushes incubated in cell culture medium for different periods of time. Bottom: Relative fluorescence intensities measured after 30 min of exposure to a 1 µM fibrinogen-Alexa fluor 647 conjugate solution for the same series of PPEGMA brushes as a function of their incubation time in cell culture medium. The PPEGMA brushes were grown from glass slides modified with 100 mol % 1 and had a thickness of 80 nm.

Figure 8. Phase contrast and fluorescence microscopy images of a glass substrate functionalized with a 80 nm thick PPEGMA brush, which was incubated for 24 h in cell culture medium at 37 °C, and subsequently immersed for 30 min in a fibrinogen–Alexa Fluor 647 conjugate containing buffer: left, phase contrast image; middle, fluorescence image; right, overlay. Scale bar: 100 µm.

samples with phase contrast and fluorescence microscopy. This is illustrated in Figure 8, which shows phase contrast and fluorescence microscopy images of an 80 nm thick PPEGMA brush, which was incubated for 24 h in cell culture medium and subsequently immersed in a fibrinogen-Alexa Fluor 647 conjugate solution for 30 min. The overlay of the phase contrast and fluorescence images indicates that the red fluorescent fibrinogen conjugate preferably adsorbs at those areas of the substrate where the brush has detached.

Conclusions In this paper, we have studied the stability and nonfouling properties of trialkoxysilane-anchored PPEGMA brushes grafted from SiOx surfaces. PPEGMA brushes were found to detach from the substrates upon prolonged exposure to cell culture medium. This process was accompanied by a drop in the (receding) water contact angle and could also be visually observed by optical and electron microscopy. Release of the PPEGMA brushes from the substrates resulted in an increase in nonspecific protein adsorption. As no detachment was observed in pure water, we hypothesize that complexation of salts from the buffer solution by the poly(ethylene glycol) side chains of the brush creates an osmotic stress, which adds to the entropically unfavorable stretched chain conformation at high brush densities and facilitates cleavage of the siloxane bond that links the polymer brush to the substrate. This cleavage process may be further facilitated by the relatively ill-defined nature of the trialkoxysilane based initiator layer. The stability of the brushes can be enhanced by controlling the brush density and, to a lesser extent, the molecular weight of the brush


molecules. By modification of a substrate with a mixture of 60 mol % of an ATRP initiator modified trimethoxysilane and 40 mol % of an inert trimethoxysilane instead of using a solution that contains only an ATRP initiator modified trimethoxysilane, the stability of the PPEGMA brushes could be improved from 1 to more than 7 days without compromising the nonfouling properties of the brushes. Acknowledgment. The authors are grateful to Dr. Frederic Juillerat for his help with the scanning electron microscopy experiments and to Professor Kai Johnsson for providing access to the microarray scanner and optical microscope. This work was financially supported by the Volkswagen Stiftung.

References and Notes (1) (2) (3) (4) (5) (6) (7)

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