Electrochemical Deposition and Surface-Initiated RAFT

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Biomacromolecules 2010, 11, 3422–3431

Electrochemical Deposition and Surface-Initiated RAFT Polymerization: Protein and Cell-Resistant PPEGMEMA Polymer Brushes Maria Celeste R. Tria, Carlos David T. Grande, Ramakrishna R. Ponnapati, and Rigoberto C. Advincula* Departments of Chemistry and Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204-5003, United States Received August 11, 2010; Revised Manuscript Received October 11, 2010

This paper introduces a novel and versatile method of grafting protein and cell-resistant poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) brushes on conducting Au surface. The process started with the electrochemical deposition and full characterization of an electro-active chain transfer agent (CTA) on the Au surface. The electrochemical behavior of the CTA was investigated by cyclic voltammetry (CV) while the deposition and stability of the CTA on the surface were confirmed by ellipsometry, contact angle, and X-ray photoelectron spectroscopy (XPS). The capability of the electrodeposited CTA to mediate surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization on both the polymethyl methacrylate (PMMA; model polymer) and PPEGMEMA brushes was demonstrated by the increase in thicknesses of the films after polymerization. Contact angles also decreased with the incorporation of the more hydrophilic brushes. Significant changes in the morphologies of the films before and after polymerization were also observed with atomic force microscopy (AFM) analyses. Furthermore, XPS results showed an increase in the O 1s peak intensity relative to C 1s after polymerizations, which confirmed the grafting of polyethyleneglycol (PEG) bearing brushes. The ability of the PPEGMEMA-modified Au surface to resist nonspecific adhesion of proteins and cells was monitored and confirmed by XPS, ellipsometry, contact angle, AFM, and fluorescence imaging. The new method presented has potential application as robust protein and cell-resistant coatings for electrically conducting electrodes and biomedical devices.

Introduction Electrically conducting metal and metal oxide surfaces are widely used for the fabrication of many biomaterial implants. For example, titanium and its alloys are used for dental implants and joint replacement for skeletal systems.1-3 Stainless steels are viable for the manufacturing of surgical instruments and bone screws.4,5 Semiconducting materials such as copper oxide and doped silicon are used in intrauterine devices and implants to regenerate nerve fibers, respectively.6-9 Platinum, on the other hand, is used for the production of electrodes in devices such as cardiac pacemakers and hearing aids.10-12 Gold, having a good malleability, is being exploited in restorative dentistry for crowns and permanent bridges and its good electrical conductivity is taken advantage for creating wires for pacemakers and other medical devices.13,14 These metals and alloys are commonly used in implants, medical device fabrication, and related accessories because of their known high stiffness, ductility, wear resistance, and thermal and electrical conductivity. However, despite the wide range of advantages offered, these biomaterials are not exempt to the most common problem of biomedical devices, namely, biofouling. This is primarily caused by the nonspecific adsorption of proteins and cells upon contact with biological tissues or fluids. The unwanted nonspecific adsorption of these biomolecules onto these biomaterials could lead to an undesirable cascade of events that eventually result in inflammation and rejection of the material or implant.15-17 The incorporation of polyethylene glycol (PEG) on surfaces as * To whom correspondence should be addressed. Phone: 713-743-1760. Fax: 713-743-1755. E-mail: [email protected].

coatings has been proven to be one of the most successful strategy in circumventing this problem. PEG is a hydrophilic and biocompatible polymer that is well-known to resist nonspecific protein, cellular adhesion, and biofilm formation.18-21 Several methods have been reported for presenting PEG chains onto these surfaces. For instance, physical adsorption of PEG on the surface is being used because it is straightforward and experimentally simple.22-24 However, it suffers drawbacks in terms of stability on the surface. Chemisorption is a technique often used to address the stability issue where the PEG chains are covalently attached onto surfaces.25,26 Nonetheless, this approach necessitates multiple processing steps, which may lead to poor reproducibility. Self-assembled monolayers (SAMs) have also became a popular technique in grafting PEG on interfaces because of its ease of preparation and production of relatively stable film on the surface.27,28 Despite the significant progress in SAM technology, several limitations are still present. To start with, SAMs are typically compatible only with specific metal or nonmetal substrates. The restricted two-dimensional arrangement also limits the conformational freedom and accessibility of the functional groups on the surface. Furthermore, the high packing densities of PEG chains produced by SAM formation leads to reduced protein resistance due to the loss of the hydrophilic interior necessary for the hydration mechanism in protein repulsion.29 One apparent solution to this restricted conformation of PEG on surfaces is to use a hyperbranched architecture of PEG units on the surface such as employing dendrimeric PEGs on surfaces to meet this kind of architecture.30 However, the main disadvantage of this approach is its tedious synthetic routes. Another approach is to use polymer

10.1021/bm1009365  2010 American Chemical Society Published on Web 10/28/2010

Protein and Cell-Resistant Brush via SI-RAFT

Biomacromolecules, Vol. 11, No. 12, 2010

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Scheme 1. General Scheme for the Preparation of Protein and Cell-Resistant PPEGMEMA Brushes from the Electrodeposited RAFT Agent

brushes containing PEG units on the surface. Studies have shown that the formation of a dense but disordered PEG is one of the factors for high protein resistance.29,31 For instance, a study showed that a polymer brush grafted with PEG has higher resistance to the protein kinesin as opposed to the OEG SAMs, which they correlated to the density of defects in the coating.32 Several studies have reported the surface-initiated polymerization (SIP) of oligo(ethylene glycol) (OEG)/poly(ethylene glycol)(PEG) methacylates to introduce an alternative macromolecular architecture of OEG or PEG units on the surface that possess a highly dense and relatively disordered structure.20,31-41 These studies showed the capability of these OEG/PEGcontaining methacrylates to resist nonspecific protein and cell adhesion on surfaces. However, these experiments necessitated different approaches on functionalizing the substrate with the initiator and involve the synthesis of initiators with different reactive groups depending on the substrate used. For example, initiators with thiol groups are synthesized to modify Au surfaces,31,33,34 while silane groups on initiators are needed for silicon substrates.31,36-40 The use of electrochemistry to introduce materials on surfaces would be advantageous because it extends surface modification on a wider variety of substrates, specifically conducting surfaces. This method does not rely on the substrate’s specific functionality but on the electrochemical activity of the material being deposited on the surface. Ignatova and co-workers reported the combination of cathodic electrografting and ATRP to graft polymers on surfaces.42 The same group also reported the direct electrografting of PEGMA on conducting surfaces to render protein-repellant properties.43 Although the latter method is a one-step process, several drawbacks are still posed by this electrografting method: (1) the process is highly sensitive to oxygen and moisture and, therefore, needs to be done in extremely anhydrous and inert conditions; (2) although demonstrated in methacrylates, cathodic electrografting is not applicable to a wide range of monomers, and other monomers may have an activity at the potential window used for direct electrografting, translating to loss of the actual chemical structure of the polymer; and (3) cathodic electrografting is also limited not only in terms of monomer but also in the solvents that can be used.44,45 In this paper, we combined electrochemistry and surfaceinitiated reversible addition-fragmentation chain transfer (SI-

RAFT) polymerization as a novel route for preparing polymer brushes, specifically, protein and cell-resistant poly(polyethylene glycol methyl ether methacrylate) (PPEGMEMA) polymer brush (Scheme 1). In this technique, a RAFT agent or chain transfer agent (CTA) containing an electrochemically active moiety was used to mediate SI-RAFT polymerization from the Au surface. The presence of the electro-active carbazole group in the RAFT agent enabled its immobilization on the electrode surface through potentiostatic and potentiodynamic methods. Reversed to cathodic electrografting, the electro-active moieties are anodically deposited on the surface. In general, this approach offers an advantage over cathodic electrografting because the process could be done under ambient conditions and can be done using a wider range of solvents. Combining this approach with surface-initiated polymerization (SIP) would also translate to a broader spectrum of polymers that can be grafted on the surface because the application of the potential is done before polymerization, which would avoid the possible chemical alterations of the monomer/polymer. Furthermore, the RAFT process is also known for its applicability over a wide variety of monomers. This tandem approach also offers a great advantage over other SIP techniques for functionalizing a variety of metal and metal oxide surfaces because of its use of a common RAFT agent. This avoids extensive synthesis of different initiators or functionalities specific for different electrodes. Although surface-initiated atom transfer radical polymerization (SI-ATRP) has been mostly demonstrated in the preparation of these POEGMEMA/PPEGMEMA brushes,31-36,39,40 the use of SI-RAFT technique for this purpose is worth studying not only because of the common advantages it shares with the ATRP (i.e., low polydispersity of brushes produced, versatility on wide range of monomers, living characteristics), but mainly because of its metal catalyst-free system,46 which is favorable for biomedical devices. To the best of our knowledge, this is the first report for preparing protein and cell-resistant polymer brushes using the combination of SIRAFT with the electrochemical deposition of the CTA.

Experimental Section Materials. Reagent chemicals were purchased from Aldrich and were used without further purification unless otherwise indicated. Tetrahydrofuran (THF) used in the synthesis and polymerization reactions was

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distilled from sodium/benzophenone ketyl. Methyl methacrylate (MMA, 99+%) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA; MW ) 300 g/mol) monomers were passed through a column with alternating layers of activated basic alumina and inhibitor remover replacement packing to remove the inhibitor and were stored at -20 °C. Au surface was prepared by deposition of a gold film (∼100 nm) on a chromium-coated (∼6-7 nm) silicon wafer by thermal vacuum evaporation. The acronym PBS designates phosphate buffered saline (Aldrich; 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2). Cell culture materials include Dulbecco’s modified Eagle’s medium (DMEM; Irvine Scientific), fetal bovine serum (FBS; Lonza Walkersville Inc.), antibiotic (penicillin-streptomycin; Irvine Scientific), 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES, 1 M; Irvine Scientific), L-glutamine (Irvine Scientific), minimum essential medium (MEM) nonessential amino acids solution 10 mM (100×; GibcoBRL), and trypsin-EDTA (Lonza Walkersville Inc.). Synthesis of Cbz-CTA. The synthesis of 3,5-bis(4-(9H-carbazol9-yl)butoxy)benzyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate (Cbz-CTA) was prepared as previously reported by our group.47 Electrodeposition of the CTA on Au Surface. Electrochemical studies were performed with a Parstat 2263 (Princeton Applied Research) instrument using PowerSuite software. All experiments were carried out using a three-electrode setup where the Au substrate was used as the working electrode, Pt wire as the counter electrode and Ag/AgCl as the reference electrode. A solution of the CTA (0.5 mM) and the supporting electrolyte, tetrabutylammonium hexafluorophosphate (TBAH; 0.1 M) in THF was used for preparing the electrogenerated CTA film. For cyclic voltammetry (CV) experiments, a scan rate of 50 mV/s was employed for the 10-cycle run in a potential window of 0-1.2 V. For potentiostatic experiments, varying constant potentials were used at a constant time and vice versa. Surface-Initiated RAFT Polymerization. In a typical run, a solution of the MMA (295.35 mg, 2.95 mmol), azobisisobutyronitrile (AIBN; 0.20 mg, 0.0012 mmol), and 5 mL of dry THF for MMA polymerization and PEGMEMA (2472.5 mg, 8.24 mmol), AIBN (0.54 mg, 0.0033 mmol), and 5 mL of dry THF for PEGMEMA polymerization were degassed in a Schlenck tube by bubbling with N2 gas for 30-45 min. The degassed solutions were transferred to another Schlenck tube backfilled with N2 gas containing the CTA-modified Au through a cannula. The tubes were placed in a preheated oil bath at 60 °C for 24 h, in the case of MMA, and 3 h, in the case of PEGMEMA. The slides were then subjected to Soxhlet extraction overnight using THF as solvent to remove any unbound polymers. Surface Characterization. Null ellipsometry was used to determine the thickness of the films after each surface functionalization. All measurements were conducted using a null ellipsometer (Multiskop, Optrel Berlin) with a He-Ne laser (λ ) 632.8 nm) as a light source. The angle of incidence was set to 60° on all measurements. A multilayer flat film model was used to calculate the thicknesses from the experimentally measured ellipsometric values ∆ and Ψ, assuming a refractive index of 1.6 for the electrochemically deposited CTA film,48 1.49 for the PMMA film,49 and 1.46 for the PPEGMEMA film.37 The film thickness was calculated using a fitting program (Elli, Optrel). Static contact angle goniometry was conducted using a KSV CAM 200 instrument (KSV Ltd.) using the bubble drop method with water. All atomic force microscopy (AFM) images were recorded in air under ambient conditions on PicoScan 2500 (Agilent Technologies formerly Molecular Imaging, Corp.) equipped with an 8 × 8 µm scanner. Intermittent contact mode was used for all imaging. The AFM tip used was a silicon-nitride AFM probe from Ted Pella Inc. A PHI 5700 X-ray photoelectron spectrometer (XPS) was equipped with a monochromatic Al KR X-ray source (hν ) 1486.7 eV) incident at 90° relative to the axis of a hemispherical energy analyzer. The spectrometer was operated both at high and low resolutions with pass energies of 23.5 and 187.85 eV, respectively, a photoelectron take off angle of 45° from the surface, and an analyzer spot diameter of 1.1 mm. The survey spectra were collected from 0 to 1400 eV, and the

Tria et al. high-resolution spectra were obtained for photoelectrons emitted from C 1s, O 1s, S 2p, and N 1s. All spectra were collected at room temperature with a base pressure of 1 × 10 -8. Electron binding energies were calibrated with respect to the C1s line at 284.8 eV. A PHI Multipak software (version 5.0A) was used for all data processing. The high-resolution data were analyzed first by background subtraction using the Shirley routine and a subsequent nonlinear fitting to mixed Gaussian-Lorentzian functions. Atomic compositions were derived from the high-resolution scans. Peak areas were obtained after subtraction of the integrated baseline and corrected for sensitivity factors. Protein Resistance Studies Using Surface Techniques. A 1 × 1 cm piece of the surfaces (PPEGMEMA-modified, CTA-modified and unmodified Au slides) was incubated with a fibrinogen solution (1 mg/ mL) in PBS buffer for 30 min. The samples were then washed with Millipore water for ∼30 s and finally dried with a flow of N2 gas. High resolution XPS was obtained for each slide where the [N]/[C] ratio was determined. Thicknesses and contact angles of the samples were also measured before and after the incubation with the fibrinogen. AFM images were also taken before and after protein adsorption on the PPEGMEMA surface. Protein Resistance Determination through Fluorescence Measurements. Nonspecific adsorption of fluorescein isothiocyanate-labeled fibrinogen (FITC-fibrinogen) on the PPEGMEMA-functionalized surface sample relative to the unmodified Au slide and CTA-modified was evaluated. A solution of FITC-fibrinogen (1.0 mg/mL) in PBS buffer was spotted on the slides using a capillary tube (Kimble Glass Inc.). The slides were placed in a closed chamber with a relative humidity of 58-60% at 25 °C for 30 min to dry the spots. The films were taken out and thoroughly rinsed with PBS (∼2 mL) for 3 times and dried under a stream of N2. The surface was scanned using a GeneTAC UC 4 Array Scanner with an excitation wavelength at 532 nm and emission wavelength at 518 nm. Fluorescence intensities were obtained using the ImagePro program. Cell Culture and Adhesion Assay. Cell adhesion assays were performed on bare Au surface, CTA-modified Au surface, and PPEGMEMA films to demonstrate the inhibition of the mammalian cell adhesion. The assays were done using three replicates per trial. NIH 3T3 fibroblasts (gift from Dr. Albee Messing of the University of Wisconsin-Madison) were cultured at 37 °C in a growth media containing 86% of Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum (FBS), 1% antibiotic (penicillin-streptomycin), 1% 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES, 1 M), 1% L-glutamine, and 1% minimum essential medium (MEM) nonessential amino acids solution 10 mM (100×; GibcoBRL). Fibroblast cells of passages 130 and 132 were harvested from culture flasks by a 10-12 min incubation with 0.25% trypsin and were resuspended in the growth media. Cell number was determined using a hemacytometer. Just prior to seeding, the surfaces (bare gold surface, CTA-modified gold slide, and PPEGMEMA film) were pretreated in a 12-well Falcon tissue culture polystyrene plate with 2 mL each of PBS for 30 min followed by another 30 min of preconditioning in 2 mL each of the growth media. Cells were seeded onto the substrates at a concentration of 60000 cells/well and incubated for 24 h in a 5% CO2 incubator at 37 °C. After 24 h, the substrates were washed with the growth media and were placed in another 12-well plate. To quantify the number of cells that adhered on the surfaces, 0.5 mL of the trypsin was added into each of the wells with the substrates and was incubated for 10 min. The samples were pipetted out into a 1.5 mL Eppendorf tube containing 0.5 mL of the media and were centrifuged for 1 min. Trypsin was aspirated and the cells were resuspended in 0.5 mL of the media for cell counting using the hemacytometer. The substrates used for imaging were washed with the media after the 24 h cell seeding. Adherent cells were stained using trypan blue and were imaged under a Nikon-80I reflected bright field microscope


Figure 1. CV diagram for the electrochemical cross-linking of 0.5 mM CTA in 0.1 M TBAH/THF with a scan rate of 50 mV/s. First cycle showed the oxidation peak of the carbazole at ∼1.15 V, which was shifted to ∼0.85 V upon the cross-linking of the carbazole units.


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Figure 2. CV diagram for the CTA-free scans of the potentiostatically deposited CTA at different voltages. All scans were carried out at a scan rate of 50 mV/s.

with the 20× objective. The digital images were taken and processed using Elements software.

Results and Discussion Scheme 1 presents the strategy employed for the fabrication of the polymer brushes on Au surfaces. The process starts with the electrodeposition of the CTA on the substrate followed by the SI-RAFT polymerization of MMA (used as a model monomer) and PEGMEMA (for protein and cell-resistant surfaces). The initial attachment of the CTA on the surface is a crucial step for the subsequent polymerization step and therefore necessitated full investigation. Electrochemical Studies for the CTA Electrodeposition. Cyclic voltammetry (CV) was used to study the electrochemical behavior of the CTA under anodic (oxidation) and cathodic (reduction) conditions. Figure 1 shows the CV diagram of the CTA ran in 10 cycles at 50 mV/s. During the first cycle, the onset of oxidation was observed at 1.0 V with the corresponding oxidation peak at ∼1.15 V. This peak is attributed to the formation of a radical cation (polaron) from the removal of an electron in the nitrogen atom of the N-substituted carbazole monomer.50 This reactive radical cation readily couples with another radical cation to form the 3,3′-bicarbazyl. Upon the next cycle, the appearance of a new anodic peak was seen at ∼0.85 V, which corresponds to the formation of a more stable dication (bipolaron) with extended π-conjugation resulting from the oxidation of the 3,3′-bicarbazyl.51,52 At this point, the peak at ∼1.15 V disappeared, signifying the complete oxidation of all the carbazole monomers. Subsequent cycles exhibited only one redox couple at ∼0.80 V (reduction) and ∼0.85 V (oxidation), which accounts for the formation of higher oligomeric species and further cross-linking of the carbazole units, as substantiated by an increase in current with each succeeding cycle at lower oxidation potentials.48 After investigating the electrochemical reactivity of the CTA using CV, the potentiostatic deposition of the CTA was also conducted. A CTA-free (without the CTA in solution) scan was done on the potentiostatically deposited CTA films to make sure that the redox signals are due to the immobilized CTA and not from the CTA present in the solution. Figure 2 shows the CTAfree CV scan of the CTA films deposited at different voltages with a constant time of 15 min. It can be observed that the effective deposition of the film occurred only at 1.2 V where the redox peaks coming from the cross-linked CTA are evident

Figure 3. XPS wide scan of the electrodeposited CTA. (Inset) Highresolution scans of (b) N 1s and (c) S 2p peaks. Table 1. XPS Elemental Composition Analysis of the Electrodeposited CTA element

atomic percentage on surface

expected atomic percentage

C 1s O 1s N 1s S 2p

78.11 5.40 8.85 7.64

78.59 5.29 8.05 8.07

even in a CTA-free solution. This result is expected because the carbazole monomer can only be activated for cross-linking upon its oxidation at a voltage equal to or higher than ∼1.15 V as depicted by the CV analysis. The XPS data of the film deposited at 1.2 V further verified the immobilization of the CTA on the surface. XPS survey scan (Figure 3a) of the film displayed the expected elemental signals of C, N, O, and S due to the CTA film. A high-resolution N 1s scan revealed an intense peak at 400.1 eV due to the nitrogen contributions from the carbazole ring53-55 and the cyano nitrogen.56,57 The appearance of a broad signal at 163.5 eV in the S 2p region was attributed to the dithio moiety of the CTA.58 The relative atomic concentrations of C, N, O, and S obtained from the XPS were in close agreement with the expected atomic percentage of the electrodeposited RAFT agent (Table 1). A parallel run on another conducting substrate, indium tin oxide (ITO), was also conducted by potentiostatic deposition of the CTA. This experiment was done to further check the successful cross-linking and deposition of the CTA on the surface as well as the feasibility of the method on other conducting surfaces (see Supporting Information). Figure S1

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Figure 4. XPS narrow scan of (a) C 1s, (b) O 1s, (c) S 2p, and (d) N 1s after immersion of the CTA-modified Au in THF for 2 h, 18 h, and 5 days. Table 2. Ellipsometric Thickness of the Electrodeposited CTA at Different Times of Immersion in THF time of immersion in THF 2h 18 h 5 days

thickness (nm) 39.75 ( 4.21 38.02 ( 2.24 38.07 ( 3.41

shows the comparison of the UV-vis spectra of the spin-coated CTA as opposed to the electrodeposited CTA. The presence of the broad maxima at around 400 and 800 nm for the electrogenerated CTA are assigned to the dicarbazyl radical cation and dicarbazyl dication, respectively,50 signifying the presence of the cross-linked carbazole moieties. On the other hand, these peaks are not present in the spin-coated CTA. In addition, no peaks were observed after rinsing the spin-coated slide, in contrast to the electrodeposited CTA, where the peaks are still present even after rinsing. The relative stability of adsorption of the film on the surface was confirmed by the XPS high-resolution scans. As shown in Figure 4, the peak intensities of all the elements present barely changed even after the immersion of the substrate in THF for long periods of time (up to 5 days), indicating that the RAFT agent is not being desorbed from the surface. Thickness measurements further supported the adsorption stability of the film as summarized in Table 2. Investigating the stability of the chemical composition of the CTA under anodic conditions is also a key step for this study. The active dithio moiety of the CTA must not be affected during the electrodeposition process to ensure its participation on the succeeding RAFT polymerization. To address this issue, the binding energies of the S 2p peaks from the electrogenerated CTA film and the spin-coated CTA film were compared. Similar spectra, which are both centered at 164 eV, were observed in the S 2p narrow scans of the electrografted and the spin-coated CTA indicating that the dithio group is unaffected during the electrodeposition (Figure 5). The absence of a peak at ∼168 eV, typical for sulfur oxides, also suggests the absence of sulfur oxides on the surface.59,60 The S 2p signal of the electrogenerated film was also compared

Figure 5. Comparison of the S 2p binding energies of the electrodeposited and spin-coated CTA on Au surface.

with the S 2p peak of a thiol, which represents a possible reduced form of the dithio moiety (Figure S2). The binding energies were also different, signifying that the dithioester is not reduced. Another issue to consider is the possible adsorption of the sulfur atom on the Au surface, which may hinder the activity of the dithio group during the polymerization. To ensure that the sulfur of the electro-immobilized CTA is not bound onto Au, a control experiment was done where the dithio moiety of the CTA was self-assembled on Au substrate61,62 instead of electrodeposition (procedure and conditions are in the Supporting Information). The S 2p narrow scan of the self-assembled CTA on the Au substrate displayed a peak at ∼162 eV (Figure 6), which is in good agreement with the reported values of a chemisorbed sulfur on Au.61,63-65 This binding energy is not observed in the electrodeposited CTA, implying that the sulfur on the electrodeposited film is not bound onto the surface. A possible reason why the adsorption of the dithio moiety onto the surface is prevented is that the rate of the electrodeposition process is faster than the rate of adsorption of the dithio group on Au. Furthermore, subjecting the self-assembled CTA to SI-RAFT polymerization of MMA for 24 h showed no brush growth based on thickness measurements, which is in contrast with the successful polymer brush growth from the electrodeposited CTA, as discussed in the next section.



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Figure 6. Comparison of the S 2p binding energies of the electrodeposited CTA and the self-assembled CTA.

SI-RAFT Polymerization from the Electrodeposited CTA on Au Surface. To investigate the capability of the electroimmobilized CTA in mediating SI-RAFT polymerization, MMA was first polymerized from the CTA-modified surface as a model monomer. The CTA-modified surface used for growing the brushes was electrodeposited for 3 min for the purpose of having thinner layers of the CTA with relatively good surface coverage based on AFM analysis. A thinner layer of the CTA is more desirable because one of the main goals of this study is to generate a PPEGMEMA-based film for protein and cell resistance studies. Thicker films of the electrodeposited CTA may affect the protein and cell resistance measurements later on. It may overpower the characteristic of the PPEGMEMA polymer brush, which would forfeit the principal focus of the paper. Ellipsometric measurements of the resulting film after 24 h of polymerization showed a pronounced change in thickness. The thickness of the film increased from ∼13 nm (initial CTA layer) to ∼99 nm (resulting film after polymerization), signifying the grafting of the PMMA brush on the surface. Changes in contact angle values were also observed before and after growing the PPMA brush on the surface (see Table S1). XPS analysis of the PMMA film showed a considerable increase in the intensity of the O 1s signal after polymerization, as compared to the electro-generated RAFT agent (Figure 7a). This increase is expected due to the PMMA growth, which presented more oxygen on the surface. The observed O/C ratio after polymerization (29:70) correlates well with PMMA composition that is 2 O atoms to 5 C atoms. In addition, the deconvolution of the C 1s peak established the presence of more O-CdO peak situated at ∼289 eV as compared to the electroimmobilized RAFT agent (Figure 7b).66 AFM analysis on the film before and after polymerization of MMA was also done. The difference in the morphologies before and after polymerization is evident, as presented in Figure 8, signifying the growth of the brush on the surface. After examining the capability of the CTA-modified gold substrate to mediate an SI-RAFT polymerization through grafting PMMA film from the surface, the method was applied to prepare the protein and cell-resistant surface. PEGMEMA, a macromonomer contains the poly(ethylene glycol) (PEG) moiety known to resist nonspecific protein and cell adhesion and the polymerizable methacrylate unit is similar to the model monomer, MMA. Similar processes were carried out for the SIP of the PEGMEMA on the gold surface. The polymerization time, however, was shorter than that of MMA as the PPEGMEMA gels out rapidly with the same conditions employed with the MMA polymerization.

Figure 7. (a) XPS narrow-scan of O 1s before (electrografted CTA only) and after SI-RAFT polymerization of MMA and (b) deconvolution of the C 1s peak after SI-RAFT polymerization of MMA.

Similar surface characterizations were performed on the film after PEGMEMA polymerization. The thickness increased from ∼11 to 26 nm, demonstrating the grafting of the PPEGMEMA polymer on the surface. Contact angle of the film also decreased due to the incorporation of the more hydrophilic PEG chains on the surface (see Table S1). XPS high-resolution scan also showed an increase in the O 1s peak intensity attributed to the growth of the PPEGMEMA brush (Figure 9a). In addition, the O/C ratio after polymerization of PEGMEMA was 30:68, close to the expected value for the PEGMEMA, that is 7 O atoms and 15 C atoms. The incorporation of the PEG chains on the surface was further evidenced by the increase of the C-O region in the deconvoluted C 1s peak as compared with the PMMA (Figure 7b). It is also noteworthy to mention the presence of the S 2p peak after SI-RAFT of PEGMEMA (Figure S3b) accounted for the sulfur in the dithioester end-group, indicating that the polymerization is still “living” up to this thickness. The growth of the PPEGMEMA brush was also supported by the AFM analysis. From Figure 10, an apparent change in the morphology is observed before and after the growth of the PPEGMEMA brush. The morphological feature in Figure 10b is typically observed for PEG brushes.67,68 Protein-Resistance Studies on the PPEGMEMA Film. The grafted PPEGMEMA brush was evaluated for nonspecific protein adsorption on the Au surface. The protein resistance was monitored using surface analytical and imaging techniques. A PPEGMEMA-modified Au substrate was incubated in the fibrinogen solution in PBS and was rinsed afterward for characterization. A CTA-modified film and a bare Au surface were also incubated in fibrinogen solution to serve as control experiments. Contact angle data of the PPEGMEMA film gave similar results before (58.05 ( 0.74°) and after protein incubation (56.26 ( 2.86°) implying a similar surface composition before and after immersion in protein. Ellipsometry data registered a very small change in thickness (∼1.3 nm) before and after protein incubation in the case of the PPEGMEMA

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Figure 8. AFM intermittent contact mode images (6 × 6 µm) of the Au surface (a) before (electrodeposited CTA only) and (b) after SI-RAFT polymerization of MMA.

Figure 11. Change in ellipsometric thicknesses before and after protein incubation of the bare Au, CTA film, and PPEGMEMA film.

Figure 9. (a) XPS narrow-scan of O 1s before (electrodeposited CTA only) and after SI-RAFT polymerization of PEGMEMA and (b) the deconvolution of the C 1s peak after SI-RAFT polymerization of PEGMEMA. Figure 12. XPS N/C ratio analysis before and after protein incubation of bare Au, CTA film and PPEGMEMA film.

Figure 10. AFM intermittent contact mode images (6 µm × 6 µm) of the Au surface (a) before (electrodeposited CTA only) and (b) after SI-RAFT polymerization of PEGMEMA.

surface (Figure 11). This observation is in contrast with the controls that gave a significant increase in thickness, suggesting the adsorption of more fibrinogen on these surfaces after incubation. To further characterize the surface after the protein resistance experiment, the N/C ratio of the XPS data was obtained. This was done to discount the N 1s and C 1s signals coming from the materials already deposited on the surface prior to protein incubation and to be more precise in accounting for the nitrogen

coming from the adsorbed protein. Presumably, an increase in the N/C ratio will be obtained only when fibrinogen is adsorbed on the surface because of the nitrogen signals coming only from the amino acids of the protein. As expected, the biggest change for the N/C ratio before and after protein adsorption was seen on bare Au substrate (Figure 12). The electrografted CTA film also showed a prominent change in the N/C ratio, giving more than a 3-fold increase as compared to the starting film. On the other hand, the PPEGMEMA film exhibited very good protein resistance, which only showed ∼2.5% increase in the N/C ratio after incubation in fibrinogen. This increase in the N/C ratio implies for ∼97.5% resistance to protein adsorption, which is comparable to the reported PPEGMA/PPEGMEMA surfaces37 and even better than the reported linear OEG SAMs on Au69 and spin-coated linear PEG.21 The excellent protein resistance of these surface-initiated PPEGMEMA brushes may be attributed to the formation of a highly dense but disordered surface. This architecture contributes to steric stabilization, lesser surface defects, and higher conformational freedom as compared to the densely packed linear PEGs/OEGs, which are necessary for the mechanism of protein resistance.70



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Figure 13. (a) Fluorescence imaging after rinsing the films spotted with FITC-labeled fibrinogen and (b) bar graph of the fluorescence intensity of the FITC-labeled fibrinogen left in the films after rinsing.

Figure 14. Microscopy images of after the cell culture on (a) bare Au, (b) electodeposited CTA, and (c) PPEGMEMA film. (d) Quantitative counting of cells on the surfaces.

The AFM data also supported the protein-resistant capability of the PPEGMEMA film grown from the electrodeposited CTA (See Supporting Information). The topographical image of the film after its immersion in protein solution is qualitatively similar prior to its exposure in the protein solution (Figure S4) verifying a notable resistance of the film to nonspecific protein adsorption. AFM analyses for the control experiments were not taken because the particle sizes for the electro-immobilized CTA and the bare Au surface may not be distinguishable from that of the fibrinogen molecule. To visually investigate the protein resistance of the PPEGMEMA film, fluorescence imaging study was employed. The FITC-tagged fibrinogen molecule was manually spotted onto the PPEGMEMA-modified surface and the control surfaces. After incubation and drying of the spots, the substrates were rinsed and visualized under a fluorescence scanner. Figure 13 clearly present the distinction of the PPEGMEMA surface over the control surfaces toward protein resistance. Very weak fluorescence intensity was seen in PPEGMEMA surface, confirming the removal of most proteins from the surface as opposed to the intense fluorescence signals observed for the adsorbed proteins on the control films. Cell-Resistance Study on the PPEGMEMA Film. Figure 14 presents the data for the NIH 3T3 fibroblast 24 h adhesion test for the prepared PPEGMEMA film and the control surfaces. Figure 14a notably showed no fibroblast adhesion on the PPEGMEMA brush after the 24 h culture test.

However, for the control surfaces (Figure 14b,c), it is apparent that cell adhesion was not inhibited. A quantitative analysis of the cell attachment on the three surfaces is given in Figure 14d. The plots corroborated with the pictures obtained under the scanning microscope where the least adhesion was obtained on the PPEGMEMA surface. The highest adsorption of the fibroblast cells was found in the CTA-modified Au surface. This result can be explained by the enhanced hydrophobic interactions between the substrate and the cell upon the incorporation of CTA on the Au surface. Hydrophobic-hydrophobic interactions were previously reported as the initial mechanism of cell adhesion onto surfaces.71,72 The capability of the PPEGMEMA-modified surface to resist cell adhesion, on the other hand, is in agreement with the results that have been previously reported by other groups.31,35-38 The resistance to cell adhesion of the PPEGMEMA surface can be explained by the same mechanism of protein resistance because of the dependence of cells on specific proteins for its anchorage to the surface.73

Conclusions A novel route for preparing polymer PPEGMEMA brushes that inhibits nonspecific adsorption of proteins and mammalian cells was demonstrated on Au surfaces. The electrochemically immobilized CTA has proven to be effective in mediating the SI-RAFT polymerization, as presented by

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different characterization techniques such as XPS, ellipsometry, contact angle, and AFM. Surface analyses also confirmed the presence of the electro-generated CTA film on the surface even after soaking for days in THF, as well as its stability under anodic conditions. The PPEGMEMA polymer brush grown from the electrodeposited CTA was verified to have very good protein and cell-resistant properties, as presented in the results established by the surface characterization and imaging techniques, for protein resistance studies and cell culture test for the investigation of cell-repellant properties. With these results, this approach should be very promising for coating any conducting materials (metal, metal alloys, metal oxides, and electrodes), which is of significance in the biomedical field. Future work in this area currently involves the use of electrodeposition with SIATRP, use of other electrochemically active groups (pyrrole, thiophene, aniline, etc.), and electropatterning and nanopatterning. Acknowledgment. The authors acknowledge funding from NSF DMR-10-06776, ARRA-CBET-0854979, CHE-1041300, Texas NHARP 01846, and Robert A. Welch Foundation, E-1551. Technical support from Agilent Technologies, Malvern Instruments, and Optrel is also acknowledged. The authors also thank Prof. Allison McDermott of the College of Optometry, University of Houston, and Dr. Catherine Santos for their kind help in the cell adhesion experiments. Supporting Information Available. Experimental procedure for SAM formation of the CTA on Au, UV-vis spectra of the electrodeposited CTA and spin-coated CTA, comparison of high-resolution S 2p peaks for thiol and CTA on Au, as well as for the films after MMA and PEGMEMA polymerization, and AFM images before and after protein incubation of PPEGMEMA brush. This material is available free of charge via the Internet at http://pubs.acs.org.

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Electrochemical Deposition and Surface-Initiated RAFT

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