Impact of Epidermal Growth Factor Tethering Strategy on Cellular

Nov 8, 2010 - Technology Group, Bioprocess Sector, Biotechnology Research Institute, ... reactive groups facilitating subsequent protein grafting, CMD...
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Bioconjugate Chem. 2010, 21, 2257–2266

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Impact of Epidermal Growth Factor Tethering Strategy on Cellular Response Benoıˆt Liberelle,† Cyril Boucher,†,‡ Jingkui Chen,† Mario Jolicoeur,† Yves Durocher,‡ and Gregory De Crescenzo*,† Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biome´dicales, Bio-P2 Research Unit, E´cole Polytechnique de Montre´al, P.O. Box 6079, succ. Centre-Ville, Montre´al (Qc), Canada H3C 3A7, and Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, Montre´al (Qc), Canada H4P 2R2. Received June 8, 2010; Revised Manuscript Received October 15, 2010

In an effort to evaluate the impact of various epidermal growth factor (EGF) grafting strategies upon cell surface receptor activation and cell adhesion, we generated low-fouling surfaces by homogeneously grafting carboxymethylated dextran (CMD) on amino-coated glass substrate. By preventing nonspecific cell adhesion while providing reactive groups facilitating subsequent protein grafting, CMD allowed achieving specific cell/tethered EGF interactions and therefore deriving unambiguous conclusions about various EGF grafting strategies. We demonstrate here that A-431 cell response to immobilized EGF is highly dependent on the bioactivity of the tagged protein being tethered, its proper orientation, and its surface density. Among all the approaches we tested, the oriented tethering of fully bioactive EGF via a de noVo-designed coiled-coil capture system was shown to be the most efficient. That is, it led to the most intense and sustained phosphorylation of EGF receptors as well as to strong A-431 cell adhesion, the latter being comparable to that observed with amino-coated surfaces in the absence of CMD.

INTRODUCTION Bioengineered implants are becoming extremely promising with respect to the development of biocompatible surfaces aiming at promoting and sustaining tissue regeneration. Within the last three decades, in an effort to mimic nature’s mechanisms that are involved in tissue development and healing processes, the immobilization of growth factors on various substrates has been extensively studied by several groups (1–3). Such an interest from the scientific community may be ascribed to the direct implication of growth factors in almost all fundamental cellular processes, including cell survival, proliferation, migration, differentiation, and apoptosis. Furthermore, growth factor immobilization is believed to limit their clearance through endocytosis/degradation pathways and their diffusive mobility, thus restraining delivery to the local implant region (4). In that context, the epidermal growth factor (EGF), a soluble 6 kDa polypeptide that mediates its biological function by promoting the oligomerization (5, 6) and the phosphorylation (7–9) of cell surface EGF receptors (EGFRs), has attracted a lot of attention and has already shown great promises for the generation of smart corneal implants promoting re-epithelialization (10, 11) as well as for ex ViVo stem cell expansion (12–15). Nonspecific adsorption of EGF on substrate has been demonstrated to drastically diminish EGF bioactivity, most likely due to the occurrence of adsorption-induced conformational changes or inappropriate orientation of EGF (4). In contrast, covalent immobilization strategies via photoimmobilization or wet chemistry route resulted in unambiguous effects upon cell adhesion, phenotypic changes, and DNA synthesis (4, 16). However, for human EGF (hEGF), amine chemistry has been acknowledged to be suboptimal since the lysine side chains of * To whom correspondence should be addressed. E-mail: gregory. [email protected]. Tel: (514) 340-4711 (#7428). Fax: (514) 3402990. † ´ Ecole Polytechnique de Montre´al. ‡ National Research Council Canada.

hEGF extensively contribute to receptor binding; their involvement in the generation of an amide bound during EGF immobilization has been shown to greatly decrease its bioactivity (4, 17, 18). To counteract this caveat, oriented and stable tethering of hEGF has been successfully achieved through the use of recombinant EGF chimeras. That is, EGF has been fused to various tags or domains including the Fc region of immunoglobulin G (19), poly histidine (12), or coil peptide tags (18), in order to subsequently tether these chimeras on surfaces on which Protein A/G, nitrilotriacetate (NTA), or complementary coil peptide had been immobilized in a covalent fashion, respectively. Although the noncovalent but stable capture of EGF via tags hold great promise, this strategy should be examined with caution. We indeed recently demonstrated that the nature and the position of the tags both influence EGF bioactivity (20). More specifically, an Fc tag was shown to have a deleterious effect on EGF bioactivity when fused at its N- or C-terminus, as deduced from the ability of these constructs to induce A-431 cell receptor phosphorylation in Vitro. In this assay, EGFR phosphorylation was not detected when the cells were treated with soluble Fc-EGF, while EC50 values of 17 nM versus 4.3 nM for EGF-Fc and EGF, respectively, were determined when these ligands were provided to the cells in their soluble forms. In stark contrast, EGF being N-terminally tagged with a short (i.e., 35 amino-acid-long) peptide (the Ecoil) was found to be as active as untagged EGF (EC50 of 5.5 nM). The effect of these various tags upon EGF signaling, when captured in an oriented fashion, has never been rigorously compared, although several independent studies have demonstrated that the EGF moiety of these chimeric proteins was able to promote a cellular response. Another point to be considered with great care in growth factor immobilization resides in the choice of the spacer arm ensuring proper display and attachment of the growth factor of interest. So far, homo- (21) and heterobifunctional (22) polyethylene glycol (PEG) chains have been extensively used. However, as outlined by Kuhl et al. (4) and Klenkler et al. (21),

10.1021/bc1002604  2010 American Chemical Society Published on Web 11/08/2010

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MATERIALS AND METHODS

99+% purity) and toluene (99.5% purity) were purchased from VWR International, Inc. (Mont-Royal, Qc, Canada). All solvents were distilled under strict anhydrous conditions prior to use. Milli-Q quality water (18.2 MΩ · cm; total organic compounds (TOC) ) 4 ppb) was generated with a Millipore Gradient A 10 purification system. Cysteine-tagged Kcoil peptides were synthesized by the peptide facility at the University of Colorado (Denver, CO) (28). Untagged hEGF and EGFRED-Fc (artificially dimerized EGFR ectodomain through the Fc portion of an IgG1) was purchased from R&D Systems (Minneapolis, MN). All the surface plasmon resonance (SPR) experiments were performed using a Biacore T100 instrument (GE Healthcare, Baie d’Urfe´, Qc, Canada). CM4 sensor chips, N-hydroxysuccinimide (NHS), ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), ethanolamine, 2-(2-pyridinyldithio)ethaneamine hydrochloride (PDEA), and borate buffer (pH 8.5) were also purchased from GE Healthcare. Dextran (70 kDa) was obtained from Pharmacosmos A/S (Holbaek, Denmark). Dextran Carboxymethylation and Characterization. Dextran carboxymethylation and characterization was performed by adapting previously reported protocols (29). Briefly, 400 mg of dextran was dissolved in 10 mL of 3 M NaOH containing 1 M monochloroacetic acid. The solution was stirred for 2 h at room temperature (RT). The reaction was stopped by the addition of 40 mg of NaH2PO4 followed by pH adjustment to neutral using 18 M H2SO4. The solution was then filtered through a 0.2 µm PTFE filter, dialyzed five times against Milli-Q water for 1 h in order to remove reagents and salts, and finally lyophilized. CMD powder was stored at 4 °C until use. The carboxymethylation degree of our product was assessed using 1H NMR spectroscopy. Spectra for CMD were recorded with a Varian Unity Inova 400 MHz NMR spectrometer equipped with a 5-mm indirect probe, operating at a frequency of 399.95 MHz. Samples were examined as solutions in D2O at 323.1 K. Water signal was suppressed by pulse sequences (72.4°). Chemical shifts are given relative to external tetramethylsilane (TMS ) 0 ppm). Ecoil-Tagged and Fc-Tagged EGF Production. Ecoil-EGF, EGF-Fc, and Fc-EGF were produced in HEK293-6E cells, purified, and quantified as previously described (20). Purified recombinant proteins were then aliquoted and stored at -80 °C until use. Surface Preparation. Glassware was carefully cleansed by overnight immersion in a bath of KOH-saturated isopropyl alcohol followed by intensive rinsing with Milli-Q water. Prior to layer grafting, silicon and amino-coated glass surfaces were washed to remove any adsorbed organic compounds. The silicon surfaces (10 × 10 mm) were cleansed according to the Piranha method, i.e., the surfaces were immersed in a 3:1 mixture of sulfuric acid and hydrogen peroxide for 10 min at 100 °C. After intensive rinsing with Milli-Q water, the surfaces were dried in air at RT and then introduced in a dried Schlenk. To ensure anhydrous conditions, two cycles of vacuum/dry argon were performed in the Schlenk. The amino-coated glass surfaces (10 × 10 mm) were cleansed in an ultrasonic bath for 1 min in distilled chloroform and then dried in air at RT.

Chemicals and Reagents. Native silicon wafers (Prime Si P/Boron) were obtained from University Wafer (South Boston, MA), and amino-coated glass slides (10 × 10 mm) were from Erie Scientific Co. (Portsmouth, NH). Anhydrous 3-aminopropyltriethoxysilane (APTES, 99% purity), cysteine (99+% purity), monochloroacetic acid (99+% purity), sodium hydroxide (98+% purity), sodium monobasic phosphate (99+% purity), and sodium chloride (99.99% purity) were purchased from Sigma-Aldrich Canada, Ltd. (Oakville, ON). Hydrogen peroxide (30% v/v), chlorhydric acid (37% v/v), chloroform (99.8% purity), sulfuric acid (98% purity), dimethyl sulfoxide (DMSO,

Grafting Procedures. APTES Grafting on Silicon Surfaces. Amino groups were generated on silicon surfaces using a silanization procedure as previously reported (18, 30). Briefly, APTES was first dissolved in anhydrous toluene at a concentration of 10 mM. The solution was injected through a 0.2 µm PTFE filter in dry Schlenk tubes each containing one silicon surface. After a reaction time of 3 h at RT, the surfaces were taken out from the Schlenk tubes, dried in air for 1 min, and placed in an oven at 120 °C for 30 min. The surfaces were then rinsed by soaking them in freshly distilled toluene for 5 min.

Figure 1. Strategies for EGF immobilization on CMD. (A) Ecoil-EGF capture on a covalently attached Kcoil peptide layer, (B) EGF-Fc capture on a covalently attached Protein A layer, and random covalent attachment of (C) EGF-Fc and (D) untagged EGF on CMD layer.

PEG grafting often results in heterogeneous surface coverage that does not totally block nonspecific adsorption of the growth factor under investigation or of other proteins present in the medium. Furthermore, as already mentioned, subsequent hEGF chemical linkage to activated PEG also leads to heterogeneous EGF display. Hence, results from in Vitro cell culture experiments performed on PEG surfaces being derivatized with growth factors may not only reflect the effect of the tethered protein but may also be attributed to more complex phenomena being triggered by the combination of suboptimally immobilized and adsorbed species. As an alternative to PEG, synthetic or natural polymers presenting functionalities such as amine (e.g., chitosan and polyvinylamine), or carboxyl (e.g., carboxymethylated dextran (CMD)) groups distributed homogeneously along their chains are often used as a linker to facilitate the attachment of specific proteins and thus further tailor material surface properties (1). In addition to introducing desired reactive groups at the material surface for subsequent protein grafting, the added polymeric layer per se can also affect the macroscopic surface properties (hydrophilicity, roughness) and modulate cell and protein affinity for the engineered material surface (22–24). The aim of this work was to compare various controlled constructions of EGF-coated surfaces within the same study (Figure 1) in order to further investigate the impact of the tags (nature and size), the immobilization strategy (i.e., random immobilization versus oriented tethering) upon EGFR activation, and cell adhesion. A-431 cells were cultured on various EGFderivatized surfaces to correlate the type of EGF display with cell adhesion and EGFR activation. In that effort, a homogeneous polymeric layer of CMD was selected as a polymeric spacer for its ability to provide sufficient reactive groups homogeneously distributed on the surface and because of its low-fouling properties (25–27). By preventing nonspecific cell adhesion, CMD allowed us to achieve specific cell interactions with immobilized EGF and therefore derive unambiguous conclusions about the impact of our various EGF grafting strategies upon receptor activation and cell adhesion.

EGF Tethering Strategies

CMD Grafting on Amino-Coated Silicon and Glass Surfaces. CMD was covalently attached onto amino-coated surfaces using water-soluble carbodiimide chemistry. CMD solutions (2 mg/ mL) were prepared in Milli-Q water. Once dissolved, NHSactivation of CMD was performed by adding 150 µL of 0.4 M EDC in Milli-Q water and 150 µL of 0.1 M NHS in Milli-Q water to the CMD solutions. The amino-coated surfaces were covered with 150 µL of NHS-activated CMD solution for 2 h at RT. The surfaces were finally washed in a 10 mM phosphate buffer solution (pH 7.4) in ultrasonic bath for 2 min, rinsed by soaking them in 10 mM phosphate buffer (pH 7.4) and rinsing three times in fresh Milli-Q water. Kcoil Peptide Grafting on CMD-Coated Surfaces. Kcoil peptides were immobilized on CMD-coated surfaces using PDEA cross-linker. CMD-coated surfaces were first activated with 100 µL of 0.2 M EDC and 0.05 M NHS in Milli-Q water for 15 min at RT and rinsed in fresh Milli-Q water for 5 min. One hundred microliters of 10 mg/mL PDEA in borate buffer (10 mM disodium tetraborate, 1 M NaCl, pH 8.5) was deposited on each surface for 15 min at RT. The surfaces were rinsed by soaking them in Milli-Q water for 5 min. The thiol-reactive surfaces were then reacted with 100 µL of 10 µM cysteinetagged Kcoil (5 heptad repeats (28)) in 10 mM acetate buffer (pH 5.5) for 2 h at RT. The surfaces were finally rinsed by soaking them in 5 M guanidium hydrochloride solution, 10 mM phosphate buffer, and in Milli-Q water for 5 min each. Unreacted PDEA sites were blocked using 100 µL of 50 mM cysteine solution (1 M NaCl in 0.1 M sodium acetate, pH 4.0) followed by rinsing with Milli-Q water for 5 min. Kcoil Peptide Grafting on Amino-Coated Glass Surfaces. Kcoil peptides were immobilized onto amino-coated surfaces via N-succinimidyl-6-(3’-(2-pyridyldithio)-propionamido)-hexanoate (LC-SPDP) linker, as previously described (18). Briefly, the surfaces were covered with 150 mL of 2 mM LC-SPDP in 100 mM phosphate buffer (10% DMSO) for 2 h at RT. The surfaces were rinsed by soaking them in Milli-Q water for 5 min. Then, the LC-SPDP-coated surfaces were covered with 150 mL of 10 mM cysteine-tagged Kcoil peptides in 10 mM phosphate buffer for 2 h at RT. The surfaces were rinsed in 10 mM phosphate buffer (pH 7.4) and in Milli-Q water. One hundred fifty microliters of 50 mM cysteine solution (1 M NaCl in 0.1 M sodium acetate, pH 4.0) was used to block unreacted LC-SPDP sites. The surfaces were rinsed by soaking them in Milli-Q water for 5 min. Ecoil-Tagged EGF Tethering and RemoVal. Surfaces harboring covalently bound Kcoil were covered with 150 µL of 600 nM Ecoil-EGF in 10 mM phosphate buffer (pH 7.4) for 2 h at RT. The surfaces were rinsed in 10 mM phosphate buffer and Milli-Q water for 5 min. When necessary, removal of the Ecoiltagged EGF was performed by soaking the surfaces in a 5 M guanidium hydrochloride solution, followed by rinsing in 10 mM phosphate buffer and Milli-Q water for 5 min each. Nonoriented EGF CoValent Immobilization on CMD-Coated Surfaces. CMD-coated surfaces were treated with 100 µL of 0.2 M EDC and 0.05 M NHS in Milli-Q water for 15 min at RT and rinsed in fresh Milli-Q water for 5 min. 100 µL of 600 nM untagged hEGF or Fc-tagged EGF in 10 mM acetate buffer (pH 4.5) were deposited on NHS-activated CMD surfaces for 2 h at RT. The surfaces were then rinsed by soaking them in 10 mM phosphate buffer and Milli-Q water for 5 min each. Fc-Tagged EGF Capture Via Immobilized Protein A. CMDcoated surfaces were activated with 100 µL of 0.2 M EDC and 0.05 M NHS in Milli-Q water for 15 min at RT and rinsed in fresh Milli-Q water for 5 min. 100 µL of 10 µM Protein A in 10 mM acetate buffer (pH 4.5) were reacted with NHS-activated CMD surfaces for 1 h at RT. The surfaces were rinsed in 10 mM phosphate buffer and Milli-Q water for 5 min each. The

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surfaces were then covered with 100 µL of 1 µM Fc-tagged EGF in 10 mM phosphate buffer (pH 7.4) for 2 h at RT. The surfaces were finally rinsed by soaking them in 10 mM phosphate buffer and Milli-Q water for 5 min each. Contact Angle and Ellipsometric Measurements. Contact angle and ellipsometric measurements were performed as previously described (18). SPR Assays. SPR experiments were conducted at 25 °C using HBS-T (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween 20) as running buffer. Kcoil Immobilization on CMD Sensorchip. All the covalent immobilization steps were carried out at a flow rate of 10 µL/ min. The carboxylic groups of the CMD matrix of biosensor chips were first activated by injecting 0.05 M NHS mixed with 0.2 M EDC (7 min). The NHS-activated carboxylic groups were then reacted with PDEA cross-linker (10 mg/mL in borate buffer, 7-min injection) to end-graft cysteine-labeled Kcoil (10 µM Kcoil in 10 mM acetate buffer (pH 5.5), 20-s injection). Finally, freshly prepared cysteine solution (50 mM in 0.1 M acetate buffer, 1 M NaCl, pH 4.0) was injected (7 min) to block all unreacted PDEA-activated sites. For each Kcoil-functionalized surface, a mock surface was also generated by applying the same procedure but replacing Kcoil peptide injection by buffer injection. Ecoil-EGF Capture and Subsequent Injections of EGFREDFc. Ecoil-EGF capture and subsequent injections of EGFREDFc to test Ecoil-EGF bioactivity were performed at a flow rate of 50 µL/min. Regeneration of the sensorchip (i.e., removal of Ecoil-EGF) was accomplished by two pulses of guanidium hydrochloride (50 µL, 100 µL/min, 5M). Immobilized EGF Quantification. Commercially available DuoSet ELISA kit (DY-236, R&D Systems, Minneapolis, MN) was used to quantify EGF and Ecoil-EGF concentrations in solution before and after immobilization/capture on surfaces according to the manufacturer recommendations and using a Victor (3) V ELISA plate reader (PerkinElmer Inc., Woodbridge, ON). Standard curves were generated with tagged and untagged EGF proteins depending on the protein to be quantified. Cellular Assays. Cell Culture. A-431 cells were maintained in 175 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO/BRL, Burlington, ON) supplemented with 10% fetal bovine serum (Invitrogen, Burlington, ON) at 37 °C with 5% CO2 until 85-90% confluence was reached. A-431 Seeding. Prior inoculations onto our various surfaces, A-431 cells were washed with phosphate buffered saline (PBS) and incubated overnight in DMEM medium without serum. After trypsinization (5 min, 37 °C), A-431 cells were washed by centrifugation and resuspended in DMEM (no serum). Then, A-431 cells were distributed at 1 × 106 cells/mL, 1 mL/well, in 24-well plates containing aminated glass surfaces, Kcoil functionalized glass surfaces where Ecoil-EGF had previously been immobilized via coiled-coil interactions, CMD-coated glass surfaces in the presence or absence of soluble EGF (4 nM), CMD-coated glass surfaces where Ecoil-EGF had been previously immobilized via coiled-coil interactions, CMD-coated glass surfaces where EGF-Fc had been previously captured via Fc/protein A interactions, and CMD-coated glass surfaces where EGF and EGF-Fc had been covalently immobilized via amine chemistry (nonoriented immobilization). Cell Adhesion. DMEM medium was removed 4 h after inoculation and adhered cells were washed three times with DMEM (serum-free). Cells were observed using inverted microscope Axiovert S100TV (Carl Zeiss Canada, North York, ON). Images were processed with a QICAM Fast 1394 camera (QImaging) and cell count was achieved using Northern Eclipse image acquisition software (Empix imaging).

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Figure 2. 70 kDa dextran carboxymethylation. Chemical reaction scheme (A) and 1H NMR spectra of dextran (B) and product of dextran carboxymethylation (C). Letters in bold correspond to the protons borne by the anomeric carbons (a and a′) or by the methyl groups (b) added by the chemical reaction.

Phosphorylation Assays and Cell Lysis. DMEM medium was removed 2, 3, 4, and 5 h after inoculation, and A-431 cells were washed three times with PBS supplemented with sodiumorthovanadate (1 mM, Sigma) and extracted with lysis buffer (50 mM HEPES pH 7.4), 150 mM NaCl, 1% Thesit, 0.5% Na Deoxycholate, Complete protease inhibitor cocktails (Roche, Laval, Qc)) supplemented with 0.1 mM Na3VO4, 20 mM β-glycerophosphate, 10 mM Na4P2O7 · 10 H2O and 0.5 µM microcystin). Insoluble material was removed by centrifugation at 10 000 g for 5 min at 4 °C. Protein phosphorylation was then analyzed by Western blotting as previously described (18).

RESULTS AND DISCUSSION Dextran Carboxymethylation and Grafting on AminoCoated Glass Surfaces. To introduce carboxylic groups, 70kDa dextran chains were first transformed into negatively charged CMD (Figure 2A). -CH2-COOH groups were introduced in the chain by reaction of monochloroacetic acid with

the hydroxyl groups held by the glucopyranose rings of dextran without degrading the polysaccharidic backbone (as opposed to methods relying on periodate oxidation) (25). Although the carboxymethylation protocol we used is known to kinetically favor the modification of hydroxyl groups held by the C2 carbon, modifications may also occur on all the -OH groups of the glucopyranose cycle (25). Dextran modification was confirmed by 1H NMR spectroscopy. As shown in Figure 2, the product of the carboxymethylation reaction exhibited two distinct peaks within the spectral region corresponding to an anomeric proton signal (Figure 2C). By comparing this spectrum to that of unmodified dextran (Figure 2B), peaks at δ ) 5.04 ppm and δ ) 5.23 ppm were attributed to the anomeric protons of glucopyranose units being unsubstituted (a) or bearing a carboxymethyl group (a′) at their C2 position, respectively. Also, the spectrum corresponding to the carboxymethylation product was characterized by the presence of new peaks within the 4.17-4.35 ppm δ region (denoted b within Figure 2C). These

EGF Tethering Strategies

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Figure 3. Ellipsometric and water contact angle characterization of CMD grafting on aminated silicon surfaces. APTES layer grafted on silicon surfaces (a) and subsequent grafting of carboxymethylated (CM) dextran in the absence of salt (b) or dissolved in 0.1 M NaCl solution (c). As a comparison, the characterization of pristine (unmodified) dextran incubation on APTES-coated surfaces is also presented. The dark and light gray bars correspond to dry thickness and contact angle measurements, respectively. The reference was set as (0 nm, 0°) for a cleaned silicon surface. The successive grafting reactions were performed as described in the Materials and Methods section and summarized in the table below the graph. Values are given as mean value ( standard deviation (n ) 5).

Figure 4. Ellipsometric characterization of Kcoil peptide covalent immobilization on CMD surfaces. PDEA grafting (b) and subsequent immobilization of cysteine-tagged Kcoil peptide (c-g) on CMD-coated silicon surfaces (a). Cysteine-tagged Kcoil was reacted on PDEAcovered surfaces at pH 3.5 (c), pH 4.5 (d), pH 5.5 (e), pH 7.4 (f) and pH 8.5 (g). The reference was set as 0 nm for a cleaned silicon surface. The grafting reactions were performed as described in the Materials and Methods section and summarized in the table below the graph. Values are given as mean value ( standard deviation (n ) 5).

peaks corresponded to the -CH2 protons of the added carboxymethyl group. The degree of carboxymethylation was thus determined from the ratio of the areas corresponding to the -CH2 signals of the CH2-COOH groups (b region within Figure 2C) to the areas corresponding to the anomeric proton signals (a and a′ peaks within Figure 2C). Under our experimental conditions, the carboxymethylation degree of our 70kDa dextran was estimated to be 22%. In a second step, silicon surfaces were chosen as substrate to investigate CMD grafting to aminated substrate. Ellipsometric and water contact angle measurements were performed to evaluate the effect of salt upon our grafting protocol. In that endeavor, amine groups were first generated on silicon surfaces via a silanization reaction occurring between APTES and silanol groups that are naturally exposed on the surfaces (18). As illustrated in Figure 3 (condition a), the layer of deposited APTES appeared to be thin and homogeneous as deduced from ellipsometric thickness (0.8 ( 0.15 nm) and water contact angle (51 ( 2°) measurements, both in excellent agreement with those we determined in a previous study (18). CMD chains were then grafted on these amino-functionalized surfaces using EDC and NHS as coupling agents. In this way, NHS-activated carboxyl groups of CMD chains were reacted with free amine groups on the surface to create a stable amide bound. In an effort to maximize CMD grafting on amino-coated surfaces, the coupling reaction was carried out both in the absence and presence of salt (0.1 M NaCl) in the CMD solution (Figure 3, conditions b and c). We reasoned that salt addition would decrease the electrostatic repulsions that may occur within and between the charged CMD chains. We thus expected to obtain more compact individual CMD chains in solution and therefore denser grafted CMD layers. Whereas a dense and hydrophilic polymeric layer was grafted (net dry thickness of 1.3 nm and water contact angle of 11 ( 2°) in the absence of salt, the addition of 0.1 M NaCl resulted in a 53% lower net dry thickness (0.7 nm). As a negative control, when the same reaction was performed with pristine dextran, only minor variations in surface characteristics were observed when compared to APTES-coated silicon surfaces (+ 0.05 nm and +0.5° for dry thickness and contact angle values, respectively). The negative impact of salt upon CMD

grafting has previously been reported by others (31). This effect is most likely related to a drastic loss of NHS-activated carboxyl groups via hydrolysis, the latter being promoted by high salt concentrations (32). EGF Grafting. The covalent immobilization as well as the stable tethering of several EGF variants were then studied on optimized CMD-grafted surfaces by applying the oriented and nonoriented strategies described in Figure 1. That is, two distinct tag systems were independently evaluated (coil and Fc tags) and compared to the nonoriented but covalent immobilization of EGF or EGF-Fc onto CMD. Coiled-Coil-Mediated EGF Tethering. The first tethering strategy consisted in covalently grafting cysteine-tagged Kcoil peptides on CMD-covered surfaces, followed by the capture of Ecoil-tagged proteins via E/K coiled-coil interactions. Cysteinetagged Kcoil peptides were first grafted on CMD using PDEA as a bifunctional linker (28). NHS-activated CMD chains were reacted with the free amine group of PDEA linker to form an amide bond. The resulting film showed an ellipsometric thickness of 2.2 ( 0.2 nm (Figure 4, condition b). The other extremity of PDEA linker, terminated by a pyridyl disulfide group, was then reacted with the thiol group of cysteine-terminated Kcoil peptide to create a covalent disulfide bond. In an effort to maximize Kcoil grafting density, the coupling reaction was studied at various pHs ranging from 3.5 to 8.5 (Figure 4, conditions c-g). Maximal effective thickness, i.e., 0.7 ( 0.1 nm (Figure 4, condition e), was obtained for Kcoil peptides reacted at pH 5.5. Cysteine was then used to block unreacted pyridyl disulfide group of PDEA linkers and therefore prevent the undesired attachment of other molecules containing thiol groups. No significant changes in surface characteristics were observed before (Figure 4, conditions b and e) and after (Figure 5, conditions a and d) cysteine-mediated PDEA deactivation. The ability of our CMD surfaces, when functionalized with Kcoil peptides, to recruit N-terminally Ecoil-tagged EGF chimera was then tested. As can be seen in Figure 5, the incubation of Ecoil-EGF on Kcoil peptides previously bound to CMD surfaces, followed by extensive rinsing to elute unbound material, led to a net increase in dry thickness of 0.8 ( 0.2 nm. Further treatment of surfaces on which Ecoil-EGF

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Figure 5. Ellipsometric characterization of Ecoil-tagged EGF tethering on Kcoil-coated CMD surfaces. Dry thickness measurements corresponding to silicon surfaces sequentially treated with APTES, CMD, PDEA, Kcoil (pH 5.5) and cysteine solutions (a) that were incubated with Ecoil-EGF (b) and then exposed to a 5 M guanidium hydrochloride (Gnd-HCl) solution (c). As negative control, the incubation of Kcoil peptides (e) and Ecoil-EGF proteins (f) on cysteine-blocked PDEA layers (d) are presented. The reference was set as 0 nm for a cleaned silicon surface. The successive grafting reactions were performed as described in the Materials and Methods section and summarized in the table below the graph. Values are given as mean value ( standard deviation (n ) 5).

had been captured, with 5 M guanidium hydrochloride, a strong chaotropic agent known to disrupt coiled-coil interactions (28), induced a decrease in dry thickness of 0.6 nm, which confirmed the reversibility of the coiled-coil interaction. Finally, when Ecoil-EGF or cysteine-tagged Kcoil were incubated on cysteine-deactivated PDEA/CMD layers (Figure 5, conditions e and f, respectively), no significant changes in dry thickness values were observed when compared to those measured prior incubation (Figure 5, condition d), hence demonstrating that nonspecific adsorption of Ecoil-EGF and Kcoil peptides on dextrancoated surfaces was negligible. Fc-Mediated EGF Tethering. The second EGF capture strategy was based on the covalent attachment of Protein A to CMD, followed by the oriented capture of C-terminally Fc-tagged EGF proteins (EGF-Fc) via Protein A/Fc interactions (33). To do so, NHS/EDC coupling agents were used to activate grafted CMD surfaces. Ten micromolar Protein A diluted in 10 mM acetate buffer (pH 4.5) was found to be the best condition among those we tested for Protein A covalent grafting on NHS-activated CMD (data not shown). The resulting film displayed a dry thickness of 3.3 ( 0.1 nm (Figure 6, conditions b). After Protein A grafting, the remaining NHS-reactive groups were deactivated using ethanolamine as blocking agent in order to prevent any undesired attachment of amine-containing molecules. The ethanolamine-mediated NHS deactivation did not induce any significant change in the layer thickness (Figure 6, compare conditions b and c). Fc-tagged EGF proteins were then tethered on the Protein A layer, giving rise to final dry thickness of 4.7 ( 0.2 nm after extensive rinsing to remove any unbound material. As negative control, Protein A and EGF-Fc were incubated on ethanolamine-deactivated CMD (Figure 6, conditions f and g, respectively). Minor variations in dry thickness

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Figure 6. Ellipsometric characterization of EGF-Fc tethering on Protein A-coated surfaces. Dry thickness measurements corresponding to silicon surfaces sequentially treated with APTES and CMD (a) on which covalent coupling of Protein A had been achieved by amine chemistry (b), and for whom remaining activated group had been blocked by ethanolamine (c). EGF-Fc capture on (c) is shown as (d). As negative control, the incubation of Protein A (f) and EGF-Fc (g) on ethanolamine-blocked CMD layers (e) are presented. The reference was set as 0 nm for a cleaned silicon surface. The successive grafting reactions were performed as described in the Materials and Methods section and summarized in the table below the graph. Values are given as mean value ( standard deviation (n ) 5).

values were noticed when compared to that of deactivated dextran layer (Figure 6, condition e). This observation suggested that Protein A was mainly attached via covalent amide bonds to CMD-coated surfaces while Fc-EGF nonspecific adsorption on CMD was negligible. Altogether, the negative controls shown in Figures 5 and 6 demonstrated that the experimental protocol we optimized to generate our low-fouling CMD layer was successful. This conclusion is in total agreement with those drawn by Monchaux and Vermette who reported very low levels of serum protein adsorption on similar CMD surfaces (31). EGF CoValent Immobilization. For the sake of comparison, covalent immobilization of EGF was also performed on CMD surfaces using untagged hEGF and EGF-Fc proteins. In that approach, available amine groups of EGF or Fc tag (displayed on lysine residue side chains or at the N-terminus of the proteins) were used to create amide bonds with NHS-activated CMD layer. One micromolar of hEGF or EGF-Fc dissolved at pH 4.5 (10 mM acetate buffer) was found to be the optimal condition to obtain thick and homogeneous grafted layers on CMD surfaces (data not shown) that were characterized by effective dry thicknesses of 1.1 and 0.9 nm, respectively. In contrast to both oriented capture approaches we described earlier, our EGF covalent immobilization protocol required the use of acetate buffer at acidic pH far from physiological values. In order to assess if such a pH difference might have induced EGF unfolding, additional SPR biosensing experiments were performed as follows. Kcoil peptide was first immobilized at the surface of commercially available biosensor chips harboring a CMD layer, using the same chemistry protocol as the one we previously described for Kcoil covalent grafting on CMD-coated silicon surfaces (650 RU of immobilized Kcoil peptide). Ecoil-EGF (43 µM) was either freshly diluted in biosensor running buffer (HBS, pH 7.4, 1/430 dilution) or diluted in acetate buffer (pH 4.5, 1/12 dilution), let for 2 h at RT then further

EGF Tethering Strategies

Figure 7. Acidic pH does not affect EGF bioactivity during immobilization procedure. Control-corrected sensorgrams corresponding to the sequential capture of Ecoil-EGF chimera that has been directly diluted in running buffer (A, pH 7.4), followed by EGFR-Fc injection (B) and 5 M Gnd-HCl pulses (C) to remove coil-tagged EGF. In order to evaluate the impact of acidic pH upon EGF bioactivity, Ecoil-EGF that had been kept for 2 h at pH 4.5, was diluted in running buffer prior biosensor surface capture (D), EGFR-Fc injection (E, same as B) and regeneration (F). Note that EGFR-Fc binding to captured Ecoil-EGFs (B and E) gave identical responses.

diluted in HBS buffer (pH 7.4, 1/36 dilution to get a final 1/432 dilution of EGF stock solution). The bioactivity of both Ecoil-EGF preparations was then assessed by capturing Ecoil-EGF onto Kcoil-functionalized biosensor surface and then injecting the soluble extracellular domain of EGFR being artificially dimerized through the Fc portion of an antibody (namely, EGFR-Fc). As can be seen in Figure 7, a 2-h incubation of Ecoil-EGF in acidic buffer did not alter its ability to bind to its receptor ectodomain since similar SPR responses were obtained when injecting EGFR-Fc over the same amount of coiled-coil captured EGF that had been either incubated in acetate or freshly dissolved at physiological pH prior to capture. It is thus most likely that the use of acetate buffer at acidic pH in our immobilization protocol does not alter per se EGF or EGF-Fc bioactivity. Quantification of EGF Surface Densities. EGF surface densities were evaluated by ELISA for all our immobilization/capture strategies. Ecoil-EGF (600 nM) incubated on Kcoil layers previously generated on amino-coated (no dextran) and on CMD-coated silicon surfaces resulted in EGF densities of 30.7 ( 3.6 and 31.5 ( 3.5 pmol/cm2, respectively. When EGF-Fc had been incubated at 600 nM on Protein A layers (previously grafted on CMD), an EGF density of 13.4 ( 2.3 pmol/cm2 was obtained. In parallel, covalent coupling of EGF proteins (i.e., untagged EGF and EGF-Fc, 600 nM) on NHS-activated CMD layers resulted in EGF grafting densities of 37.5 ( 1.6 and 9.3 ( 2.4 pmol/cm2, respectively. Dry thickness measurements were also performed to evaluate the impact of soluble tagged EGF concentration upon resulting amount of captured EGF. As shown in Figure 8, EGF surface density could be easily controlled over a wide range by varying soluble concentrations during incubation. Similar apparent thermodynamic dissociation constant (KD) of 15 and 30 nM were deduced for Ecoil-EGF binding to immobilized Kcoil peptide and EGF-Fc binding to immobilized Protein A, respectively, assuming that Ecoil-EGF/Kcoil and EGF-Fc/Protein A interactions are well-depicted by a Langmuirian isotherm. These results thus indicate that the compact coil tag is an interesting alternative to bulkier Fc tag since the former allowed higher EGF surface density to be reached without any compromise on affinity.

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Figure 8. The amount of tagged EGF being captured on CMD layers via coiled-coil or Fc-Protein A interactions is controllable. Variation of the effective dry thickness as a function of tagged EGF concentration when Ecoil-EGF was incubated on Kcoil layers grafted on CMD (A) or when EGF-Fc was incubated on Protein A layers grafted on CMD (B). Surface density (pmol/cm2) was deduced by ELISA assay for the maximal Ecoil-EGF or EGF-Fc concentrations. Each dot corresponds to values obtained for three independent measurements while the dotted line corresponds to data fitting using a simple Langmuirian isotherm (KD ) 15 and 30 nM for panels A and B, respectively).

EGFR Phosphorylation Assays. EGF-dependent cellular response is mediated through EGF binding to its cell surface receptors, which in turn lead to receptor intracellular kinase activation via a trans-autophosphorylation mechanism (6). Phosphorylation levels of A-431 cell EGFR were thus assayed at different time points by Western blot analysis (Figure 9). As expected, no phosphorylation of EGFR was observed for aminated glass surfaces that were coated or not with CMD. Also, Fc-EGF (a chimeric protein in which EGF is fused to the C-terminus of the Fc portion of an IgG), when captured onto Protein A surfaces did not lead to any significant EGFR phosphorylation. This result was expected since this chimeric construct was shown to poorly interact with EGFR ectodomain (20). When the cells were treated with soluble EGF, EGFR phosphorylation was found to be transient (maximal phosphorylation levels occurred after 2 h and no more phosphorylation could be observed after 4 h). These results sharply contrasted with those related to covalently immobilized or stably captured EGF conditions, for which sustained phosphorylation (i.e., at least 5 h after cell seeding) was always observed. Of interest, the stable capture of EGF in an oriented fashion (via Fc-Protein A or coiled-coil interactions) resulted in increased EGFR activation when compared to all the nonoriented immobilization strategies we tested. That is, phosphorylation signals were found to be more intense for surfaces where Ecoil-EGF or EGF-Fc had been captured via coiled-coil or Fc-Protein A interactions than for surfaces where EGF or EGF-Fc had been covalently immobilized in a random fashion (compare “nonoriented immobilization” to “oriented capture” panels in Figure 9). Since comparable levels of coil-tagged EGF (31.5 ( 3.5 pmol/cm2) or untagged EGF (37.5 ( 1.6 pmol/ cm2) had been captured or covalently immobilized, respectively, these results unambiguously confirmed that hEGF grafting strategies relying on covalent coupling through amine chemistry negatively impact its bioactivity, as previously reported (17, 18). This loss of bioactivity cannot be attributed to the acidic

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Figure 9. Phosphorylation of A-431 cell surface EGFRs. The phosphorylation of EGFRs on A-431 cells was monitored at different times by Western blot. The 180 kDa band immunoreactive to anti-pTyr antibody (corresponding to EGFR) was captured using a Kodak 440cf imager. Surface type and mode of EGF supply is indicated for each panel.

conditions that were used for covalent grafting (see previous SPR results, Figure 7), but most likely results from the involvement of the EGF lysine side-chains in the grafting reaction. This conclusion holds true when examining the phosphorylation levels (Figure 9) for EGF-Fc that was captured via Protein A/Fc interactions (13.4 ( 2.3 pmol/cm2) to those related to randomly immobilized EGF-Fc (9.3 ( 2.4 pmol/ cm2). Finally, Ecoil-EGF tethering via coiled-coil interactions on surfaces previously coated with CMD was the approach that induced the most intense activation of EGFR. In that specific case, phosphorylation levels were comparable to those obtained when Ecoil-EGF was directly tethered on amino-coated surfaces via coiled-coil interactions (Figure 9). This maximal response can be attributed to the fact that (i) the EGF moiety within the Ecoil-EGF chimera is fully bioactive, as opposed to EGF-Fc (20), (ii) that the coiled-coil strategy allowed capturing almost 3-fold more EGF when compared to strategies relying on Protein A/Fc interactions (most likely due to the compactness of the coiled-coil structure), and (iii) that the use of a coil tag linked to the EGF moiety thanks to a glycine linker, enhanced EGF mobility when compared to that of untagged EGF being directly coupled. Tethered EGF Increases A-431 Cell Adhesion on LowFouling CMD. The effect of CMD layer, combined to our various strategies aiming at immobilizing/tethering EGF, upon A-431 cell adhesion was then investigated. For this purpose, A-431 cells were directly seeded on aminated glass surfaces (control), on Kcoil functionalized glass surfaces to which Ecoil-EGF had been captured via coiled-coil interactions, on CMD-coated glass surfaces in the presence or absence of soluble EGF (4 nM), on CMD-coated glass surfaces on which Ecoil-EGF had been immobilized via coiled-coil interactions or via Fc-Protein A interactions and on CMD-coated glass surfaces where EGF and EGF-Fc were covalently immobilized via amine chemistry (nonoriented immobilization) (Figure 10). As expected from the low-fouling properties of dextran (26), CMD layers drastically limited A-431 cell adhesion when compared to aminated surfaces (110 versus 2177 cells/mm2). The nonoriented immobilization of EGF (native EGF or EGF-Fc) on CMD only resulted in a less than 3-fold increase of A-431 cell adhesion (299 and 277 cells/mm2, respectively) and to levels comparable to those determined when signalingincompetent Fc-EGF had been tethered via its interaction with Protein A (283 cells/mm2). In stark contrast, a marked increase of A-431 cell adhesion was observed for Ecoil-EGF being tethered via coiled-coil interaction on CMD-coated surfaces (2182 cells/mm2). In that specific case, adhesion was determined

Figure 10. Cell adhesion assays. A-431 cells were incubated for 4 h on glass surfaces being amino-coated only (a), or coated with CMD both in the absence (b) or presence of soluble EGF (c). Conditions (d-h) correspond to approaches leading to EGF covalent immobilization (d,e) or to its tag-driven capture (g,h). Condition f corresponds to the negative control for condition g. The successive grafting reactions were performed as described in the Materials and Methods section and summarized in the table below the graph. Values are given as mean value ( standard deviation (n ) 5). In order to compare each condition to condition b, statistical analysis was performed by independent twosample t test with equal variances. Values of p < 0.05 and p < 0.001 were considered to be statistically significant and are identified by * and **, respectively.

to be as strong as for amino-coated surfaces (no CMD, Figure 10). Statistically significant but lower increases of cell adhesion were also observed for EGF-Fc being tethered via Fc/Protein A interactions (716 cells/mm2) or with soluble EGF supply (522 cells/mm2). These results are in excellent agreements with those from Kempiak et al. demonstrating that local activation of EGFR with EGF-coated beads caused a rapid actin polymerization response and lamellipod extrusion (34), two traits of A-431 cellular adhesion. Additionally, EGF grafting has also been shown to increase 3T3 Swiss (19) and human corneal epithelial

EGF Tethering Strategies

(35) cell adhesion, most likely due to the sustained activation of various downstream signaling pathways (e.g., ERK 1/2 and Akt), in turn leading to increased production of extracellular matrix proteins. Altogether, the results presented in Figures 9 and 10 demonstrate that a strong EGF-induced A-431 cell adhesion on CMD surfaces can only be achieved when EGF is presented to the cells in a nondiffusible way, while preserving its biological activity. Indeed, EGF tethering on CMD surfaces via coiledcoil interactions (which corresponded to the most intense and sustained phosphorylation of A-431 cell surface receptors on CMD coating; Figure 9), was the only experimental condition that lead to adhesion levels similar to those obtained on control aminated surfaces (Figure 10). In stark contrast, only limited, but yet significant, increase in cell adhesion was observed (Figure 10, conditions c-g) when the grafting strategies altered EGF bioactivity (as demonstrated by low levels of EGFR phosphorylation, Figure 9) or when only transient phosphorylation of EGFR was triggered by diffusible EGF (Figure 9).

CONCLUSION In the context of tissue engineering and regenerative medicine, EGF immobilization at the surface of implants has attracted a lot of attention. Here we show that fully bioactive EGF being tethered on carefully engineered surfaces may play, per se, a significant role in promoting cell adhesion, in addition to cell survival and proliferation as already demonstrated by several research groups. Among all the approaches we tested, the oriented tethering of EGF via coiled-coil interactions was shown to be the most efficient on low-fouling CMD surfaces. This most likely resulted from the many advantages of this capture system. First, it permitted the adequate display of captured EGF, and in turn the maximal biological effect as opposed to random covalent coupling. While we previously showed that the Ecoil peptide had no impact on EGF bioactivity (in contrast to the Fc tag), the relatively small size of the coiled-coil structure allowed achieving levels of captured EGF 3-fold higher than those obtained with a bulkier Fc/Protein A capture system. Altogether, coiled-coil tethered EGF led to the most intense and sustained phosphorylation of A-431 cell EGFRs as well as to strong cell adhesion. Of interest, the latter was comparable to that observed with an amino-coated surface. Our results thus highly suggest that CMD may be an interesting alternative to PEG for the generation of surfaces limiting nonspecific adhesion, and that these surfaces could be further tailored through growth factor capture via coiled-coil interactions.

ACKNOWLEDGMENT This work was supported by the Canada Research Chair on Protein-Enhanced Biomaterials (G.D.C.), the Canada Research Chair in Applied Metabolic Engineering (M.J.), by the Natural Sciences and Engineering Research Council of Canada (G.D.C., M.J.) and by GRSTB (postdoctoral fellowship to B.L. and financial help to J.C.).

LITERATURE CITED (1) Goddard, J. M., and Hotchkiss, J. H. (2007) Polymer surface modification for the attachment of bioactive compounds. Prog. Polym. Sci. 32, 698–725. (2) Ito, Y. (2008) Covalently immobilized biosignal molecule materials for tissue engineering. Soft Matter 4, 46–56. (3) Ma, Z. W., Mao, Z. W., and Gao, C. Y. (2007) Surface modification and property analysis of biomedical polymers used for tissue engineering. Colloids Surf. B: Biointerfaces 60, 137– 157.

Bioconjugate Chem., Vol. 21, No. 12, 2010 2265 (4) Kuhl, P. R., and Griffith-Cima, L. G. (1996) Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat. Med. 2, 1022–1027. (5) Ogiso, H., Ishitani, R., Nureki, O., Fukai, S., Yamanaka, M., Kim, J. H., Saito, K., Sakamoto, A., Inoue, M., Shirouzu, M., and Yokoyama, S. (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110, 775–787. (6) Schlessinger, J. (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110, 669–672. (7) Yarden, Y., and Sliwkowski, M. X. (2001) Untangling the ErbB signalling network. Nat. ReV. Mol. Cell Biol. 2, 127–137. (8) Zhang, X., Gureasko, J., Shen, K., Cole, P. A., and Kuriyan, J. (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137– 1149. (9) Hubbard, S. R., and Miller, W. T. (2007) Receptor tyrosine kinases: mechanisms of activation and signaling. Curr. Opin. Cell Biol. 19, 117–123. (10) Klenkler, B. J., Dwivedi, D., West-Mays, J. A., and Sheardown, H. (2009) Corneal epithelial cell adhesion and growth on EGF-modified aminated PDMS. J. Biomed. Mater. Res. A 93, 1043–1049. (11) Klenkler, B. J., Griffith, M., Becerril, C., West-Mays, J. A., and Sheardown, H. (2005) EGF-grafted PDMS surfaces in artificial cornea applications. Biomaterials 26, 7286–7296. (12) Kato, K., Sato, H., and Iwata, H. (2005) Immobilization of histidine-tagged recombinant proteins onto micropatterned surfaces for cell-based functional assays. Langmuir 21, 7071–7075. (13) Fan, V. H., Tamama, K., Au, A., Littrell, R., Richardson, L. B., Wright, J. W., Wells, A., and Griffith, L. G. (2007) Tethered epidermal growth factor provides a survival advantage to mesenchymal stem cells. Stem Cells 25, 1241–1251. (14) Nakaji-Hirabayashi, T., Kato, K., Arima, Y., and Iwata, H. (2007) Oriented immobilization of epidermal growth factor onto culture substrates for the selective expansion of neural stem cells. Biomaterials 28, 3517–3529. (15) Nakaji-Hirabayashi, T., Kato, K., and Iwata, H. (2009) Surface-anchoring of spontaneously dimerized epidermal growth factor for highly selective expansion of neural stem cells. Bioconj. Chem. 20, 102–110. (16) Ogiwara, K., Nagaoka, M., Cho, C. S., and Akaike, T. (2006) Effect of photo-immobilization of epidermal growth factor on the cellular behaviors. Biochem. Biophys. Res. Commun. 345, 255–259. (17) O’Connor-McCourt, M. D., De Crescenzo, G., Lortie, R., Lenferink, A., Grothe, S. (1998) The analysis of surface plasmon resonance-based biosensor data using numerical integration: The epidermal growth factor receptor-ligand interaction as an example. In QuantitatiVe Analysis of Biospecific Interactions (Lundahl, P., Lundqvist, A., Greiger, E., Eds.), pp 175-190, Harwood Academic Publishers, Australia. (18) Boucher, C., Liberelle, B., Jolicoeur, M., Durocher, Y., and De Crescenzo, G. (2009) Epidermal growth factor tethered through coiled-coil interactions induces cell surface receptor phosphorylation. Bioconj. Chem. 20, 1569–1577. (19) Ogiwara, K., Nagaoka, M., Cho, C.-S., and Akaike, T. (2005) Construction of a novel extracellular matrix using a new genetically engineered epidermal growth factor fused to IgGFc. Biotechnol. Lett. 27, 1633–1637. (20) Boucher, C., St-Laurent, G., Loignon, M., Jolicoeur, M., De Crescenzo, G., and Durocher, Y. (2008) The bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags. Tissue Eng. Part A 14, 2069–2077. (21) Klenkler, B. J., and Sheardown, H. (2006) Characterization of EGF coupling to aminated silicone rubber surfaces. Biotechnol. Bioeng. 95, 1158–1166.

2266 Bioconjugate Chem., Vol. 21, No. 12, 2010 (22) Klenkler, B. J., Chen, H., Chen, Y., Brook, M. A., and Sheardown, H. (2008) A high-density PEG interfacial layer alters the response to an EGF tethered polydimethylsiloxane surface. J. Biomater. Sci. Polym. Ed. 19, 1411–1424. (23) Jo, S., and Park, K. (2000) Surface modification using silanated poly(ethylene glycol)s. Biomaterials 21, 605–616. (24) Massia, S. P., Stark, J., and Letbetter, D. S. (2000) Surfaceimmobilized dextran limits cell adhesion and spreading. Biomaterials 21, 2253–2261. (25) Heinze, T., Liebert, T., Heublein, B., and Hornig, S. (2006) Functional polymers based on dextran. Polysaccharides II 205, 199–291. (26) Monchaux, E., and Vermette, P. (2007) Bioactive microarrays immobilized on low-fouling surfaces to study specific endothelial cell adhesion. Biomacromolecules 8, 3668–3673. (27) Monchaux, E., and Vermette, P. (2008) Cell adhesion resistance mechanisms using arrays of dextran-derivative layers. J. Biomed. Mater. Res. A 85A, 1052–1063. (28) De Crescenzo, G., Litowski, J. R., Hodges, R. S., and O’Connor-McCourt, M. D. (2003) Real-time monitoring of the interactions of two-stranded de noVo designed coiled-coils: Effect of chain length on the kinetic and thermodynamic constants of binding. Biochemistry 42, 1754–1763. (29) Surugiu, L., Dey, E. S., Svitel, J., Pirvutoiu, S., and Danielsson, B. (2001) Dextran-modified surface for highly sensitive chemiluminescent ELISA. Analyst 126, 1633–1635.

Liberelle et al. (30) Liberelle, B., Banquy, X., and Giasson, S. (2008) Stability of silanols and grafted alkylsilane monolayers on plasma-activated mica surfaces. Langmuir 24, 3280–3288. (31) Monchaux, E., and Vermette, P. (2007) Development of dextran-derivative arrays to identify physicochemical properties involved in biofouling from serum. Langmuir 23, 3290–3297. (32) Tobiesen, F. A., and Michielsen, S. (2002) Method for grafting poly(acrylic acid) onto nylon 6,6 using amine end groups on nylon surface. J. Polym. Sci., Part A1: Polym. Chem. 40, 719– 728. (33) Saha, K., Bender, F., and Gizeli, E. (2003) Comparative study of IgG binding to proteins G and A: Nonequilibrium kinetic and binding constant determination with the acoustic waveguide device. Anal. Chem. 75, 835–842. (34) Kempiak, S. J., Yip, S. C., Backer, J. M., and Segall, J. E. (2003) Local signaling by the EGF receptor. J. Cell. Biol. 162, 781–787. (35) Boucher, C., Ruiz, J.-C., Thibault, M., Buschmann, M. D., Wertheimer, M., Jolicoeur, M., Durocher, Y., and De Crescenzo, G. (2010) Human corneal epithelial cell response to epidermal growth factor tethered via coiled-coil interactions. Biomaterials 31, 7021–7031. BC1002604