Protein Adhesion on Silicon-Supported Hyperbranched Poly(ethylene

May 17, 2007 - Department of Chemical Engineering, 204 Fenske Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802-4420,...
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Langmuir 2007, 23, 7018-7023

Protein Adhesion on Silicon-Supported Hyperbranched Poly(ethylene glycol) and Poly(allylamine) Thin Films Maureen A. Dyer,† Kristy M. Ainslie,† and Michael V. Pishko*,†,‡,§ Department of Chemical Engineering, 204 Fenske Laboratory, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802-4420, Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802-4420, and Department of Materials Science and Engineering, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802-4420 ReceiVed February 20, 2007. In Final Form: April 4, 2007 Hyperbranching poly(allylamine) (PAAm) and poly(ethylene glycol) (PEG) on silicon and its effect on protein adhesion was investigated. Hyperbranching involves sequential grafting of polymers on a surface with one of the components having multiple reactive sites. In this research, PAAm provided multiple amines for grafting PEG diacrylate. Current methodologies for generating PEG surfaces include PEG-silane monolayers or polymerized PEG networks. Hyperbranching combines the nanoscale thickness of monolayers with the surface coverage afforded by polymerization. A multistep approach was used to generate the silicon-supported hyperbranched polymers. The silicon wafer surface was initially modified with a vinyl silane followed by oxidation of the terminal vinyl group to present an acid function. Carbodiimide activation of the surface carboxyl group allowed for coupling to PAAm amines to form the first polymer layer. The polymers were hyperbranched by grafting alternating PEG and PAAm layers to the surface using Michael addition chemistry. The alternating polymers were grafted up to six total layers. The substrates remained hydrophilic after each modification. Static contact angles for PAAm (32-44°) and PEG (33-37°) were characteristic of the corresponding individual polymer (30-50° for allylamine, 34-42° for PEG). Roughness values varied from ∼1 to 8 nm, but had no apparent affect on protein adhesion. Modifications terminating with a PEG layer reduced bovine serum albumin adhesion to the surface by ∼80% as determined by ELISA and radiolabel binding studies. The hyperbranched PAAm and PEG surfaces described in this paper are nanometer-scale, multilayer films capable of reducing protein adhesion.

Introduction Modification of metal surfaces has application in a variety of areas ranging from anticorrosion1 to antibiofouling treatments.2,3 Much of the research into biofouling prevention focuses on using thin films to reduce adhesion of biological entities.4-10 Poly(ethylene glycol) (PEG) modifications are most widely employed to reduce protein adhesion while maintaining a hydrophilic, biocompatible surface. Using in vitro protein and cell assays, Zhang et al. showed that increasing the amount of PEG decreased protein and cell adhesion to chitosan-PEG blended materials.11 In in vivo rabbit studies, Rogero et al. demonstrated biocompatibility of hydrogels incorporating PEG. The hydrogels were * To whom correspondence should be addressed. Phone: (814) 8652574. Fax: (814) 865-7846. E-mail: [email protected]. † Department of Chemical Engineering. ‡ Department of Chemistry. § Department of Materials Science and Engineering. (1) Bethencourt, M.; Botana, F. J.; Calvino, J. J.; Marcos, M.; RodrIguezChacon, M. A. Corros. Sci. 1998, 40, 1803-1819. (2) Vermette, P.; Meagher, L. Colloids Surf., B 2003, 28, 153-198. (3) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Curr. Opin. Colloid Interface Sci. 2001, 6, 3-10. (4) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1071410721. (5) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 50595070. (6) Park, S.; Padeste, C.; Schift, H.; Gobrecht, J. Microelectron. Eng. 2003, 67-68, 252-258. (7) Takahara, A.; Hara, Y.; Kojio, K.; Kajiyama, T. Colloids Surf., B 2002, 23, 141-152. (8) Sharma, S.; Johnson, R. W.; Desai, T. A. Appl. Surf. Sci. 2003, 206, 218229. (9) Faucheux, N.; Schweiss, R.; Lutzow, K.; Werner, C.; Groth, T. Biomaterials 2004, 25, 2721-2730. (10) Branch, D. W.; Wheeler, B. C.; Brewer, G. J.; Leckband, D. E. Biomaterials 2001, 22, 1035-1047. (11) Zhang, M.; Li, X. H.; Gong, Y. D.; Zhao, N. M.; Zhang, X. F. Biomaterials 2002, 23, 2641-2648.

not cytotoxic to cells and did not irritate the rabbit skin.12 Commercially available products also utilize PEG modifications to reduce protein adhesion. Nektar Therapeutics (www.nektar.com) has collaborated with several pharmaceutical companies (such as Schering-Plough, Roche) to generate six PEGylated products currently FDA approved for therapeutic use. In a review by Vermette and Meagher,2 the available data on what aspects of PEG are involved in reducing protein adhesion are analyzed. The main characteristics of PEG contributing to its protein resistance are (1) high polymer surface density, (2) the need to overcome a potential energy barrier to adhere to PEG as compared to no barrier to adhere to a bare surface, (3) the generation of a repulsive energy due to the approaching protein compressing the PEG chains, and (4) limitation of PEG chain movement by protein proximity.2 To introduce protein-resistant PEG coatings on surface, there is a variety of methods available. An early method involved polymer adsorption to glass or silica substrates.13 This method has more recently been used to adsorb PEG-containing copolymers to target surfaces. Silicon wafers have also been modified with PEG by treating surfaces with PEG-silane molecules. Such treatment results in a covalently grafted PEG monolayer that reduces protein attachment.8-10,14,15 PEG-silane monolayers are useful for substrates of homogeneous composition. However, monolayers may not be able to form on surfaces of heterogeneous composition due to nonuniform availability of reactive substrate. (12) Rogero, S. O.; Malmonge, S. M.; Lugao, A. B.; Ikeda, T. I.; Miyamaru, L.; Cruz, A. S. Artif. Organs 2003, 27, 424-427. (13) Maechling-Strasser, C.; Dejardin, P.; Galin, J. C.; Schmitt, A. J. Biomed. Mater. Res. 1989, 23, 1384-1393. (14) Lan, S.; Veiseh, M.; Zhang, M. Biosens. Bioelectron. 2005, 20, 16971708. (15) Sharma, S.; Johnson, R. W.; Desai, T. A. Biosens. Bioelectron. 2004, 20, 227-239.

10.1021/la7004997 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/17/2007

Protein Adhesion to Hyperbranched PAAm and PEG

The incomplete, nonuniform layer would allow for proteins to adhere in the gaps between PEG molecules. Another approach to introduce PEG modifications is through polymerization of PEG derivatives forming a barrier layer on the surface of interest.16-18 Local polymerization of amine-terminated PEG using a focused electron beam,16 UV-induced PEGmethacrylate polymerization on stainless steel substrates, and photolithographic PEG patterning are current methods of forming a PEG barrier layer.18 Unlike monolayer formation, polymerization methods do not require a uniform substrate to be modified. Whether PEG is patterned for protein resistance on specific areas or polymerized for complete coverage, the surface to be modified can be more heterogeneous than surfaces used for monolayer formation. Here we explore hyperbranching poly(allylamine) (PAAm) and PEG on silicon wafers and its effect on protein adhesion. Hyperbranching polymers has several potential advantages over the currently available methods for improving biocompatibility. The covalent grafting of each layer to the previous modification enhances stability compared to electrostatic adsorption of PEG polymers. Grafting several defined nanoscale layers also provides for fine control over the ultimate thickness of the modifications. Compared to monolayers, branching several layers of polymer would allow for complete surface coverage even when coupling chemistry at the surface may be inefficient. With multiple layers, loss of coverage from the outermost layer does not lead to surface exposure. Instead, the underlying polymer layer is exposed. Also, loss of biocompatibility due to polymer degradation would be minimized. In studies using one layer of covalently linked PEG, biofilm growth was prevented for up to 26 days in cell culture, but film thickness decreased after 24 days in PBS leading to total film failure by day 27.10 Additional layers as proposed with polymer hyperbranching could provide additional protein resistance by increasing the number of protein resistant layers in the film. Loss of one layer from a hyperbranched substrate would leave several underlying layers to resist protein adsorption, whereas a similar loss from a monolayer would result in an unmodified surface no longer able to prevent protein adhesion. Developing and characterizing hyperbranching and its effects on protein adhesion to model homogeneous silicon substrates provides the basis for further studies involving heterogeneous, nonuniform surfaces, as well as film stability studies. Materials and Methods Silicon wafers (100) were purchased from Wafer World (West Palm Beach, FL). Ultrapure water with a specific resistance of greater than 18 MΩ/cm (Barnstead Nanopure Diamond RO system; npH2O) was used for all aqueous solutions and washing steps. Chemicals were purchased from Aldrich (Milwaukee, WI; poly(ethylene glycol) diacrylate (PEGDA, MW 575), anhydrous carbon tetrachloride (CCl4), n-heptane), Gelest, Inc. (Morrisville, PA; allyltrichlorosilane (allylTCS), 7-octenyltrimethoxysilane (OTMS)), Polysciences, Inc (Warrington, PA; Poly(allylamine) hydrochloride (PAH, MW ∼60 000), Sigma (St. Louis, MO; potassium permanganate (KMnO4), sodium periodate (NaIO4), potassium carbonate (K2CO3), sodium bisulfite (NaHSO3), hydrochloric acid (HCl), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), 2-(Nmorpholino)ethanesulfonic acid (MES), sodium hydroxide (NaOH), ethanol (EtOH), bovine serum albumin (BSA), chicken egg albumin (OvA), 3,3′,5,5′-tetramethylbenzidine (TMB), Tris-buffered saline with Tween (TBST; 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0)), VWR (Bristol, CT; sulfuric acid (H2SO4), 30% hydrogen (16) Hong, Y.; Krsko, P.; Libera, M. Langmuir 2004, 20, 11123-11126. (17) Zhang, F.; Kang, E. T.; Neoh, K. G.; Wang, P.; Tan, K. L. Biomaterials 2001, 22, 1541-1548. (18) Revzin, A.; Tompkins, R. G.; Toner, M. Langmuir 2003, 19, 9855-9862.

Langmuir, Vol. 23, No. 13, 2007 7019 peroxide (H2O2)), and Bethyl labs (Montgomery, TX; sheep antibovine albumin-affinity purified capture antibody, sheep anti-bovine albumin-HRP conjugated detection antibody). PEG and PAAm Hyperbranching. The procedure for hyperbranching on gold was adapted for use on silicon.19 The series of modifications is outlined in Scheme 1. After each modification, wafers were rinsed thoroughly with npH2O then EtOH, dried under an N2 stream and measured for thickness unless otherwise noted. Silicon wafers (∼1 cm2) were cleaned in “piranha” solution (3:1 concentrated H2SO4/30% H2O2) for 1 h. Ns and Ks values were obtained for ellipsometry on each wafer. Cleaned silicon surfaces were silanized in 3:1 heptane/CCl4 (1 µL silane/20 mL solvent) for 1 h, rinsed with CCl4 then EtOH. Initial silane modifications were done using allylTCS. Later trials used OTMS for increased stability due to the less labile methoxysilyl groups compared to chlorosilyl groups. The terminal vinyl group was oxidized to an acid function by immersing wafers in permanganate solution (1.8 mM K2CO3, 19.5 mM NaIO4, 0.5 mM KMnO4) for 24 h. Excess permanganate solution was neutralized by sequential exposure to 0.3 M NaHSO3, npH2O, 0.1 N HCl. Carboxyls were activated by 90 min incubation with 15 mM EDAC in 0.1 M MES. Immediately following activation, wafers were rinsed with npH2O and immersed in 15 µM PAH containing 100 mM NaOH for 1 h. PEG was grafted to the amine surface by incubating PAAm-modified wafers in 10% (v/v) aqueous PEGDA containing 100 mM NaOH for 2 h. Alternating PAH and PEGDA incubations resulted in hyperbranched substrates with terminal PAAm or PEG surfaces, depending on the final layer grafted. Modifications ranged from one to six layers in the sequence of PAAm, PEG, PAAm, PEG, PAAm, and PEG. Ellipsometry. Thickness of surface modifications was measured by ellipsometry. Measurements were taken on a Gaertner LSE singlewavelength ellipsometer. The light source had an angle of incidence of 70° and was generated by a helium-neon laser. Nine measurements were taken per substrate and averaged to give the thickness grafted to the silicon substrate. Averaging thickness of three substrates gave the average thickness reported for each modification. Quartz Crystal Microbalance (QCM). Silicon-plated QCM crystals were modified with PAAm and PEG similar to the silicon wafers above. The flow cell and silicon-plated QCM crystals (ATcut, 10 MHz base resonant frequency, electrode area of 0.2 cm2) were purchased from ICM Co, Inc. (Oklahoma City, OK). Frequency was measured using a 10 MHz lever oscillator (ICM), a digital multimeter (Agilent, Palo Alto, CA), and frequency counter (Agilent). The silicon is layered directly on the quartz on the crystal, similar to the commonly used gold-plated crystals. The clear acrylic liquid flow cell accommodates 70 µL of liquid in its reservoir, sealed with an O-ring seal. After the first two steps of the hyperbranching procedure (silanization and oxidation), the crystal was mounted in the liquid flow cell. npH2O was flushed through the cell, and a baseline frequency for the crystal was obtained. EDAC activation and subsequent polymer additions were all performed while the crystal was mounted in the holder. After each polymer addition, frequency was monitored while the cell was flushed with npH2O and allowed to come to equilibrium. Frequency was not monitored continuously through all solution addition steps due to the jump in frequency due to turbulence. Contact Angle Measurement. Contact angles were measured using a locally assembled device similar in design to the Rame´Hart Model 100 contact angle goniometer with a camera to record images. npdH2O (10 µL) was delivered to the surface via a flattipped micrometer syringe. Liquid was either dispensed or withdrawn to measure advancing or receding angles, respectively. For static contact angles, 10 µL of liquid was dispensed and the surface approached until the drop made contact and transferred to the surface. The angles were measured relative to the surface using software (ImageJ) available from the National Institute of Health (NIH). Atomic Force Microscopy. Surface roughness was measured using atomic force microscopy. Images were obtained for 10 µm × 10 µm areas on each wafer at each modification using a Digital (19) Russell, R. J.; Sirkar, K.; Pishko, M. V. Langmuir 2000, 16, 4052-4054.

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Scheme 1. Hyperbranching PAAm and PEG on Silicona

a Cleaned silicon wafers were silanized with 1 µL/mL OTMS in 3:1 heptane/carbon tetrachloride (1). The terminal vinyl group was immersed in permanganate solution (i) for 24 h, yielding an acid function (2). The acid group was activated (ii, 15 mM EDAC in 0.1M MES) then amine coupled to PAAm (iii; 15 µM PAH, 100 mM NaOH; 1 h). This surface is referred to as 4, PAAM. The PAAm-modified substrate (3; 4, PAAm) was grafted with PEG (iv; 10% v/v, 100 mM NaOH; 2 h), generating a PEG-terminated substrate (4; 5, PEG). Subsequent polymer additions repeated steps iii and iv to the desired degree of hyperbranching, yielding four more hyperbranched structures (6, PAAm; 7, PEG; 8, PAAm; 9, PEG).

Instruments Nanoscope IIIa atomic force/scanning probe microscope (Veeco, NY) with a silicon nitride probe in tapping mode. rms roughness values for each area were calculated by the NanoImage v 4.24r4 software provided with the instrument. Monitoring of Protein by ELISA. Protein adsorption was measured by a colorimetric ELISA and quantified using a calibration standard run in a 96-well immuno-plate. All incubations were at room temperature. After each incubation, wells were rinsed 3-5 times with TBST containing 1% OvA and the remaining wash solution was aspirated from each well. For the standard curve, 100 µL of the capture antibody, diluted in PBS to a concentration of 0.01 mg/µL, was placed in each well of a 96-well plate and incubated for 1 h. Wells were then incubated at room temperature with OvA blocking solution followed by washing and aspiration. Blocked wells were incubated with 100 µL of protein standards (6.25-400 ng/mL) for 1 h. One hundred microliters of secondary antibody (1 µL/30 mL in TBST) was incubated in each well for 1 h at room temperature. Wells were incubated with TMB (100 µL) solution for 5 min, after which the reaction was quenched with an equal volume of 0.2 N sulfuric acid to stop color development. For protein adherence to silicon wafers, bare and hyperbranched silicon wafers were placed at the bottom of a 24-well tissue culture plate. Surfaces were incubated for 15 min in 500 µL of BSA in PBS (1 mg/mL). Wells were incubated in blocking solution (1 mL) for 1 h. Five hundred microliters of secondary antibody (1 µL/30 mL in TBST) was incubated in each well for 1 h at room temperature. Wells were incubated with TMB (500 µL) solution for 5 min, after which the reaction was quenched with 0.2 N sulfuric acid (500 µL) to stop color development. Quenched solutions from standards and protein adhesion samples were transferred in triplicate (100 µL each) to a new 96-well plate for analysis. Absorbance at 450 nm was measured on a Thermo Labsystem Multiskan Spectrum plate reader (Milford, MA).

Monitoring of Protein with [125I]-BSA. Iodinated bovine serum albumin ([125I]-BSA) was purchased from Perkin-Elmer in the amount of 500 µCi. An initial stock solution of [125I]-BSA was created of 125 µCi/mL with PBS (10 µCi per 4 mg BSA/mL).20 Dilutions were made of the stock solution to create a 400 ng/mL, 1 mg/mL, or 5 mg/mL stock solution of BSA labeled with radioligand. After protein adsorption, the raw count of emitted γ rays was detected on a Wallac 1470 Wizard automatic gamma counter (Perkin-Elmer) courtesy of Dr. Craig Baumrucker, Department of Dairy and Animal Science.

Results and Discussion PEG and PAAm Hyperbranching. Ellipsometry. PAAm and PEG were hyperbranched on silicon substrates as outlined in Scheme 1. This method differs from the previously published hyperbranching on gold19 in that a vinyl-terminated silane was used for initial modification of a silicon surface and the vinyl group was oxidized to generate the acid function for subsequent PAAm addition. Another modification was the inclusion of NaOH to each polymer addition step to ensure a basic environment for the Michael addition between the PEG’s acrylate and PAAm’s amine groups. Layers formed from polymer solutions lacking NaOH washed off after a water rinse but not after only an ethanol rinse (data not shown). With these modifications to the procedure, PAAm and PEG were alternately added, resulting in increasing thickness (Figure 1). Average PAAm thickness was about 12 Å, and the average PEG layer thickness was 18.7 Å, both of which are in good agreement with average values for hyperbranching on gold.19 The overall thickness after all six polymer additions (20) Hardin, J. A.; Kimm, M. H.; Wirasinghe, M.; Gall, D. G. Gut 1999, 44, 218-225.

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Figure 1. Ellipsometry data displaying the surface modifications (as outlined in Scheme 1) on the silicon wafer. Layers are cumulative on the silanized and oxidized surfaces. Labels indicate the terminal modification on the wafers. PAAm (poly(allylamine)), and PEG (poly(ethylene glycol)). Error bars represent a 95% confidence interval (n ) 3).

Figure 2. Resonant frequency shift of silicon-plated QCM crystal after each polymer addition. Polymer additions were performed on silicon-plated crystals that were silanized with OTMS and oxidized with permanganate solution. Treated crystals were mounted in the liquid flow cell then immersed in water. All measurements were taken in an aqueous environment. The initial PAAm layer required carbodiimide activation of the surface carboxyl for conjugation. Subsequent polymer layers were added using a Michael addition between terminal acrylate groups on PEGDA and pendant amine groups on PAH. Frequency shifts reported are for cumulative polymer layers.

was under 10 nm. Modifying the hyperbranching procedure from a gold substrate to a silicon substrate suggests that polymer additions should be the same regardless of the underlying substrate. QCM. PAAm and PEG were hyperbranched on silicon-plated QCM crystals as outlined in Scheme 1. QCM was used to confirm that the thickness change seen for each addition was caused by polymer addition, not just rearrangement of the polymers to give thicker films. QCM monitors changes in mass load on a siliconplated crystal by detecting changes in the resonant frequency of the crystal. For rigid, thin films, the Sauerbrey equation relates mass change directly to frequency change.21 Qualitatively, this means that a decrease in frequency represents an increase in adherent mass. For each of the PAAm and PEG layers, frequency decreased with each subsequent addition, indicating that polymer was added to the surface. Figure 2 shows the magnitude of the frequency shift after each modification. The increase in mass load for polymer addition agrees with the increase in thickness observed by ellipsometry and confirms the addition of polymer to the surface. All layers increased in thickness and all layers (21) Sauerbrey, G. n. Eur. Phys. J. A 1959, 155, 206-222.

Table 1. Contact Angles for Cumulative PAAm and PEG Modifications to Silicon Wafers 4, PAAm 5, PEG 6, PAAm 7, PEG 8, PAAm 9, PEG

static

advancing

receding

hysteresis

37 ( 5 33 ( 3 42 ( 6 37 ( 1 39 ( 5 36 ( 1

27 ( 7 50 ( 6 39 ( 6 38 ( 7 35 ( 12 43 ( 3

12 ( 3 27 ( 9 15 ( 8 13 ( 4 7(4 13 ( 3

16 ( 6 24 ( 8 24 ( 13 25 ( 9 28 ( 11 29 ( 2

displayed an increase in frequency change after each addition. Taken together these results support the stepwise addition of alternating polymer layers onto silicon substrates. Contact Angle. PAAm and PEG hyperbranched surfaces were also characterized by contact angle analysis. The sessile drop method was used to obtain static contact angles, whereas the captive bubble technique was used for advancing and receding contact angles. At all stages of modification, the static contact angles (θ < 45°, Table 1) indicate a hydrophilic surface. Regardless of the terminal polymer graft, the surface remains hydrophilic throughout the modification sequence. Static contact angle measurements for PAAm-terminated surfaces were between

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Table 2. rms Roughness Values (nm) for Cumulative PAAm and PEG Modifications to Silicon Wafers surface

rms, nm

1, unmodified 4, PAAm 5, PEG 6, PAAm 7, PEG 8, PAAm 9, PEG

0.24 1.36 1.11 4.13 2.30 7.94 4.82

32° and 44°. PAAm deposition on glass plates was reported to have static contact angles within the range of 30-50°.22 The observed range of PAAm contact angles during hyperbranching falls within the literature range for a comparable allylamine surface indicating complete surface coverage by PAAm. PEG-terminated hyperbranched substrates gave static contact angles ranging from 33° to 37°. Sharma et al. report static contact angles ranging from 34° to 42° for ultrathin PEG films after 2 h of incubation.8 The static contact angles for PEG modifications throughout hyperbranching are comparable to those reported for the PEG ultrathin films supporting PEG modification. Despite different underlying supports, i.e., one, three, or five polymer layers, the PEG modifications in both cases exhibit similar hydrophilicity. Static contact angle measurements for both PAAm- and PEGterminated substrates are in agreement with reported contact angles for PAAm and PEG films individually. Complete coverage by PEG was evaluated using advancing contact angles. The second PEG modification had advancing contact angle of 38° ( 7°, while the first and third PEG modifications were slightly higher (Table 1). The expected range for complete PEG coverage was 36-39°.23 The second PEG layer (7, PEG) fell within this range. Layers 5, PEG and 9, PEG had slightly higher advancing angles, suggesting that PEG coverage was not complete. For 9, PEG, the measured advancing contact angle range was 40-46°. While the advancing contact angle for the final PEG layer is close to the expected range, it is slightly higher, suggesting incomplete PEG coverage of the underlying layers, 4,PAAm through 8, PAAm. Since the PEG modifications are on one to five layers of alternating PAAm and PEG, the underlying layers could be influencing the observed advancing contact angle compared to a uniform, homogeneous PEG surface. The contact angle hysteresis or difference between advancing and receding contact angles can also be used as an indicator of surface roughness or heterogeneity. For the polymer additions presented here, the average contact angle hysteresis increases with each subsequent layer. The increase could result from changes in surface roughness, surface heterogeneity, number of polymer layers, or a combination thereof. The static contact angle data for all PAAm additions show completely covered surfaces. This coverage reduces the likelihood of surface heterogeneity being the cause for hysteresis. For PEG-terminated substrates, the variation in advancing contact angles supports some heterogeneity for the first (5, PEG) and final (9, PEG) additions. To address roughness as an explanation for increasing contact angle hysteresis, the trend in rms roughness values (Table 2) was compared to the trend in hysteresis values. While the hysteresis values increase slightly during hyperbranching, rms roughness values do not show a corresponding increase. Instead, roughness values appear to be more dependent upon the terminal modification whereas hysteresis values are influenced by the number of (22) Harsch, A.; Calderon, J.; Timmons, R. B.; Gross, G. W. J. Neurosci. Methods 2000, 98, 135-144. (23) Papra, A.; Gadegaard, N.; Larsen, N. B. Langmuir 2001, 17, 1457-1460.

polymer layers. Adding more hyperbranched layers increases the capacity of the film to adsorb water. AFM. Surface topology and roughness for PAAm and PEG hyperbranched substrates was imaged using AFM analysis. The roughness values obtained from AFM images (Table 2) show that the surface roughness varies through the different modifications. PEG additions reduce roughness compared to the PAAm modifications they are layered on. In general, surfaces with terminal PEG modifications are smoother than those with terminal PAAm modifications. PEGDA has only two sites, one acrylate at each end of the molecule, available for grafting to the PAAm surface. PAAm has many pendant amines that are available for addition to free acrylates on the underlying PEG layer. The ability of the PAAm molecule to bind to the PEG at any point within the molecule could lead to greater variety in the length of polymer extending from the surface. It is also possible for one PAAm molecule to bind to several acrylate groups, thereby having one PAAm molecule tethered to several PEG molecules. Both of these properties of PAAm grafting to PEGDA could result in the observed variation in roughness. PEG-silane films grafted on silicon, ranging from 3 to 32 Å in thickness, were reported to have rms roughness values were between 0.4 and 1 nm.8 For PEG-copolymer-silane monolayers, 11 Å films exhibited 1.3 Å rms roughness.24 Hyperbranched polymers terminating in PEG have higher roughness and thickness values than PEG monolayers. However, the rms roughness values for the hyperbranched PEG-terminated substrates are within ∼10% of the overall film thickness, which is comparable to that seen for the monolayers. While surface roughness can alter protein adhesion,25 the effects of roughness on protein adsorption are expected to be minimized in the ELISAs. In an aqueous environment, surface roughness decreases and coverage increases because PEG swells resulting in reduced protein adhesion.26 Protein Adhesion to PAAm- and PEG-Modified Silicon. PEG modification is known to reduce protein and cell adhesion to surfaces.8-10,14,15 Grafting surfaces with PEG derivatives such as using PEG-silanes to generate polymer monolayers is a common approach to inhibiting protein adsorption.8,24,27 Another approach is to incorporate PEG with other molecules such as chitosan28 or poly(L-lysine) to form a copolymer29 capable of reducing protein adhesion. PEG-copolymer-silane-modified silicon surfaces reduced insulin, lysozyme, and fibrinogen adsorption by 98% as detected by X-ray photoelectron spectroscopy24 Zhu et al. used surface plasmon resonance measurements to demonstrate reversibility of FBS binding to PEGsilane-modified gold surfaces.27 ELISA and radiolabel techniques were used to monitor protein adsorption to bare and hyperbranched silicon wafers. BSA adsorption to hyperbranched polymers was less than adsorption to bare silicon using both methods. Reduction of protein adherence due to terminal PEG modifications averages 89% ( 8% by ELISA and 80% ( 6% by radiolabel study for the three PEG layers studied (Figure 3). BSA adherence is reduced by 58% ( 32% by ELISA and 51% ( 14% by radiolabel for the PAAmterminated substrates (Figure 3). The extent of inhibition is comparable using both detection methods. (24) Jon, S.; Seong, J.; Khademhosseini, A.; Tran, T.-N. T.; Laibinis, P. E.; Langer, R. Langmuir 2003, 19, 9989-9993. (25) Deligianni, D. D.; Katsala, N.; Ladas, S.; Sotiropoulou, D.; Amedee, J.; Missirlis, Y. F. Biomaterials 2001, 22, 1241-1251. (26) Jo, S.; Park, K. Biomaterials 2000, 21, 605-616. (27) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56, 406-16. (28) Zhang, J.; Au, K. H.; Zhu, Z. Q.; O’Shea, S. Opt. Mater. 2004, 26, 47-55. (29) Tosatti, S.; Paul, S. M. D.; Askendal, A.; VandeVondele, S.; Hubbell, J. A.; Tengvall, P.; Textor, M. Biomaterials 2003, 24, 4949-4958.

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Conclusions

Figure 3. Protein adhesion to silicon wafers with surface modifications (as outlined in Scheme 1). Labels indicate the last modification performed on the wafers measured. Error bars represent the standard error of the mean. Columns represent bovine serum albumin adhesion to the modified surfaces in (a) ELISA studies and (b) 125I-labeled BSA adherence studies.

It is not surprising that BSA adheres better to PAAm surfaces than to PEG surfaces. BSA is negatively charged at neutral conditions, whereas PAAm-terminated hyperbranched polymers are cationic. This interaction of oppositely charged molecules has been used to preferentially bind a variety of anionic molecules to PAAm.30,31 Because of this favorable interaction, more BSA adherence to the surface is expected compared to PEG-terminated surfaces. Interestingly, PAAm-terminated substrates exhibit less BSA adsorption than bare substrates. Protein adhesion to the second- and third-generation PAAm surfaces (6, PAAm; 8, PAAm) was less than adhesion on first generation PAAm (4, PAAm) surfaces by ELISA but were comparable by quantified using radiolabeled protein. The ability of PAAm-terminated substrates to inhibit BSA adsorption suggests that PAAm can inherently repel proteins, but to a lesser extent than PEG. It also suggests that the underlying PEG layers may be affecting the ability of proteins to adhere to the PAAm layer. The implication of surface heterogeneity from the high hysteresis values from contact angle data suggests that the PEG may also be at or near the surface and able to repel proteins that would otherwise adsorb to PAAm. Masking the positive character of a substance was also observed for PEG-grafted poly(L-lysine) copolymers. The polycationic poly(L-lysine) backbone electrostatically adhered to the titanium surface, while the PEG grafts extended away from the surface. This arrangement was sufficient to reduce protein adhesion.29,32 (30) Nabok, A. V.; Tsargorodskaya, A.; Hassan, A. K.; Starodub, N. F. Appl. Surf. Sci. 2005, 246, 381-6. (31) Kreke, M. R.; Badami, A. S.; Brady, J. B.; Michael, Akers, R.; Goldstein, A. S. Biomaterials 2005, 26, 2975-2981.

Hyperbranching polymers on substrates offers several potential advantages over current coating technologies (e.g., polymerization, PEG-silane monolayer grafting, and PEG-copolymer adsorption). Hyperbranching combines the benefit of covalently anchored modifications with the ability to increase surface coverage of nonuniform substrates. This paper describes a procedure to hyperbranch PAAm and PEG on silicon. Preparing silicon wafers for polymer addition involved silanization followed by oxidation of the terminal vinyl group to present an acid function. Carbodiimide activation of the acid group allowed for coupling to PAAm through its pendant amine groups. Further modification was accomplished by Michael addition between PAAm amine groups and PEGDA acrylate groups. Alternating PAAm and PEG additions resulted in surfaces modified with hyperbranched polymer containing up to a total of six layers, i.e., 4, PAAm; 5, PEG; 6, PAAm; 7, PEG; 8, PAAm; 9, PEG. This hyperbranching procedure could easily be transferred to other surfaces receptive to silane chemistry, such as titanium and stainless steel. Surface modification was characterized using ellipsometry, QCM, contact angle measurements, and roughness analysis by AFM. PAAm and PEG additions gave uniform thickness increases of 12 and 18.7 Å, respectively. Thickness increases corresponded to the addition of mass load, as determined by decreasing resonant frequency of the QCM crystal with each modification. All surfaces were hydrophilic, and the PAAm- and PEG-terminated substrates exhibited static contact angle measurements within the ranges expected for films of the individual polymers. AFM analysis showed that PEG-terminated substrates were smoother than PAAm-terminated substrates. Once characterized, the ability of hyperbranched polymers to affect BSA adsorption was assessed. All polymer additions reduced protein adhesion compared to unmodified surfaces, as demonstrated by both ELISA and radiolabel analysis. PEGterminated substrates were better inhibitors of protein adhesion than PAAm-terminated surfaces, reducing protein adhesion by 89% ( 8% (ELISA) and 80% ( 6% (radiolabel). While PEGterminated substrates were better inhibitors of protein adhesion than PAAm-terminated substrates, both were able to reduce the amount of protein adsorbed to the surface compared to bare silicon. On the uniform silicon wafers used here, hyperbranching generates multiple layers of protein resistant polymers. On a nonuniform surface, hyperbranching could also provide protein resistance. The ability of multiple PEGDA molecules to add to one PAAm molecule allows for many PEG molecules to be introduced at the surface. By repeating PAAm and PEG additions, many PEG branches are created on PAAm molecules. The hyperbranching would result in surface coverage despite an irregular underlying silane layer. Monitoring protein adhesion to a modified substrate, such as stainless steel, is needed to address the protein resistance of hyperbranched polymers on nonuniform surfaces. Also of interest is the stability of these films for providing long-term antifouling capability. Acknowledgment. This work was funded by the National Institutes of Health (5R01EB000684-02). We thank Dr. David Allara for use of his goniometer and Dr. Craig Baumrucker for the use of his lab and equipment for the radiolabeled BSA studies. LA7004997 (32) Harris, L. G.; Tosatti, S.; Wieland, M.; Textor, M.; Richards, R. G. Biomaterials 2004, 25, 4135-4148.