Self-Assembled Monothiol-Terminated Hyperbranched Polyglycerols

Langmuir 2008, 24, 4907-4916

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Self-Assembled Monothiol-Terminated Hyperbranched Polyglycerols on a Gold Surface: A Comparative Study on the Structure, Morphology, and Protein Adsorption Characteristics with Linear Poly(ethylene glycol)s Po-Ying J.Yeh,† Rajesh K. Kainthan,‡ Yuquan Zou,‡ Mu Chiao,*,† and Jayachandran N. Kizhakkedathu*,‡ Department of Mechanical Engineering, Centre for Blood Research, and Department of Pathology & Laboratory Medicine, UniVersity of British Columbia, VancouVer, British Columbia V6T 1Z4, Canada ReceiVed September 14, 2007. In Final Form: February 5, 2008 Monothiol-terminated hyperbranched polyglycerols (HPGs) were synthesized by ring-opening polymerization of glycidol from partially deprotonated 2,2′-dihydroxyethane disulfide as the initiator and subsequent reduction of the disulfide group. Two molecular weights of HPG thiols were synthesized. The molecular weights of the polymers were determined by MALDI-TOF analysis, and the presence of thiol was verified by Ellman’s assay. The self-assembly of HPG thiols on gold was studied and compared with that of linear poly(ethylene glycol) (PEG) thiols utilizing various surface analysis techniques. Monothiol-functionalized HPGs readily adsorbed to a gold surface and formed highly uniform thin films on the surface. The graft density of the HPG layer decreased with an increase in the molecular weight of the polymer. The amount of polymer on the surface increased with increasing incubation concentration and saturated above 6 g/L polymer concentration. Generally, HPG thiols gave lower graft density compared to linear PEG thiols of similar molecular weight. AFM morphological studies showed that HPG thiols form more uniform and smooth surface films compared to PEG thiols. Incubation of a polymer-coated surface (HPG thiols and PEG thiols) with bovine serum albumin and immunoglobulin showed that the high molecular weight hyperbranched polyglycerol was more resistant to protein adsorption than linear PEG of similar molecular weight or lower molecular weight HPG. The protein adsorption decreased with increasing graft density of the HPG chains on the surface. Our results show that HPG could be a good alternative to PEG in the development of nonfouling functional surfaces.

Introduction Protein adsorption at the biomaterial-tissue interface is the first and critical event that initializes a cascade of host responses, including platelet activation, blood coagulation, and complement activation.1 Many approaches have been used to prevent such nonspecific biological interactions.2-5 Hydrophilic polymer-based coatings6-14 have been used as an antibiofouling agent for a number of applications including coatings to biomedical devices such as BioMEMs,15 biosensors,16 and drug delivery systems.17 * To whom correspondence should be addressed. (M.C.) E-mail: [email protected]. (J.N.K.) E-mail: [email protected]. Phone: (604) 822-7085. † Department of Mechanical Engineering. ‡ Centre for Blood Research and Department of Pathology & Laboratory Medicine. (1) (a) Gorbet, M. B.; Sefton, M. V. Biomaterials 2004, 25, 5681. (b) Ratner, B. D.; Bryant, S. J. Annu. ReV. Biomed. Eng. 2004, 6, 41. (2) Kitching, K. J.; Pan, V.; Ratner, B. D. In Plasma Polymer Films; Biederman, H., Eds.; Imperial College Press: London, 2004; pp 325-377. (3) Nath, N.; Hyun, J.; Ma, H.; Chilkoti, A. Surf. Sci. 2004, 570, 98. (4) Bohringer, K. F. J. Micromech. Microeng. 2003, 13, S1. (5) Mao, C.; Qiu, Y.; Sang, H.; Mei, H.; Zhu, A.; Shen, J.; Lin, S. AdV. Colloid Interface Sci. 2004, 110, 5. (6) Ma, H.; Hyun, J.; Stiller, P.; Chilkoti, A. AdV. Mater. 2004, 16, 338. (7) Zhang, Z.; Chen, S.; Chang, Y.; Jiang, S. J. Phys. Chem. B 2006, 110, 10799. (8) Szleifer, I. Physica A 1997, 244, 370. (9) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Biointerphases 2006, 1, 50. (10) Cho, W. K.; Kong, B.; Choi, I. S. Langmuir 2007, 23, 5678. (11) Sofie, L.; Salvage, J. P.; Lobb, E. J.; Armes, S. P.; Billingham, N. C.; Lewis, A. L.; Hanlon, G. W.; Lloyd, A. W. Biomaterials 2003, 25, 1195. (12) Tosatti, S.; De Paul, S. M.; Askendal, A.; VandeVondele, S.; Hubbell, J. A.; Tengvall, P.; Textor, M. Biomaterials 2003, 24, 4949. (13) Pasche, S.; De Paul, S. M.; Voeroes, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216. (14) Schlapak, R.; Armitage, D.; Saucedo-Zeni, N.; Hohage, M.; Howorka, S. Langmuir 2007, 23, 10244.

Such coatings frequently extend the life span of biomedical devices18,19 and the circulation half-life of drug delivery systems.20,21 Several factors affect the protein-repelling properties of such polymer thin films on the surface including the similarity of interfacial free energies of the polymer to that of water, interaction of proteins with polymers through hydrophobic or charge interactions, and environmental factors such as temperature and pH.22-24 In the case of neutral hydrophilic polymer brushes, the steric barrier due to high conformational entropy of anchored chains is the main contributing factor toward protein repulsion.25-31 (15) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55. (16) Sharma, S.; Johnson, R. W.; Desai, T. A. Biosens. Bioelectron. 2004, 20, 227. (17) Klibanov, A. L.; Torchilin, V. P.; Zalipsky, S. In Liposomes, 2nd ed.; Torchilin, V. P., Weissig, V., Eds.; Oxford University Press: Oxford, U.K., 2003; pp 231-265. (18) Tsujino, I.; Ako, J.; Honda, Y.; Fitzgerald, P. J. Expert Opin. Drug DeliVery 2007, 4, 287. (19) Burt, H. M.; Hunter, W. L. AdV. Drug DeliVery ReV. 2006, 58, 350. (20) Kim, D.; Park, S.; Lee, J. H.; Jeong, Y. Y.; Jon, S. J. Am. Chem. Soc. 2007, 129, 7661. (21) Caliceti, P.; Veronese, F. M. AdV. Drug DeliVery ReV. 2003, 55, 1261. (22) Lilian, E.; van Vlerken, Tushar, K. V.; Mansoor, M. A. Pharm. Res. 2007, 24, 1405. (23) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Biomaterials 2005, 26, 5927. (24) Zdyrko, B.; Varshney, S. K.; Luzinov, I. Langmuir 2004, 20, 6727. (25) McNamee, C. E.; Yamamoto, S.; Higashitani, K. Biophys. J. 2007, 93, 324. (26) Bartucci, R.; Pantusa, M.; Marsh, D.; Sportelli, L. Biochim. Biophys. ActasBiomembranes 2002, 1564, 237. (27) Steels, B. M.; Koska, J.; Haynes, C. A. J. Chromatogr., B: Biomed. Sci. Appl. 2000, 743, 41. (28) Singh, N.; Cui, X.; Boland, T.; Husson, S. M. Biomaterials 2007, 28, 763. (29) Carignano, M. A.; Szleifer, I. Colloids Surf., B 2000, 18, 169. (30) Satulovsky, J.; Carignano, M. A.; Szleifer, I. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9037.

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Other factors include the structure of the polymer on the surface (linear vs branched) and the molecular weight of the grafted chains. One of the commonly used polymers for making such films is poly(ethylene glycol) (PEG) because of its properties such as low toxicity, low immunogenicity, and the ability to prevent nonspecific protein adsorption and cell adhesion.32-34 Covalent coupling and chemisorption are the two common methods used for the attachment of poly(ethylene glycol) onto surfaces, and such methods have been studied extensively on various substrates.35,36 Though this is one of the widely used polymers for biomedical applications, one of the disadvantages is its susceptibility to oxidation and subsequent degradation.37 Another disadvantage is the availability of only two functionalities per chain because of its linear nature, which limits further modification. Also when attached at one end, the apparent surface density of functionalities decreases with an increase in molecular weight owing to the inherent low graft density of such assemblies on the surface.38 This is a huge disadvantage when developing functional surfaces for the immobilization of biomolecules. Thus, development of surface modification techniques which could make the surface not only nonfouling but also functional will be of great importance. Such developments will potentially increase the sensitivity and detection limit of immunoassays and protein arrays due to the decreased nonspecific protein interaction and increased coupling capacity.39 The use of dendritic polymers with multiple surface functional groups has gained increased attention in recent years for such applications, and it has been demonstrated that such surfaces can be modified with a broad range of functional groups and biomolecules such as peptides, antibodies, and DNA for various biomedical applications.40-42 We have recently shown that hyperbranched polyglycerols are highly biocompatible43,44 and can potentially be functionalized to various degrees due to the presence of a large amount of reactive hydroxyl groups.45 A recent report of Haag and coworkers demonstrated that surfaces coated with hyperbranched polyglycerols are resistant to protein adsorption.46 The thiolfunctionalized polyglycerols used in their study were synthesized by the postmodification of hydroxyl groups which could potentially generate multiple attachment points on the surfaces due to the presence of more than one thiol group per molecule.46 On a similar note, Sunder et al. reported the synthesis of hyperbranched polyglycerol dendron analogues from an alkene(31) Halperin, A. Langmuir 1999, 15, 2525. (32) Harris, J. M. In Poly(ethylene glycol) Chemistry, Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992; Chapter 1. (33) Cima, L. G. J. Cell. Biochem. 1994, 56, 155. (34) Tessmar, J. K.; Go¨pferich, A. M. Macromol. Biosci. 2007, 7, 23. (35) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L. Langmuir 2003, 19, 6912. (36) Unsworth, L. D.; Tun, Z.; Sheardown, H.; Brash, J. L. J. Colloid Interface Sci. 2005, 281, 112. (37) Sharma, S.; Johnson, R. W.; Desai, T. A. Appl. Surf. Sci. 2003, 206, 218. (38) Milner, S. T. Science 1991, 251, 905. (39) Stadler, V.; Beyer, M.; Koenig, K.; Nesterov, A.; Torralba, G.; Lindenstruth, V.; Hausmann, M.; Bischoff, F. R.; Breitling, F. J. Proteome Res. 2007, 6, 3197. (40) Ajikumar, P. K.; Ng, J. K.; Tang, Y. C.; Lee, J. Y.; Stephanopoulos, G.; Too, H. P. Langmuir 2007, 23, 5670. (41) Pathak, S.; Singh, A. K.; McElhanon, J. R.; Dentinger, P. M. Langmuir 2004, 20, 6075. (42) Park, J. W.; Jung, Y.; Jung, Young, H.; Seo, J. S.; Lee, Y. Bull. Korean Chem. Soc. 2004, 25, 1667. (43) (a) Kainthan, R. K.; Brooks, D. E. Biomaterials 2007, 28, 4779. (b) Kainthan, R. K.; Hester, S. R.; Levin, E.; Devine, D. V.; Brooks, D. E. Biomaterials 2007, 28, 4581. (44) Kainthan, R. K.; Janzen, J.; Levin, E.; Devine, D. V.; Brooks, D. E. Biomacromolecules 2006, 7, 703. (45) (a) Kainthan, R. K.; Gnanamani, M.; Ganguli, M.; Ghosh, T.; Brooks, D. E.; Maiti, S.; Kizhakkedathu, J. N. Biomaterials 2006, 27, 5377. (b) Frey, H.; Haag, R. ReV. Mol. Biotechnol. 2002, 90, 257. (46) Siegers, C.; Biesalski, M.; Haag, R. Chem.sEur. J. 2004, 10, 2831.

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functionalized initiator. The terminal double bonds were quantitatively converted to thioethers by reaction with thiols.47 In this paper, we report the synthesis of monothiol-functionalized hyperbranched polyglycerols using dihydroxyethane disulfide as the initiator for the anionic ring-opening multibranching polymerization of glycidol, followed by disulfide reduction. The polymers were characterized, and their selfassembly on a gold surface was studied in detail. Being functionalized with a single thiol group within the polymer, these polymers are expected to form only one attachment point on the surface. We studied the properties of the thin films formed by these polymers with respect to their molecular weight, graft density, thickness, and morphology and protein adsorption characteristics from single protein solutions. Further, we compared the results with those of thin polymer films formed by linear PEG thiols of similar molecular weights under identical conditions. Materials and Methods Materials. Bovine serum albumin-fluorescein isothiocyanate (BSA-FITC, A9771) and anti-mouse goat immunoglobulin G-FITC (IgG-FITC, F5265) were purchased from Sigma and used without further purification. Methoxypoly(ethylene glycol) thiol 5000 (mPEG-5000, CH3O(CH2CH2O)127CH2CH2CH2CH2SH; average molecular weight 5703) was purchased from Nektar Therapeutics. Methoxypoly(ethylene glycol) thiol 2000 (mPEG-2000) was purchased from Laysan Bio, Inc. (lot no. 103-56, average molecular weight 2000). 2,2′-Dihydroxyethane disulfide was purchased from Aldrich and used as such. Glycidol purchased from Aldrich was purified by distillation. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was obtained from Sigma. Silicon substrate with a thickness of 400 µm was purchased from Helitek Co. Ltd. Phosphate-buffered saline (PBS) (1×, 0.067 M PO4 and 0.15 M NaCl, pH 7.4) was purchased from HyClone. Polymer Synthesis. Disulfide-Containing Hyperbranched Polyglycerol (HPG-S-S-HPG). Hyperbranched polyglycerols containing a disulfide group were synthesized by anionic ring-opening multibranching polymerization of glycidol using partially deprotonated 2,2′-dihydroxyethane disulfide as the initiator as described earlier.47 Briefly, 0.44 mL of the disulfide initiator was taken in a three-neck round-bottom flask equipped with a mechanical stirrer. Approximately 10% of the hydroxyl groups were deprotonated using (diphenylmethyl)potassium (K+CH-Ph2) (0.25 M solution in THF). The mixture was stirred for 30 min, and the reaction flask was immersed in an oil bath maintained at a temperature of 100 °C. Glycidol (10 mL, corresponding to a monomer to initiator ratio of 40) was added dropwise over a period of 12 h using a syringe pump. The polymerization mixture was stirred for an additional 2 h. Another polymer was synthesized using a higher glycidol to initiator ratio. The product was dissolved in methanol and neutralized by passing three times through a column containing cation-exchange resin (Amberlite IRC-150). Both HPG disulfide polymers were then precipitated three times from acetone and dried. Monothiol-Functionalized HPG. Monothiol-functionalized HPGs (HPG-SH) were synthesized by reduction of the disulfide bond present in HPG-S-S-HPG polymers.48 The polymer (200 mg) was dissolved in 10 mL of ethanol, and an excess of dithioerythritol (100 mg) was added to this solution. The pH was adjusted to 10 by adding ammonium hydroxide solution. The solution was stirred for 1 h, and the resulting HPG-SH was precipitated three times from acetone. The molecular weight of the polymers was characterized by MALDI-TOF mass spectrometry analysis (DHB matrix), and the presence of thiol in the polymers was tested by UV-vis spectroscopy following their reaction with DTNB.49 (47) Sunder, A.; Mulhaupt, R.; Haag, R.; Frey, H. Macromolecules 2000, 33, 253. (48) Chen, S.; Zheng, J.; Li, L.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473. (49) (a) Ellman, G. L. Arch. Biochem. Biophys. 1958, 74, 443. (b) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.

Self-Assembled HPGs on a Gold Surface Gold Substrate Preparation. Silicon substrate was treated with RCA1 clean (NH4OH:H2O2:H2O ) 1:1:5) for 15 min to remove any organic contamination, rinsed thoroughly with DI water, and dried under a stream of nitrogen. Then a chromium layer (99.95% purity, Kurt J. Lesker Co., EVMCR35D) 5 nm thick was deposited as an adhesion layer for Au layer deposition. A Au layer (99.999% purity, Kurt J. Lesker Co., EVMAUXX50G, diameter 1/8 in., length 1/8 in.) 50 nm thick was then deposited as a functional layer for PEG coating. Chromium and Au were deposited by an evaporator with an electron beam source. The substrate was characterized by AFM topography analysis. Preparation of a Monolayer on Gold Substrates. Before polymer coating, the substrates were further cleaned by freshly prepared piranha solution (H2SO4:H2O2 ) 7:3 (v/v)) to remove any organic contamination. The substrates were rinsed with DI water thoroughly and dried with a nitrogen gun. The mPEG thiol solutions were prepared by dissolving powdered polymer in ethanol at 40 °C. The HPG-SH solutions were prepared by dissolving HPG-SH in ethanol at room temperature. The gold-coated plates were immersed in the mPEG thiol and HPG-SH solutions of different concentrations (from 2 × 10-5 to 20 g/L) at room temperature for polymer coating. We have also investigated the adsorbed mPEG thickness at different incubation times (9, 16, 24, and 48 h) in 6 g/L mPEG-5000 solution. The polymer-coated surfaces were washed thoroughly with 100% ethanol to remove unbound polymer and dried. Surface Characterization. Ellipsometry. The variable-angle spectroscopic ellipsometry (VASE) spectra were collected on an M-2000V spectroscopic ellipsometer (J.A. Woolham Co. Inc., Lincoln, NE) at 50°, 55°, 60°, and 65°, at wavelengths from 370 to 1000 nm with an M-2000 50 W quartz tungsten halogen light source. The VASE spectra were then fitted with the multilayer model (ambient-film-substrate) on the basis of the WVASE32 analysis software, using the optical properties of a generalized Cauchy layer to obtain the “dry” ellipsometric thickness of the adsorbed polymer layer. This dry thickness is also known as the dehydrated thickness, measured under ambient conditions. For evaluating the thickness of the polymer layer on the gold surface, the refractive indices of mPEG and HPG were determined. Transparent and relatively thick layers (∼50 nm) of polymer (mPEG thiol and HPG thiol) were prepared (to avoid surface interference) on glass slides with a coarse surface on the back side. Then the optical constant (n) curve of the polymer with multiple wavelengths (λ ) 400-1000 nm) was measured at incident angles of 65°, 70°, and 75° at different places on the surface, which was repeated for at least two different polymer films, and the data were averaged. The standard homogeneous multilayer model was used to interpret the experimental data. This model contains two unknowns: the thickness of the polymer layer and refractive indices of the adsorbed polymer. Since the polymer films used in this study were thin and transparent (i.e., κ ) 0), we used the Cauchy layer,50-52 in which n ) A + Bλ-2 (A and B are constants), to model the polymer layer. At high λ (700-1000 nm), the optical constant approximates to A, so we can fix B ) 0 and fit the experimental data by changing A and the thickness of the polymer layer. Once the thickness is decided, we fitted all the experimental data (370-1000 nm) for calculating A and B. For mPEG-5000, the calculated refractive index for A is 1.474 and that for B is 0.0081, which are close to the reported values for crystalline PEG (1.4650,51 to 1.46524 for A and 0.0151 for B). These values are good estimates and have been commonly used by other researchers.50,53 The calculated refractive indices of HPG were 1.473 for A and 0.0082 for B and were used for evaluating the polymer film thickness on the gold surface. To further characterize the polymer films, several other parameters were evaluated. The polymer surface coverage (adsorbed amount), Γ (mg/m2), was calculated from the ellipsometry thickness of the layer, h (nm):24,54 (50) Sharma, S.; Johnson, R. W.; Desai, T. A. Langmuir 2004, 20, 348. (51) Xiaowu, F.; Lijun, L.; Phillip, B. M. Biomacromolecules 2006, 7, 2443. (52) Tang, C.; Feller, L.; Rossbach, P.; Keller, B.; Vo¨ro¨s, J.; Tosatti, S.; Textor, M. Surf. Sci. 2006, 600, 1510. (53) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164.

Langmuir, Vol. 24, No. 9, 2008 4909 Γ ) Fh

(1)

where F is the density of the attached molecules. The density of mPEG-SH (1.08 g/cm3) was provided by the supplier, and that of HPG-SH was assumed to be the same. The chain density, Σ (chains/ nm2), i.e., the inverse of the average area per adsorbed chain, was determined by24,54 Σ ) 10-21ΓNA/MW ) 602.3Γ/MW

(2)

where NA is Avogadro’s number and MW is the molecular weight of the grafted polymer. ATR-FTIR Spectroscopy. ATR-FTIR absorption spectra were collected on a Nexus 670 FT-IR ESP (Nicolet Instrument Corp., Waltham, MA) with an MCT/A liquid nitrogen cooled detector, a KBr beam splitter, and an MkII Auen Gate single-reflection attenuated total reflectance (ATR) accessory (Specac Inc., Woodstock, GA). The sample stage contained a diamond window and a sapphire anvil on a torque-limiting screw set to deliver 80 lb of pressure. IR spectra of the surfaces were recorded from 600 to 4000 cm-1 at room temperature. X-ray Photoelectron Spectroscopy (XPS) Analysis. The surface chemical analysis of reacted samples was done with the XPS (Leybold MAX200 X-ray photoelectron spectrometer, XPS/ESCA dual anode (Mg KR and Al KR) achromatic X-ray source) method using an Al KR X-ray source (1486.6 eV). The samples (0.5 cm × 1 cm) were mounted onto the XPS stage. The XPS spectra of all studied elements such as C1s, O1s, S2p, and Au4f were measured with a constant analyzer with a 48 eV pass energy. All binding energies (BEs) were referenced to the Au4f peak (BE ) 84 eV). Atomic Force Microscopy. The atomic force microscope used for this experiment was a commercially available multimode system with an atomic head of 100 × 100 µm2 scan range which used a NanoScope IIIa controller (Digital Instruments, Santa Barbara, CA). AFM was performed in air by contact mode using a commercially manufactured V-shaped silicon nitride (Si3N4) cantilever with gold on the back for laser beam reflection (Veeco, NP-S20). The typical tip radius and spring constant of the cantilever were 5-40 nm and 0.06N/m, respectively. The raw data were processed with NanoScope IIIa software by first-order flattening. Force measurements were performed using the same AFM instrument equipped with a Nanoscope IIIa controller and a fluid cell. The experiments were performed in force mode under 0.1 M NaCl. All measurements were taken at an approach speed of 1.0 Hz. The raw AFM force data (cantilever deflection vs displacement data) were converted into the reduced force vs separation following the principle of Ducker et al.55 The onset of the region of constant compliance was used to determine the zero distance, and the region in which force was unchanged was used to determine the zero force. The hydrated thicknesses of the grafted polymer layers in 0.1 M NaCl were determined by two points, zero distance and zero force, from force-distance curves. The average thickness obtained from several force curves at various locations was taken as the hydrated thickness. Protein Adsorption Studies. The substrates were initially equilibrated with PBS buffer for 3 h, and then the protein adsorption experiments were conducted by incubating substrates with or without PEG modification in 2 mg/mL BSA and 0.1 mg/mL IgG solutions in 48-well polystyrene plates at room temperature for 1 h. Following the incubation, the substrates were rinsed thoroughly with PBS (>10 times) to remove loosely adsorbed proteins. The images of the proteinadsorbed surfaces were taken by a fluorescence microscope (Nikon Eclipse TE 2000-U with an X-Cite 120 fluorescence illumination system, an FITC filter, and a DS-U1 suit digital camera). The green fluorescence intensity of the images was transferred to gray scale by Adobe Photoshop 6.0, which is linear with the relative quantity of adsorbed proteins on the surface.56 The calibration scale was set (54) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059. (55) Ducker, W. A.; Sendan, T. J.; Pashley, R. M. Langmuir 1992, 8, 1831. (56) Yeh, P. Y.; Kizhakkedathu, J. N.; Madden, J. D.; Chiao, M. Colloids Surf., B 2007, 59, 67.

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Scheme 1. Synthetic Route for Monothiol-Funtionalized Hyperbranched Polyglycerols

by two points: the gray scale intensity of the bare surface was set as 0%, and that of the protein-adsorbed bare surface was set as 100%. The relative adsorption of proteins on various surfaces is normalized on the basis of the calibration scale.

Results and Discussion Polymer Synthesis and Characterization. The synthetic strategy for the monothiol-functionalized polyglycerols is given in Scheme 1. The polymerization of glycidol was initiated from 2,2′-dihydroxyethane disulfide. Two polymers were synthesized at different initiator to glycidol ratios to achieve high and low molecular weights. 1H NMR showed the presence of disulfide initiator moieties (peak at 2.7 ppm for -CH2S-) in the purified polymer (Figure 1S, Supporting Information). Reduction of the disulfide group with dithioerythritol produced a monothiolfunctionalized HPG. The formation of thiol is evident from the formation of a yellow-colored solution upon reaction with DTNB and absorbance at λ ) 420 nm49 (Figure 2S, Supporting Information). The disulfide polymers did not produce any color

after being mixed with DTNB. The synthesis of the disulfide precursor polymer was very simple, robust, and quantitative. The molecular weight of the polymer can easily be adjusted by employing a suitable glycidol to initiator ratio, and this method could be an attractive alternative to using similar dendritic PEGbased polymers. Also the presence of several reactive hydroxyl groups in the polymer makes it an ideal candidate for coupling biomolecules. The monothiol-functionalized HPG polymers designated as HPG-SH-L and HPG-SH-H have molecular weights of 1586 and 4261 (the molecular weight of the highest intensity peak in the MALDI-TOF spectra, Figure 3S, Supporting Information), respectively. Surface Modification with Polymers. Self-assembly of thiolfunctionalized polymers on a metallic surface is a widely studied and versatile method for surface modification.57 In this study we used four monothiol-functionalized polymers, HPG-SH-L (low (57) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. ReV. 2005, 105, 1103.

Self-Assembled HPGs on a Gold Surface

Figure 1. Representation of the structures of polymeric films on a gold surface: (a) linear mPEG monothiol and (b) HPG monothiol.

molecular weight), HPG-SH-H (high molecular weight), linear mPEG-5000 thiol (high molecular weight), and mPEG-2000 thiol (low molecular weight). Since the linear and hyperbranched polymers have a single thiol group within the polymer, we expect to get a single gold-sulfur attachment point per polymer chain

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on the surface (Figure 1). Thus, these systems could be a good model to study and compare the adsorption characteristics and properties of linear and branched polymers on the surface. Although one could expect a more ordered monolayer of PEG with a higher carbon spacer (e.g., C11) as reported previously,57 our intention here was to compare the surface adsorption characteristics of HPGs with a two-carbon spacer and PEGs having a similar chemical structure. Therefore, in the present case, we used mPEG-5000 with a four-carbon spacer (commercially available) and mPEG-2000 (two-carbon spacer) for our studies. The gold surfaces were incubated with polymer solutions of different concentrations. Unlike the thiol-functionalized polymers, the nonreduced disulfide polymers and a control HPG polymer (without a thiol group) did not adsorb significantly to the surfaces (see Table 1S, Supporting Information). The poor adsorption of disulfide-containing HPG on the gold surface might be due to the inaccessibility of the “deeply buried” disulfide groups to the gold surface. The presence of HPG on both sides of the S-S bond may be acting as a barrier to stable bond formation between gold and sulfur (see the structure, Scheme 1). Surface Characterization: FTIR and XPS. To investigate the chemical characteristics of the HPG- and mPEG-coated

Figure 2. ATR-FTIR spectra of gold surfaces coated with (a) monothiol-functionalized linear mPEG-5000 at (1) 6 g/L, (2) 2 × 10-2 g/L, (3) 2 × 10-3 g/L, and (4) 2 × 10-5 g/L polymer incubation concentration and (b) monothiol-functionalized HPG-SH-L at (1) 6 g/L, (2) 1 g/L, (3) 2 × 10-2 g/L, and (4) 2 × 10-3 g/L polymer concentration. (c) Comparison of linear mPEG-5000, HPG-SH-L, and HPG-SH-H at 6 g/L solution concentration. Polymer films were produced by incubating the gold surface in polymer solution for 16 h.

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Figure 3. High-resolution C1s scan from XPS spectra of surfacegrafted HPG-SH-L at (1) 6 g/L, (2) 1 g/L, and (3) 2 × 10-3 g/L polymer incubation concentration and (4) the bare gold-coated substrate.

surfaces, ATR-FTIR spectroscopy was employed. As shown in Figure 2, the presence of polymer on the surface is evident from the characteristic peaks for HPG and PEG. The C-O-C stretching around 1200 cm-1 and C-H stretching at 2875 cm-1 showed the presence of HPG and PEG on the gold surface. An additional broad peak around 3400 cm-1 in the case of HPG is from the hydroxyl stretching (Figure 2b,c; see also Figure 4S, Supporting Information).58,59 The polymer grafting on the surface increased with an increase in polymer incubation concentration as evident from the increased intensity of the IR bands (Figure 2a,b). The intensity of the C-O-C peaks was higher and sharp in the case of the linear PEG-coated surface compared to the HPG-coated surface. Even at low polymer incubation concentration (0.02 g/L), the C-O-C peaks were clearly visible in the ATR-IR spectra of the polymer-coated gold surface for both types of polymers. The presence of HPG on the surface was further demonstrated by measuring the XPS spectra of the gold substrates coated with the polymers (Figure 5S, Supporting Information). Figure 3 shows the high-resolution C1s spectra of the HPG-SH-L-coated surface. The presence of a peak at 286.4 eV in the C1s spectra is indicative of carbon attached to oxygen, which is different from adventitious carbon.16 With an increase in the HPG-SH-L solution concentration, the C1s peak shifted to higher energy. The atomic composition (ratio of C to O) and thickness of the HPG-SH-L films were calculated from the XPS spectra54 and are given in Figure 6S (Supporting Information). Results show that the dry thickness measured by ellipsometry (see the next section) and those calculated from XPS intensities (Au peak) were comparable. The C:O ratios of the grafted HPG-SH-L films decreased with increasing polymer incubation concentration. Polymer Film Thickness and Graft Density. The dry thickness of the polymer film on the gold surface was calculated from the ellipsometric measurements. Figure 4 shows the dependence of adsorption of linear mPEG-5000 thiol on the incubation time. The measured polymer film thicknesses of PEG films at 6 g/L polymer incubation concentration showed no significant change after 9 h. Therefore, we decided to keep a time of 16 h for further experiments. (58) Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N. J. Biomed. Mater. Res. 2000, 51, 345. (59) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56, 406.

Yeh et al.

Figure 4. Effect of the incubation time on the dry thickness of the linear mPEG-5000 film measured by ellipsometry on the gold surface at 6 g/L polymer concentration.

Figure 5. Effect of the incubation concentration and type of polymer on the (a) thickness and (b) graft density of polymer films on the gold surface. Polymer films were produced by incubating the gold surface in polymer solution for 16 h.

The dry thickness of HPGs and linear PEG films obtained with different incubation concentrations is given in Figure 5a. The results show that the polymer concentration in solution significantly influences the film thickness. The dry thickness increased gradually with polymer incubation concentration in

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Table 1. Characteristics of Polymer-Grafted Surfaces Obtained from AFM Analysis polym incubation concn 2 × 10-5 g/L particle height (nm)

rms roughness (nm)

particle height (nm)

rms roughness (nm)

polym incubation concn 2 × 10-2 g/L particle height (nm)

rms roughness (nm)

polym incubation concn 6 g/L particle height (nm)

rms roughness (nm)

hydrated thicknessa (nm)

gold 3.87 ( 0.23 1.05 ( 0.12 mPEG-5000 5.05 ( 0.5 1.02 ( 0.15 10.23 ( 1.89 1.66 ( 0.16 12.98 ( 1.42 1.95 ( 0.55 14.79 ( 1.69 HPG-SH-L 5.23 ( 0.29 1.17 ( 0.11 5.58 ( 0.52 1.23 ( 0.10 7.74 ( 0.42 1.69 ( 0.25 7.57 ( 1.14 HPG-SH-H 4.75 ( 0.35 1.05 ( 0.3115 5.08 ( 0.13 1.49 ( 0.23 7.92 ( 0.45 1.58 ( 0.12 11.8 ( 1.35 a

Measured in 100 mM NaCl at 22 °C.

the case of HPGs compared to linear PEG and became almost constant above 6 g/L polymer concentration (Figure 5a). Linear mPEG-5000 gave the maximum thickness (∼7.3 nm) compared to HPG-SH-L and HPG-SH-H (∼3.8 nm) or mPEG-2000 (∼4.2 nm). In the case of mPEG-5000, there is a jump in the film thickness at 0.02 g/L concentration. The film thickness reported for very low polymer solution concentrations (at or below 2 × 10-3 g/L) may have some uncertainty due to the surface roughness of gold films, which could possibly influence the ellipsometric analysis. Moreover, the thickness values are very small ( HPG-SH-L ≈ mPEG-2000 > mPEG-5000 > HPGSH-H. Figures 7f and 8e show the relative fluorescence intensity of the polymer-coated gold surface after the adsorption of BSA and IgG at different graft densities. The data given are the average from six photos for one sample done in duplicate. Approximately 75% reduction in the adsorption of SA on the HPG-SH-Hmodified surface (graft density ∼0.57 chain/nm2) compared to ∼62% for linear mPEG-5000 (∼0.83 chain/nm2), ∼50% for mPEG-2000 (∼1.38 chains/nm2), and ∼49% for HPG-SH-L (∼1.6 chains/nm2) (Figure 7f). In the case of IgG, the percentage reduction in the protein adsorption under identical conditions was 72%, 51%, and 58%, respectively, for HPG-SH-H, HPGSH-L, and linear mPEG-5000 (Figure 8e). Although HPGs showed a decrease in protein adsorption with an increase in the graft density in the case of IgG, mPEG-5000 did not show any significant dependence on the graft density. The total protein adsorption decreased upon mPEG-5000 grafting. The relative magnitude of protein adsorption was different for linear PEG and HPGs. For a given graft density, the high molecular

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Figure 8. Fluorescence photographs of IgG-adsorbed (a) bare gold and (b) linear mPEG-5000-, (c) low molecular weight HPG-SHL-, and (d) high molecular weight HPG-SH-H-grafted surfaces. Polymer films were produced by incubating the gold surface in polymer solution at 6 g/L for 16 h. (e) Effect of the graft density on the IgG adsorption of mPEG-, HPG-SH-L-, and HPG-SHH-grafted surfaces.

Figure 7. Fluorescence photographs of BSA-adsorbed (a) bare gold and (b) linear mPEG-2000-, (c) linear mPEG-5000-, (d) low molecular weight HPG-SH-L-, and (e) high molecular weight HPG-SH-Hgrafted surfaces. Polymer films were produced by incubating the gold surface in polymer solution at 6 g/L for 16 h. (f) Effect of the graft density on the BSA adsorption of mPEG-, HPG-SH-L-, and HPG-SH-H-grafted surfaces.

weight HPG-SH-H is more protein resistant than linear mPEG5000 or low molecular weight HPG-SH-L. Even at very low polymer incubation concentration (i.e., at low graft densities) there is some reduction in protein adsorption compared to that of the bare surface. IgG adsorption also followed a pattern similar to that of BSA (Figures 7e and 8e) in the case of HPGs but not for mPEG-5000. The difference between HPG-SH-H and linear mPEG-5000 was smaller in this case compared to BSA adsorption. Our results demonstrated that higher molecular weight HPG has a better ability to resist protein adsorption than linear mPEG5000. Both mPEG-2000 and low molecular weight HPG-SH-L behaved similarly. Our results also proved that the molecular weight of the branched polymer is important in the development of nonfouling surfaces. Although the low molecular weight HPGSH-L-grafted surface has a higher graft density than the high molecular weight HPG-SH-H-grafted surface, more proteins adsorbed to that surface. This suggests that, along with surface coverage, the flexibility of the chains also contributes to the

protein repulsion characteristics. Increased chain extension and flexibility of this surface film is evident from the larger hydrated thickness of HPG-SH-H (at 0.57 chain/nm2) compared to HPGSH-L (∼1.6 chains/nm2) under aqueous conditions (Table 1).

Conclusions We have reported a facile and robust method for the synthesis of monothiol-functionalized hyperbranched polyglycerols. Two different molecular weights of HPG thiols were synthesized and characterized. The surface adsorption characteristics of newly synthesized HPG thiols on gold were studied and compared with those of linear PEG thiols. Our results show that HPG thiols readily adsorbed to the gold surface and gave a uniform coating as evident from ellipsometric, ATR-FTIR, and AFM topography measurements. Low molecular weight HPG thiol gave a higher graft density compared to higher molecular weight HPG thiol or linear PEG thiols. The graft density of the chains on the surface increased with an increase in polymer incubation concentration and became almost constant above 6 g/L polymer concentration. The dry film thickness of HPG thiol-coated surfaces was lower compared to that of linear PEG thiol-coated surfaces at all the polymer solution concentrations studied. There are finite differences in the topography of the polymer layer formed by mPEG5000 and HPG thiols. The branched polymers produced more uniform structures on the surface and low surface roughness. The high molecular weight HPG thiol-coated surface is more resistant to protein adsorption than the low molecular weight

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HPG thiol-coated or linear mPEG thiol-coated surface. The protein repulsion of HPG-coated surfaces increased with increasing graft density of chains on the surface. Our results show that HPGs with a single thiol group could be a good alternative to PEGs in the development of nonfouling surfaces with an additional advantage of further surface functionalization.

Research (CIHR) new investigator award in transfusion science. M.C. is supported by the Canada Research Chair, Tier 2 program and a discovery grant from the Natural Science and Engineering Research Council of Canada (NSERC). We thank Dr. Johan and Dr. Kin Chung Won for their help with ellipsometry and XPS analysis, respectively.

Acknowledgment. J.N.K. acknowledges financial support from the University of British Columbia, Canada Foundation for Innovation (CFI). The LMB Macromolecular hub at the Centre for Blood Research is supported by the CFI and Michael Smith Foundation for Health Research (MSFHR). J.N.K. is the recipient of a Canadian Blood Services (CBS)/Canadian Institutes of Health

Supporting Information Available: 1H NMR spectra of HPG disulfide, MALDI-TOF spectra of HPG thiol, UV-vis spectra of the DTNB-reacted thiol polymer, and ellipsometric characterization of HPG disulfide adsorption on the gold surface. This material is available free of charge via the Internet at http://pubs.acs.org. LA702867T