pubs.acs.org/Langmuir © 2010 American Chemical Society
Multilayer Buildup and Biofouling Characteristics of PSS-b-PEG Containing Films Christina Cortez,† John F. Quinn,† Xiaojuan Hao,‡,§ Chakravarthy S. Gudipati,‡ Martina H. Stenzel,‡ Thomas P. Davis,‡ and Frank Caruso*,† † Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australia, and ‡Centre for Advanced Macromolecular Design, The University of New South Wales, Sydney, NSW 2052, Australia. § Current address: CSIRO Molecular and Health Technologies, Bayview Avenue, Clayton, Private Bag 10, Clayton South MDC, Victoria 3169, Australia.
Received January 28, 2010. Revised Manuscript Received February 17, 2010 Thin films exhibiting protein resistance are of interest in diverse areas, ranging from low fouling surfaces in biomedicine to marine applications. Herein, we report the preparation of low protein and cell binding multilayer thin films, formed by the alternate deposition of a block copolymer comprising polystyrene sulfonate and poly(poly(ethylene glycol) methyl ether acrylate) (PSS-b-PEG), and polyallylamine hydrochloride (PAH). Film buildup was followed by quartz crystal microgravimetry (QCM), which showed linear growth and a high degree of hydration of the PSS-b-PEG/PAH films. Protein adsorption studies with bovine serum albumin using QCM demonstrated that multilayer films of PSS/PAH with a terminal layer of PSS-b-PEG were up to 5-fold more protein resistant than PSS-terminated films. Protein binding was dependent on the ionic strength at which the terminal layer of PSS-b-PEG was adsorbed, as well as the pH of the protein solution. It was also possible to control the protein resistance of the films by coadsorption of the final layer with another component (PSS), which showed an increase in protein resistance as the proportion of PSS-b-PEG in the adsorption solution was increased. In addition, protein resistance could also be controlled by the location of a single PSS-b-PEG layer within a PSS/PAH film. Finally, the buildup of PSS-b-PEG/PAH films on colloidal particles was demonstrated. PSS-b-PEG-terminated particles exhibited a 6.5-fold enhancement in cell binding resistance compared with PSS-terminated particles. The stability of PSS-bPEG films combined with their low protein and cell binding characteristics provide opportunities for the use of the films as low fouling coatings in devices and other surfaces requiring limited interaction with biological interfaces.
Introduction The layer-by-layer (LbL) assembly of interacting materials on a substrate provides a facile and versatile approach to the preparation of thin films.1-3 The versatility of the technique arises from the wide range of substrates and materials that can be used in the process, and includes both planar and colloidal substrates, polymers,4 DNA,5,6 polysaccharides,7,8 and polypeptides9-11 as film materials. In addition, the buildup can be facilitated by electrostatic interaction, hydrogen bonding, covalent bonding, and/or hybridization, further broadening the scope of materials that can be prepared using the approach.12 LbL-assembled multilayered thin films can therefore be designed with nanoscale precision and with tailored functionality to suit a wide range of applications, including sensing, surface coatings, and therapeutic delivery. *To whom correspondence should be addressed. E-mail: fcaruso@ unimelb.edu.au. (1) Decher, G.; Hong, J. D. Makromol. Chem., Macromol. Symp. 1991, 46, 321. (2) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831. (3) Ariga, K.; Hill, J. P.; Ji, Q. M. Phys. Chem. Chem. Phys. 2007, 9, 2319. (4) Decher, G. Polyelectrolyte Multilayers, An Overview. In Multilayer Thin Films; Decher, G., Schlenoff, J. B., Eds.; Wiley-VCH Verlag: Weinheim, 2003. (5) Johnston, A. P. R.; Read, E. S.; Caruso, F. Nano Lett. 2005, 5, 953. (6) Johnston, A. P. R.; Mitomo, H.; Read, E. S.; Caruso, F. Langmuir 2006, 22, 3251. (7) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Langmuir 2004, 20, 448. (8) Picart, C.; Schneider, A.; Etienne, O.; Mutterer, J.; Schaaf, P.; Egles, C.; Jessel, N.; Voegel, J.-C. Adv. Funct. Mater. 2005, 15, 1771. (9) Richert, L.; Arntz, Y.; Schaaf, P.; Voegel, J.-C.; Picart, C. Surf. Sci. 2004, 570, 13. (10) Boulmedais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J.-C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003. (11) Boulmedais, F.; Bozonnet, M.; Schwinte, P.; Voegel, J.-C.; Schaaf, P. Langmuir 2003, 19, 9873. (12) Quinn, J. F.; Johnston, A. P. R.; Such, G. K.; Zelikin, A. N.; Caruso, F. Chem. Soc. Rev. 2007, 36, 707.
9720 DOI: 10.1021/la100430g
Multilayer film formation by LbL assembly has traditionally involved the use of two interacting materials to produce a twocomponent film, whereby polyelectrolyte A is assembled in alternation with polyelectrolyte B.4 Depending on the constituents of the film, film stability, thickness, and morphology can be highly influenced by assembly or postassembly conditions such as pH, ionic strength, or temperature. Recently, three-component or multicomponent systems have been investigated as a means to further tailor the properties and functionality of multilayer films.13-32 (13) Dierich, A.; Le Guen, E.; Messaddeq, N.; Stoltz, J.-F.; Netter, P.; Schaaf, P.; Voegel, J.-C.; Benkirane-Jessel, N. Adv. Mater. 2007, 19, 693. (14) Benkirane-Jessel, N.; Schwinte, P.; Falvey, P.; Darcy, R.; Haikel, Y.; Schaaf, P.; Voegel, J.-C.; Ogier, J. Adv. Funct. Mater. 2004, 14, 174. (15) Schultz, P.; Vautier, D.; Richert, L.; Jessel, N.; Haikel, Y.; Schaaf, P.; Voegel, J.-C.; Ogier, J.; Debry, C. Biomaterials 2005, 26, 2621. (16) Caruso, F.; Caruso, R. A.; Mohwald, H. Science 1998, 282, 1111. (17) Schuetz, P.; Caruso, F. Chem. Mater. 2002, 14, 4509. (18) Gittins, D. I.; Susha, A. S.; Schoeler, B.; Caruso, F. Adv. Mater. 2002, 14, 508. (19) Yoo, P. J.; Nam, K. T.; Qi, J.; Lee, S.-K.; Park, J.; Belcher, A. M.; Hammond, P. T. Nat. Mater. 2006, 5, 234. (20) Cho, J. H.; Quinn, J. F.; Caruso, F. J. Am. Chem. Soc. 2004, 126, 2270. (21) Quinn, A.; Such, G. K.; Quinn, J. F.; Caruso, F. Adv. Funct. Mater. 2008, 18, 17. (22) Yap, H. P.; Quinn, J. F.; Johnston, A. P. R.; Caruso, F. Macromolecules 2007, 40, 7581. (23) Johal, M. S.; Ozer, B. H.; Casson, J. L.; St. John, A.; Robinson, J. M.; Wang, H.-L. Langmuir 2004, 20, 2792. (24) Johal, M. S.; Chiarelli, P. A. Soft Matter 2007, 3, 34. (25) Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203. (26) Schuetz, P.; Caruso, F. Colloids Surf., A 2002, 207, 33. (27) Cho, J.; Caruso, F. Macromolecules 2003, 36, 2845. (28) Khopade, A. J.; Caruso, F. Chem. Mater. 2004, 16, 2107. (29) Tjipto, E.; Quinn, J. F.; Caruso, F. Langmuir 2005, 21, 8785. (30) Tjipto, E.; Quinn, J. F.; Caruso, F. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 4341.
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The ability to tailor the properties of the film in this manner is important for enhancing the suitability of the film for many existing and novel applications. For example, films incorporating biomolecules make suitable bioactive coatings, such as for bone regeneration13 and as anti-inflammatory coatings,14,15 while nanoparticle-16-18 or virus-infused films19 are among the many smart materials that can be prepared from multicomponent systems. There are various approaches available for obtaining threecomponent systems. Blending of adsorbing species in one of the adsorption solutions is a common method for obtaining multicomponent films. This approach includes blending of like-charged polyelectrolytes,20-22 surfactants and polyelectrolytes,23,24 and proteins and polyelectrolytes.25 Other methods for obtaining threecomponent systems involve the use of precomplexed polyions,26 intercalated stacks of different polyion pairs,27 and the incorporation of degradable core materials.28 Additionally, the use of copolymers with two distinct monomer moieties provides a facile approach for the preparation of multifunctional, multicomponent thin films.29-32 In the example of poly(4-styrene sulfonic acid-comaleic acid) copolymer (PSSMA) layered with poly(allylamine hydrochloride) (PAH), the prepared films exhibited greater responsiveness to pH and ionic strength compared with the traditional PSS/PAH films.29 Further, the MA moieties provide the functionality of carboxyl groups that can be utilized for further functionalization of the film, for example, for conjugation with biomolecules. In another example, the adsorption of an amphiphilic block copolymer, polystyrene-block-poly(acrylic acid) (PS-PAA) on PAA/PAH multilayer films, improved the hydrophobicity of the film, as demonstrated by an increase in the advancing contact angle by about 40°.31 This demonstrates that surface and film properties can be easily modified with the use of copolymers in multicomponent systems. A useful property of films or coatings, especially in many biomedical applications where minimal protein adsorption is desirable, is low fouling. This property is typically imparted by hydrophilic polymers such as poly(vinylpyrolidone),33,34 poly(acrylamide),35 poly(hydroxyethyl methacrylate),36 hyaluronic acid,37 and the widely studied poly(ethylene glycol) (PEG).38,39 PEG is believed to impart low fouling properties to surfaces due to the screening of interfacial charges, repulsion (entropic and osmotic), and excluded volume effects.40,41 A high surface density of PEG, which forms a brushlike structure on the surface, acts to shield the underlying area from the penetration and subsequent adsorption of proteins. PEG can be attached to various surfaces by hydrophobic interactions, by hydrogen bonding, by covalent attachment, as self-assembled monolayers, or by adsorption via electrostatic binding of a PEG-conjugated charged molecule. In LbL films, PEGylated multilayers can be prepared by inserting copolymers incorporating PEG in the same mechanism as in (31) Choi, J.; Rubner, M. F. J. Macromol. Sci., Part A: Pure Appl. Chem. 2001, 38, 1191. (32) Wang, F.; Li, L. H.; Gao, C. Y. Macromol. Chem. Phys. 2009, 210, 2022. (33) Roesink, H. D. W.; Beerlage, M. A. M.; Potman, W.; van den Boomgaard, T.; Mulder, M. H. V.; Smolders, C. A. Colloids Surf. 1991, 55, 231. (34) Robinson, S.; Williams, P. A. Langmuir 2002, 18, 8743. (35) Musale, D. A.; Kulkarni, S. S. J. Membr. Sci. 1996, 111, 49. (36) Ulbricht, M.; Matuschewski, H.; Oechel, A.; Hicke, H. G. J. Membr. Sci. 1996, 115, 31. (37) Morra, M.; Cassineli, C. J. Biomater. Sci. 1999, 10, 1107. (38) Harris, J. M., Ed. Poly(ethyleneglycol) Chemistry: Biotechnical and Biomedical Applications; Plenum Press: New York, 1992. (39) Bailey, F. E., Jr.; Koleske, J. Y. Poly(Ethylene Oxide); Academic Press: New York, 1976. (40) Feldman, K.; Hahner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134. (41) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426.
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conventional LbL films. This provides an easy approach for rendering surfaces low fouling and is useful for PEGylating a wide range of structures or surfaces. In the past, grafted copolymers of either poly(L-glutamic acid) (PGA-g-PEG)10 or poly(L-lysine) (PLL-g-PEG)42,43 have been incorporated in LbL films and have demonstrated reduced adhesion of proteins and biological cells.10,42,43 Challenges with the stability of polypeptide multilayers bearing PEG under certain conditions (such as extreme pH and high ionic strength) remain a limitation due to the weak, or pH-sensitive, nature of PLL and PGA. In this study, the multilayer buildup of PSS-b-PEG and PAH on both planar and colloidal surfaces was investigated. This system is particularly interesting as it couples the ability of PSS/ PAH to form highly stable multilayers with the low fouling feature of PEG. In this case, PSS serves as the anchoring block and attaches the copolymer to the multilayer surface (PAH). While the interaction will predominantly arise from the strong electrostatic interaction between PSS and PAH, the presence of the uncharged PEG is expected to influence not only the fouling behavior of the film but also its assembly, morphology, integrity, and responsiveness. The PSS-b-PEG block copolymer was produced by living radical polymerization via reversible additionfragmentation chain transfer (RAFT).44-48 Being synthesized from an acrylate ester of PEG, the polymer has a brush structure, as shown in Scheme 1. The widely studied protein bovine serum albumin (BSA) was used as a model protein to investigate the fouling behavior of PSS-b-PEG-containing films. The results demonstrate the ability to control the fouling behavior of PSSb-PEG-containing films by varying the ionic strength of the adsorption solution or the pH of the protein solution. In addition, “blending” or the coadsorption of PSS-b-PEG with a nonresistant, like-charged polymer (such as PSS), as well as the location of a single PSS-b-PEG layer within a film otherwise prepared from PAH and PSS can influence the protein resistance of the film. These results are complemented by the reduced cell binding observed for PSS-b-PEG-terminated colloidal particles (compared with PSS-terminated particles), as measured by flow cytometry. These films may be useful in applications where control of protein adsorption is important, such as coatings for biomedical devices or marine biofouling applications, where the stability of the film would ensure the integrity and durability of the nonfouling layer.
Experimental Section Materials. Poly(allylamine hydrochloride) (PAH), Mw = 70 000 g mol-1, poly(sodium 4-styrenesulfonate) (PSS), Mw = 70 000 g mol-1, polyethyleneimine (PEI), Mw = 25 000 g mol-1, and poly(ethylene glycol) methyl ether acrylate (PEGMEA) (Mn = 480 g mol-1) were purchased from Sigma-Aldrich and were used as received. Poly(sodium 4-styrenesulfonate)-b-poly(poly(ethylene glycol) methyl ether acrylate) (PSS-b-PEG) was synthesized as described below. Polystyrene (PS) particles (0.98 μm diameter) were purchased from Microparticles GmbH (Germany). Bovine serum albumin, 2-(N-morpholino)ethanesulfonic (42) Heuberger, R.; Sukhorukov, G.; Voros, J.; Textor, M.; Mohwald, H. Adv. Funct. Mater. 2005, 15, 357. (43) Bl€attler, T. M.; Pasche, S.; Textor, M.; Griesser, H. J. Langmuir 2006, 22, 5760. (44) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 1998, 31, 5559. (45) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58, 379. (46) Boyer, C.; Bulmus, V.; Davis, T. P.; Ladmiral, V.; Liu, J.; Perrier, S. Chem. Rev. 2009, 109, 5402. (47) Barner, L.; Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C. Macromol. Rapid Commun. 2007, 28, 539. (48) Stenzel, M. H. Chem. Commun. 2008, 30, 3486.
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Article Scheme 1. Structures of Poly(allylamine hydrochloride) (PAH), Poly(sodium 4-styrenesulfonate) (PSS), and Poly(sodium 4styrenesulfonate)-b-Poly(poly(ethylene glycol) methyl ether acrylate) (PSS-b-PEG)
acid (MES), and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased from Sigma-Aldrich, while sodium acetate and sodium chloride were obtained from BDH. Silicon wafers (oxidized, monocrystalline) were purchased from Micro Materials and Research Consumables (MMRC) Pty Ltd. (Melbourne, Australia). Quartz crystal microgravimetry with dissipation (QCM-D) sensor electrodes (frequency = 5 mHz, AT-cut) were purchased from Q-Sense Corp. (Q-Sense AB, G€ oteberg, Sweden). Substrates (silicon wafers and QCM electrodes) were cleaned with Piranha solution (concentrated sulfuric acid/hydrogen peroxide (30%), 7:3 (v/v)). Caution! Piranha solution is highly corrosive. Extreme care should be taken when handling Piranha solution and only small quantities should be prepared. The water used in all experiments was purified with a Millipore Rios/Synergy 2-stage purification system and had a resistivity greater than 18 MΩ cm. Synthesis of PSS-b-PEG. The macroRAFT agent, PSSRAFT, was prepared in the presence of a 4-cyanopentanoic acid dithiobenzoate (CPADB) chain transfer agent. A water-soluble thermal initiator, 4,40 -azobis-4-cyanopentanoic acid (ACPA), was used for polymerization, which was conducted in a mixture of ethanol and water at 70 °C. The monomer sodium 4-styrenesulfonate (6 g, 0.029 mol) was dissolved in 20 mL of distilled water. The CPADB RAFT agent (0.1387 g, 0.0005 mol) and the initiator, ACPA (28.6 mg, 0.0001 mol), were dissolved in 10 mL of ethanol. The two solutions were mixed together and degassed on a Schlenk line by three cycles of freeze-pump-thaw, after which they were blanketed with nitrogen. The polymerization was initiated by immersing the glass vial in an oil bath set at 70 °C, and the polymerization was allowed to proceed for 16 h. The polymer was purified by precipitation into acetone, filtering, and drying in a fume hood overnight. Close to 100% conversion was achieved. The Mn determined by NMR was 14 600 g mol-1, which is close to the calculated Mn of 12 400 g mol-1. The PDI determined by aqueous GPC was 1.2. The block copolymer PSS-b-PEG was synthesized by chain extension from PSS-RAFT. ACPA was used for polymerization, which was conducted in water at 70 °C. The monomer PEGMEA (2.2704 g, 0.005 mol), macroRAFT agent PSS-RAFT (1.24 g, 0.0001 mol), and initiator ACPA (5.6 mg, 0.00002 mols) were weighed into a glass vial. Distilled water (13 mL) was added to dissolve all of the components. The mixture was then degassed on a Schlenk line and blanketed with nitrogen after three degassing cycles. The polymerization was carried out by immersing the glass vial in an oil bath set at 70 °C for 60 min. The resulting polymer was purified by dialysis followed by freeze-drying. Conversion of 86.7% was determined by 1H NMR (D2O as solvent). The NMR determined Mn of the second block (PEGMEA) was 9722 DOI: 10.1021/la100430g
Cortez et al. estimated to be 25 600 g mol-1, which is close to the calculated Mn 19 680 g mol-1. The Mn determined by aqueous GPC was 70 340 g mol-1, and the PDI = 1.13, which deviates from the theoretical value due to the relative calibration of the GPC with commercially available linear standards (see Supporting Information, Figure S1). LbL Buildup of PSS-b-PEG and PAH. LbL thin film formation using PSS-b-PEG and PAH was followed by QCMD (Q-Sense AB, G€ oteberg, Sweden). All polyelectrolytes were prepared at 1 mg mL-1 with 0.5 M NaCl, unless stated otherwise. PEI was deposited on the electrode as the first layer (15 min incubation from a solution containing 0.5 M NaCl). Excess polymer was removed by rinsing for 3 min with water. Sequential deposition of PSS-b-PEG and PAH (15 min adsorption followed by washing) was repeated until 21 layers (i.e., 10.5 bilayers) of PSS-b-PEG and PAH were adsorbed with the outer layer being PSS-b-PEG, giving a film structure of [PEI-(PSS-b-PEG/PAH)10.5]. For comparison, PSS was used instead of PSS-b-PEG to produce PSS-terminated films, PEI-(PSS/PAH)10-PSS films.
Fourier Transform Infrared Reflection-Absorption (FTIR-RA) Spectroscopy. PEI-(PSS-b-PEG/PAH)10.5 films were also prepared on gold-coated silicon wafers for FTIRRAS measurements. Measurements were taken using a Varian 7000 FTIR spectrometer with a variable angle reflectance attachment. The incident angle for the measurements was 15°. Films were deposited on highly reflective substrates (silicon wafers coated with chromium (10 nm) and then gold (150 nm)) using an Edwards Auto 306 thermal deposition chamber. Atomic Force Microscopy (AFM). PEI-(PSS-b-PEG/ PAH)10.5 films were also prepared on silicon wafers for AFM measurements. AFM images were taken on air-dried films with an MFP-3D Asylum Research instrument in noncontact mode using silicon cantilevers with a resonance frequency of about 290 kHz (Budget Sensors BSTop300). Image processing (first-order flattening and plane fitting) was carried out with Igor Pro 5.04B software. Advancing Contact Angle Measurements. Samples for contact angle measurements were prepared on hydrophilized glass slides. Films were formed with the terminal layer assembled from a solution containing various ratios of PSS/PSS-b-PEG. The specific ratios investigated were 0:100, 25:75, 50:50, 75:25, and 100:0. Eight measurements were made on each film, and the results averaged to determine the mean contact angle. Protein Adsorption on PSS-b-PEG Multilayers. Protein adsorption measurements were carried out using QCM-D. The frequency change (ΔF) after protein adsorption was measured and converted to mass (m) using the Sauerbrey equation.49 For a 5 MHz quartz crystal, the Sauerbrey equation simplifies to Δm = -[(CΔF)/n], where C = 17.7 ng Hz-1 cm-2 and n is the overtone number (n = 1, 3, 5, 7). The dissipation change corresponding to the frequency change was such that the Sauerbrey equation could be reliably used to calculate the adsorbed mass. Effect of pH. To investigate the effect of the pH of the BSA solution on adsorption, BSA solutions (0.5 mg mL-1) were prepared from acetate buffer (50 mM) at pH 4.0, MES buffer (50 mM) at pH 6.0, or MOPS buffer (50 mM) at pH 7.2. These were introduced to PEI-(PSS/PAH)5-PSS-b-PEG films, with the terminal PSS-b-PEG layers adsorbed from 0.01 M NaCl. All protein solutions were filtered immediately prior to introduction into the QCM chamber. Effect of Ionic Strength. To investigate the effect of ionic strength, the terminal layer of PSS-b-PEG was deposited at various salt concentrations: 0.01, 0.1, 0.5, 1, and 2 M. The frequency change upon PSS-b-PEG deposition was measured. A BSA solution at 0.5 mg mL-1 (pH 6) was then introduced to these PEI-(PSS/PAH)5-PSS-b-PEG films. The frequency change (49) Sauerbrey, G. Z. Phys. 1959, 155, 206.
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upon protein deposition was measured after washing with MES buffer at pH 6.0. Effect of Coadsorption of PSS-b-PEG with PSS. Blends of PSS-b-PEG and PSS (giving a total polymer concentration of 1 mg mL-1) were prepared at the following ratios (PSS-b-PEG: PSS): 0:100, 25:75, 50:50, 75:25, and 100:0. The blended polymers were deposited on a PAH-terminated film [PEI-(PSS/PAH)5(PSS-PSS-b-PEG blend)], and BSA at 0.5 mg mL-1 (pH 7.2 in MOPS buffer) was then introduced.
Effect of PSS-b-PEG Localization within Multilayer Films. BSA was introduced to films terminating with PSS-bPEG [PEI-(PSS/PAH)5-PSS-b-PEG] and films terminated with one to two additional bilayers of PAH/PSS on top of PSS-b-PEG, that is, PEI-(PSS/PAH)4-PSS-b-PEG-(PAH/PSS) or PEI-(PSS/ PAH)3-PSS-b-PEG-(PAH/PSS)2, respectively. The PSS-b-PEG layer was adsorbed from 0.5 M NaCl. Adsorption of BSA was from MES buffer (50 mM) at pH 6.0. Multilayer Formation on Colloidal Particles. The LbL deposition of PAH and PSS-b-PEG on particles to produce core-shell particles was performed using 0.98 μm diameter polystyrene particles as templates. In the first adsorption step, 500 μL of PAH was added to 200 μL of a 0.5 wt % PS particle suspension in water. Adsorption was carried out for 20 min followed by centrifugation (10 000g, 6 min). The supernatant was removed, and the particles were washed by redispersion in 500 μL of water, followed by centrifugation. The washing step was repeated a further two times before redispersing in 500 μL of water. An equal volume of PSS-b-PEG was then allowed to adsorb for 20 min and washed as described above. The process was repeated with alternating layers of PAH and PSS-b-PEG until eight layers of polyelectrolyte were deposited with the outermost layer being PSS-b-PEG [(PAH/PSS-b-PEG)4]. For comparison, PSS was used instead of PSS-b-PEG to form eight layers of PAH and PSS on a colloidal template [(PAH/PSS)4], as well as a film comprising (PAH/PSS)3(PAH/PSS-b-PEG), that is, a PAH/PSS multilayer with only the outer layer being PSS-bPEG. Microelectrophoresis. Microelectrophoresis was employed to follow the buildup of the multilayers on colloidal particles. The ζ-potential of the particles (1 in 1000 dilution in water) after each adsorption step was measured using a ZetaSizer 2000 instrument (Malvern Instruments). Binding of Particles to Cells. LIM1215 human colorectal cancer cells50 were grown to subconfluency in RPMI 1640 medium supplemented with additives (10.8 μg mL-1 R-thioglycerol, 0.025 U mL-1 insulin, 1 μg mL-1 hydrocortisone, 60 μg mL-1 penicillin, and 12.6 μg mL-1 streptomycin) and 10% heatinactivated fetal calf serum at 37 °C in a 5% CO2 humidified atmosphere. Cells were washed with phosphate buffered saline (PBS) before being detached from the surface with 10 mM EDTA in PBS at 37 °C for 10 min. Cells were mixed by gentle pipetting to produce a single cell suspension. They were washed by centrifugation (400g for 3 min) and resuspended in PBS at a concentration of 106 cells mL-1. Fluorescently labeled PSS-b-PEG-terminated particles [(PAH/PSS)2(PAH-FITC/PSS)(PAH/PSS-b-PEG)] were incubated with 106 cells at the following concentrations: 100, 200, and 400 particles cell-1. For comparison, PSS-terminated particles (100 particles cell-1) were also incubated with cells. After 1 h incubation at 4 °C, excess (unbound) particles were removed by centrifugation (400g, 3 min) and the cell pellet was resuspended in PBS containing 5 μg mL-1 propidium iodide (PI). The degree of particle binding to cells was analyzed by flow cytometry (FACSCalibur, Becton Dickinson). Forty thousand events were acquired. Flow cytometry data analysis was performed with Summit v3.1 software (Cytomation, Inc., CO) taking into account live cells only (PI negative). (50) Whitehead, R. H.; Macrae, F. A.; St. John, D. J.; Ma, J. J. Natl. Cancer Inst. 1985, 74, 759.
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Figure 1. Buildup of PSS-b-PEG/PAH and PSS/PAH multilayer films, as measured by QCM-D. The polyelectrolyte concentrations were 1 mg mL-1 in an aqueous solution also containing 0.5 M NaCl.
Results and Discussion Formation of PSS-b-PEG/PAH Multilayers. The growth of PSS-b-PEG/PAH multilayer films was followed by QCM with dissipation capability, QCM-D (see Figure 1). QCM-D allows real-time analysis of the LbL deposition of each polyelectrolyte. It measures the frequency of an oscillating piezoelectric crystal, which decreases as the mass adsorbed is increased. The change in frequency can therefore be converted to the amount of mass adsorbed using the Sauerbrey equation, assuming a rigid layer with low dissipation change.49 In all of the experiments described, low dissipation levels were observed during deposition, which validates the use of the Sauerbrey equation for the estimation of mass adsorbed. The QCM-D data in Figure 1 show the linear buildup of PSS-b-PEG/PAH, which is similar to the buildup of the well-studied PSS/PAH system.51 The mass adsorbed is greater for PSS-b-PEG/PAH than for PSS/PAH, with approximately 50% additional mass after 10 bilayers. This is predominantly due to the water associated with PEG within the layers. An AFM image of a PSS-b-PEG/PAH multilayer film showed a morphology similar to that of PSS/PAH films (see Supporting Information, Figure S2). FTIR spectra of PSS/PAH and PSS-b-PEG/PAH films are shown in Figure 2. The bulk of the PEG peaks are weak and in the range of 800-1600 cm-1. They overlap peaks from PSS and PAH, causing shape changes in the 1000-1300 and 13001400 cm-1 ranges. An obvious carbonyl peak arising from the PEG acrylate ester is evident at 1700-1800 cm-1, confirming the presence of PSS-b-PEG in the film. Protein Adsorption on Films Terminating with PSS-bPEG. The fouling behavior of films terminating with PSS-b-PEG was investigated by the adsorption of BSA. Adsorption of the protein, resuspended at different pH values, on PEI-(PSS/PAH)5PSS-b-PEG films was monitored by QCM-D. Figure 3a shows a decrease in the amount of BSA adsorbed as the pH of the BSA solution increased; this was on films in which the PSS-b-PEG is adsorbed from solutions incorporating 0.01 M NaCl. High protein adsorption was observed at pH 4, which is close to the isoelectric point of BSA (4.7).52 Previous adsorption studies with BSA and the structurally similar human serum albumin (HSA) have also reported the highest binding at around the isoelectric (51) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422. (52) Hu, J.; Li, S.; Liu, B. Biochem. Eng. J. 2005, 23, 259.
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Figure 2. FTIR-RAS of (a) (PSS/PAH)10.5 and (b) (PSS-b-PEG/ PAH)10.5 multilayer films assembled from polyelectrolytes at a concentration of 1 mg mL-1 with 0.5 M NaCl.
point of the protein.53,54 The protein would be less charged at pH 4 (slightly positive) and therefore could potentially be attracted to PSS-b-PEG, which despite the presence of the PEG moieties, imparts a net negative charge to a surface due to the SS groups (see later). As the pH of the protein solution is increased, BSA becomes more negatively charged and would hence be repelled by the PSS-b-PEG layer. Electrostatic interaction between the protein and the surface therefore plays a role in protein binding. The effect of the ionic strength of PSS-b-PEG assembly on subsequent BSA adsorption is shown in Figure 3b. PSS-b-PEG assembled at the higher ionic strength (0.5 M) attracted more protein than PSS-b-PEG assembled at 0.01 M, although the trend of BSA adsorption as a function of pH is similar at both ionic strengths. Even at high ionic assembly conditions, BSA binding is minimized by the presence of a terminating PSS-b-PEG layer. For example, at 0. 5 M ionic strength, the BSA surface coverage (at pH 6) is reduced to 1.25 mg m-2 (when the terminal layer is PSS-bPEG ) from about 7.5 mg m-2 (when the terminal layer is PSS). The effect of ionic strength of PSS-b-PEG assembly on BSA adsorption was investigated further using a wider range of ionic strengths. Figure 4a clearly shows an increase in the amount of BSA adsorbed as the ionic strength of the PSS-b-PEG adsorption solution is increased. This correlates with a seemingly smaller mass of PSS-b-PEG adsorbed on the final PAH layer at higher ionic strengths (Figure 4b). Increased salt concentration is (53) Joon, T. O.; Kim, J. H. Enzyme Microb. Technol. 2000, 27, 356. (54) Gergely, C.; Bahi, S.; Szalontai, B.; Flores, H.; Schaaf, P.; Voegel, J.-C.; Cuisinier, F. J. G. Langmuir 2004, 20, 5575.
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believed to result in the reduction in the hydration volume of PEG.55 This loss of water is indicated by a reduction in mass increment as the ionic concentration of the PSS-b-PEG solution increases (Figure 4b). As a result of the reduced hydration of the PSS-b-PEG layer, protein adsorption becomes entropically and enthalpically favorable, leading to a higher amount of protein adsorbed on the film.38,39 Blended PSS/PSS-b-PEG Surfaces. The blending of likecharged polyelectrolytes (e.g., PSS with poly(acrylic acid)20-22 and PSS with DNA56) has previously been shown to result in multilayer films with improved and unique properties, including controlled responsiveness. The effect of blending PSS with PSS-bPEG in the final layer of the film was investigated in terms of the mass of the final layer adsorbed and the degree of fouling as the proportion of PSS-b-PEG in the blend is varied. Figure 5a shows that as the concentration of PSS-b-PEG in the PSS-b-PEG/PSS adsorption solution is increased, the mass of the blend deposited as the final layer increases. With each increment of 25% in PSS-bPEG concentration, the frequency measured increases by ∼11 ( 3 Hz. The water associated with PSS-b-PEG is likely to represent a significant fraction of this mass, as the QCM-D measurement is performed in aqueous conditions. Further, with increasing concentration of PSS-b-PEG in the blend solution, the amount of BSA that adsorbs on the final film decreases (see Figure 5b). Protein resistance is improved in the blended films by ∼32% ( 17% for each 25% increment in PSS-b-PEG concentration. This decrease in fouling is consistent with a higher percentage of PEG being available on the surface as the amount of PSS-b-PEG in the blend solution is increased. The observed relationship is close to a linear correlation between the PSS-b-PEG in the adsorption solution and the amount of protein adsorbed on the final film. AFM data showed little morphological variation of the blended films (regardless of composition) and no obvious variation in the morphology of the film before and after protein adsorption (data not shown). This suggests that PSS-b-PEG behaves like a strong polyelectrolyte, as far as the film morphology is concerned. In addition, contact angle measurements showed little variance in the advancing contact angles for the different films prepared from the blend solutions (0, 25, 50, 75, and 100% PSS-b-PEG in the blend solution), which were approximately 33° (data not shown). The effect of the location of the PSS-b-PEG layer within the film, or rather its distance from the surface (top), on the fouling behavior of the film was also investigated. This was studied to determine whether protein resistance is also exhibited from within the film and not only when the film terminates with a PSS-b-PEG layer, that is, whether some of the PEG chains effectively “bloom” at the surface of the multilayer. The amount of protein bound to the films terminating with PSS-b-PEG was compared to the amount bound on films with one or two PSS/PAH bilayers adsorbed on the underlying PSS-b-PEG layer (see Scheme 2). Figure 6 shows that protein resistance is reduced by approximately 50% as each protective PSS/PAH bilayer is deposited. Effectively, with two covering bilayers, the PEG effect is substantially diminished as the film becomes at least 4 times more fouling than a film terminating with PSS-b-PEG. Nevertheless, the results suggest that some PEG chains are able to penetrate through to the surface and exhibit some protein resistance even after additional bilayers are adsorbed. Even with two protecting bilayers, the PSS-b-PEG-containing films still display lower (55) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 2043. (56) Quinn, J. F.; Yeo, J. C. C.; Caruso, F. Macromolecules 2004, 37, 6537.
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Figure 3. BSA adsorption on PEI/(PSS/PAH)5/PSS-b-PEG films as a function of the pH of the BSA solution. The final PSS-b-PEG layer was adsorbed from (a) 0.01 M NaCl or (b) 0.5 M NaCl. The BSA binding data for PSS-b-PEG adsorption from 0.01 M NaCl are also shown in (b).
Figure 4. (a) Effect of the ionic strength of PSS-b-PEG assembly on BSA adsorption. The film composition was PEI/(PSS/PAH)5/PSS-bPEG. (b) QCM frequency increment as a result of PSS-b-PEG deposition on PEI/(PSS/PAH)5 films from different ionic strength solutions.
Figure 5. (a) Layer increment as a result of the deposition of PSS-b-PEG/PSS blend final layers at various polyelectrolyte compositions. The final layer was adsorbed at 0.01 M NaCl. (b) Adsorption of BSA (pH 7.2) onto blended PSS-b-PEG/PSS surfaces. The PSS-b-PEG/PSS blend layers were deposited on PEI/(PSS/PAH)5 films.
protein coverage (4.5 mg m-2) compared with a PSS/PAH film under the same conditions (7.5 mg m-2). Formation of PSS-PEG/PAH Multilayers on Colloids. It was shown above that PSS-b-PEG/PAH films can be successfully prepared on planar surfaces. This was extended to colloidal supports by the sequential deposition of PSS-b-PEG and PAH on spherical (1 μm diameter) polystyrene particles. Figure 7 shows microelectrophoresis data on the reversal of charge of the particles as PSS-b-PEG and PAH are sequentially deposited. A gradual decrease in the positive charge is observed as PAH is Langmuir 2010, 26(12), 9720–9727
deposited on PSS-b-PEG layers, which was not observed with PSS/PAH particles. The cell binding of particles terminating with PSS-b-PEG (as opposed to PSS) was investigated using flow cytometry. LIM1215 colorectal cancer cells were incubated with an increasing number of fluorescently labeled PSS-b-PEG-terminating particles for 1 h at 4 °C. After removal of excess, unbound particles by centrifugation, the cells were analyzed by flow cytometry to quantify the number of cells with bound particles. Although particles terminated with PSS alone showed limited interaction with the DOI: 10.1021/la100430g
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Scheme 2. Location of PSS-b-PEG within the Film (PSSPEG = PSS-b-PEG)
inherently negatively charged cells, the interaction was further reduced with the presence of PSS-b-PEG on the surface. Figure 8 shows the reduced cell binding of particles terminating with PSSb-PEG compared with PSS, when incubated with the same amount of particles (∼100 particles cell-1) (columns 1 and 2). This represents approximately an 85% reduction in fouling or a 6.5-fold increase in cell binding resistance and is in close agreement with the degree of protein resistance exhibited by PSS-bPEG-terminated planar films (4-5-fold enhanced resistance). Increasing the number of PSS-b-PEG particles incubated with the cells increased cell binding, which is consistent with our previous work.57 Interestingly, even at 400 particles cell-1, the PSS-b-PEG particles still showed lower cell binding than PSS particles incubated at 100 particles cell-1. Similar results were obtained with 500 nm particles (see Supporting Information, Figure S3). This demonstrates the lower affinity of the PSS-bPEG surface to bind to biological cells, which is important for surfaces requiring limited interaction with biointerfaces, including proteins and cells. Overall, surface modification of planar and colloidal surfaces with PSS-b-PEG resulted in reduced protein and cell binding. In some cases, the protein binding was as low as 14 ng cm-2. For comparison, serum protein binding to PEG grafted on poly-Llysine (PLL-g-PEG) can be reduced to
