Poly(oligo(ethylene glycol)acrylamide) Brushes by Surface Initiated

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Langmuir 2009, 25, 3794-3801

Poly(oligo(ethylene glycol)acrylamide) Brushes by Surface Initiated Polymerization: Effect of Macromonomer Chain Length on Brush Growth and Protein Adsorption from Blood Plasma Jayachandran N. Kizhakkedathu,*,† Johan Janzen,† Yevgeniya Le,† Rajesh K. Kainthan,† and Donald E. Brooks*,†,‡ Center for Blood Research and Department of Pathology and Laboratory Medicine, and Department of Chemistry, 2350 Health Sciences Mall, UniVersity of British Columbia, VancouVer, Britsh Columbia V6T 1Z3, Canada ReceiVed NoVember 6, 2008. ReVised Manuscript ReceiVed December 19, 2008 Three hydrolytically stable polyethyleneglycol (PEG)-based N-substituted acrylamide macromonomers, methoxypolyethyleneglycol (350) acrylamide (MPEG350Am) methoxypolyethyleneglycol (750) acrylamide(MPEG750Am) and methoxypolyethyleneglycol (2000)acrylamide (MPEG2000Am) with increasing PEG chain length were synthesized. Surface-initiated aqueous atom transfer radical polymerization (ATRP) using CuCl/1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA) catalyst was utilized to generate dense polymer brushes from these monomers via an ester linker group on the surface of model polystyrene (PS) particles. The molecular weight, hydrodynamic thickness, and graft densities of the grafted polymer layers were controlled by changing the reaction parameters of monomer concentration, addition of Cu(II)Cl2, and sodium chloride. The graft densities of surface-grafted brushes decreased with increasing PEG macromonomer chain length, 350 > 750 . 2000, under similar experimental conditions. The molecular weight of grafts increased with increase in monomer concentration, and only selected conditions produced narrow distributed polymer chains. The molecular weight of grafted polymer chains differs significantly to those formed in solution. The hydrodynamic thicknesses of the grafted polymer layers were fitted to the Daoud and Cotton model (DCM) for brush height on spherical surfaces. The results show that the size of the pendent groups on the polymer chains has a profound effect on the hydrodynamic thickness of the brush for a given degree of polymerization. The new PEG-based surfaces show good protection against nonspecific protein adsorption from blood plasma compared to the bare surface. Protein adsorption decreased with increasing surface density of grafted polymer chains. Poly(MPEG750Am) brushes were more effective in preventing protein adsorption than poly(MPEG350Am) even at low graft densities, presumably due to the increase in PEG content in the grafted layer.

Introduction Initial contact between blood and biomaterials involves the adsorption of plasma proteins and frequently the activation of plasma enzyme cascades such as the complement, coagulation, and kallikrein systems.1-3 Understanding the nature of protein adsorption and other activation events upon contact with a welldefined surface will enable the development of improved surfaces and materials for blood-contacting applications. Recent theoretical and experimental developments show that high density hydrophilic polymer brushes can resist nonspecific protein adsorption due to entropic repulsion of macromolecules.4-14 * Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected] (J.N.K.);[email protected] (D.E.B.). Phone: 604 822 7085 (D.E.B.). Fax: 604 822 7742 (D.E.B.). † Center for Blood Research and Department of Pathology and Laboratory Medicine. ‡ Department of Chemistry.

(1) Ratner, B. D.; Bryant, S. J. Annu. ReV. Biomed. Eng. 2004, 6, 41. (2) Gorbet, M. B.; Sefton, M. V. Biomaterials 2004, 25, 5681–5703. (3) Shard, A. G.; Tomlins, P. E. Regener. Med. 2006, 1, 789–800. (4) Halperin, A. Langmuir 1999, 15, 2525. (5) Halperin, A.; Fragneto, G.; Schollier, A.; Sferrazza, M. Langmuir 2007, 23, 10603–10617. (6) Steels, B. M.; Koska, J.; Haynes, C. A. J. Chromatogr. B 2000, 743, 41. (7) Satulovsky, J.; Carignano, M. A.; Szleifer, I. Pro. Nat. Acad. Sci. 2000, 97, 9037–9041. (8) Kingshott, P.; Griesser, H. J. Cur. Opin. Solid State Mater. Sci. 1999, 4, 403–412. (9) Leckband, D.; Sheth, S.; Halperin, A. J. Biomater. Sci., Polym. Ed. 1999, 10, 1125–1147. (10) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6, 2427–2448. (11) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Biointerphases 2006, 1, 50.

There is considerable interest in the development of polymer grafted surfaces with minimal nonspecific protein adsorption to surfaces of biomedical materials. For many years, surface coatings based on poly(ethylene glycol) (PEG), a nontoxic and nonimmunogenic polymer, have been used with a variety of biomedical surfaces to reach this goal.8-11,13 Surface-initiated polymerization (SIP) has proven to be a facile method for producing dense polymer layers on surfaces,15,16 but this approach is so far not successful in developing PEG brushes; the grafting of preformed PEG is the most common method used. SIP of PEG macromomers (mostly with acrylate and methacrylate monomers) is another approach used to produce PEG-based surface coatings.17-25 Unlike the grafting of end-functionalized PEG chains onto a (12) Fukuda, T.; Tsujii, Y.; Ohno, K. Macromol. Eng. 2007, 2, 1137–1178. (13) Boyes, S. G.; Granville, A. M.; Baum, M.; Akgun, B.; Mirous, B. K.; Brittain, W. J. Surf. Sci. 2004, 570, 1–12. (14) Singh, N.; Cui, X.; Boland, T.; Husson, S. M. Biomaterials 2007, 28, 763–771. (15) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Biomaterials 2005, 26, 5927–5933. (16) Feng, W.; Brash, J. L.; Zhu, S. Biomaterials 2006, 27, 847–855. (17) Yu, W. H.; Kang, E. T.; Neoh, K. G. Langmuir 2005, 21, 450–456. (18) Ma, H.; Hyun, J.; Stiller, P.; Chilkoti, A. AdV. Mater. 2004, 16, 338–341. (19) Lutz, J. J. Poly. Sci., Part A: Polym. Chem. 2008, 46, 3459–3470. (20) Fu, L.; Chen, X.; He, J.; Xiong, C.; Ma, H. Langmuir 2008, 24, 6100– 6106. (21) Tugulu, S.; Klok, H. Biomacromolecules 2008, 9, 906–912. (22) Fan, X.; Lin, L.; Messersmith, P. B. Biomacromolecules 2006, 7, 2443– 2448. (23) Ma, H.; Wells, M.; Beebe, T. P., Jr.; Chilkoti, A. AdV. Funt. Mater. 2006, 16, 640–648. (24) Brown, A. A.; Khan, N. S.; Steinbock, L; Huck, W. T. S. Eur. Poly. J. 2005, 41, 1757–1765. (25) Iguerb, O.; Bertrand, P. Surf. Interface Anal. 2008, 40, 386–390.

10.1021/la803690q CCC: $40.75  2009 American Chemical Society Published on Web 02/13/2009

Polymer Brushes by Surface-Initiated Polymerization

surface, PEG macromonomers produce comblike polymer with PEG side chains and a hydrophobic backbone (-CH2-CH-) which is initiated from the surface. These surface structures may have properties that differ from linear PEG layers. However, the important advantages of this method is that one can potentially produce high density brushes and PEG content on the surface can be adjusted by changing the length of the PEG side chain in the macromonomers.18,22 Although SIP is now a well-developed technique for smallmolecular-weight monomers,12,13 there is only limited information available for macromonomers.17-25 SIP in principle allows the synthesis of denser polymer surface layers than can be produced by grafting end-functionalized linear chains.15,16 However, one can anticipate that the accessibility of polymerizing groups on large macromonomers to surface initiator groups could be inhibited, preventing the formation of a densely grafted polymer brushes from such monomers. Also, it will be difficult to analyze the absolute molecular weight and density of the grafted chains by cleaving it from the surface when the grafted polymer itself contains hydrolyzable groups as described in the case of PEG acrylates and methacrylates.17-25 To overcome this difficulty and to study SIP in PEG macromonomer systems, we have designed and synthesized three new hydrolytically stable acrylamide-functionalized PEG macromonomers with different side chain length and carried out SIP to produce well-characterized poly(MPEGnAm) grafted surfaces. The polymer chains are attached to the surface via an ester linker group to facilitate the absolute characterization of both surface-grafted chains and those formed in solution. We have established a correlation between the length of the macromonomer and its ability to form surfacegrafted chains. Furthermore, we have shown that the amounts of adsorbed proteins from blood plasma on newly developed PEG surfaces depended both on the chain density and PEG content of the grafted chains and the protein species.

Experimental Section Materials. All the reagents and chemicals were purchased from Aldrich Chemicals. Narrowly dispersed polystyrene seed latex (PS seed latex) was synthesized by surfactant free emulsion polymerization of styrene and characterized by reported procedures.26,27 Instrumentation. Gel permeation chromatography (GPC) for molecular weight determinations was carried out using multiangle laser light scattering quantitation (MALLS; DAWN-EOS, Wyatt Tech. Corp.) and refractive index (Optilab DSP, Wyatt Tech. Corp.) detection both at a wavelength of 690 nm. Aqueous 0.1 N NaNO3 solution was used as the mobile phase at a flow rate of 0.8 mL/min. Aliquots of 200 µL of the polymer solution were injected through Ultrahydrogel columns at 22 °C connected in series, guard column, Ultrahydrogel Linear with bead size 6-13 µm, elution range 103-5 × 106 Da and Ultrahydrogel 120 with bead size 6 µm, elution range 150-5 × 103 Da from Waters Corp. The refractive index (RI) increments, (dn/dc), values for the PEG-based polymers were determined in 0.1 N NaNO3 at λ ) 690 nm and were used for the molecular weight calculations. Nuclear magnetic resonance (NMR) spectra were collected on a Brucker Avance 300 spectrometer using the solvent peak as reference. Particle size (i.e., the hydrodynamic diameter distribution of particle suspensions) was measured by dynamic light scattering (DLS) on a Beckman Coulter N4 Plus particle size analyzer. Dispersed particles were thermally equilibrated to 20 °C for measurements. All analysis was performed using software supplied by the manufacturer. Synthesis of PEG Macromonomers. Synthesis of MPEG350-Acrylamide. Methoxy PEG350-acrylamide was synthesized by the amidation (26) Jayachandran, K. N.; Takas-Cox, A.; Brooks, D. E. Macromolecules 2002, 35, 4247. (27) Kizhakkedathu, J N.; Brooks, D E. Macromolecules 2003, 36, 591–598.

Langmuir, Vol. 25, No. 6, 2009 3795 of MPEG350 amine with acryloyl chloride. MPEG350 amine was synthesized following the procedure reported by Kang et al.28 Briefly, MPEG-OH was first converted to MPEG-mesylate and then to MPEG-azide followed by reduction to amine with Zn/HCl. Purity of all the compounds was checked by 1H NMR and thin layer chromatography (TLC). MPEG350-acrylamide was synthesized from MPEG350-amine (10 g, 0.0285 mol) by reacting with acryloyl chloride (3.2 g, 0.035 moles) in the presence of triethyl amine (3.6 g, 0.035 mol) and dichloromethane (DCM) (100 mL). The mixture was stirred at 0 °C for 6 h. After the reaction the DCM was evaporated and the residue was dissolved in dilute NaHCO3 (0.1 N). The product was extracted with DCM and the organic layer evaporated. The final product was purified by column chromatography using DCM/methanol as eluent. 1 H NMR (DMSO-d6) 3.20 (s, CH3O-(CH2-CH2-O-)n-, 3.25-3.35 (t, -CH2-CH2-NH-CO-CHdCH2), 3.4-3.8 (m, CH3O-(CH2-CH2-O-)n-CH2-CH2-), 5.5-5.55 (d, -NH-COCHdCH2), 6.05-6.15 (q, -NH-CO-CH)CH2), 6.20-6.25 (d, -NH-CO-CHdCH2), 6.45-6.80 ppm (broad peak, -NH-CO-CHdCH2). MPEG750-acrylamide and MPEG2000-acrylamide were synthesized by similar methods using MPEG750-amine and MPEG2000-amine, respectively, and had similar 1H NMR spectrum except for differences in the peak intensities. Synthesis of ATRP Initiator-Functionalized Surface and Surface-Initiated Aqueous ATRP of MPEG Macromonomers. 2-(Methyl-2′-chloropropionato) ethyl acrylate (HEA-Cl) and ATRP initiator incorporated PS latex particles were synthesized and characterized as described in our earlier report.26,27 Briefly, an aqueous suspension of PS seed latex particles (3.33 wt %, 265 g) was heated to 70 °C with stirring at 350 rpm, degassed, and purged with argon. Styrene (0.025 mol) and HEA-Cl (0.0078 mol) were added successively to the suspension 10 min apart and shell polymerization initiated with potassium persulfate (0.37 mmol in 20 mL of water, degassed) 5 min later. The reaction was continued for 6 h, and then the latex was cleaned by dialysis against water for 1 week with frequent changes in water followed by five cycles of centrifugation and resuspension. The solid content was determined by freeze-drying. All ATRP reactions were performed in a glovebox under argon atmosphere. A suspension of ATRP initiator-incorporated PS latex in water (22 g, 3 wt % solids) was degassed for 2.5 h by continuous vacuum and argon cycles and transferred to the glovebox. Brij-35 (0.035 g, 0.16 wt %) was added to the suspension and stirred for 5 min. For a typical reaction, CuCl (13 µmol), CuCl2 (3 µmol), HMTETA (33 µmol), Cu (19 µmol), and MPEG350Am (0.002 mol) were used; 3.5 g of Brij-35 stabilized latex was added with stirring, and the reaction was continued for 24 h. Different sets of experiments were performed by varying the monomer concentration, addition of Cu(II)Cl2, sodium chloride, and employing different monomers. Purification of the grafted latex was done by repeated centrifugation and resuspension in filtered water (Milli-Q) following our earlier report.26 Grafted chains were cleaved from the surface by saponification and analyzed for molecular weight, molecular weight distribution, mass per unit area of surface, and radius of gyration, Rg. A calibrated RI detector was used for calculating the mass of cleaved chains from the surface. Hydrodynamic thicknesses (HT) of polymer-grafted layers are calculated as the differences in the sizes of the bare and polymer-grafted lattices measured by DLS. Plasma Protein Adsorption to Polymer-Grafted Surface. Plasma was isolated from fresh, EDTA-anticoagulated human blood from a single donor by centrifugation (IEC Centra-8R) at room temperature and 3000 rpm for 15 min. Polymer-grafted latex was washed with 50 mM EDTA several times (minimum four washes) and with water (three washes) to remove any adhered copper species before incubating with plasma. A 400 µL aliquot of plasma was added to ∼400 µL of water-latex suspension with a total latex surface area of 500 cm2. Latex-plasma suspensions were equilibrated for 1 h at room temperature. Plasma protein adsorption determined (28) Kang, Y.; Seo, Y.; Lee, C. Bull. Kor. Chem. Soc. 2000, 21, 241.

3796 Langmuir, Vol. 25, No. 6, 2009 Scheme 1. MPEGnAm Monomer Synthesis

by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was found to be unchanged between 1 and 24 h incubation. PS latex particles with adsorbed protein was separated from solution protein by repeated centrifugation at 13 000 rpm in an Eppendorf microcentrifuge, removal of a portion of the supernatant solution, and resuspension with an equal volume of protein-free buffer. After being washed (typically six cycles), latex pellets were transferred to a clean Eppendorf tube and subjected to a desorption “wash” in 50 µL of PAGE sample buffer (SB) concentrate (4-fold SB concentrate: 2% SDS solution, 0.0625 M Tris buffer, 10% glycerol, 0.01% bromophenol blue, and 150 µL of water). These final suspensions were thoroughly mixed and brought to boiling for 1 min. After cooling, the suspensions were centrifuged for 3 min and the supernatants were transferred into new, clean Eppendorf tubes. Desorbed protein and control solutions were loaded onto 4% 1-mmthick SDS-PAGE gels. The following controls were present on each gel: 1xSB for background OD measurements; size marker (SM) for molecular weight estimation (BioRad prestained broad-range Precision Protein Standards 250, 150, 100, 75, 50, 37, and 25 kDa); human plasma 1000 times diluted in 1xSB and protein desorbed from ungrafted shell latex. Gels were silver stained following Morrissey29 modified as follows. Fixed in 12% trichloroacetic acid for 30 min, development stopped with 1 mL of acetic acid in 100 mL of water, background destained in 10% Rapidfix(Kodak), and stored in water at 4 °C for subsequent evaluation. It was estimated from OD calibration curves that 4 min and 30 s was the optimal time for silver stain development. Gels were photographed using a JVC color CCD camera fitted with a Hoya neutral filter for transmission illumination provided by an X-ray light box. Camera software white balance control of the red, blue, and green (RGB) signals was set so that a nominal 1 optical density (OD) neutral density (ND) filter (5 × 5 cm2, Newport) gave equal 8-bit R, G, and B values. Line profiles (40 pixels wide) of the gel lanes were obtained using image analysis software (Northern Eclipse 6.0, Empix Imaging, Mississauga, Canada). The sample buffer lane was used for background correction. Lateral scans of ND filters showed that illumination by the light box was uniform on the scale of the gel holder and provided a calibration to convert RGB bit values to OD. The OD value of each ND filter was determined by measuring the average OD of the filter over 535-735 nm using a holmium oxide calibrated spectrophotometer (HP 8450A UV-vis spectrophotometer, Hewlett-Packard, Palo Alto, CA). Radio-labeled human serum albumin (HSA) was used to calibrate the R, G, and B OD values from silver staining in terms of protein amount.43

Results and Discussion Macromonomers, MPEG350 acrylamide, MPEG750 acrylamide, and MPEG2000 acrylamide, were synthesized from the corresponding amine by an amidation reaction with acryloyl chloride (Scheme 1). The macromonomers have ∼8, 17, or 45 ethylene (29) Morrissey, J. H. Anal. Biochem. 1981, 117, 307.

Kizhakkedathu et al. Scheme 2. Representation of Different Brushes (a) Small Monomer Linear Polymer Brush (b) Poly(MPEGnAm) Brushes with Different Side Chain Length

oxide units attached to an acrylamide group and are highly soluble in water. Upon polymerization these macromonomers, MPEG350 acrylamide, MPEG750 acrylamide, and MPEG2000 acrylamide, produce PEG-based polymers with ∼ 84, 92, and 97 wt % PEG content in the polymer, respectively, and are expected to form comblike polymer on the surface (Scheme 2). Since these monomers have N-substituted amide groups, they are expected to produce hydrolytically stable polymers under the conditions studied. It is known that N-substituted acrylamides are stable under a wide range of pH conditions and stable to base hydrolysis under our experimental conditions.30 So we have used these monomers to produce polymers that are stable to saponification which is necessary since we have used one such protocol for the characterization of surface-grafted polymers in the current work. We have used latex particles as a model surface to investigate the experimental parameters that control growth of PEG-based polymers from surfaces. These particles have proven to provide a good model system for study of the mechanism of surfaceinitiated polymerization due to the large surface area, narrow size distribution, and the synthetic control over surface properties offered by surfactant-free latex synthesis.26,27 ATRP initiators are attached to the latex surface through an ester group and can be cleaved by ester hydrolysis for the characterization of the initiator surface, as well as for polymer analysis once the polymer is grafted to the surface. Thus, the molecular weight distribution and total mass of the chains grafted on the surface can be determined without ambiguity. This enables us to correlate the effect of various reaction parameters on polymer chain growth from a surface. Initiator is incorporated on the surface of seed PS particles by shell growth polymerization using styrene and HEA-Cl.26 The surface concentrations of initiator accessible from the aqueous phase was measured by conductometric titration (1.56 × 10-6 mol/m2) after saponification.26 The hydrodynamic diameter of the latex was 650 ( 10 nm as determined from DLS. Surface-Initiated Aqueous ATRP: Effect of Monomer Concentration and Molecular Weight of Macromonomers. In this set of experiments we varied the concentration of monomers from 0.036 to 0.392 mol/L for the three MPEG acrylamide macromonomers; the results are given in Table 1 and Figures 1 and 2. The values given are the average of two independent experiments from the same initiator-functionalized latex surface. The molecular weight of the grafted polymer increased with increase in monomer concentration, except in the few cases where the graft density was very low. These results are similar to those found for other hydrophilic monomers.12,14,17,18,26 The number average molecular weight (Mn) of the polymer increased from 54 800 at 0.036 mol/L to 213 000 at 0.392 mol/L for MPEG350Am (30) Barton, D. ComprehensiVe Org. Chem. 1979, 2, 1004.

Polymer Brushes by Surface-Initiated Polymerization

Langmuir, Vol. 25, No. 6, 2009 3797

Table 1. Characteristics of Poly(MPEGnAm) Brushes and Polymers Formed in Solution for Different Macromonomers solution polymerb

surface polymer monomer concentration (mol/L) MPEG350Am

MPEG750Am

MPEG2000Am

a

0.036 0.124 0.181 0.276 0.392 0.091 0.127 0.181 0.272 0.362 0.051 0.109 0.145

Mn

Mw/Mn

HTa (nm)

54800 ( 5200 95700 ( 4300 96000 ( 7000 188000 ( 7300 213000 ( 9800 ND 227000 ( 12500 210000 ( 9800 501000 ( 15500 602000 ( 8500 149000

1.34 ( 0.02 1.90 ( 0.08 2.50 ( 0.1 3.0 ( 0.08 3.3 ( 0.12 ND 1.27 ( 0.03 1.29 ( 0.05 1.76 ( 0.08 2.22 ( 0.1 NDc NDc NDc

29 ( 3 174 ( 10 345 ( 25 551 ( 21 735 ( 35 26 ( 2 55 ( 5 117 ( 4 233 ( 20 270 ( 10 10 ( 2 ND 24 ( 4

82400

HT ) hydrodynamic thickness of the polymer layer.

b

Rg (nm)

Mn

Mw/Mn

14.6 17.7 29.7 43.6

168 800 372 400 650 600 1 086 000 1 108 000

1.73 3.44 2.76 2.02 1.82

17.2 11.4 24.0 30.0

From one of the experiments. c Very little polymer grafted; ND ) not determined.

Figure 1. Effect of monomer concentration on the molecular weight and molecular weight distribution of grafted polymer chains. Experimental conditions: 3.5 g of 3% by weight suspension of latex, 1,1,4,7,10,10hexamethyl triethylene tetramine (HMTETA 30 µmol), CuCl (13 µmol) CuCl2/HMTETA (3 µmol), copper powder (19 µmol) at 22 °C. Surface initiator concentration: 1.56 × 10-6 mol/m2.

Figure 2. Effect of macromonomer chain length and monomer concentration on the graft density of polymer chains on the surface. Experimental conditions are the same as given in Figure 1.

and from 210 000 at 0.181 mol/L to 602 000 at 0.362 for MPEG750Am. The amount of MPEG2000Am polymer grafted on

the surface was small and did not show a regular trend. The molecular weight distribution of the grafted chains increased with increase in monomer concentration for all PEG macromonomers. The polydispersity (Mw/Mn) of chains increased from 1.34 to 3.3 for MPEG350Am and from 1.27 to 2.22 for MPEG750Am, and MPEG2000Am did not produce enough surfacegrafted polymers for an unambiguous determination at any monomer concentration. Due to the increase in polydispersity of grafted chains, the molecular weight (Mn) increase was not regular, but the Mw of the polymers increased with monomer concentration. PEG macromonomers produced polydisperse brushes compared to other hydrophilic monomers like N,N-dimethylacrylamide (DMA) and N-isopropylacylamide (NIPAM) under aqueous ATRP conditions.26,27,31 In the present case, a limited range of reaction conditions (only low monomer concentration) produced narrow molecular weight dispersed polymers on the surface. Our data show that the polymerizations became uncontrolled with increase in monomer concentration presumably due to increase in the amount of soluble polymer formed along with grafted chains. In the case of MPEG350Am only one monomer concentration produced grafts with low molecular weight distribution. The surface density of grafted polymer chains (Figure 2), calculated from Mn and the amount of polymer grafted per unit area of latex increased with increasing monomer concentration in all cases. The highest value observed was ∼0.52 chains/nm2 for MPEG350Am at 0.392 mol/L. Since the graft density was calculated from the mass of the grafts cleaved from unit area of the latex surface, assuming a flat surface, these values may be overestimated due to the surface roughness of the latex surface. A striking feature of these polymerization reactions done under identical reaction conditions is seen upon comparison of the graft density obtained from three macromonomers. The number of chains grown on the surface decreased with increasing chain length of PEG macromonomers (Figure 2), i.e., MPEG350Am gave a higher graft density than MPEG750Am or MPEG2000Am. This behavior is likely due to the decrease in accessibility of the reactive monomer end to the surface radicals as the excluded volume of the macromonomer increases. This observation is quite different from polymerization of macromonomers in homogeneous solution32,33 and, to the best of our knowledge, is (31) Kizhakkedathu, J. N.; Norris-Jones, R.; Brooks, D. E. Macromolecules 2004, 37, 734. (32) Neugebauer, D.; Zhang, Y.; Pakula, T.; Sheiko, S. S.; Matyjaszewski, K Macromolecules 2003, 36, 6746–6755. (33) Wang, X.-S.; Armes, S. P. Macromolecules 2000, 33, 6640–6647.

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Kizhakkedathu et al.

Table 2. Effect of Cu(II)Cl2 and Sodium Chloride on the Characteristics of Surface Grafted Poly(MPEnAm) Brushes

sample MPEG350Am MPEG750Am

graft polymer

monomer concn (mol/L)

Cu(II)Cl2 (mol % wrt to Cu(I))

0.276 0.276 0.276 0.127 0.127

15 100 15 + 100 mM NaCl 15 100

Mn 188 300 91 600 81 200 226 000 166 000

the first observation of its kind. Our results show that it is difficult to achieve high graft densities on a surface using SIP of large macromonomers. Another experimental parameter that may be contributing to this behavior is the presence of Brij-35 in polymerization medium as a stabilizing agent for functionalized latex. The adsorbed surfactant may also be influencing the observed results. It was essential to use Brij-35 for producing a stable latex suspension during the polymerization and uniform polymer growth. Since the graft density which can obtained by the adsorption surfactant is low, this factor may not hugely influence on the observed results. The increase in graft density with increase in monomer concentration may be due to the change in polymer growth. We believe this is due to the change in accessibility of hydrophilic monomer to the hydrophobic initiator on latex surface in aqueous conditions. The observation was similar to our previous results26,27 on the polymerization of DMA on latex surface. Along with surface polymerization, we also observed solution polymerization. This is typical of SIP from latex surfaces under aqueous conditions.26,27,31 The molecular weights of polymer produced under selected conditions are given in Table 1. The amount of solution polymer formed compared to grafted polymer was significant for MPEG750Am and MPEG2000Am relative to MPEG350Am (data not shown). In the case of MPEG2000Am most of the polymer formed was in solution. The molecular weight of the solution polymer increased with monomer concentration, similar to the SIP reaction products. In all cases, the polymer formed in solution was of higher molecular weight than that grown from the surface in contrast to the observation we have reported for low-molecular-weight monomers.26,27,31,34,35 In our earlier experiments with DMA26,27 and NIPAM,31 we have seen that both solution and surface polymer have similar molecular weights, albeit with some difference in molecular weight distribution which was consistent with other reports.34,35 Thus, assuming equivalence between surface-grafted and solution polymer characteristics may lead to incorrect conclusions in the case of macromonomers. In general, the HT of the grafted layer increased with increase in monomer concentration (Table 1). This is due to the increase in both the molecular weight and graft density of chains on the surface. HT increased from 29 to 730 nm for MPEG350Am and from 26 to 270 nm for MPEG750Am with increase in monomer concentration. The lower HT values for poly MPEG750Am brushes are due to the lower graft densities of these brushes. In most of the cases HT is greater than the average size of the polymer in solution determined by light scattering (Table 1). Effect of Cu(II)Cl2 Concentration and Addition of NaCl. It is known that addition of a deactivator (Cu(II) complex in the present case) in ATRP reactions decreases the propagation rate (34) Ejaz, M.; Yamamoto, S.; Ohno, K.; Tsujii, Y.; Fukuda, T. Macromolecules 1998, 31, 5934–5936. (35) Matyjaszewski, K.; Miller, P. J.; Shukla, N.; Immaraporn, B.; Gelman, A.; Luokala, B. B.; Siclovan, T. M.; Kickelbick, G.; Vallant, T.; Hoffmann, H.; Pakula, T. Macromolecules 1999, 32, 8716–24.

Mw/Mn 3.01 2.32 2.37 1.27 1.32

solution polymer

graft density (chains/nm2)

HT (nm)

0.38 0.62 0.79 0.0023 0.003

345 ( 20 420 ( 32 384 ( 25 55 ( 9 49 ( 7

Rg (nm)

17.2 15.6

Mn 1086 000 576 000 456 000 ND ND

Mw/Mn 2.02 3.00 3.07 ND ND

Scheme 3

of polymerization and improves polymerization control.36,37 We added Cu(II)Cl2/HMTETA complex along with Cu(I)Cl/ HMTETA in the reaction medium; the results are given in Table 2. In the case of MPEG350Am the molecular weights and polydispersities of grafted chains decreased with an increase in the concentration of Cu(II)Cl2/HMTETA at two monomer concentrations, consistent with reports in the literature.36,37 The higher MW monomer reactions also produced a lower Mn at the higher Cu(II) concentration; however, the polydispersity was unaffected. Due to the much lower amount of polymer produced, this result is less certain however. The addition of a nonreactive salt with a common ion could favor the ATRP equilibrium toward the dormant side by stabilizing the active Cu(II)/HMTETA complex as shown in Scheme 3.38,39 An increase in NaCl concentration is expected to decrease propagation of the active chain end due to the increased concentration active copper(II) complex in the medium compared to the case where there are no salt present, could result in lower molecular weights. We have increased the concentration of NaCl from 0 to 200 mM in the reaction medium for two monomers; the results are given in Table 2 and Figure 3. The molecular weight of the grafted chains decreased with the addition of NaCl. In the case of MPEG350Am the molecular weight decreased from 188 300 to 81 200 in presence of 100 mM NaCl. As shown in Figure 3 MPEG750Am also follows the same trend, Mn decreasing from ∼225 000 to ∼110 000 with increase in salt concentration from 0 to 200 mM. For both monomers the graft density increased considerably as the salt concentration was increased (Table 2 and Figure 3). The increase in graft density is a surface effect, likely due to the screening of the surface charges by NaCl40 with accompanying effects on initiation and/or chain transfer by the charged Cu catalyst complex. Thus, addition of salt to the reaction medium gives another experimental tool with which surface initiation and subsequent growth of grafted chains can be manipulated to modify the properties of the resulting polymer brushes. (36) von Werne, T.; Patten, T. E. J. Am. Chem. Soc. 2001, 123, 7497. (37) Teodorescu, M.; Matyjaszewski, K. Macromolecules 1999, 32, 4826. (38) Tsarevsky, N. V.; Pintauer, T.; Glogowski, E.; Matyjaszewski, K. Polym. Prep. 2002, 43, 201–202. (39) Jewrajka, S. K.; Mandal, B. M. Macromolecules 2003, 36, 311–317. (40) Hiemenz, P. C. Principles of Colloid and Surface Chemistry: Marcel Dekker, Inc.: New York, 1977; Chapter 9.

Polymer Brushes by Surface-Initiated Polymerization

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Figure 3. Effect of sodium chloride concentration on molecular weight and graft density of poly(MPEG750Am) chains grown on surface at a constant monomer concentration of 0.127 mol/L. Mw/Mn is given besides the molecular weight values. Experimental conditions: 3.5 g of 3% by weight suspension of latex, 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA 30 µmol), CuCl (13 µmol) CuCl2/ HMTETA (3 µmol), copper powder (19 µmol) at 22 °C. Surface initiator concentration: 1.56 × 10-6 mol/m2. The values given are average of two experiments; deviations in molecular weights of two repeats are smaller than the size of the symbol.

Figure 4. Comparison of the hydrodynamic thickness of spherical brushes of poly(PEG350Am), PDMA, and PNIPAM. Linear regressions are shown. The hydrodynamic thickness, molecular weight, and graft density values for PDMA and PNIPAM brushes were taken from refs 26, 27, and 31.

Hydrodynamic Thickness of the Polymer Brushes Synthesized from Macromonomers. The Daoud and Cotton (DC) model for brush height corrected for curvature of the anchoring surface can be written in the form41,42

[

( )

5⁄3 HT Nσ1⁄3 + 1 - 1 ) const R R

]

(1)

where HT is the hydrodynamic thickness of the grafted polymer layer, R is the radius of the spherical particle on which the brushes are grafted, N is the degree of polymerization of grafted chains, and σ is the graft density. This model was tested for poly(MPEG350Am) and compared with data from poly(N,Ndimethylacrylamide) (PDMA) and poly(N-isopropylacrylamide) (PNIPAM) brushes reported earlier.26,27,31 Since these polymers have the same backbone, we believe that this model can be applied to test the effect of polymer side chain length on brush height. The plots of [((HT/R) + 1)5/3 - 1] vs [Nσ1/3/R] are given in Figure 4. Our usage of the DC model differs from that described by Biver et al.42 in that the const on the right-hand-side of eq 1 carries units rather than being nondimensional. By comparison with eq 2 given by Biver et al. 42 the const used here is given by

[ RL + 1]

5⁄3

const ) l5⁄3

( )

Nl σν R l3 ν 1⁄3 k l3

-1)k

()

1⁄3

(2) (3)

where l and ν are the monomer segment length and excluded volume, respectively, and k is a dimensionless constant. Additionally, σ in eq 2 in ref 42 is the dimensionless chain density. Independent measures of these parameters for poly(MPEG350Am) are not presently available, so a dimensionless master curve42 can not be plotted. The macromonomer-based polymer produces grafted layers whose HT values fit the model very well, but the slope of the line is obviously much steeper for poly(MPEG350Am) (41) Daoud, M.; Cotton, J. P. J. Phys. (Paris) 1982, 43, 531–538. (42) Biver, C.; Hariharan, R.; Mays, J.; Russel, W. B. Macromolecules 1997, 30, 1787–1792. (43) Janzen, J.; Le, Y.; Kizhakkedathu, J. N.; Brooks, D. E. J. Biomater. Sci., Polym. Ed. 2004, 15, 1121.

Figure 5. SDS-PAGE of plasma proteins desorbed from poly(MEG350Am) brushes with different graft densities; Lane 1, sample buffer; Lane 2, protein size markers; Lane 3, diluted plasma; Lane 4, 0.00054 chains/nm2; Lane 5, 0.118 chains/nm2; Lane 6, 0.380 chains/ nm2; Lane 7, 0.414 chains/nm2; Lane 8, 0.52 chains/nm2; Lane 9, 0.79 chains/nm2; Lane 10, bare latex surface (no polymer).

than for PDMA or PNIPAM (4.73 ( 0.12 (R2 ) 0.99) vs 0.79 ( 0.02 (R2 ) 0.99) or 0.95 ( 0.04 (R2 ) 0.95), respectively). Presumably, the higher slope is due to the combined effect of increases in monomer segment length and excluded volume. Hence, the hydrodynamic thickness of the brushes is strongly affected by the size of the pendant chain on the monomer. This shows that the molecular weight of the side chain, which extends from the backbone of the polymer (56, 69, and 437, respectively, for DMA, NIPAM, and MPEG350Am), plays an important role in the chain extension from the surface. Although we tested the theory with poly(MPEG750Am), due to the absence of samples with wide variation of graft density in the data set, we have not included those in the Figure 4. The values of Nσ1/3/R for poly(MPEG750Am) varied only a little (0.09-0.17) compared other types of brushes. Thus, the length of the monomer side chain provides another parameter by which the physical structure of the grafted brushes can be controlled, through choice of monomers of different sizes. Plasma Protein Adsorption onto Brushes. Figure 5 is a representative image of an SDS PAGE gel analysis of proteins desorbed from polymer-grafted surfaces after incubating different

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Kizhakkedathu et al.

Figure 6. Effect of graft density (chains/nm2) (A) and and surface PEG content (mg/nm2), (B, D) poly(MEG350Am) (A, B), and poly(MEG750Am) (C, D) brushes on protein adsorption from whole blood plasma. Protein absorption is given as relative to bare surface and is determined from the integrated staining intensity of the whole gel lane. The measurements of standard deviations are obtained from measurements on three different gels. Molecular weight of the poly(MEG350Am) chains varied from ∼50 000 to 230 000 and poly(MEG750Am) from ∼200 000 to 600 000.

brush-bearing latex suspension in blood plasma. In the absence of grafted polymer, radiolabeled HSA recovery in the gel was 77% ( 7% from the unmodified latex surface (data not shown). One would expect a larger desorption of proteins from the surface upon SDS wash when polymer is grafted to surface.43 The degree to which this occurs with the macromonomer polymers used here was examined by measuring the average staining density per gel in a series of experiments in which the graft density was varied. The total protein adsorption to surface decreased considerably in the presence of polymer grafts, the bare surface giving maximum adsorption (Figures 5 and 6). In the case of poly(MPEG350Am) brushes at high density (0.381 chains/nm2) the total protein adsorption was estimated to be 37 ng/cm2 compared to 352 ng/cm2 on the bare surface, as calculated from the band staining intensities. Changes in the pattern of protein adsorption on grafted brushes (lanes 4-9) compared to bare latex (Figure 5, lane 10) included both the appearance and removal of certain protein bands on the gel. In most cases, low-graftdensity surfaces behaved similarly to the bare surface but as the graft density increased the pattern of protein adsorption changed; however, it should also be noted that compared to blood plasma (Figure 5, lane 3) some proteins are concentrated on some grafted surfaces. The difference is due to the selective adsorption of certain plasma proteins to either bare surface or polymer-grafted surface with different polymers. In considering interactions between proteins and neutral hydrophilic polymer brush layers, one can anticipate a number of effects on the basis of thermodynamics. It is now well-known

that the change in entropy of the protein-polymer systems plays a major role in determining the degree to which a protein is rejected from a polymer brush layer with which it comes in contact.4-7,44 The strong dependence of protein adsorption with graft density is believed to be due to the steric repulsion caused by the compression of stretched chains. This is due to the unfavorable entropic contribution to the total free energy of polymer brush-protein system. Therefore, such systems tend to minimize the adsorption of proteins and the adsorption decreases with increase in conformational restriction, i.e., increase in graft density. However, as the graft density increases secondary increactions between the protein and polymer (weak enthalpic contribution) might play a role in adsorption or rejection of proteins. The chemistry of the polymers could influence this behavior. Such enthalpic interactions between protein and monomer can enhance or reduce rejection, although the effects are not predicted to be as large as those due to entropy changes. If the enthalpy change is favorable for adsorption, high-molecularweight components could adsorb more strongly than lower MW species as is observed on solid surfaces. In the present case, the entropic effect evidently dominates, as seen by the roughly exponential decrease in protein adsorption with increase in chain graft density on the surface (Figure 6A and C). This is true for both poly(MPEG350Am) and Poly(MPEG750Am) brushes, but the grafting density required to attain a given degree of protein rejection is much less for poly(MPEG750Am) brushes than for poly(MPEG350Am). Since (44) Brooks, D. E.; Haynes, C. A.; Hritcu, D.; Steels, B. M.; Muller, R. H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7064.

Polymer Brushes by Surface-Initiated Polymerization

the graft density of the brushes is the major factor in preventing the protein adsorption as suggested by other researchers4,6,12,14 and our own experience with PDMA brushes,45 in the present case we have correlated the protein adsorption with the graft density of brushes, although there are changes in molecular weight and polydispersity through this data set. An absolute comparison may not be possible here due to the changes in both molecular weight and polydispersity. It is also apparent from these results that the relative protein adsorptiononpoly(MPEG350Am)isgreaterforpoly(MPEG750Am)grafted surfaces. This might be due to the higher PEG content in the case of poly (MPEG750Am). This argument is supported by correlation of mass of PEG grafted versus relative protein adsorption presented in Figure 6B and D. Even though mass of PEG present in poly(MPEG350Am) brushes is higher than poly(MPEG750Am), the graft density required to achieve minimum protein adsorption is much lower in the poly(MPEG750Am). This might be due the weak interactions between the polymer chains and proteins (enthalpic contributions). The high graft density requirement for poly(MPEG350AM) compared to poly(MPEG750AM) to achieve lower protein adsorption is pointing to fact that such interactions are more dominant in this case. We attribute the residual protein adsorption at high graft densities for both poly(MPEG350AM) and poly(MPEG750AM) to the weak interactions between the polymer chains and proteins. At higher graft densities the average distance between the chains (∼1.1-1.5 nm in the case of poly (MPEG350Am)) is much smaller than the hydrodynamic size of the plasma proteins observed on our gels. We expect that it would be difficult for proteins to penetrate through the thick polymer layer (HT ) 174-735 nm for poly (MPEG350Am) and 55-270 nm for poly (MPEG750Am)) to adsorb to the underlying PS surface. Thus, the protein adsorption is likely due to the interaction of proteins with polymer chains themselves. Since the polymer backbone is more hydrophobic (-CH2-CH-) it may be that interactions at this level are involved. This is supported by the lower residual protein concentration on poly(MPEG750Am)-grafted surfaces than on poly(MPEG350Am)-grafted surfaces where the PEG content is higher (Figure 6B and D). Our results show both sterric repulsion (45) Kizhakkedathu J. N. 2008. Unpublished results.

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(entropic factor) and weak polymer-protein interactions (enthalpic) contributing to overall reduction or enhancement of protein adsorption. We plan to investigate the detailed thermodynamic aspects of protein adsorption to grafted brushes of different chemical composition to shed light on this possibility.

Conclusions Synthesis of hydrolytically stable, PEG acrylamide-based polymer brushes having different molecular weight, graft density, PEG composition, and HT from three new PEG acrylamide macromonomers with increasing chain length has been reported. Apart from monomer concentration, we have identified several different experimental manipulations such as the presence of salt or Cu(II) concentration which influence the controlled growth of polymer chains from latex surfaces. We found that the chain length of the macromonomer influences the graft density of chains on the surface, presumably due to the limited accessibility and steric influence of larger side chains. Also, it was found that hydrodynamic thickness of macromonomer brushes strongly depend on the side chain length for a given degree of polymerization. Protein adsorption studies from whole blood plasma shows that the grafted polymer layers reduce protein adsorption as a function of graft density of chains on the surface. Although the PEG based comblike polymer brushes studied here greatly reduced the protein adsorption from whole blood plasma compared to adsorption to the underlying bare surface, the total protein adsorption to PEG-based brushes depends on the PEG content (or the type of the monomer used) of grafted polymer layer. Thus, PEG-based comb polymer grafts should not be considered as equivalent to linear PEG coatings. Acknowledgment. We thank the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada, and the Canadian Blood Services (CBS) for financial support. The authors thank the LMB Macromolecular Hub at the UBC Centre for Blood Research for the use of their research facilities; the infrastructure facility is supported by the Canada Foundation for Innovation (CFI) and the Michael Smith Foundation for Health Research (MSFHR). J.N.K. is the recipient of a CBS/CIHR new investigator award in transfusion science. LA803690Q