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Langmuir 2006, 22, 2222-2226
Highly Protein-Resistant Coatings from Well-Defined Diblock Copolymers Containing Sulfobetaines Yung Chang, Shengfu Chen, Zheng Zhang, and Shaoyi Jiang* Department of Chemical Engineering, UniVersity of Washington, Seattle, Washington 98195 ReceiVed NoVember 3, 2005. In Final Form: December 26, 2005 Three well-defined diblock copolymers of poly(sulfobetaine methacrylate) [poly(SBMA)] and poly(propylene oxide) (PPO) were synthesized by the sequential addition of SBMA monomer to fixed amounts of PPO using an atom transfer radical polymerization method and varying poly(SBMA) lengths. These copolymers were characterized by 1H NMR and aqueous gel permeation chromatography. These copolymers were physically adsorbed onto a surface plasmon resonance (SPR) sensor surface covered by methyl-terminated self-assembled monolayers, followed by the in situ evaluation of protein adsorption on the adsorbed copolymers. It is found that the behavior of the protein adsorption depends on the molecular weight of the copolymers. Results show that the diblock copolymers containing poly(SBMA) can be highly protein resistant when surface SBMA densities are well controlled. Thus, copolymers containing zwitterionic groups are ideal for resisting protein adsorption when the surface density of zwitterionic groups is controlled.
Introduction Synthetic polymers have been widely used as biocompatible materials in biomedical fields.1 Poly(ethylene glycol) (PEG)based materials are the most commonly used nonfouling materials. However, PEG is a polyether that autoxidizes relatively rapidly, especially in the presence of oxygen and transition metal ions, and most biochemically relevant solutions contain transition metal ions.2 Lipid components that constitute the outside surface of a cell membrane are mainly zwitterionic phospholipids and are believed to be nonthrombogenic. The majority of work relating to phosphorylcholine (PC)-based materials is on methacryloyloxyethyl phosphorylcholine (MPC)-based copolymers with the PC group located in the side chains, such as MPC-co-BMA (butyl methacrylate). MPC-based copolymers have been used commercially in contact lenses.3 Over the past few years, several approaches have been used to prepare zwitterionic materials, including self-assembled monolayers (SAMs),4-6 polymer blends,7 graft polymerization,8-12 and interpenetrating polymer networks.13,14 Besides phosphobetaine, other zwitterionic groups, including sulfobetaine and carboxybetaine, have been studied. * To whom correspondence
[email protected].
should
be
addressed.
E-mail:
(1) Tsuruta, T.; Hayashi, T.; Kataoka, K.; Kimura, Y.; Ishihara, K. Biomedical Application of Polymeric Materials; CRC Press: Boca Raton, FL, 1993. (2) Lewis, A. L. Colloids Surf., B 2000, 18, 261. (3) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841. (4) Tegoulia, V. A.; Cooper, S. L. J. Biomed. Mater. Res. 2000, 50, 291. (5) Holmlin, P. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841. (6) Kane, R. S.; Deschatelets, Pascal.; Whitesides, G. M. Langmuir 2003, 19, 2388. (7) Ishihara, K.; Shibata, N.; Tanaka, S.; Iwasaki, Y.; Kurosaki, T.; Nakabayashi, N. J. Biomed. Mater. Res. 1996, 32, 401. (8) Feng, W.; Brash, J.; Zhu, S. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 2931. (9) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. Langmuir 2005, 21, 5980. (10) Jun, Z.; Youling, Y.; Kehua, W.; Jian, S.; Sicong, L. Colloids Surf., B 2003, 28, 1. (11) Yuan, Y. L.; Ai, F.; Zhang, J.; Zang, X. B.; Shen, J.; Lin, S. C. J. Biomater. Sci. Polym. Ed. 2003, 13, 1081. (12) Yuan, Y.; Zang, X.; Ai, F.; Zhou, J.; Shen, J.; Lin, S. Polym. Int. 2004, 53, 121. (13) Iwasaki, Y.; Aiba, Y.; Morimoto, N.; Nakabayashi, N.; Ishihara, K. J. Biomed. Mater. Res. 2000, 52, 701. (14) Iwasaki, Y.; Shimakata, K.; Morimoto, N.; Kurita, K. J. Polym. Sci. Polym. Chem. 2003, 41, 68.
These zwitterionic-based materials have received more and more attention because of their biocompatibilities10-12,15 and interesting solution properties.16 There are only a few studies on poly(sulfobetaine methacrylate) [poly(SBMA)]. However, it was shown that poly(SBMA)-based materials were not as effective as MPC-based materials at resisting protein adsorption and cell adhesion.17 On the basis of previous studies of molecular-level nonfouling mechanisms using experimental and molecular approaches,18-20 we believe that zwitterionic poly(SBMA) should be able to achieve superlow fouling if its surface packing can be well controlled. In this work, we design three well-defined diblock copolymers containing poly(SBMA) with poly(propylene oxide) (PPO) as a hydrophobic moiety. These copolymers were synthesized via the atom transfer radical polymerization (ATRP) method to control their poly(SBMA) chain lengths to a narrow molecular weight distribution, while the chain length of the hydrophobic PPO segment is fixed. One of the copolymers has poly(SBMA) and PPO of similar segment size, while the other two contain poly(SBMA) that is larger than PPO. These copolymers are adsorbed onto methyl (CH3)-terminated SAMs, where the hydrophobic segment will bind to the hydrophobic surface and the hydrophilic poly(SBMA) will be exposed to the solution. The effect of PPO-b-poly(SBMA) molecular weights [or the ratio of poly(SBMA) to PPO] on protein adsorption was compared. In a recent study by Park and co-workers, a triblock copolymer of poly(ethylene oxide)-block-poly(propylene oxide)block-poly(ethylene oxide) (PEO-b-PPO-b-PEO) was adsorbed on a modified glass surface.21 It was found that this surface could reduce fibrinogen adsorption to 20 ng/cm2 with an optimized control. Results from this work will compare with that of PEOb-PPO-b-PEO. (15) Iwasaki, Y.; Ishihara, K. Anal. Bioanal. Chem. 2005, 381, 534. (16) Laschewsky, A.; Touillaux, R.; Hedlinger, P.; Vierengel, A. Polymer 1995, 36, 3045. (17) West, S. L.; Salvage, J. P.; Lobb, E. J.; Armes, S. P.; Billingham, N. C.; Lewis, A. L.; Hanlon, G. W.; Lioyd, A. W. Biomaterials 2004, 25, 1195. (18) Li, L.; Chen, S.; Zheng, J.; Ratner, B.; Jiang, S. J. Phys. Chem. B 2005, 109, 2934. (19) Zheng, J.; Li, L.; Chen, S.; Jiang, S. Langmuir 2004, 20, 8931. (20) Chen, S.; Zheng, J.; Li, L.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473 (21) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176.
10.1021/la052962v CCC: $33.50 © 2006 American Chemical Society Published on Web 01/31/2006
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Figure 1. Reaction scheme for (a) the reaction of monohydroxy-capped PPO with 2-bromoisobutyryl bromide in THF at 20 °C and (b) the block copolymerization of poly(SBMA) with PPO via ATRP in methanol at 20 °C.
Experimental Section Chemicals. [2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide (sulfobetaine methacrylate, SBMA) monomer, copper(I) bromide [Cu(I)Br], 2,2′-bipyridine (bpy), 2-Bromoisobutyrylbromide, and triethylamine were purchased from Aldrich and used as received. Poly(propylene glycol) monobutyl ether (PPOOH) with an average molecular weight of 1200 (Dpn ) 20), was also purchased from Aldrich. Tetrahydrofuran (THF; Aldrich, 99%) was distilled from sodium to keep it anhydrous. Preparation of SBMA Block Copolymerization in Aqueous Solution. The controlled polymerization is achieved via the ATRP method (Figure 1). The copolymerization of a diblock copolymer is a reversible redox process through which a transition metal compound acts as a carrier of a halogen atom to sequentially link a monomer to a monofunctional macroinitiator. PPO with a macroinitiator (PPO-Br) was synthesized by reacting monohydroxybased poly(propylene glycol) with 2-bromoisobutyrylbromide in THF. The product was purified by extraction with brine three times. For the polymerization of SBMA with 11200 molecular weight, SBMA (2.0 g, 6.77 mmol) was polymerized in 10 mL of methanol using [SBMA]/[PPO-Br]/[CuBr]/[bpy] ) 50:1:1:2 under nitrogen at 20 °C. After 24 h, the resulting reaction solution was passed through an aluminum oxide column, precipitated into ethanol, and redissolved into water repeatedly to remove residue catalysts. After solvent evaporation, the copolymer was dried in a vacuum oven at room temperature to yield a white powder. Characterization of the Copolymers. The structure of PPOb-poly(SBMA) diblock copolymers was characterized by 1H NMR spectra using a Bruker 300 MHz spectrometer and D2O as solvent. A typical spectrum for PO20-b-SBMA35 is shown in Figure 2. Results showed that a pure PPO-b-poly(SBMA) diblock copolymer was obtained. Molecular weights and molecular weight distributions of prepared diblock copolymers were determined by aqueous gel permeation chromatography (GPC), using 2 columns of Ultrahydrogel 1000 and Ultrahydrogel 250 (the range of molecular weight was from 586 Da to 885 kDa) connected to a model VE3580 Viscotek differential refractometer detector from Waters. For GPC experiments, the flow rate was 0.7 mL/min, and the column temperature was 25 °C. The eluent was an aqueous solution composed of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 at pH 8.0. PEG standards from Scientific Polymer Products (Ontario, NY) were used for calibration. Typical aqueous data of the three synthesized PPO-b-poly(SBMA) copolymers from GPC are shown in Figure 3. Protein Adsorption Measurements by a Surface Plasmon Resonance (SPR) Sensor. A custom-built SPR biosensor based on wavelength interrogation with a dual-channel Teflon flow cell was
used to monitor protein adsorption on surfaces coated with copolymers. In this work, optical glass substrates were used as sensor chips and coated with a 2 nm adhesion-promoting chromium layer and a 50 nm surface plasmon active gold layer by electron beam evaporation under vacuum. CH3-terminated SAMs were formed by overnight soaking of UV ozone-cleaned, gold-coated substrates in a 1.0 mM ethanolic solution of HS(CH2)8CH3. The modified chip was attached to the base of the prism, and optical contact was established using refractive index matching fluid (Cargille). For protein adsorption measurements, the SPR was first stabilized with a 2mM phosphate-buffered saline (PBS) solution. PPO-b-poly(SBMA) diblock copolymer solution was then flowed into the SPR cell for 20 min, followed by flushing with 2 mM PBS solution for 15 min to remove loosely adsorbed copolymers. A 1.0 mg/mL protein was flowed for 20 min, followed by flushing with 2 mM PBS solution for 15 min. In this work, fibrinogen was used as a model system to evaluate protein adsorption on surfaces covered with physically adsorbed copolymers. All SPR experiments were conducted at room temperature (∼25 °C) and at a flow rate of 0.05 mL/min. The amount of protein adsorption is defined as the difference between the two baselines established before and after protein adsorption. Figure 4 shows a typical SPR sensorgram for the adsorption of copolymer A, followed by the in situ evaluation of fibrinogen adsorption.
Results and Discussion In this work, the physical adsorption of well-defined diblock copolymers PPO-b-poly(SBMA) onto hydrophobic CH3-SAM surfaces was performed to study the nonfouling properties of poly(SBMA). PPO was used as the hydrophobic moiety of the diblock copolymers. To control the surface packing density of physically adsorbed copolymers, three different SBMA-based block copolymers (PO20-b-SBMA20, PO20-b-SBMA35, and PO20b-SBMA50) were synthesized. The chain lengths of poly(SBMA) were controlled by sequential monomer addition via ATRP at ambient temperature, while the chain length of PPO was kept constant. The synthesis parameters and average molecular weights for the three PPO-b-poly(SBMA) copolymers are summarized in Table 1. PO20-b-SBMA20, PO20-b-SBMA35, and PO20-bSBMA50 are denoted as copolymers A, B, and C, respectively. When the poly(SBMA) chain is longer, the chain-length ratio of poly(SBMA)/PPO is higher, and the structure of diblock copolymer is less symmetric. This two-step reaction route (Figure 1) provided PPO-b-poly(SBMA) copolymers with controlled molecular weights (Mn) and polydispersities (Mw/Mn ) 1.21.35). Low polydispersities indicate well-controlled polymeri-
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Figure 2. 1H NMR spectrum of the PO20-SBMA35 diblock copolymer in D2O.
Figure 3. Aqueous GPC curves for three PPO-b-poly(SBMA) diblock copolymers (A: Mn ) 6490, Mw/Mn ) 1.232; B: Mn ) 11183, Mw/Mn ) 1.255; C: Mn ) 15114, Mw/Mn ) 1.353, determined with PEG as a reference) prepared via ATRP at 20 °C.
zation accuracy. Figure 3 shows that the molecular weight of the three copolymers obtained ranges from 6500 to 15000 with low polydispersity. It is expected that the three SBMA-based block copolymers have different packing densities and protein adsorption behaviors. The adsorbed amounts of both copolymers and proteins were obtained from SPR. In this work, we studied the effect of PPO-b-poly(SBMA) solution concentration from 0.005 to 1 mg/mL on surface packing densities and thus protein adsorption. As can be seen from Figure 5, protein adsorption depends on the chemistry and structure of the layer, namely SBMA surface density and (SBMA)/PPO ratio,
Figure 4. A typical SPR sensorgram for the adsorption of copolymer A (PO20-SBMA20), followed by the in situ evaluation of fibrinogen adsorption.
which are determined by (a) the concentration of PPO-b-poly(SBMA) in solution (CPPO-b-poly(SBMA)) and (b) the volume fraction of poly(SBMA) [f poly(SBMA)]. At low CPPO-b-poly(SBMA) (e.g., CPPO-b-poly(SBMA) < 0.02 mg/mL), protein adsorption is lower for PPO-SBMA diblock copolymers of higher fpoly(SBMA) because of the higher surface SBMA coverage. By contrast, at higher CPPO-b-poly(SBMA), protein adsorption on PPO-b-poly(SBMA) diblock copolymers of lower fpoly(SBMA) quickly decreases. For copolymer A, which has a lower molecular weight, protein adsorption is very low (3 ng/cm2) when CPPO-b-poly(SBMA) > 0.03 mg/mL. As compared to the work reported previously by Park and co-workers, protein adsorption on the PPO-b-poly(SBMA)-covered surface is 1 order of magnitude lower than that on the optimized PEO-b-PPO-b-PEO surface.21 For copolymer
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Table 1. Reaction Conditions and Average Molecular Weights of Three PPO-b-poly(SBMA) Copolymers Synthesized via ATRP at 20 °Ca sample
composition (DPn)b
solvent (10 mL)
[SBMA] (g)
[PPO-Br] (mg)
reaction time (hr)
Mn,GPCc
Mw/Mnc
A B C
PO20-SBMA20 PO20-SBMA35 PO20-SBMA50
MeOH MeOH MeOH
2.0 2.0 2.0
358.0 143.2 71.6
24 24 24
6490 11183 15114
1.232 1.255 1.353
a The ratio of initiator/Cu(I)Br/bpy was 1:1:2. b DPn is the degree of polymerization. c Mn is the average molecular weight, and Mw/Mn is the polydispersities of the prepared copolymers.
Figure 7. Illustration showing the adsorption of copolymer C onto the CH3-terminated SAM surface and back-filling by copolymer A to achieve higher SBMA surface densities, thus increasing its effective resistance to protein adsorption.
Figure 5. Fibrinogen adsorption on the surfaces covered with physically adsorbed PPO-b-poly(SBMA) as a function of PPO-bpoly(SBMA) concentration in solution (CPPO-b-poly(SBMA)) for the three copolymers at 25 °C from SPR measurements.
Figure 8. Adsorption of fibrinogen, BSA, and lysozyme on copolymer A-coated surfaces from SPR. Final wavelength shift for each case is indicated inside the blankets. A 1 nm wavelength shift in the SPR response is equivalent to 15 ng/cm2 of adsorbed proteins.20
Figure 6. SPR sensorgrams for fibrinogen adsorption at 25 °C onto copolymer-coated surfaces with CPPO-b-poly(SBMA) ) 1.0 mg/mL (A: copolymer A; B: copolymer B; C: copolymer C; C+A: copolymer C back-filled with copolymer A). Final SPR wavelength shift for each case is indicated inside the blankets. A 1 nm wavelength shift in the SPR response is equivalent to 15 ng/cm2 of adsorbed proteins.20
C, protein adsorption remains at a higher level (20.3 ng/cm2) over a wide range of CPPO-b-poly(SBMA). This is because the larger SBMA segments create cavities among themselves and cannot fully cover the surface, leading to protein adsorption. Figure 6 shows SPR sensorgrams for fibrinogen adsorption on various PPO-b-poly(SBMA)-coated surfaces for CPPO-b-poly(SBMA) ) 1.0 mg/mL. Fibrinogen adsorption is very low on surfaces covered with copolymers A and B and higher on copolymer C surfaces. This is due to increased surface packing defects formed from the large molecular size of copolymer C. When the surface covered with copolymer C was back-filled
with the smaller molecular weight copolymer A (illustrated in Figure 7), very low protein adsorption was also achieved. This result indicates that higher fibrinogen adsorption is due to higher surface vacancies caused by the adsorption of the copolymer with higher molecular weight, and these cavities can be backfilled with copolymers of smaller molecular weights. Results also show that the surface packing density of PPO-b-poly(SBMA) plays a significant role in surface resistance to protein adsorption. A similar idea was also demonstrated by Nagasaki et al. to further reduce protein adsorption on a surface tethered with longer PEG chains by adding shorter PEG chains onto the surface.22 To further evaluate SBMA-based copolymers’ resistance to the adsorption of various proteins, we screened the adsorption of three proteinss fibrinogen, bovine serum albumin (BSA), and lysozymeswith varying molecular weights (14.3-340 kD) and pI values (4.810.9) on copolymer A. Figure 8 shows that the adsorption of all three proteins on copolymer A is lower than 0.25 nm (∼3.75 ng/cm2). These results together suggest that copolymers containing SBMA are ideal for resisting protein adsorption if the surface SBMA density is high. (22) Uchida, K.; Otsuka, H.; Kaneko, M.; Kataoka, K.; Nagasaki, Y. Anal. Chem. 2005, 77, 1075.
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Conclusions It is demonstrated in this work that physically adsorbed diblock copolymers containing sulfobetaines [PPO-b-poly(SBMA)] on a hydrophobic surface are highly resistant to protein adsorption if the surface SBMA densities are well controlled. The direct adsorption of PPO-b-poly(SBMA) copolymers with smaller molecular weights onto a hydrophobic surface has very low protein adsorption. The PPO-b-poly(SBMA) copolymer with a higher molecular weight has high protein adsorption after direct adsorption onto a hydrophobic surface, but this can be reduced
Chang et al.
to a very low value after the surface is back-filled with a copolymer of small molecular weight. Thus, copolymers containing zwitterionic groups are ideal for highly resisting protein adsorption if the surface density of the zwitterionic groups is controlled to a high level. Acknowledgment. This work is supported by the Office of Naval Research (N000140410409). GPC experiments were performed in Allan Hoffman’s laboratory with help from Scott M. Henry. LA052962V
