© Copyright 2004 American Chemical Society
DECEMBER 21, 2004 VOLUME 20, NUMBER 26
Letters Characterization of Aldehyde-PEG Tethered Surfaces: Influence of PEG Chain Length on the Specific Biorecognition Hidenori Otsuka,†,§ Yukio Nagasaki,*,‡,| and Kazunori Kataoka*,† Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Department of Materials Science & Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan Received July 3, 2004. In Final Form: September 18, 2004 A functionalized poly(ethylene glycol) (PEG) layer possessing a reactive aldehyde group at the free end of the tethered PEG chain was constructed by simple coating on the substrate, using the acetal-PEG/ poly(DL-lactide) block copolymer, followed by the hydrolysis of the acetal end group by an acid treatment. The reactivity of the aldehyde group at the distal end of the PEG tethered chain was evaluated via a reductive amination using 4-amino-2,2,6,6-tetramethylpiperidinyloxy as the model compound. Further conjugation of the aldehyde group with sugar moieties has demonstrated an increased recognition ability with lectins with an increasing PEG chain length, which was attributable to the mobility of the chain end. These results provide a novel idea for highly sensitive biorecognition, suggesting a method to create highly selective biosensing surfaces that are able to prevent the undesired nonspecific adsorption of biocomponents.
Recently, the analysis of specific biomolecular interactions, such as antigen-antibody, sugar-lectin, and protein-DNA, has become increasingly important. To create a high-performance biosensor, the surface of the sensor chip must have a suitable structure. For biosensing with high sensitivity, the biospecific recognition on nonfouling substrates must be optimized. Surface modification with poly(ethylene glycol) (PEG) is a well-established strategy to improve the biocompatibility of the surface, which reduces complicated responses such as biofouling and thrombus formation on contact with biological milieus.1 We have previously reported2 that an AB-type block co†
The University of Tokyo. Tokyo University of Science. § Present address: Biomaterials Center, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan. | Present address: Tsukuba Research Center for Interdisciplinary Materials Science, University of Tsukuba, Tennoudai 1-1-1, Tsukuba 305-8573, Japan. ‡
polymer composed of PEG as the hydrophilic segment and polylactide (PLA) as the hydrophobic segment possessing an acetal group at the PEG chain end was synthesized and used to construct a functionalized PEG layer by simple coating on various substrates. In this way, a PEG-brushed layer with a terminal aldehyde group was readily prepared via the hydrolysis of the acetal end group, which may have both nonfouling and ligand-binding properties. (1) (a) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 1043. (b) Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365. (c) Qie, Y. X.; Klee, D.; Pluster, W.; Severich, B.; Hocker, H. J. Appl. Polym. Sci. 1996, 61, 2373. (d) Elbert, D. L.; Hubbell, J. A. Chem. Biol. 1998, 5, 177. (e) Deible, C. R.; Beckman, E. J.; Russell, A. J.; Wagner, W. R. J. Biomed. Mater. Res. 1998, 41, 251. (f) Suggs, L. J.; West, J. L.; Mikos, A. G. Biomaterials 1999, 20, 683. (g) Jo, S.; Park, K. Biomaterials 2000, 21, 605. (2) (a) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Biomacromolecules 2000, 1, 39. (b) Otsuka, H.; Nagasaki, Y.; Kataoka, K. In Polymers from Renewable Resources: Biopolyesters and Biocatalysis; Gross, R., Scholz, C., Eds.; ACS Symposium Series 764; American Chemical Society: Washington, DC, 2000; pp 311-327. (c) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Curr. Opin. Colloid Interface Sci. 2001, 6, 3.
10.1021/la0483414 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/16/2004
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Table 1. Molecular Weights of PEG/PLA Block Copolymers PEG sample
Mn
Mw
Mw/Mn
PLA Mn
PLA PEG/PLA(0.65/11.5) PEG/PLA(1.8/7.0) PEG/PLA(3.3/5.4) PEG/PLA(5.0/4.6) PEG/PLA(8.7/6.9)
650 1880 3340 5050 8730
720 1930 3470 5210 8810
1.10 1.03 1.04 1.03 1.01
20000 11470 7020 5410 4640 6940
Scheme 1. Synthetic Procedure of r-Acetal-PEG/PLA Block Copolymer and the Schematic Representation for the Construction of Sugar-Functionalized PEGylated Surfaces Figure 1. Specific and nonspecific lectin recognition on the sugar-functionalized PEGylated surfaces as a function of PEG molecular weight.
Using dynamic contact angle measurements,2,4 we have revealed that the mobility of the tethered PEG chains, which contributes to the steric exclusion, plays an extremely important role in reducing nonspecific adsorption of biocomponents such as proteins and lipids.3 One of the other important points is to estimate the effect of a surface-dynamic characteristic on the specific biorecognition. Especially in the case of the reactive group at the distal end of the tethering PEG chain, the dynamic information is very important. From our intensive investigation, however, no detailed information on the mobility or the reactivity of the tethering PEG chain end was available. In this study, electron spin resonance (ESR) spectroscopy was utilized for the measurement of both the reactivity and mobility of the PEG free end on the tethering surface. Biospecific recognition of the sugarinstalled PEG tethered chain surface with lectin proteins was also examined. Acetal-PEG/PLA block copolymers were synthesized according to our previous paper.2 The molecular weight (MW) of each segment can be controlled by changing the initial monomer/initiator ratio, resulting in the preparation of five PEG/PLA block copolymer samples with varying compositions, which are abbreviated as follows: PEG/PLA (0.65/11.5, 1.8/7.0, 3.3/5.4, 5.0/4.6, 8.7/6.9) where the numbers in parentheses denote the number-average MW of the PEG segments and PLA segments in kg/mol, respectively (Table 1). The PEGylated samples were prepared by spin coating of the acetal-PEG/PLA block copolymers at 2000 rpm onto PLA-spin-coated silanized glass substrates from a 20 mg/mL toluene solution and dried at room temperature under a vacuum for 24 h.2 Furthermore, the conversion of the acetal group into an (3) Jeon, S. L.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (4) Takahara, G. A.; Jo, N. J.; Kajiyama, T. J. Biomater. Sci., Polym. Ed. 1989, 1, 17.
aldehyde group was smoothly conducted by immersing the PEGylated substrates into aqueous media adjusted to pH 2 using hydrochloric acid for 5 h. The lactose (Lac) and mannose (Man) groups were successfully used in this study as the sugar moiety installed into the distal PEG chain ends on the glass substrates through a reductive amination reaction of the aldehyde group at the PEG terminal and corresponding sugar derivatives having p-aminophenyl moieties at the C-1 position (p-aminophenyl-β-D-lactopyranoside or mannopyranoside, Sigma Chemical Co., St. Louis, MO) as shown in Scheme 1. Since the reactivity of the aminophenyl group with the aldehyde is likely to be equal between the p-aminophenyl-Lac and p-aminophenyl-Man, the amounts of the two sugar groups on the glass substrate are assumed to be equal.5 The reaction of these Lac-derivatized surfaces with the FITC-labeled bivalent galactose-binding lectin (Ricinus communis agglutinin, RCA120; Vector Laboratories, Burlingame, CA) was estimated as a function of PEG MW through the fluorescence intensity normalized by that on the PLA surface. The fluorescence intensity shown in Figure 1 became more significant with increasing MW of PEG and was maximized at the highest PEG (8.7K). Contrary to this treatment, when the Lac-functionalized PEGylated surfaces were treated with FITC-labeled concanavalin A, which is known as glucose and mannose binding lectin (ConA, Vector Laboratories), only a slight fluorescence was observed probably due to nonspecific adsorption of ConA on the surface, especially with the rather shorter PEG tethered chain surface. When the Manfunctionalized PEGylated surfaces were treated with RCA120, a slightly nonspecific interaction with the surfaces was observed. These results demonstrate that the increasing fluorescence observed upon addition of RCA120 to Lac-functionalized PEGylated surfaces was indeed the result of specific interactions between the immobilized lactose moieties on the glass surface and the RCA120 lectin without nonspecific interaction. To obtain information on the reactivity and the mobility of the aldehyde group at the free end of the tethered PEG chain, an ESR study was carried out using an aminofunctionalized ESR probe, 4-amino-2,2,6,6-tetramethylpiperidinyloxy (4-amino-TEMPO, Aldrich Chemical Co., Inc., Milwaukee, WI) as a model compound.2a The 4-aminoTEMPO was installed in the same manner as the aminosugar via the reductive amination reaction. The ESR was measured at 25 °C on an ESR spectrometer (JEOL JES(5) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226.
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Langmuir, Vol. 20, No. 26, 2004 11287
Figure 2. Rotational correlation times and the concentration of TEMPO at the free end of PEG chains as a function of PEG molecular weight.
RE2X) using Mn2+ as the standard signal, operating at the X-band frequency of 9.15 GHz, scanning from 3240 to 3340 G. Experiments were done in quartz ESR sample tubes. Since the spin-probe is covalently conjugated at the distal end of the PEG chain, the relative anisotropy observed in the ESR spectrum is directly related to the rotational mobility of the probe. The rotational correlation time τc (s), which is shown as the time taken for an axis of the nitroxide group to travel through one radian,6,7 is highly sensitive to the motion of the chain and is calculated8,9 from
τc ) 6.6 × 10-10∆H[(h-1/h+1)1/2 - 1]
(1)
where ∆H is the peak-to-peak line width of the low-field line (in G) and h-1 and h+1 are peak-to-peak heights of the (6) (a) Otsuka, H.; Esumi, K. Langmuir 1994, 10 (1), 45. (b) Ji, J.; Feng, L. X.; Qiu, Y. X.; Yu, X. J. Makromol. Rapid. Commun. 1998, 19, 473. (c) Otsuka, H.; Ring, T. A.; Li, J.-T.; Caldwell, K. D.; Esumi, K. J. Phys. Chem. B 1999, 103 (36), 7665. (7) Cameron, G. G. ESR Spectroscopy. In Comprehensive Polymer Science: Polymer Characterization; Allen, S. G., Ed.; Pergamon Press: New York, 1989; p 517. (8) Martinie, J.; Michon, J.; Rassat, A. J. Am. Chem. Soc. 1975, 97, 1818. (9) Ji, J.; Feng, L. X.; Qiu, Y. X.; Yu, X. J. Polymer 2000, 41, 3713.
low- and high-field lines, respectively. As shown in Figure 2, the τc decreased with the increasing MW of the PEG, indicating that the mobility of the PEG chain end increased with its chain length. Actually, the τc of the probe at the PEG(8.7K) was close to that of the free probe in PBS buffer; viz., when the MW was greater than ca. 9K, the Brownian motion of the distal end of the PEG tethered chain become almost equal to that of the free molecule in solution. In Figure 2, the spin concentration is also plotted as a function of the MW of PEG. As can be seen in the figure, the spin concentration increased with the increasing MW of the PEG, indicating the increased reactivity of the end aldehyde group, which is strongly attributed to the mobility of the aldehyde group at the PEG chain end. This phenomenon corresponds very well with the specific lectin recognition (lactose-RCA120 system). At the same time, sugar-functionalized PEGylated surfaces adsorb less nonspecific lectins with the increasing PEG MW (Figure 1), which is also attributed to the increasing chain mobility. The high surface mobility of the PEG chain may be the key effect for both the specific biological interaction and the protein-resistant properties of the PEGylated surfaces. Note that the PEG chain density studied by X-ray photoelectron spectroscopy measurement, estimated from the ratio of PEG ether (C-O)% over PLA methine [Od C-C*(-C)-O]% from C1s high-resolution spectra, suggests a slight increase with PEG MW. In conclusion, the functionalized PEG layer on substrates serves as the basis for the design of a biointerface having both nonfouling and ligand-binding properties and may provide a convenient and promising platform for creating engineered biomaterials including highly selective biosensors and drug delivery and tissue engineering scaffolds. Acknowledgment. This study was partly supported by Special Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). LA0483414
