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Langmuir 1999, 15, 482-488
Self-Assembled Monolayer of Sugar-Carrying Polymer Chain: Sugar Balls from 2-Methacryloyloxyethyl D-Glucopyranoside† Akira Yoshizumi, Naoki Kanayama, Yukari Maehara, Makoto Ide, and Hiromi Kitano* Department of Chemical and Biochemical Engineering, Toyama University, Toyama, 930-8555 Japan Received April 6, 1998. In Final Form: November 2, 1998
A sugar-carrying polymer chain with a disulfide group (DTPA-PMEGlc) was prepared by the coupling of poly(2-methacryloyloxyethyl D-glucopyranoside) (PMEGlc), which carried an amino group at its end, with 3,3′-dithiodipropionic acid di-p-nitrophenyl ester. The polymer obtained was incubated with colloidal silver, and a self-assembled monolayer (SAM) of the polymer chain (PMEGlc) was formed on the surface of the colloid as evidenced by surface-enhanced Raman spectroscopy, cyclic voltammetry, dynamic light scattering, and ellipsometry. The silver colloid was largely stabilized by the modification with PMEGlc chains due to the formation of a thick diffuse layer on the surface. The critical flocculation concentration (CFC) of the modified colloid was not detectable, which is a contrast to the presence of CFC for the starting silver colloid dispersion (0.1 M NaCl at 25 °C). The polymer-coated colloids obtained (“sugar balls”) were aggregated when a solution of lectin (concanavalin A (Con A) from Canavalia ensiformis) was added to the dispersion, due to a specific binding of D-glucopyranoside residues on the colloid particles by a tetrameric lectin molecule. The association constant (Kasn) for glucose residues on the colloid with Con A (7.1 × 105 M-1) was much larger than those for the complexation of Con A with small molecular weight sugars such as R-methyl D-glucopyranoside (4.9 × 103 M-1) due to the so-called “cluster effect”. Thermodynamic parameters for the binding of Con A to sugar residues in the SAM of PMEGlc clearly showed that the binding is governed by entropy change (∆S° ) 108 J/K‚mol). The usability of polymeric SAM in the biomedical field was strongly suggested.
Introduction Polymer colloids, which are industrially very important materials for paints, adhesives, and diagnoses, have been prepared by various methods including macroinitiator and macromonomer methods.1,2 For example, temperatureresponsive polymer microspheres were prepared by emulsion copolymerization of styrene and divinylbenzene with a macroinitiator carrying poly(N-isopropylacrylamide) (PIPA) chains at its ends,3 or a macromonomer carrying PIPA chain as a side group.4 The colloidal stability of the microspheres obtained was strongly affected by the temperature. Meanwhile, polymer compounds having glyco-conjugates are of interest because of their usefulness in biomedical fields (carrier for drug delivery system, substratum for cultivation of various cells, etc.).5 Using a lipophilic radical initiator, we previously prepared liposome-forming amphiphiles, which have a sugar-carrying polymer chain in their polar heads, and examined recognition of pendent sugar residues on the liposome surfaces by lectin or enzyme.6-8 Sugar-carrying block † Presented at the 46th Regional Meeting of the Society of Polymer Science, Japan, at Nagaoka in August, 1997. * To whom all correspondence should be addressed.
(1) Tauer, K. Polym. Adv. Technol. 1995, 6, 435. (2) Barakat, I.; Dubois, P.; Je´roˆme, R.; Teyssie´, P.; Mazurek, M. Macromol. Symp. 1994, 88, 227. (3) Kitano, H.; Kawabata, J. Macromol. Chem. Phys. 1996, 197, 1721. (4) Takeuchi, S.; Oike, M.; Kowitz, C.; Shimasaki, C.; Hasegawa, K.; Kitano, H. Makromol. Chem. 1993, 194, 551. (5) Lee, Y. G., Lee, R. T., Eds. Neoglycoconjugates; Academic Press: New York, 1994 (Methods in Enzymology, Vols. 242 and 247). (6) Kitano, H.; Ohno, K. Langmuir 1994, 10, 4131. (7) Kitano, H.; Sohda, K.; Kosaka, A. Bioconjugate Chem. 1995, 6, 131.
polymers were also obtained by the macroinitiator method, and the interfacial recognition of sugar residues by lectin was investigated by using the multiple internal reflection fluorescence method.9 In this report we examined preparation of colloidal particles that have sugar-carrying polymer chains on their surfaces (sugar balls)10 by using a self-assembled monolayer (SAM) technique.11,12 SAM is a monolayer which is spontaneously formed from organo-sulfur compounds such as aromatic and alkanethiols and disulfides on gold or silver surfaces via chemisorptive S-Au or S-Ag bonds, respectively. Therefore, its super thin structure has the controlled arrangement and orientation like cell membranes. To obtain sugar-carrying polymer colloids, 6-(2-methylpropenoyloxy)hexyl β-D-cellobioside or a liposaccharide monomer, 11-(N-p-vinylbenzyl)amido undecanoyl maltobioamide, was previously introduced into polymer microspheres by copolymerization with methyl methacrylate or styrene.13 Recently sugar-carrying microspheres were prepared by using a macroinitiator which had poly(2-methacryloyloxyethyl D-glucopyranoside) (PMEGlc) (8) (a) Ohno, K.; Sohda, K.; Kosaka, A.; Kitano, H. Bioconjugate Chem. 1995, 6, 361. (b) Ohno, K.; Kitano, H. Bioconjugate Chem. 1998, 9, 548. (9) Kitano, H.; Maehara, Y.; Matano, M.; Sugimura, M.; Shigemori, K. Langmuir 1997, 13, 5041. (10) Aoi, K.; Ito, K.; Okada, M. Macromolecules 1995, 28, 591. (11) Ulman, A. An Introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (12) (a) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (b) Wollman, E. W.; Fresbie, C. D.; Wrighton, M. S. Langmuir 1993, 9, 1517. (13) (a) Charreyre, M. T.; Boullanger, P.; Delair, T.; Mandrand, B.; Pichot, C. Colloid Polym. Sci. 1993, 271, 668. (b) Revilla, J.; Elaissari, A.; Pichot, C.; Gallot, B. Polym. Adv. Technol. 1995, 6, 55.
10.1021/la980374u CCC: $18.00 © 1999 American Chemical Society Published on Web 12/18/1998
SAM of Sugar-Carrying Polymer Chain Scheme 1
chains.14 Using the SAM technique,15 we can expect to introduce glyco-conjugate polymer chains, which are definitely characterized, onto colloidal silver particles (sugar balls) and vacuum-evaporated gold plates. Experimental Section Materials. 2-Methacryloyloxyethyl D-glucopyranoside (MEGlc, R:β ) 2.3:1) was kindly donated by Nippon Fine Chemicals, Osaka, Japan. 2-Methacryloyloxyethyl β-D-galactopyranoside (MEGal) was prepared from lactose and 2-hydroxyethyl methacrylate as previously reported.7,8 2,2′-Azobis(isobutyronitrile) (AIBN) from Wako Pure Chemicals, Osaka, Japan, was recrystallized from methanol. 3,3′-Dithiodipropionic acid di-p-nitrophenyl ester (DTPA-ONp) was prepared by the conventional synthetic procedure from 3,3′-dithiodipropionic acid, p-nitrophenol, and dicyclohexylcarbodiimide (DCC) in chloroform and purified by a silica gel column chromatography (mobile phase, chloroform) and subsequent recrystallization from chloroform-diethyl ether. Concanavalin A (Con A) from Canavalia ensiformis was purchased from Sigma, St. Louis, MO. Other reagents were commercially available. Deionized water was distilled just prior to use for preparation of sample solutions. Preparation of Poly(2-methacryloyloxyethyl D-Glucopyranoside) (PMEGlc) (Scheme 1). MEGlc (2.5 g, 8.6 × 10-3 mol), β-mercaptoethylamine hydrochloride (MEA, 49 mg, 4.3 × 10-4 mol, chain transfer reagent), and AIBN (14 mg, 8.5 × 10-5 mol, initiator) were dissolved in a methanol-water mixture (1: 3, 15 mL) in a three-necked flask with a condenser. After deoxygenation of the reaction solution by passing a N2 gas for 20 min, the solution mixture was incubated at 70 °C for 24 h. After evaporation of the solvent, the oily mixture was precipitated by acetone, and the precipitation procedure was repeated several times. The precipitate was dried in vacuo, dissolved in water, and finally lyophilized (PMEGlc, 2.43 g, yield 97%). The numberaverage molecular weight (Mn) of the polymer was evaluated as 5100 (degree of polymerization (DP), 17) by the conductometric titration of amino group at the end of the polymer with a N/100 aqueous NaOH solution. The tacticity of PMEGlc was analyzed (14) Kitano, H.; Kawabata, J.; Muramoto, T. Macromol. Chem. Phys. 1996, 197, 3657. (15) (a) Maeda, Y.; Kitano, H. J. Phys. Chem. 1995, 99, 487. (b) Yamamoto, H.; Maeda, Y.; Kitano, H. J. Phys. Chem. B 1997, 101, 6855.
Langmuir, Vol. 15, No. 2, 1999 483 with a 1H NMR apparatus (DX-400, JEOL, Tokyo, Japan) by comparing the proton signals of R-CH3 group in three kinds of poly(methyl methacrylate)s whose congfigurations (isotactic, syndiotactic, and atactic) had been clarified by X-ray analysis.16 Using a similar procedure, poly(2-methacryloyloxyethyl β-Dgalactopyranoside) (PMEGal, DP ) 17) was also prepared. Preparation of SAM-Forming Polymers (Scheme 1). PMEGlc (1.47 g, 2.9 × 10-4 mol) was dissolved in dry DMF (30 mL) in a round-bottomed flask, and DTPA-ONp (65 mg, 1.4 × 10-4 mol) and anhydrous triethylamine (TEA, 30 µL, 2.2 × 10-4 mol) were added. The solution mixture was continuously stirred at room temperature for 3 days. After evaporation of the solvent, the oily mixture was dissolved in water, and after filtration (to remove unreacted DTPA-ONp), the yellow-colored solution was dialyzed (seamless cellulose tube, Wako Pure Chemicals; exclusion limit, 12 000) against water for 6 days to remove unreacted PMEGlc (Mn ) 5100) and other small imparities. The PMEGlc linked by a disulfide bond was finally lyophilized (DTPAPMEGlc, 1.17 g, yield, 76%). The disappearance of free amino group at the end of PMEGlc after incubation with DTPA-ONp was confirmed by the conductometric titration. The amide bands (ν(CdO), δ(N-H)) newly appeared around 1640 cm-1 in IR spectra of the polymer product (KBr, FT-IR System 2000, Perkin-Elmer), while the bands attributable to the p-nitrophenyl group (νas(N-O) at 1520 cm-1, νs(N-O) at 1346 cm-1, and νs(C-N) at 868 cm-1) were absent. These results showed that the amino group at the end of PMEGlc completely reacted with DTPA-ONp (we could not completely exclude a possibility of the presence of 3,3′-dithiodipropionic acid linked with one PMEGlc chain). Similarly, a polymer DTPAPMEGal was also prepared from DTPA-ONp and PMEGal. Preparation of Silver Colloids and Gold Substrates Covered with SAM of PMEGlc. Silver colloid was prepared by the reduction of AgNO3 (6 mg) with NaBH4 (10 mg) in water (100 mL) at 0 °C as previously reported.15 The concentration of silver colloid was evaluated to be 360 µM by the conductometric titration with a N/1000 HNO3 aqueous solution. The colloidal dispersion (5 mL) was incubated with DTPA-PMEGlc (5.6 mg/ mL) for 1 day at room temperature and ultrafiltrated with an Amicon YM-30 membrane (exclusion limit for globular proteins, 30 000) (PMEGlc-Ag, Scheme 1). The average hydrodynamic diameters (dhd) of the silver colloids obtained were estimated to be 39.6 ( 5.8 nm (bare Ag colloid) and 51.5 ( 8.1 nm (PMEGlcAg) in 10-5 M NaCl by the dynamic light scattering (DLS) technique (DLS-7000, Otsuka Electronics, Hirakata, Osaka, Japan; light source, He-Ne laser 632.8 nm). For ellipsometry, gold substrates were prepared by vacuum evaporating deposition of chromium (thickness, 5 nm) followed by gold (thickness, 45 nm) onto the surface of a slide glass (18 × 18 × 0.9 mm). The freshly prepared gold substrates were incubated with a solution of DTPA-PMEGlc (10 mg/mL, ethanol: water ) 3:2) for 1 day at room temperature. The substrates were vigorously rinsed with water and ethanol and finally dried in vacuo. For infrared reflection-absorption spectroscopy (IRRAS), a SAM of PMEGlc chain was formed on a vacuumevaporated gold thin layer (thickness, 50 nm; deposited on a cover glass 12.5 × 19.0 × 0.32 mm; Japan Laser Electronics, Nagoya, Japan) under the same conditions as those for ellipsometry. Absorption Measurements. The sugar-carrying colloid dispersion (0.8 mL) was mixed with a lectin (Con A) solution (0.2 mL, dissolved in a HEPES (2-hydroxyethylpiperazine-2′-ethanesulfonic acid) buffer, 10 mM, pH 8.2, [MnCl2] ) [CaCl2] ) 0.1 mM) of various concentrations using a polyethylene mixing rod. The turbidity change (actually decadic absorbance) at 350 nm due to the aggregation of silver colloids was followed by using a UV-visible spectrophotometer (Ubest 35, Japan Spectroscopic Co., Tokyo, Japan). The observation cell was thermostated at 25 °C by a Peltier device (EHC-363, Japan Spectroscopic Co.). For the salt-induced coagulation experiments, the colloidal dispersion (0.5 mL) was mixed with an aqueous sodium chloride solution (0.5 mL) of various concentrations using the mixing rod, and the change in absorbance of the silver colloid at 395 nm was followed. (16) Bovey, F. A. High-Resolution NMR of Macromolecules; Academic Press: New York, 1972.
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To avoid the orthokinetic effect,17 the way of mixing was always kept constant (the rate of particle collision can be enhanced by the hydrodynamic forces created by the rapid stirring) in each experiment. Raman Scattering Measurements. The dispersion of silver colloid modified with PMEGlc chains (PMEGlc-Ag) was examined by a Raman spectrophotometer (NR-1100, Japan Spectroscopic Co.) with an argon laser at 488.0 nm. The observation cell was thermostated at 25 °C by a Peltier device (Model RT-IC, Japan Spectroscopic Co.). Electrochemical Measurements. Cyclic voltammetric (CV) measurements were performed with a potentiostat (HA-301, Hokuto-Denko, Tokyo, Japan) and function generator (HA-104, Hokuto-Denko). Outputs of the potentiostat were converted by an analog to digital converter and collected by a microcomputer (PC-486 SE, Epson, Suwa, Japan). Data analyses were carried out with a homemade program. Gold, Pt, and KCl saturated calomel electrodes (SCE) were used as working, counter, and reference electrodes, respectively. An electrochemical cell was thermostated at 25 °C by a circulating water bath (RM6, Lauda, Postfach, Germany). The gold electrode (area, 0.020 cm2) was incubated with DTPA-PMEGlc or PMEGlc solution (30 mg/mL) for 48 h and rinsed with water several times before the voltammetric experiment. The electroactive probe used was 2.5 mM K3[Fe(CN)6] dissolved in a 0.1 M Na2SO4 analyte solution. The scan rate was 10 mV/s. The electrochemical reductive desorption of DTPA-PMEGlc from the electrode was performed in a 0.5 M KOH solution at a scan rate of 100 mV/s.18-21 The corresponding process is represented by the following equation:
R-S-Au + e- ) Au + R-S-
(1)
Ellipsometry Measurements. Thickness of SAMs was measured using a DVA ellipsometer (Mizojiri Optical Co., Ltd., Tokyo, Japan) employing a 632.8 nm He-Ne laser at an incident angle of 70°. Data at five points were averaged for a given sample. The refractive index for the SAM of PMEGlc was assumed to be 1.489, in accordance with the value for poly(methyl methacrylate).22 Infrared Reflection-Absorption Spectroscopy (IRRAS) Measurements. The SAM of PMEGlc chain formed on a vacuum-evaporated gold thin layer was examined by the infrared reflection-absorption spectroscopic (IR-RAS) technique using the infrared spectrophotometer. Each spectrum was obtained by 304 scans at 8 cm-1 resolution, with a light incident on the metal substrate at 80°. Calculation of Association Constant (Kasn). The Kasn values for complexation of PMEGlc-Ag, PMEGlc, and MEGlc with Con A were estimated by the Scatchard plot ( eq 2).23
[PL]/[L] ) Kasn(n[P]0 - [PL])
(2)
where n is the number of binding site in the protein molecule. [PL], [L], and [P]0 are the concentration of protein-ligand complex, that of ligand, and the initial concentration of protein, respectively. The plot of [PL]/[L] versus [PL] gives a straight line with a slope of -Kasn and x-intercept of n[P]0Kasn. The initial concentration of sugar residues contained in the PMEGlc solution or PMEGlc-Ag dispersion was determined by a phenol-H2SO4 method beforehand.24 The solution of glucose derivative (PMEGlc or PMEGlc-Ag) was incubated with Con A at 25 °C for 1 h, and the Con A-sugar complex in the mixture was (17) Everett, D. H. Basic Principle of Colloid Science; Royal Society of Chemistry: London, 1988. (18) Walczak, M. M.; Popenone, D. D.; Deinhammer, R. S.; Lamp, B. D.; Chung, C.; Porter, M. D. Langmuir 1991, 7, 2687. (19) Widrig, C. A.; Chung, C.; Porter, M. D. J. Erectroanal. Chem. 1991, 310, 335. (20) Zhong, C. J.; Porter, M. D. J. Am. Chem. Soc. 1994, 116, 11616. (21) Rojas, M. T.; Ko¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336. (22) Bohn, L. in Polymer Handbook, 3rd ed.; Brandrup, J., Immerght, E. H., Eds.; John Wiley & Sons: New York, 1989; pp VI/459. (23) Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 60. (24) Dubois, M.; Gilles, K.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem. 1956, 28, 350.
Figure 1. Raman and SERS spectra for various systems excited at 488.0 nm: (a) aqueous solution of DTPA-PMEGlc (1 mM); (b) SAM on silver colloids, [DTPA-PMEGlc] ) 50 µM; (c) aqueous solution of 3,3′-dithiodipropionic acid (1 mM). precipitated by a centrifugation (15 000 rpm, 15 min) at 25 °C. The concentration of free Con A in the supernatant was determined from the absorbance at 280 nm by using the UVvisible spectrophotometer. To check a possibility of physical adsorption of Con A, the bare Ag colloid (365 µM) was incubated with a Con A solution (1.02.5 mg/mL) and subsequently centrifuged. Only the bare Ag colloid was precipitated, and Con A completely remained in the supernatant at any initial concentration (99.7 ( 4.1% of the initial concentration of Con A). Therefore, it can be said that the interaction between the bare colloidal silver and Con A is negligibly small, and the Kasn value obtained for the PMEGlc-Ag system is reliable. In the MEGlc system, the unbound MEGlc in its solution mixture with Con A was recovered by an ultrafiltration using a syringe-type filter (USY-5, Advantec Toyo, Tokyo, Japan; exclusion limit for globular proteins, 50 000) and quantified by the phenol-H2SO4 method.
Results and Discussion A. Chemisorption of PMEGlc on Silver Colloids. Figure 1a shows a Raman spectrum of aqueous DTPAPMEGlc solution (1 mM). There seems to be no distinct peak attributable to either DTPA nor chain transfer reagent moieties in the polymer. After incubation of DTPA-PMEGlc with the silver colloid, on the other hand, a Raman spectrum of the dispersion mixture showed a new distinct peak at 647 cm-1 corresponding to a stretching vibration of C-S bond (ν(C-S)) in the polymer due to the so-called “surface-enhanced Raman (SER) effect” (Figure 1b).15 The concentration of DTPA-PMEGlc in the dispersion (50 µM) was too low to be detected without the SER effect. When the SAM is formed on the metal surface, the C-S bond exists in the closest neighborhood of the metal surface. Since the intensities of Raman scattering by molecular moieties neighboring the metal surface are most largely enhanced by the SER effect, the C-S band should be most clearly observed in comparison with other bands. Figure 1b is consistent with this explanation and shows that the SAM is formed on the silver colloid. It should be mentioned here that the concentration of DTPA-PMEGlc examined in Figure 1a (1 mM) was large enough, because all the distinct bands in the figure could be definitely attributed to glucose residues (34 mequiv‚L-1). In Figure 1c a Raman spectrum of 3,3′-dithiodipropionic acid (the essential moiety in the DTPA-PMEGlc molecule for the formation of SAM on the silver colloid) at 1 mM is also shown. From these spectra, it can be said that the relative intensities of both ν(C-S) and ν(S-S) bands in the polymer were negligibly weak in comparison with those of the bands for glucose residues in the same wavenumber region. Consequently, although the C-S band for alkanethiols adsorbed on Ag was frequently observed to
SAM of Sugar-Carrying Polymer Chain
shift to smaller wavenumber region by about 5-10 cm-1,25 we could not compare the C-S band of PMEGlc on the colloidal Ag with those in solid state and aqueous solution of DTPA-PMEGlc. In Figure 1b, a weak peak at 721 cm-1 could be also observed in addition to the distinct peak at 647 cm-1. Previously, Joo et al. and Bryant et al. extensively studied the ν(C-S) bands of solid, liquid, and adsorbed alkanethiols (CnH2n+1SH, n ) 2-17) on silver and reported that the bands of trans and gauche conformers (ν(C-S)T and ν(C-S)G) exist at 729-737 cm-1 and 651-655 cm-1 in solid and liquid states, respectively.25 Therefore, the two peaks at 721 and 647 cm-1 in Figure 1b could be attributed to ν(C-S)T and ν(C-S)G, respectively, which means that the gauche conformer was dominant in the SAM of the sugar-carrying polymers. The 1H NMR spectra showed that PMEGlc had an atactic configuration (R-CH3 mm:mr:rr ) 1:30:64). It is known that the tacticity of poly(methyl mathacrylate) is affected by various factors at the polymerization.26 For example, an isotactic configuration appears when catalyzed by BuLi, whereas the atactic configuration appears in ordinary radical polymerization.26 Because of the atactic configuration of the polymer chains, and the influence of steric hindrance by pendent sugar residues, therefore, the conformation of PMEGlc chains in the vicinity of the C-S bond at the surface of Ag colloids might not be in a highly ordered manner, which is a contrast to twodimensional crystalline structure of disulfides and thiols with small molecular weight on gold(111).27 Recently, it has been reported that thioethers (dodecyl sulfide, thiophene, etc.) also form monolayer on the metal surface (Ag or Au) as well as disulfides and thiols.28-30 Since DTPA-PMEGlc has two thioether groups and a disulfide group, there is a possibility that the polymer made adsorption on the metal surface with the thioether groups which are attributable to the chain transfer reagent. We could not exclude this possibility by using Raman scattering measurements. This point will be discussed in detail in the next section using cyclic voltammetry and ellipsometry. B. Chemisorption of PMEGlc on Gold Substrates. DTPA-PMEGlc was also chemisorbed on the gold substrate and characterized by using both cyclic voltammetry and ellipsometry. The gold electrode treated with DTPAPMEGlc exhibited a very small redox activity for [Fe(CN)6]3- (potential difference between cathodic and anodic peaks ∆Ep ) 435 mV), whereas the electrode treated with PMEGlc (which has a thioether group) exhibited a much larger redox activity (∆Ep ) 83 mV). This value is not far from that for bare electrode (∆Ep ) 60 mV), which suggests that the DTPA-PMEGlc forms a well-packed monolayer structure while the PMEGlc forms a much lesspacked one. In addition, the gold electrode treated with DTPAPMEGlc exhibited an irreversible cathodic peak in the 0.5 M KOH solution (cathodic peak potential Epc ) -0.92 (25) (a) Joo, T. H.; Kim, K.; Kim, M. S. J. Phys. Chem. 1986, 90, 5816. (b) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 3629. (c) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 8284. (26) Hatada, K. In Experimental Procedures in Polymer Science Vol. 1; Ed. by the Society of Polymer Science, Japan; Kyoritsu: Tokyo, 1979. (27) Nelles, G.; Scho¨nherr, H.; Jaschke, M.; Wolf, H.; Schaub, M.; Ku¨ther, J.; Treme, W.; Bamberg, E.; Ringsdorf, H.; Butt, H.-J. Langmuir 1998, 14, 808. (28) Motesharei, K.; Ghadiri, M. R. J. Am. Chem. Soc. 1997, 119, 11306. (29) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G. Langmuir 1988, 4, 365. (30) Dishner, M. H.; Hemminger, J. C.; Feher, F. J. Langmuir 1996, 12, 6176.
Langmuir, Vol. 15, No. 2, 1999 485 Chart 1
V versus SCE) which can be assigned to the reductive desorption of chemisorbed DTPA-PMEGlc. On the other hand, the electrode treated with PMEGlc did not exhibit the irreversible cathodic peak at all. These results support that DTPA-PMEGlc was adsorbed to the gold electrode with the cleavage of the S-S bond and following Au-S bond formation. By integration of the cathodic peak, a surface coverage of DTPA-PMEGlc was estimated as 7.2 × 10-11 mol/cm2, and using this value, an occupied area of DTPA-PMEGlc was evaluated as 2.3 nm2. Assuming that DTPA-PMEGlc forms a monolayer only with disulfide cleavage and following formation of Au-S bond at a tilt angle of 30°,12b the occupied area is calculated to be about 2.25 nm2 (the radius of the PMEGlc chain is assumed to be equal to the distance between the main chain and the C1 of glucose residue at the all-trans configuration, 0.75 nm), which is in agreement with the experimental result. The occupied area of DTPA-PMEGlc having bulky side chains is very large, and no space seems to exist for the Au-thioether bond between the metal surface and DTPA-PMEGlc. Moreover, the thickness of the PMEGlc layer on the gold substrate was measured to be 4.5 nm by ellipsometry. This value was slightly smaller than the calculated value of DTPA-PMEGlc at the all-trans configration (5.3 nm). Therefore, a well-packed SAM with more or less stretched conformation seemed to exist at the flat gold substrate (Chart 1). Taking account of the previous report that the rate constant for adsorption of didecyl sulfide was 2500 times as small as that of didecyl disulfide,31 we consider that both thioether and disulfide groups in DTPAPMEGlc can adsorb on the metal surface, but the disulfide group was much more effective than the thioether for the adsorption in the present system. As mentioned in the Experimental Section, the DLS data showed that the hydrodynamic diameter of the silver colloid was increased after the incubation with DTPAPMEGlc (39.6 ( 5.8 f 51.5 ( 8.1 nm), which is in agreement with the value obtained by the ellipsometry (total length of two DTPA-PMEGlc layers, 9 nm) and not inconsistent with the value calculated for DTPA-PMEGlc chemisorbed on the silver colloid mainly via Ag-S-C linkage. Consequently, we consider that DTPA-PMEGlc formed a SAM on the Au flat substrate via Au-S-C linkage in a manner similar to the globular silver colloids. C. Stability of Silver Colloids. To examine a colloidal stability of the sugar-carrying silver colloids (“sugar balls”), the salt-induced coagulation of the colloids was examined. (31) Jung, Ch.; Dannenberger, O.; Xu, Y.; Buck, M.; Grunze, M. Langmuir 1998, 14, 1103.
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Figure 2. Relationship between the initial absorption change and the concentration of sodium chloride at 25 °C: (b) bare Ag colloids; (O) PMEGlc-carrying Ag colloids (PMEGlc-Ag). Wavelength ) 395 nm. [Ag colloid] ) 360 µM.
By the addition of electrolyte the stability of silver colloids was largely reduced because of the shielding of electrostatic repulsion between the colloid particles.17 Figure 2 shows a plot of the initial change in absorbance (dAbs/dt)o of bare silver colloid versus concentration of NaCl added. The DLS data showed that the hydrodynamic diameter of the colloid increased correspondingly to the absorption change. The bending point in Figure 2 (0.1 M) is called a “critical flocculation concentration” (CFC), above which there is no effective electrostatic repulsion between the particles, which results in a rapid coagulation where the process is diffusion-controlled.17 As for the silver colloid modified with PMEGlc chains (PMEGlc-Ag), on the contrary, there was no significant change in absorbance after the addition of electrolyte (even at 3 M of NaCl). The absence of CFC in the case of PMEGlcAg is due to the enormous steric stabilization effect of PMEGlc layer stretching outward from the surface of the colloids, which drastically decelerates the coagulation. Previously Tamai et al. reported that the introduction of polyacrylamide diffuse layer on polymer microspheres diminished the salt-induced coagulation of the microspheres.32 Recently we also showed that the introduction of poly(2-methacryloyloxyethyl D-glucopyranoside) chains onto poly(styrene-co-divinylbenzene) microspheres diminished the CFC.14 These results are consistent with the present data. Consequently, PMEGlc-Ag could be used in buffer solutions to examine the recognition of sugar residues on its surface by Con A as discussed in following sections. D. Recognition of Sugar Residues on Colloids by Lectin. Figure 3 shows plots of the rate of turbidity change, (dAbs/dt)o, versus the concentration of Con A added to the dispersion of silver colloid which had been modified with two kinds of sugar-carrying polymer chains (PMEGlc and PMEGal) at various concentration ratios beforehand. The tetrameric protein, Con A, can bind sugars located on different colloid particles at the same time and induce the aggregation of the colloids. The occurrence of the aggregation of silver colloids corresponding to the turbidity increase was confirmed by the DLS measurements. Con A is well-known to have a strong interaction with D-glucopyranosides but not with β-D-galactopyranosides,33 which is consistent with the result that the rate of turbidity change increased with a reduction in the concentration ratio of PMEGal (Figure 3). The turbidity of PMEGlc-Ag-Con A mixture was largely reduced by the subsequent addition of R-methyl Dmannopyranoside (R-Me-Man) which has a strong affinity for Con A (Figure 3 inset). In addition, the rate of turbidity (32) Tamai, H.; Fujii, A.; Suzawa, T. J. Colloid Interface Sci. 1987, 118, 176. (33) Sharon, N.; Lis, H. Lectins; Chapman & Hall: New York, 1989.
Yoshizumi et al.
Figure 3. Effect of concentration of Con A on the rate of turbidity change after the addition of Con A into the dispersion of sugar-carrying Ag colloids at 25 °C: (b) Ag colloid covered with DTPA-PMEGlc (DP ) 17), (9) DTPA-PMEGal (DP ) 17); DTPA-PMEGlc:DTPA-PMEGal (O) 2:1, (2) 1:1, (3) 1:2. Wavelength ) 350 nm. [Ag colloid] ) 290 µM. Inset: R-MeMan (20 mM) was added at the arrow. Abscissa, time (s); Ordinate, absorbance (OD).
Figure 4. Inhibitory effect of sugar on the rate of turbidity change at 350 nm in the agglutination of PMEGlc-Ag by Con A. PMEGlc SAM, DP ) 17. [Con A] ) 0.6 mg/mL. In a HEPES buffer (10 mM, pH 8.2). [CaCl2] ) [MnCl2] ) 0.1 mM. [Ag colloid] ) 290 µM.
change was extremely decreased with the increase in the concentration of coexisting R-Me-Man (Figure 4), which definitely supports the attribution. E. Estimation of Association Constant (Kasn ). The association constant (Kasn) for the MEGlc-Con A system was determined by the Scatchard plot as 4.9 × 104 M-1 at 25 °C, which is more or less larger than those for the complexation of Con A with small sugar derivatives (Rmethyl D-mannopyranoside, 2.1 × 104 M-1; R-methyl D-glucopyranoside, 4.9 × 103 M-1).34 This may be attributed to the presence of nonpolar groups (methyl or vinyl, etc.) in the MEGlc molecule. It was previously reported that the Kasn value of 4-methylumbelliferyl R-D-mannopyranoside (MUM) with Con A was about 5-fold larger than that of R-methyl D-mannopyranoside due to the presence of large nonpolar aglycon.35 The Kasn values for PMEGlc-Con A and PMEGlc-AgCon A systems were 2.7 × 105 M-1 and 7.1 × 105 M-1, respectively. It has been frequently reported that the Kasn values of sugar residues in the polymers are much larger than those for sugars with small molecular weight due to the so-called “cluster effect” (both the number of sugar residues together with the respective propinquity confer to the glycosylated clusters their improved overall binding affinity).36 The Kasn value for the PMEGlc-Ag system was, however, smaller (ca. 1/3 times) than that for the com(34) So, L. L.; Goldstein, I. J. Biochim. Biophys. Acta 1968, 165, 398. (35) Frank, G. L.; Robert, M. C.; Thomas, M. J. Biochemistry 1977, 16, 159. (36) Lee, R. T.; Lee, Y. C. In Neoglycoconjugates: Preparation and Application; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, CA, 1994; p 23.
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Figure 5. van’t Hoff plots for the complexation of Con A with various sugar derivatives: (O) PMEGlc-Ag; (b) PMEGlc, and (4) MEGlc. Table 1. Thermodynamic Parameters for the Complexation of Con A with Various Sugar Derivativesa temp Kasn ∆H° ∆G° b ∆S° (°C) (105 M-1) (kJ/mol) (kJ/mol) (J/K‚mol) PMEGlc-Ag (SAM) PMEGlc (homopolymer) MEGlc (monomer)
15 25 35 15 25 35 15 25 35
7.20 7.05 6.96 4.43 2.71 2.13 0.60 0.49 0.24
-1.0
-33.4
108
-27.0
-31.0
13
-33.9
-26.7
-24
a In a HEPES buffer (10 mM, pH 8.2). [CaCl ] ) [MnCl ] ) 0.1 2 2 mM. b At 25 °C.
plexation of Con A with a triblock copolymer, PMEGlcb-PSt-b-PMEGlc (DP of blocks: 23 (MEGlc block), 5 (styrene block) and 23 (MEGlc block)), deposited on the PMMA surface (Kasn ) 2.0 × 106 M-1).9 This is probably because of the difference in the evaluation methods (the static method in the former and the kinetic method (Kasn ) k forward/k backward) in the latter). The effective concentration of sugar residues to which Con A can have access in the PMEGlc-Ag dispersion might be much smaller than the total concentration because of the large steric hindrance of the SAM (only the outermost sugar residues on the PMEGlc-Ag might be recognized by Con A), resulting in the small Kasn value in the static method. F. Thermodynamic Parameters for the Kasn Values. Temperature dependence of the association constants for three systems was examined by UV-vis spectroscopy, and van’t Hoff plots for the determination of ∆H° are shown in Figure 5. The other thermodynamic parameters (∆S° and ∆G°) were calculated by eqs 3 and 4
∆G° ) -RT ln Kasn
(3)
∆S° ) (∆H° - ∆G°)/T
(4)
As shown in Table 1, ∆H° values were all negative (PMEGlc: ∆H° ) -27.0 kJ/mol, MEGlc: ∆H° ) -33.9 kJ/mol, PMEGlc-Ag: ∆H° ) -1.0 kJ/mol), and the absolute value of ∆H° for the PMEGlc-Ag-Con A system was the smallest among them. The changes in enthalpy can be interpreted in terms of differences in the interactions between the solvent and lectin and between the ligand and lectin. These interactions explicitly involve hydrogen bonding, and electrostatic and van der Waals interactions which can be identified from both knowledge of the positioning of the ligand at the binding site of lectin and energy minimization calculations.37
The positive ∆S° value for PMEGlc and PMEGlc-Ag could be mainly attributed to the release of water from the binding site of Con A and the sugar residues into bulk solvent.38 Only the ∆S° value for MEGlc was negative (-24 J/K‚mol), probably because the structured water on the surface of Con A is not almost broken during the binding processes of Con A to sugars with small molecular weight. It has been reported that neither Con A nor its sugar ligands with small molecular weight undergo any appreciable alternation during their interaction.39 Furthermore, a recent microcalorimetric study showed that the specific binding of Con A to different sugars is accompanied by a differential uptake of water molecules during the binding process.40 The positive ∆S° for the PMEGlc-Con A system examined here could be attributed to the break of water cluster structures around the solutes with large size (both PMEGlc and Con A). This tendency was more conspicuous in the PMEGlc-Ag-Con A system due to the existence of gigantic and hydrophilic colloids. Therefore, the driving force estimated by the thermodynamic analysis is as follows. As for the MEGlc-Con A system, the driving force of the association is the large negative ∆H° (-33.9 kJ/mol), so is in the PMEGlc-Con A system (∆H° ) -27.0 kJ/mol). On the other hand, the association of Con A with PMEGlc-Ag is clearly governed by the entropy change (∆S° )108 J/K‚mol). This is probably because, at the binding of pendent sugars to Con A, the release of water around Con A and sugar residues on the gigantic carrier colloid is more dominant than the interaction between the binding site of Con A and sugar residues. Previous investigations of the binding thermodynamics of galactose-binding lectin (L-14) with various R-D-galactopyranosides showed that the reaction was enthalpically driven (for example, ∆H° ) -48 kJ/mol and ∆S° ) -78 J/K‚mol for thiodigalactopyranoside).41 However, the binding of 4-methylumbelliferyl-R-galactopyranoside, which has a bulky nonpolar aglycon, displays relatively favorable entropic contribution (∆H° ) -10 kJ/ mol and ∆S° ) 42 J/K‚mol),41 supporting our results. G. IR-RAS Observation of Recognition of Sugar Residues on the SAM Bound to Gold Layer by Lectin. To observe the specific recognition of sugars by Con A directly, we examined IR-RAS of the PMEGlc SAM. There appeared absorption bands corresponding to O-H(ν) at 3200-3500 cm-1, CH2(νas) at 2936 cm-1, CH2(νs) at 2885 cm-1, CdO(νs) at 1730 cm-1, and C-O(νs) at 1148 cm-1 (Figure 6a), all of which are attributable to the PMEGlc chains introduced. Figure 6b shows the absorption spectra of PMEGlc-SAM incubated in a solution of Con A (1 mg/ mL) for 1 h at 25 °C. The newly appeared bands (N-H(ν) at 3298 cm-1, CdO(ν) at 1645 cm-1, and N-H(δ) at 1534 cm-1) refer to amide bonds in the protein, Con A. Figure 6c shows the spectra for the SAM of PMEGlc incubated first with the Con A solution for 1 h and subsequently with a solution of R-Me-Man (10 mM) for 1 h at 25 °C. These spectra clearly show that the specific recognition of sugar residues in the PMEGlc-SAM by Con A occurred, and the Con A bound to the SAM was completely dissociated by the incubation with the sugar with low (37) Imberty, A.; Hardman, K. D.; Carrer, J. P.; Perez, S. Glycobiology 1991, 1, 631. (38) Creighton, T. E. Proteins: Structures and Molecular Properties, 2nd ed.; W. H. Freeman & Co.: New York, 1993. (39) Naismith, J. H.; Field, R. A. J. Biol. Chem. 1996, 271, 972. (40) (a) Swaminathan, C. P.; Gupta, D.; Sharma, V.; Surolia, A. Biochemistry 1997, 36, 13428. (b) Swaminathan, C. P.; Surolia, N.; Surolia, A. J. Am. Chem. Soc. 1998, 120, 5153. (41) Ramkumar, R.; Surolia, A.; Podder, S. K. Biochem. J. 1995, 308, 237.
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it quite easy to control the stability of dispersion of the particles.14,32,42,43 In this regard, the usage of polymeric SAM for preparation of stable colloids with various surface morphologies and functions in the biomedical field would be very hopeful.
Figure 6. IR-RAS spectra of (a) SAM of DTPA-PMEGlc (DP ) 17) on gold. (b) SAM of DTPA-PMEGlc after incubation with Con A solution (1 mg/mL). (c) SAM of (b) after further incubation with R-Me-Man solution (10 mM).
molecular weight (R-Me-Man) because of its strong affinity for Con A, supporting the discussion in sections D, E, and F. It is well-known that the introduction of polymer chains (in other words, diffuse layers) onto the surface of colloidal particles by graft reaction or physical adsorption makes
Acknowledgment. This work was supported by the Grant-in-Aid for Scientific Research on Priority Areas (08246222, 09240213, 09232223, 10126221) from the Ministry of Education, Science, Sports and Culture. The authors are grateful to Dr. Y. Tsujii and Mr. K. Ohno, Institute for Chemical Research, Kyoto University, for their kind measurements of ellipsometry. The authors also wish to thank Nippon Fine Chemicals for their kind donation of MEGlc. LA980374U (42) Napper, D. In Polymeric Stabilization of Colloidal Dispersions; Academic Press: New York, 1983. (43) Kitano, H.; Fukui, N.; Ohhori, K.; Maehara, Y.; Yoshizumi, A. To be submitted.
