Langmuir 2005, 21, 797-799
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Sugar-Induced Disintegration of Layer-by-Layer Assemblies Composed of Concanavalin A and Glycogen Katsuhiko Sato, Yoko Imoto, Jun Sugama, Shunsuke Seki, Hiroyuki Inoue, Tsuyoshi Odagiri, Tomonori Hoshi, and Jun-ichi Anzai*
Figure 1. Complexation between Concanavalin A and sugar.
Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan Received August 1, 2004. In Final Form: October 9, 2004
1. Introduction Recently, a layer-by-layer deposition technique has been widely used for developing multilayer thin films.1 This technique usually relies on an alternate deposition of polycationic and polyanionic materials on the surface of a solid substrate through electrostatic force of attraction. It is also possible to use biological affinity such as antibody-antigen and avidin-biotin interactions to deposit materials to prepare multilayer films. Thus, the materials used for preparing multilayer films include synthetic polymers,2 proteins,3 DNA,4 nanoparticles,5 etc. Optical and electrical devices,6 biosensors and bioreactors,7 separation and purification,8 and the controlled release of drugs9 are typical applications of the multilayer thin films. Heretofore, much attention has been devoted to the development of durable multilayer films that are stable in the media where the films are used because multilayer thin films are often fragile due to their thin nature. In contrast, Sukhishvili and Granick recently showed that multilayer films composed of poly(methacrylic acid) and poly(vinylpyrrolidone) can be disintegrated by a change in the environmental pH.10 They suggested a possible use of the film for the controlled release of dyes and drugs. We report here another example of stimuli-sensitive multilayer films that can be disintegrated by being exposed to sugars. The stimuli-sensitive multilayer films were prepared using concanavalin A (Con A) and glycogen. Con * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Decher, G. Science 1997, 277, 1232. (2) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213. (3) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨wald, H., Eds.; Marcel Dekker: New York, Basel, 1999; p 125. (4) Sukhorukov, G. B.; Montrel, M. M.; Petrov, A. I.; Shabarchina, L. I.; Sukhorukov, B. I. Biosens. Bioelectron. 1996, 11, 913. (5) Caruso, F. In Colloids and Colloid Assemblies; Caruso, F., Ed.; Wiley-VCH: Weinheim, 2003; p 246. (6) (a) Lesser, C.; Gao, M.; Kristein, S. Mater. Sci. Eng. C 1999, 8-9, 159. (b) Lee, S. H.; Kumar, J.; Tripathy, S. K. Langmuir 2000, 16, 10482. (c) McShane, M. J.; Brown, J. Q.; Guice, K. B.; Lvov, Y. M. J. Nanosci. Nanotechnol. 2002, 2, 1. (d) Liu, A.; Anzai, J. Langmuir 2003, 19, 4043. (7) (a) Chen, T.; Friedman, K. A.; Lei, I.; Heller, A. Anal. Chem. 2000, 72, 3757. (b) Hoshi, T.; Saiki, H.; Kuwazawa, S.; Tsuchiya, C.; Chen, Q.; Anzai, J. Anal. Chem. 2001, 73, 5310. (c) Liu, A.; Ehara, M.; Takahashi, S.; Hoshi, T.; Anzai, J. Electrochemistry 2003, 71, 508. (d) Hoshi, T.; Saiki, K.; Anzai, J. Talanta 2003, 61, 363. (e) Onda, M.; Ariga, K.; Kunitake, T. J. Biosci. Bioeng. 1999, 87, 69. (f) Schuler, C.; Caruso, F. Macromol. Rapid Commun. 2000, 21, 750. (8) (a) Katayama, H.; Ishihama, Y.; Asakawa, N. Anal. Chem. 1998, 70, 2254. (b) Meier-Haack, J.; Lenk, W.; Lehmann, D.; Lunkwiz, K. J. Membr. Sci. 2001, 184, 223. (c) Krasemann, L.; Toutianoush, A.; Tieke, B. J. Membr. Sci. 2001, 181, 221. (9) (a) Tiourina, O. P.; Sukhorukov, G. B. Int. J. Pharm. 2002, 242, 155. (b) Radtchenko, I. L.; Sukhorukov, G. B.; Mo¨hwald, H. Int. J. Pharm. 2002, 242, 219. (c) Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. J. Controlled Release 2003, 86, 56. (10) Sukhishvili, S. A.; Granick, S. J. Am. Chem. Soc. 2000, 122, 9550.
Figure 2. A schematic illustration of a sugar-induced disintegration of a Con A-glycogen multilayer film.
A is a lectin protein (molecular weight 104 000) found in Jack bean and is known to contain four identical binding sites to sugars such as mannose and glucose (the binding between Con A and sugar is not a covalent binding but a reversible one, Figure 1).11 On the other hand, glycogen is a branched polysaccharide composed of D-glucose units. Thus, it should be possible to construct a multilayer thin film by depositing Con A and glycogen alternately on the surface of a solid. In fact, Lvov and co-workers have reported that the multilayer thin films can be prepared using Con A and glycogen through the biological affinity.12 However, they have not reported the stability or sensitivity of the films to sugars. It is reasonable to assume that the Con A-glycogen multilayer films are sensitive to sugars because the multilayer films rely on the reversible binding between Con A and D-glucose units in glycogen. Recently, glucose-sensitive multilayer films composed of Con A-dextran have been studied for developing optical glucose sensors.13 The present paper reports that the Con A-glycogen multilayer films are sensitive to sugars and can be fully disintegrated in the presence of D-glucose, D-mannose, and derivatives as schematically illustrated in Figure 2. 2. Experimental Section Materials. Con A and glycogen were purchased from Funakoshi Co. (Kyoto, Japan) and Tokyo Kasei Co. (Tokyo, Japan), respectively. Other reagents used were of the highest grade available and used without further purification. Apparatus. UV-visible absorption spectra were measured using a Shimadzu UV-3100PC spectrophotometer (Kyoto, Japan). Preparation of the Con A-Glycogen Multilayer Films. The multilayer films were prepared on the surface of a quartz slide (5 × 1 × 0.1 cm) according to the reported procedure with a minor modification.12 The quartz slide was first treated in diclorodimethylsilane (10% solution in toluene) overnight at room temperature, to make the surface hydrophobic, and was washed with toluene, acetone, and distilled water. For the formation of Con A-glycogen multilayer films, the silylated quartz slide was immersed in an Con A solution (100 µg mL-1, in 0.1 M Tris-HCl buffer containing 1 mM MnCl2 and 1 mM CaCl2, pH 7.4) for 30 min to deposit the first Con A through a hydrophobic force of attraction. In this study, Con A was dissolved in the buffer containing MnCl2 and CaCl2 for avoiding possible deactivation of Con A. After being rinsed in water for 5 min to remove any weakly adsorbed Con A, the quartz slide was immersed in a glycogen solution (100 µg mL-1 in 0.1 M Tris(11) Becker, J. W.; Reeke, G. N., Jr.; Edelman, G. M. Nature 1976, 259, 406. (12) (a) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Chem. Soc., Chem. Commun. 1995, 2313. (b) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 284-285, 797. (13) Chinnayelka, S.; McShane, J. M. J. Fluorescence 2004, 14, 585.
10.1021/la048059x CCC: $30.25 © 2005 American Chemical Society Published on Web 12/17/2004
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Figure 3. UV-visible absorption spectra of Con A-glycogen multilayer films on the surface of a quartz slide. (Inset) Absorbance of the films at 280 nm as a function of the number of depositions (plot a). Plot b is the data for the film prepared using a Con A solution containing 10 mM D-glucose. HCl buffer, pH 7.4) for 30 min to deposit the glycogen through a biological affinity between Con A and sugar residues in glycogen. This process provides both sides of the quartz slide with a Con A-glycogen bilayer. The second Con A layer was deposited similarly on the surface of the slide modified with a Con A-glycogen bilayer film through the biological affinity. The deposition was repeated to build up multilayer films, and absorption spectrum of the film-deposited quartz slide was recorded after each deposition of Con A to evaluate the loading of Con A in the film. To check the effects of D-glucose on the deposition of the Con A-glycogen films, a Con A solution (100 µg mL-1, in 0.1 M Tris-HCl buffer containing 1 mM MnCl2, 1 mM CaCl2 and 10 mM D-glucose, pH 7.4) was used for the deposition of Con A-glycogen film. Disintegration of the Con A-Glycogen Films. A sugarinduced disintegration of the Con A-glycogen films was evaluated by measuring absorption spectra of the films. After immersing the films in a large volume of buffer solution for an appropriate time in the presence and absence of sugar, the absorbance of the film was measured at 280 nm in the working buffer to estimate the remaining amount of Con A in the film. Tris-HCl (0.1 M) was used for preparing pH 8 and 7 buffers, while pH 6 buffer was prepared using 0.1 M phosphate. All experiments were carried out at room temperature (ca. 20 °C).
3. Results and Discussion Spectroscopic Characterization of the Con A-Glycogen Multilayer Films. The preparation of Con A-glycogen multilayer films has already been reported by Lvov and co-workers.12 They found that Con A-glycogen multilayer films can be prepared by an alternate deposition of Con A and glycogen on the surface of quartz resonator, by which the loadings of Con A and glycogen in the films were studied gravimetrically using a quartz crystal microbalance. In the present study, we prepared the Con A-glycogen multilayer films on the surface of a quartz slide to evaluate the films by means of UV-visible absorption spectroscopy. Figure 3 shows the UV-visible absorption spectra of the layers 1-10 of the Con A/glycogen multilayer films. The intensity of the 280 nm band in the spectra originating from Con A increased in proportion to the number of depositions, suggesting the formation of a multilayer structure on the quartz slide. The inset shows the absorbance of the film at 280 nm as a function of the number of depositions, confirming a linear growth of the multilayer film. On the other hand, when the Con A solution that contained 10 mM D-glucose was used for the deposition, the absorbance at 280 nm did not increase, as shown in the inset, because the binding sites of Con A were preferentially occupied by the D-glucose added in the solution. This explicitly shows that the driving force of the multilayer formation is not a nonspecific adsorption of Con A but a Con A-glycogen complexation.
Notes
Figure 4. Disintegration of the Con A-glycogen multilayer film (10 bilayers) in the presence of 10 mM D-glucose at pH 6 (b), 7 (2), and 8 (9).
From the slope of the plot in Figure 3, the loading of Con A in the Con A/glycogen film was calculated to be ca. 1.4 × 10-11 mol cm-2 per deposition using the molar extinction coefficient () of Con A ( ) 1.6 × 105 M-1 cm-1 at 280 nm). If Con A forms a close-packed monomolecular layer in the film, the density of Con A on the surface is calculated to be (0.5-1.1) × 10-11 mol cm-2 layer-1, depending on the orientation of Con A molecule (the molecular dimensions of Con A are reported to be 8.4 × 7.8 × 4.0 nm11). Consequently, the loading of Con A in the multilayer film is slightly higher than the calculated value for the monomolecular layer of Con A. This may originate from the randomly branched structure of glycogen. The adsorbed glycogen probably assumes a shaggy surface because of the branched backbone, where a few residues of glucose units are able to locate along with the polymeric chains that protrude vertically from the surface. Our results based on the spectroscopic measurements are basically in line with the gravimetric data reported by Lvov and co-workers, who reported the formation of a monomolecular layer of Con A in the Con A-glycogen films.12 Stability of the Con A-Glycogen Films. The stability of the Con A-glycogen multilayer films was studied in the sugar-free buffer solutions at pH 6, 7, and 8. The 10 layers of Con A-glycogen multilayer film were immersed in a large volume of buffer solutions at ca. 20 °C for 3 h, and the absorbance of the film was monitored at 280 nm every 20 min. Figure 4 shows that the absorbance of the film at 280 nm remained almost unchanged at pH 7 and 8, though ca. 20% decrease in the absorbance was observed at pH 6. Thus, the Con A-glycogen multilayer film was stable at pH 7-8. The instability of the film in the pH 6 buffer may originate from the fact that Con A tends to assume a dimeric form at pH 5-6.7 in contrast to the tetramer (see Figure 1) at higher pH.14 It can be envisaged that the Con A-glycogen film composed of dimeric Con A is less stable because the degree of crosslinking in the film should be lower than that in the films containing tetramer Con A. Thus, the Con A molecules on the outermost layer of the film may easily dissociate from the film in the pH 6 buffer. Disintegration of the Con A-Glycogen Films by Sugars. The response of the film to D-glucose was evaluated at pH 6, 7, and 8. For this purpose, the absorbance of the Con A-glycogen multilayer film was monitored at 280 nm for 3 h in the buffer solutions after adding 10 mM D-glucose (Figure 4). The absorbance of the film decreased in the presence of D-glucose in all buffer solutions tested, suggesting that the Con A-glycogen film is disintegrated in the presence of D-glucose as illustrated (14) Kalb, A.; Lustig, A. Biochim. Biophys. Acta 1968, 168, 11209.
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Figure 5. Disintegration of the Con A-glycogen multilayer film (10 bilayers) in the presence of 10 mM D-galactose (a), D-glucose (b), D-mannose (c), Me-R-Glu (d), and Me-R-Man (e) at pH 7.4.
in Figure 2.15 This should be ascribed to the preferential binding of the added D-glucose to the binding sites of Con A, followed by the expulsion of the D-glucose residues of glycogen from the binding sites, resulting in the dissociation of Con A from the multilayer film. It is likely that D-glucose penetrates the film smoothly. It is interesting to know the sensitivity of the Con A-glycogen film to different kinds of sugars. Figure 5 shows the changes in the absorbance of the 10 bilayers of Con A-glycogen film in the buffer solutions (pH 7.4) containing D-galactose, D-glucose, D-mannose, methyl R-Dglucopyranoside (Me-R-Glu), and methyl R-D-mannopyranoside (Me-R-Man). The absorbance of the film was recorded in the sugar-free buffer every 20 min. All compounds except for D-galactose facilitated the disintegration of the film more or less depending on the binding affinity to Con A. The film was decomposed nearly completely within 10 min in the presence of Me-R-Man and Me-R-Glu because of the high affinity of the methylated sugars to Con A (the binding constants of Me-RMan and Me-R-Glu are reported to be 2.1 × 104 and 4.9 × 103 M-1, respectively16). D-Mannose was also more effective than D-glucose for replacing the glycogen from the binding sites of Con A, resulting in a rapid decomposition of the Con A-glycogen film. It is known that D-mannose binds to Con A more strongly than D-glucose; the binding constants of D-mannose and D-glucose are reported to be 2.2 × 103 and 0.8 × 103 M-1, respectively.17 On the other hand, the Con A-glycogen multilayer film was scarcely decomposed upon addition of D-galactose. This is reasonable because D-galactose is known not to bind to Con A. These results clearly show that the disintegration of the film originates from a specific binding of the sugars to Con A. Figure 6 shows the disintegration of the Con A-glycogen multilayer film as a function of the concentration of sugars. For all cases tested, the disintegration of the film was facilitated when the film was exposed to higher concentration of sugars. A 50 mM D-glucose was required for the complete decomposition of the film, while the film was decomposed upon addition of 20 mM D-mannose or MeR-Glu. On the other hand, only 2 mM Me-R-Man sufficed to decompose the film completely. The effects of the different sugars can be rationalized by taking into account (15) The fraction that remained/% in Figures 4, 5, and 6 was calculated for Con A in the second to tenth layers because the first Con A layer was adsorbed to the quartz slide through hydrophobic force of attraction and, consequently, this layer cannot be desorbed upon addition of sugar. (16) Schwarz, F. P.; Puri, K. D.; Bhat, R. G.; Surolia, A. J. Biol. Chem. 1993, 268, 7668. (17) Mandel, D. K.; Kishore, N.; Brewer, C. F. Biochemistry 1994, 33, 1149.
Figure 6. Effects of the concentraion of D-glucose (b in panel A), D-mannose (9 in panel A), Me-R-Glu (panel B), and MeR-Man (panel C) on the rate of disintegration of the Con A-glycogen multilayer film (10 bilayers). The concentration of the sugar was as follows: (panel A) (a) 5 mM, (b) 10 mM, (c) 20 mM, (d) 50 mM, and (e) 100 mM; (panel B) (a) 5 mM and (b) 20 mM; (panel C) (a) 1 mM and (b) 2 mM.
of the different binding constants of the sugars to Con A as described above. In this context, Danielsson and coworkers reported that a monomolecular layer of Con A immobilized on a glucose-modified surface could be removed by being treated with 1 mg mL-1 (or 4 mM) p-aminophenyl R-D-glucopyranoside completely.18 They proposed a renewable biosensing surface based on the sugar-Con A interactions. 4. Conclusions We have demonstrated that Con A-glycogen multilayer films can be disintegrated upon exposure to sugars in the aqueous solution at neutral pH. This behavior originated from the competitive binding of the free sugars to the binding sites of Con A in the film, resulting in the expulsion of glycogen from the binding sites. The Con A-glycogen films decomposed nearly completely in the presence of 50 mM D-glucose, 20 mM D-mannose or Me-R-Glu, or 2 mM Me-R-Man, depending on the binding affinity of the sugars to Con A. The Con A-glycogen films would be useful for designing the sugar-sensing systems and sugar-sensitive delivery systems. Acknowledgment. This work was supported in part by a Grant-in-Aid (No. 16390013) from Japan Society for Promotion of Sciences (JSPS). LA048059X (18) Svitel, J.; Dzgoev, A.; Ramanathan, K.; Danielsson, B. Biosens. Bioelectron. 2000, 15, 411.
