Biomacromolecules 2005, 6, 27-29
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Disintegration of Layer-by-Layer Assemblies Composed of 2-Iminobiotin-Labeled Poly(ethyleneimine) and Avidin Hiroyuki Inoue, Katsuhiko Sato, and Jun-ichi Anzai* Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan Received July 20, 2004; Revised Manuscript Received October 14, 2004
A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4′-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules. Introduction The layer-by-layer (LbL) deposition technique has been widely used for preparing nanometer-sized multilayer thin films by an electrostatic force of attraction, hydrogen bonding, and biological affinity.1-3 The materials used for preparing LbL films include synthetic polymers,4 proteins,5 nanoparticles,6 etc. The LbL thin films have found applications in optical devices,7 biosensors,8 bioreactors,9 and the controlled release of drugs.10 Heretofore, much attention has been devoted to the development of durable LbL films that are stable in the media where the LbL films are used because LbL thin films are often fragile due to their thin nature. In contrast to this concept, it has been recently shown that LbL films composed of poly(methacrylic acid) and poly(vinylpyrrolidone) can be disintegrated by a change in the environmental pH.11 A possible use of the films for the controlled release of dyes and drugs is suggested. We report here another example of a stimuli-sensitive LbL assembly that can be disintegrated by changing the environmental pH or by being exposed to biotin. The stimuli-sensitive LbL assembly was prepared using 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) and avidin. Avidin is a glycoprotein (molecular weight, 68 000) found in egg white and is known to contain four identical binding sites to biotin. The binding constant between avidin and biotin is reported to be ca. 1015 M-1.12 Due to this strong affinity, the avidin-biotin system has been employed for surface modification and the preparation of LbL assemblies.13 It is also known that avidin binds 2-iminobiotin less strongly than biotin and the affinity is pH dependent (the binding constant of 2-iminobiotin to avidin is 2.9 × 1010 M-1 in a * To whom correspondence should be addressed. E-mail: junanzai@ mail.pharm.tohoku.ac.jp.
basic solution while the value of the protonated form in acidic media is ca. 103 M-1).14 It is thus reasonable to assume that the LbL multilayer films prepared with ib-PEI and avidin are sensitive to the environmental pH and biotin. In fact, we have found that the avidin/ib-PEI multilayer films can be completely decomposed by changing the environmental pH or by adding biotin to the solution (Figure 1). Experimental Section Materials. Avidin was purchased from Calzyme Lab. Inc. ib-PEI was prepared by the reaction of poly(ethyleneimine) (PEI) (from Nakalai Tesque Co., Japan, molecular weight, 60 000-80 000) and 2-iminobiotin N-hydroxysuccinimide ester hydrobromide (Sigma Co.). PEI (333 mg) and 2-iminobiotin N-hydroxysuccinimide ester hydrobromide (42 mg) were dissolved in dry methanol (20 mL) and the mixture was stirred at room temperature overnight. The product was purified by dialysis against water. The PEI used has a random branched structure (the ratio of primary, secondary, and tertiary amino groups is nominally ca. 1:2:1). The content of the 2-iminobiotin residues in the ib-PEI was determined to be 2.3 mol % (based on the total primary and secondary amino groups) by the sulfur % from an elemental analysis. 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 Avidin/ib-PEI Multilayer Assemblies. To prepare the avidin/ib-PEI LbL assemblies, avidin and ib-PEI were alternately deposited on a quartz slide. The quartz slide (5 × 1 × 0.1 cm) was first treated in a 10% dichlorodimethylsilane solution in toluene overnight to make
10.1021/bm0495856 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/30/2004
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Biomacromolecules, Vol. 6, No. 1, 2005
Communications
Figure 3. Disintegration of 10-bilayer of avidin/ib-PEI film in buffer solutions (pH 5.0-8.0).
Figure 1. Disintegration of avidin/ib-PEI assembly by a pH change or by adding biotin.
Figure 2. UV-visible absorption spectra of avidin/ib-PEI 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 an avidin solution containing 10 mM biotin. All the avidin and ib-PEI solutions were prepared in 10-2 M borate buffer (pH 12).
the surface hydrophobic. The silylated quartz slide was immersed in an avidin solution (0.1 mg mL-1 in 10 mM borate buffer, pH 12) for 30 min at room temperature to deposit the first layer of avidin. After being rinsed with the buffer for 10 min, the quartz slide was immersed in an ib-PEI solution (0.1 mg mL-1 in 10 mM borate buffer, pH 12) for 30 min to deposit the ib-PEI. This deposition was repeated in order to build up the multilayer assemblies. Disintegration of the Avidin/ib-PEI Multilayer Assemblies. The disintegration of the avidin/ib-PEI multilayer assemblies was evaluated by measuring absorption spectra of the film assemblies. After immersing the films in a large volume of buffer solution for an appropriate time in the presence or absence of biotin and analogues, the absorbance of the film was measured at 280 nm to estimate the remaining amount of avidin in the film. All experiments were carried out at room temperature (ca. 20 °C). Results and Discussion Figure 2 shows the UV-visible absorption spectra of the layers 1-10 of the avidin/ib-PEI LbL films. The intensity of the 280 nm band in the spectra increased in proportion to the number of depositions, suggesting the formation of an LbL multilayer structure on the quartz slide. The inset shows the absorbance of the film at 280 nm (plot a), confirming a
linear growth of the LbL multilayer film after 4th deposition. The inset also contains data for the film prepared using the avidin solution that contained 10 mM biotin (plot b), where the binding sites of avidin should be preferentially occupied by the added biotin. The absorbance did not increase in the presence of biotin, explicitly showing that the driving force of the multilayer formation is not the nonspecific adsorption of avidin but an avidin/ib-PEI complexation. From the slope of the a, the loading of avidin in the LbL assembly was calculated to be ca. 0.6 × 10-10 mol cm-2 per deposition using the molar extinction coefficient () of avidin ( ) 97 400 at 280 nm). In other words, the loading of avidin in the LbL assembly is nearly 10-times higher than the calculated value for the monomolecular layer of avidin. This may originate from the randomly branched structure of the ib-PEI. The adsorbed ib-PEI probably assumes a shaggy surface because of the branched backbone, where several residues of 2-iminobiotin are able to locate along with the polymer chains that protrude vertically from the surface. Thus, each deposition may form a rather thicker layer. The binding constant of 2-iminobiotin to avidin is 2.9 × 1010 M-1 in a basic solution and is reduced to ca. 103 M-1 in an acidic media as the result of protonation.14 Consequently, the avidin/ib-PEI LbL assembly should be also sensitive to the environmental pH. The assembly may be unstable in an acidic environment due to the lower binding constant and the electrostatic repulsion between the protonated PEI chains and positively charged avidin (the isoelectric point of avidin is pH 9).12 The pH response of the assembly was evaluated by immersing the 10 bilayers of the avidin/ib-PEI LbL film in the buffer solutions at pHs 5, 6, 7, and 8. Figure 3 shows the avidin percent retained in the film as a function of the immersion time. The LbL assembly was stable in the pH 8 buffer, where no deterioration was observed for up to 30 min. More basic buffers (pHs 9, 10, and 11) also induced no deterioration in the film (data not shown). On the contrary, at pHs 5 and 6, the assembly was completely decomposed within a few minutes due to the reduced binding constant between 2-iminobiotin and avidin. This is not due to the denaturation (or deactivation) of avidin at pHs 5 and 6, because it is reported that avidin can strongly bind biotin in the pH 3-7 range.12 The electrostatic repulsion between the protonated ib-PEI and avidin is also responsible for the disintegration in the weakly acidic media. Thus, the avidin/ib-PEI LbL assembly can be disintegrated by changing the environmental pH from basic to weakly acidic. The pH response of the
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Biomacromolecules, Vol. 6, No. 1, 2005 29
exposure to 10-3 M lipoic acid or HABA. Thus, it is possible to arbitrarily control the decomposition rate of the avidin/ ib-PEI film by changing the type of stimulant and its concentration. Conclusion
Figure 4. Disintegration of 10-bilayer of avidin/ib-PEI film at pH 8.0 upon exposure to 10-5 M biotin and analogues.
present system is in clear contrast to that of the LbL films reported by Sukhishvili and Granick, where the films are stable in acidic media and decomposed in neutral and basic solutions.11 More interesting is the fact that the avidin/ib-PEI assembly can be disintegrated by adding biotin or analogues to the solution without changing the environmental pH. Figure 4 shows the disintegration of the avidin/ib-PEI film upon the addition of 10-5 M biotin and analogues in the buffer solution at pH 8. The avidin/ib-PEI assembly instantly decomposed upon exposure to the biotin solution. The decomposition of the assembly should be ascribed to the preferential binding of biotin to the binding sites of avidin due to the higher affinity of biotin, resulting in the expulsion of the 2-iminobiotin residues in the ib-PEI from the binding sites. It is known that biotin analogues such as desthiobiotin, lipoic acid, and 2-(4′-hydroxyphenylazo)benzoic acid (HABA) can be bound by avidin less strongly than biotin.15 These compounds may induce the decomposition of the avidin/ ib-PEI assembly. Figure 4 shows the decomposition of the assembly induced by 10-5 M of these compounds and 2-iminobiotin. All compounds tested facilitated the decomposition of the assembly more or less depending on the binding affinity to avidin. Desthiobiotin bound to the film effectively decomposed the assembly because of the high affinity to avidin (the binding constant, 2 × 1012 M-1).15 2-Iminobiotin was also effective for replacing the ib-PEI from the binding sites, suggesting that the binding constant of the 2-iminobiotin residues in the ib-PEI may be lower than that of the free 2-iminobiotin due to polymeric effects. On the other hand, the effects of lipoic acid and HABA were weak due to their lower affinity to avidin (the binding constants of lipoic acid and HABA are 1.4 × 106 M-1 and 1.7 × 105 M-1, respectively).15 We separately ascertained that the decomposition rates significantly depend on the concentration of the analogues. For example, the avidin/ib-PEI assembly was completely decomposed within a few minutes upon
We have found that the avidin/ib-PEI multilayer films can be completely decomposed by changing the environmental pH or by adding biotin to the solution. The present work strongly suggests a possible use of other binding proteins such as antibody and lectin to construct stimuli-sensitive LbL films that can be disintegrated by antigens and sugars. These types of films would be useful for designing the nanometersized devices that can release drugs and other functional molecules in response to environmental stimuli. Acknowledgment. This work was supported in part by a Grant-in-Aid (No. 16390013) from Japan Society for Promotion of Sciences (JSPS). References and Notes (1) Decher, G. Science 1997, 277, 1232. (2) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717. (3) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Chem. Soc., Chem. Commun. 1995, 2313. (4) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213. (5) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mowald, H., Eds.; Marcel Dekker: New York, 1999; p 125. (6) Caruso, F. In Colloids and Colloid Assemblies; Caruso, F. Ed.; WileyVCH: Weinheim, Germany, 2003; p 246. (7) (a) Lee, S.-H.; Kumar, J.; Tripathy, S. K. Langmuir 2000, 16, 10489. (b) McShane, M. J.; Brown, J. Q.; Guice, K. B.; Lvov, Y. M. J. Nanosci. Nanotechnol. 2002, 2, 1. (8) (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. (9) (a) Schuler, C.; Caruso, F. Macromol. Rapid Commun. 2000, 21, 750. (b) Disawal, S.; Qiu, J.; Elmore, B. B.; Lvov, Y. M. Colloids Surf. B: Biointerfaces 2003, 32, 145. (10) Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. J. Controlled Release 2003, 86, 59. (11) Sukhishvili, S. A.; Granick, S. J. Am. Chem. Soc. 2000, 122, 9550. (12) Wilchek, M., Bayer, E. A., Eds.; Methods in Enzymology; Academic Press: San Diego, CA, 1990; Vol. 184. (13) (a) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443. (b) Cosnier, S.; Pellec, A. L.; Marks, R. S.; Perie, K.; Lellouche, J. P. Electrochem. Commun. 2003, 5, 973. (c) Anzai, J.; Nishimura, M. J. Chem. Soc., Perkin Trans. 2 1997, 1887. (b) Anzai, J.; Hoshi, T.; Nakamura, N. Langmuir 2000, 16, 6306. (d) Hoshi, T.; Saiki, H.; Anzai, J. J. Chem. Soc., Perkin Trans. 2 1999, 1293. (e) Hoshi, T.; Saiki, H.; Anzai, J. Biosens. Nioelectron. 2000, 15, 623. (14) Hofmann, K.; Judith, G. T.; Montibeller, J. A.; Finn. F. M. Biochemistry 1982, 21, 978. (15) Masson, M.; Yun, K.; Haruyama, T.; Kobatake, E.; Aizawa, M. Anal. Chem. 1995, 67, 2212.
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