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Liquid Crystalline Gel with Refractive Index Gradient of Curdlan Toshiaki Dobashi,* Masahiro Nobe, and Hiromi Yoshihara Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
Takao Yamamoto Department of Physics, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan
Akira Konno Department of Food and Nutrition, Faculty of Human Life Sciences, Senri Kinran University, Suita, Osaka 565-0873, Japan Received September 30, 2003. In Final Form: June 1, 2004 Curdlan dissolved in aqueous sodium hydroxide was dialyzed to aqueous calcium chloride to form a gel. Transparent and turbid concentric layers observed in the gel cross section perpendicular to the long axis of the dialysis tube were identified as liquid crystalline gels with refractive index gradient and amorphous gels, respectively. The thickness of each layer was proportional to the diameter of the dialysis tube, and the gelation proceeded in proportion to the root of time. The unique pattern formation was attributed to the change of curdlan conformation and calcium-induced cross-linking resulting from a diffusion of calcium cations and hydroxide anions through the dialysis tube. It is suggested that the orderedness of the curdlan molecules decreases by the increase of the curvature of the concentric liquid crystal layers as the layer comes toward the center of the dialysis tube.
Introduction Since the well-known Graham’s work with vegetable parchment and the membrane from an ox bladder,1 the membrane technology using selective diffusion (or dialysis) has been developed in a variety of fields. Typically, the hemodialysis is now commonly used for removing the end products of nitrogen metabolism and the bicarbonate deficit of the metabolic acidosis.2 The dialysis is also useful to change the structure of macromolecules mildly. For example, the equilibrium dialysis is one of the most general strategies to crystallize biological macromolecules in biotechnology.3,4 In this kind of dialysis, the biopolymers confined in a semipermeable tubing form a homogeneous ordered structure that is induced by the change of the interaction between the biopolymers and the dispersing medium transferred from the outside. The orderedness of the structure depends on the affinity among the components of the medium at the both sides, and it is expected that the specific interactions between the biopolymers and the medium components could result in multiform unique structures. In the present paper, we report a new finding of the structure, liquid crystalline gel (LCG) with refractive index gradient, consisting of one of the polysaccharides, * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: +81-277-30-1477. (1) Graham, T. Philos. Trans. R. Soc., London 1861, 151, 183-224; The Bakerian lecture - on osmotic force. Philos. Trans. R. Soc., London 1854, 144, 177-228. (2) Bland, L. A.; Favero, M. S. Microbiologic aspects of hemodialysis systems. In AAMI Standards and Recommended Practices; Association for the Advancement of Medical Instrumentation: Arlington, VA, 1993; Vol. 3, pp 257-265. (3) McPherson, A. Crystallization of Biological Macromolecules; CSHL Press: New York, 1999. (4) Bergfors, T. M. Protein Crystallization: Techniques, Strategies and Tips; IUL Biotechnology Series, La Jolla, CA, 1999.
curdlan, induced by a simple dialysis. LCG has attracted recent interest in the field of electrooptics.5-7 One of the most useful preparation techniques utilizes the selfassembly of amphiphilic rodlike molecules. Synthetic thermally reversible gelators such as cholesteryl derivatives, gluconamide complexes, and glycolipids are typical examples of the amphiphilic molecules.8-10 Such LCGs are also interesting as green recyclable alternatives to the traditional organic compounds.10 The methodology to prepare the curdlan LCG reported here enables us to obtain another green LCG, with functionally gradient characteristics. Curdlan, a bacterial polysaccharide, consists entirely of linear β-1,3-glucan.11,12 The specific chain conformation of curdlan in aqueous solutions has been studied by light scattering, dynamic viscoelasticity, static viscosimetry, X-ray analysis, NMR, etc.13-17 Curdlan forms a triple helix (5) Kato, T. Science 2002, 295, 2414-2418. (6) Kato, T. In Molecular Self-Assembly: Organic vs Inorganic Approaches; Fujita, M., Ed.; Springer-Verlag: New York, 2000. (7) Firestone, M. A.; Thiyagarajan, P.; Tiede, D. M. Langmuir 1998, 14, 4, 4688-4698. (8) Lin, Y.-C.; Kachar, B.; Weiss, R. G. J. Am. Chem. Soc. 1989, 111, 5542-5551. (9) Hafkamp, R. J. H.; Kokke, B. P. A.; Danke, I. M.; Geurts, H. P. M.; Rowan, A. E.; Feiters, M. C.; Nolte, R. J. M. Chem. Commun. 1997, 545-546. (10) Kimizuka, N.; Nakashika, T. Langmuir 2001, 17, 6759-6761. (11) Harada, T.; Fujimori, K.; Hirose, S.; Masada, M. Agric. Biol. Chem. 1967, 31, 1184. (12) Harada, T.; Misaki, A.; Saito, H. Arch. Biochem. Biophys. 1968, 124, 292-298. (13) Nakata, M.; Kawaguchi, T.; Kodama, Y.; Konno, A. Polymer 1998, 39, 1475-1481. (14) Takeda, H.; Yasuoka, N.; Kasai, N.; Harada, T. Polym. J. 1978, 10, 365-368. (15) Saito, H.; Ohki, T.; Sasaki, T. Biochemistry 1977, 16, 908-914. (16) Tada, T.; Matsumoto, T.; Masuda, T. Carbohydr. Polym. 1999, 39, 53-59.
10.1021/la035822z CCC: $27.50 © 2004 American Chemical Society Published on Web 07/09/2004
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and random coils in aqueous solutions of low and high sodium hydroxides, respectively. The critical concentration of sodium hydroxide for the conformation change is around 0.23 N, where the densest structure is constructed.13,18 The specific optical rotation angle of curdlan is around +30-35° in dilute sodium hydroxide and is very small in concentrated sodium hydroxide (given by the manufacturer (Wako Pure Chemical Ind. Ltd., Japan)). The triple helix structure is believed to relate with the inhibitory effects of curdlan against HIV virus and malicious tumors, and a lot of scientific studies have been performed from this aspect.19-21 Curdlan is also known to yield various types of gel, depending on the chain conformation.15,22-25 Basic properties of calcium-induced gelation of curdlan were reported by Konno et al. in a series of papers.18,25,26 The gel is prepared simply by a dialysis of curdlan in aqueous sodium hydroxide into aqueous calcium chloride without any heating. Interestingly, the gel has multiple cylindrical structure parallel to the long axis of the cylindrical dialysis tube.26 To investigate the mechanism is of academic interest, since the characteristic pattern could be only constructed by inhomogeneity of constituent molecules, and the interaction force between the layers would be very weak. The task is even more challenging for developing a new methodology to prepare functionally gradient LCG, as shown below. However, the mechanism of this unique phenomenon has not been clarified yet. To obtain insight into the mechanism, we traced the gelation process, estimated the calcium content in each layer, and measured the birefringence of the gel prepared with the dialysis tube of various sizes. It is strongly suggested that the unique pattern formation results from the change of the orderedness of the curdlan molecules by the increase of the curvature of concentric liquid crystal layers as the layer comes toward the center of the dialysis tube. Experimental Results Curdlan was purchased from Wako Pure Chemical Co. Ltd. and used without further purification. The molecular weight of the curdlan was determined as 5.9 × 105 from intrinsic viscosity in 0.3 M NaOH solution at 25 °C using the viscosity-molecular weight relation9 [η] ) 0.0079Mw0.78 cm3 g-1. Reagent grade sodium hydroxide and calcium chloride and Milli-Q water were used for preparation. A desired amount of curdlan was dissolved in 0.4 M NaOH at 7 wt %. One hundred milliliters of a curdlan solution poured into seamless cellulose tubing with 28.8 mm diameter (UC-36-32, Sanko Junyaku, Japan) was dialyzed to 2 L of 10 g/dL calcium chloride bath at 25 °C, which was completely wrapped to prevent any effects of air. No supernatant calcium carbonate was observed during the experiment at this condition. We note that without (17) Watase, M.; Nishinari, K. Rheology of curdlan-DMSO-water system. In Food hydrocolloids: structures, properties and functions; Nishinari, K., Doi, E., Eds.; Plenum Press: New York, 1993; pp 125129. (18) Konno, A. Kinran Tanki Daigaku Kenkyushi 1997, 28, 185-192 (in Japanese). (19) Sasaki, T.; Takasuka, N. Carbohydr. Res. 1976, 47, 99-104. (20) Sasaki, T.; Abiko, N.; Sugano, Y.; Nitta, K. Cancer Res. 1978, 38, 379-383. (21) Yoshioka, Y.; Tabeta, R.; Saito, H.; Uehara, N.; Fukuoka, F. Cancer Res. 1985, 140, 93-100. (22) Harada, T. ACS Symp. Ser. 1977, No. 45, 265-283. (23) Harada, T. In Polysaccharides in Food; Blanshard, J. M. W., Mitchell, J. R., Eds.; Butterworths: London, 1979; pp 283-300. (24) Saito, H.; Yoshioka, Y.; Yokoi, M.; Yamada, J. Biopolymers 1990, 29, 1689-1698. (25) Konno, A.; Kimura, H. Kinran Tanki Daigaku Kenkyushi 1992, 23, 173-182. (26) Konno, A.; Tsubouchi, M. Kinran Tanki Daigaku Kenkyushi 1998, 29, 89-95 (in Japanese).
Figure 1. (a) Illustration of a round slice of curdlan gel and two strips excised from the slice. The observed directions are shown by the arrows. (b) The upper view (in the direction of A in Figure 1a) of the slice of the curdlan gel observed under natural light. The figures at the side and the bottom show the length scale in millimeters.
wrapping, increasing amounts of calcium carbonate supernatants were produced on the surface of the calcium chloride bath with time. The curdlan solution gelled within 4 h, and a rodlike gel with the diameter of 25.6 mm was formed. Thus, the size reduction by gelation was 10% in diameter and 29% in volume. A round slice of the gel with the thickness of 5 mm and its two cross sections were excised out as shown in Figure 1a. The upper view of the slice (A) and the side views of the cross sections shown by the arrows B and C were observed. The gel consists of concentric layers, as shown in Figure 1b observed under natural light. The transparent and turbid layers appear alternatively from the rim to the center. Those rings were observed from one end of the dialysis tube to another end continuously to make concentric pipes. The outermost transparent pipe was easily peeled off at the boundary with the turbid layer by a weak force. In the upper view (A in Figure 1a) observed under crossed nicols, two orthogonal lines appear except near the center, as shown in Figure 2a. The outermost multiple concentric layers with various colors in Figure 2a correspond to the first (outermost) transparent layer observed in Figure 1. The thickness of each step with the same color is around 10 µm at the rim and gradually increases on approach to the center. The average thickness of the step with the same color is several hundred micrometers. On approach to the center, a narrow black layer with the
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Figure 2. The curdlan gel observed under polarized light from the direction of A and B in Figure 1a. The figures at the side and the bottom show the length scale in millimeters.
radius of 5.1 mm, a wide white layer, and the second black layer with the radius of 1.8 mm, are observed sequentially, as shown in Figure 2a. These concentric layers correspond to the first turbid layer, the second transparent layer and the second turbid layer in Figure 1b, respectively. The central line observed in the innermost layer was swirled by rotating the sample around the center under crossed nicols, indicating a large optical rotation. Figure 2b shows the front view observed from the direction of the arrow B in Figure 1a. The lines corresponding to the different colors in Figure 2a are observed in Figure 2b. On the other hand, no transmitted light was observed under the crossed nicols in the direction of the arrow C in Figure 1a. The birefringence measurements showed that the average retardation is ∆n ) 2.0 × 10-4 at the first transparent layer and ∆n ) 3.2 × 10-5 at the second transparent layer. The similar experiment was performed with dialysis tubes of different diameters, and the thickness of each layer was measured for each gel. The same pattern with the same average retardation at each layer was observed. Figure 3 shows the thickness of the outermost transparent layer δo, the first turbid layer δm and the second transparent layer δi, as a function of the diameter of the dialysis tube d. All the observed thicknesses were proportional to d. To estimate the calcium content of each concentric layer of the gel, 0.02 g of gel sheet was excised from each layer and dissolved in 20 mL of 0.02 M EDTA-4Na. The solution was mixed with 2 mL of ammonium buffer at pH 10, 1 mL of 0.1 M Mg-EDTA, 2 drops of eriochrome black T (EBT) as the indicator, and an appropriate amount of pure water to make 50 mL of solution. Calcium content of the gel layer was determined by the back-titration with 0.02 M Ca+ standard solution. The average calcium concentrations of the outermost transparent layer, the turbid layer, and the second transparent layer were 0.66, 0.70 and 0.59 mol/g, respectively. The standard deviation was 0.01 mol/g.
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Figure 3. Thickness of outer transparent layer δ0 (a), turbid layer δm (b), and inner transparent layer δi (c).
Figure 4. Gel thickness as a function of dialysis time.
To trace the gelation process, we assembled a sample cell as follows. Two PMMA plates of 20 mm diameter, the upper one of which had a small pit for pouring the sample, were arranged face to face, connected with a central Teflon rod that was 1 mm in diameter and 5 mm long and surrounded with a dialysis membrane. An appropriate amount of 5 wt % curdlan in 0.3 M sodium hydroxide was poured into the cell from the small pit on the upper plate, and then the pit was sealed. The cell was settled in a large amount of 8 g/dL calcium chloride solution at 20 °C. The front line of the gel was traced with time, and the distance from the dialysis tube to the gel front line was measured with a cathetometer. No difference in the rates between gelation and liquid crystal growth was detected. As shown in Figure 4, the thickness of the growing gel was proportional to the root of time. It shows that the growth process is dominated by a diffusion. We tried to find a similar calcium-induced gel for different polysaccharides such as κ-carrageenan, carboxymethylcellulose, and curdlan dissolved in dimethyl sulfoxide, but no concentric rings were observed. Curdlan aqueous solution gelled with a variety of divalent cations but did not gel with any monovalent cations. We did not observe the rings in curdlan gels prepared with divalent cations except for calcium cations. Thus, the unique
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Figure 5. Illustration of curdlan molecules in the gel from the upper view. R0 and R1 are the radii of the gel and the outer surface of the turbid layer, respectively. The orderedness of the molecules is drawn exaggerated as a guide for the eyes.
pattern formation observed in this study is, at least for the present, unique to the calcium-induced gel of curdlan dissolved in sodium hydroxide. The characteristic phenomena appeared in a wide range of concentrations of curdlan, calcium chloride, and sodium hydroxide, although the birefringence decreased with decreasing sodium hydroxide and disappeared below the critical value for the triple helix formation at 0.23 N. Therefore, these factors can only modify the quantitative properties of the gel, such as the number of layers and the time required for liquid crystal growth and/or gel formation. For example, the number of layers increased with increasing temperature. The polydispersity of curdlan is not significant for the phenomena, since essentially the same pattern formation was observed using fractionated and unfractionated curdlans. Discussion The colored patterns observed in Figures 1 and 2 suggest liquid crystal formation (probably nematic) generated in the outermost transparent layer in the gel, and the different colors in the layer show a gradient of orderedness, as illustrated in Figure 5. The thickness of each colored step of several hundred micrometers is comparable to that of common polymer liquid crystals. The turbid layer in Figure 1b could be assumed to consist of curdlan molecules at amorphous state. The lower birefringence indicated by no color observed in the second transparent layer might be attributed to a lower degree of crystallinity. The swirling of the central line suggests a high optical rotation near the center of the gel. The observation that each layer was peeled off by a weak force is consistent with our model of curdlan conformational change at the interface of each layer. It is interesting to speculate the mechanism of this liquid crystal growth coupled with gel formation, since no such findings have been reported on hydrogels. When curdlan in aqueous 0.3 M sodium hydroxide is brought in contact with aqueous calcium chloride through the dialysis tube, hydroxide ions are transferred to the dispersing medium and calcium ions are transferred into the dialysis tube simultaneously by diffusion. According to the experimental result that the gel thickness is proportional to the root of
dialysis time, the gelation coupled with liquid crystal growth arises predominantly from the diffusion. Since the curdlan molecules have lower affinity to the outside calcium chloride solution than the inside sodium hydroxide solution, the molecules might take a conformation with low contact area to the outside solution. The direction of the curdlan molecules in the ordered structure is perpendicular to the circumference of the circle on which the molecules sit. Therefore, the radial arrangement of the curdlan molecules illustrated in Figure 5 matches this requirement. The angle between the directions of the neighboring molecules becomes large as the positions of the molecules move closer to the center. Since the interaction energy between the molecules increases with the angle, the orderedness of the radial arrangement structure lessens as the position moves closer to the center. The sequential change in color in the outermost layer is attributed to the slight change in the orderedness. That is, since the color corresponds to the wavelength satisfying the diffraction condition, the slight change of the orderedness is consistent with the sequential color variation. The ordered liquid crystal structure is supposed to be formed through the nucleation-growth process. On the basis of the nucleation-growth picture, we can derive the relationship between the thickness of the outermost liquid crystal layer R1 and the radius of the dialysis tube R0. First, the nucleation points of the liquid crystal structure appear on the inside of the dialysis tube. The average distance between two adjacent nucleation points is independent of the radius R0. We denote the distance between two adjacent nucleation points along the crosssectional surface as l. From the nucleation points, the liquid crystal grows toward the center of the dialysis tube. The growth process is expected to stop when the growth fronts collide. An amorphous structure grown there resets the growth condition. The shortest distance between the growth fronts is taken to be lc when the growth fronts collide. The number of the nucleation points and that of the growth fronts should be the same. Therefore, we have
2πR0 2πR1 ) l lc
(1)
Thus, the thickness of the outermost liquid crystal layer δo is expressed as
( )
δ 0 ≡ R0 - R 1 ) 1 -
lc R l 0
(2)
Since l and lc should only depend on the thermodynamic condition of “the curdlan solution” but not on the radius of the dialysis tube, δ0 is proportional to R0. Therefore, the experimental result of the proportionality, δ0 ∝ R0, is consistently derived on the basis of our model. Although the roles of calcium ions and hydroxide ions (or pH change) have not been clarified with a characterization of the gel on a microscopic or mesoscopic level, we can discuss them using the experimental results reported in previous papers and the current study. Konno assumed25 the dissociated hydroxide anions yielded at 6C carbons to cross-link with calcium anions on the analogy of gelation of amylose.27 As the gelation of curdlan is induced only by divalent cations and not by monovalent cations, it is natural to consider that the calcium ions cross-link the curdlan molecules by ionic bonding or coordinate bonding. From a calcium content of around 6.6 × 10-4 mol/g in the gel and the monomer molar weight of (27) Rao, V. S.; Foster, J. F. Biopolymers 1963, 1, 527-544.
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curdlan, 161, it is estimated that there exist two calcium ions per three curdlan monomers in the gel. From this result it may be assumed that the calcium ions in the gel only partly contribute to the cross-linking. However, since the gel shrank very slowly still after 1 month from the start of the dialysis, the mechanism of the whole gelation process may not be so simple. Curdlan takes two conformations: random coil at high sodium hydroxide and triple helix at low sodium hydroxide. In the dialysis process the hydroxide anions are released from the inside curdlan solution and then the curdlan conformation changes from random coil to triple helix. Since the calcium ions induce cross-links between curdlan molecules at both random coil state and triple helical state, the competition of liquid crystal growth and gelation might determine the degree of orderedness. The strong birefringence at lower sodium hydroxide results from this situation. The relatively slow diffusion of calcium ions could be attributed to Coulombic repulsion between extra calcium cations in the curdlan gel surface and calcium ions coming later. Full support of our conclusions awaits studies of LCG formation using small-angle scattering (and/or small-angle neutron scattering) and extended X-ray absorption fine structure for mesoscopic and molecular-level characterization, respectively, that will be carried out soon. Since curdlan is one of FDA approved polysaccharides,28 the curdlan gel having the unique structure could be utilized as a drug delivery carrier. The effective drugs (28) Jezequel, V. Cereal Foods World 1998, 43, 361-364.
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often produce harmful side effects. In such a case, the sustained release of various types such as stepwise release and constant release are required, while the common exponential type release is not preferred.29 If we prepare curdlan gels by the current method from a curdlan solution containing such drugs, they distribute inhomogeneously in the gel. For example, we can localize the drugs near the center of the gel by considering the size of the drug and the affinity between the drug and curdlan. In this case, a constant release from the gel is realized. When the drugs are localized between the steps, a stepwise release might be obtained. As we can prepare a microspherical Curdlan LCG using the same principle as developed in the current study, it is applicable to some oral prescriptions. In conclusion, we prepared a curdlan LCG with refractive index gradient, based on an advantage of the specific interaction of curdlan in sodium hydroxide with calcium chloride only by means of a simple dialysis. The interface of the dialysis tube partition between the calcium solution and the curdlan solution plays an essential role to yield unique LCG. Acknowledgment. We are grateful to Professor Mitsuo Nakata in Hokkaido University for his continuous encouragement. LA035822Z (29) Langer, R. Science 1990, 249, 1527-1533.
