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Langmuir 2005, 21, 2-4
Letters Liquid Crystalline Gel Beads of Curdlan Toshiaki Dobashi,* Hiromi Yoshihara, Masahiro Nobe, and Michiru Koike 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 August 23, 2004. In Final Form: October 30, 2004 Curdlan beads consisting of liquid crystalline gel (LCG) and amorphous gel (AG) in alternating layers in a wide range of diameters were newly prepared by interfacial insolubilization reactions using calcium chloride as the setting reagent. The thickness of the liquid crystalline layer was proportional to the diameter of the gel bead, and the proportional constant agreed with that determined for the cylindrical gel prepared by a dialysis method. The proportional constant initially increased with increasing calcium concentration of the dispersing medium and saturated at a high concentration limit. These results suggest that the mechanisms for forming the alternating LCG/AG structures prepared with different boundary conditions are the same. The LCG/AG structure could be controlled by calcium concentration.
Chemical reactions and physical associations of macromolecular species are coupled to generate a variety of mesoscale structures that attracted recent attention in nano/microtechnology. We showed in a previous paper that even simple dialysis could help polymers form a unique ordered structure with a refractive index gradient: In the process of the dialysis of Curdlan dissolved in aqueous sodium hydroxide into aqueous calcium chloride, the outflux of hydroxide anions changes the conformation of the Curdlan molecules from random coil to triple helix due to the pH change and the influx of calcium cations cross-links the helical Curdlan molecules intermolecularly, resulting in cylindrical liquid crystalline gel (LCG) and amorphous gel (AG) in alternating layers.1 The method for preparing the unique LCG/AG could be useful for manufacturing biodegradable optical components such as a polarized lens. The aim of this paper is to apply this method to prepare and characterize LCG/AG beads, which could be utilized for a new type of drug delivery carriers consisting of Curdlan, one of the U.S. Food and Drug Administration (FDA) approved polymers.2 The drugs loaded at the interstitial layers of LCG could be released in proportion to time, in contrast to the unfavorable exponential release in conventional systems. Ample knowledge of microencapsulation techniques is helpful for finding the optimal method for preparing spherical gel beads.3 In this study, we use the insolubilization * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: +81-277-30-1477. (1) Dobashi, T.; Nobe, M.; Yoshihara, H.; Yamamoto, T.; Konno, A. Langmuir 2004, 20, 6530-6534. (2) Jezequel, V. Cereal Food World 1998, 43, 361-364.
reaction with calcium chloride at various concentrations as a setting reagent.4 When a droplet of viscous polymer solution is dipped into the reactive solvent, an insolubilization reaction occurs on the spherical surface of the droplet. If the interfacial layer formed by the initial reaction plays the role of the dialysis membrane, the dialysis from the spherical surface is realized. The task is also interesting from an academic aspect, since the results give us information on the similarity of two- and three-dimensional LCG/AGs and the effect of calcium ions on the structure. 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 aqueous solution at 25 °C. Reagent grade sodium hydroxide and calcium chloride and Milli-Q water were used for preparation. A desired amount of Curdlan was dissolved in 0.3 M NaOH at 5 wt %. An aliquot of the solution was sprayed into 8 g/dL calcium chloride aqueous solution in a beaker through injection nozzles with different apertures to set gels at room temperature around 25 °C. The beaker was completely wrapped to prevent it from any effects of air and left at rest for 24 h to reach an equilibrium state. Gel beads with a wide distribution of radius from 0.16 to 3.2 mm were prepared in this way. For the experimental study on the effect of calcium concentration, the Curdlan solution was dipped drop by drop into calcium chloride aqueous solution with various concentrations to prepare gel beads (3) Kondo, T. In Surface and Colloid Science; Matijevic, E., Ed.; Plenum: New York, 1978; Vol. 10, pp 1-43. (4) Kondo, T. J. Oleo Sci. 2001, 50, 1-11.
10.1021/la047901w CCC: $30.25 © 2005 American Chemical Society Published on Web 12/08/2004
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Langmuir, Vol. 21, No. 1, 2005 3
Figure 2 shows the proportional relationship between the averaged radius and the averaged thickness of the LCG layer as shown by open circles in the whole experimental range of radius from 160 µm to 3.2 mm. The proportional coefficient was determined by a least-squares fit to be 0.37 ( 0.01. The proportional line is extrapolated to large radii to successfully express the corresponding data for the cylindrical gels prepared by dialysis (closed circles), as shown in the inset of Figure 2. On the basis of the nucleation-growth (NG) picture, the relationship between the thickness of the outer liquid crystal layer, δ, and the radius of the dialysis tube, R, is determined by the growth dynamics of multi-LCG nuclei and is expressed as1 Figure 1. Cross section of Curdlan gel beads observed under natural light (a) and crossed nicols (b).
δ ) CR
(1)
with
C)1-
Figure 2. Relationship of the thickness of the LCG layer of the cross sections and the diameter of the gel bead (O) or the cylindrical gel (b).
with a similar radius of 2.3 ( 0.1 mm. The gel particles were then incised to obtain the cross section passing through the center. The optical photographs of the cross section observed under natural light and crossed nicols are shown in parts a and b of Figure 1, respectively. The outer transparent layer, the turbid ring, and the inner transparent core in Figure 1a correspond to the outer transparent layer with high birefringence, the entirely dark ring, and the inner slightly transparent core with low birefringence in Figure 1b, respectively. The birefringence of LCG at the external surface is larger than that at the boundary with AG. These results suggest that the beads consist of LCG with high orderedness, AG, and LCG with low orderedness from the external spherical surface to the center. The Curdlan molecules or their aggregates are radially arranged in the LCG layer, and the orderedness in each LCG layer decreases as the position approaches the center. The crossed dark lines observed in Figure 1b also support this model. The radius of the gel beads, R′, and the thickness of the LCG layer, δ′, were measured with a caliper and averaged for more than 10 beads, except that we could not measure δ′ in the range R′ < 150 µm because of low birefringence. For comparison, 30 mL of the Curdlan solution was poured into seamless cellulose tubing with a diameter in the range from 6.4 to 28.6 mmφ (UC-36-32, Sanko Junyaku, Japan) and dialyzed to 300 mL of 8 g/dL calcium chloride bath at 25 °C. The cylindrical gels prepared by dialysis were cut out perpendicular to the long direction. The pattern similar to the beads was observed in the cross section perpendicular to the long axis of the dialysis tubing. The radius of the gel cross section, R, and the thickness of the LCG layer, δ, were measured.
lc l
(2)
In the above equation, l is the distance between two adjacent nucleation points along the dialysis tube for cylindrical gels or the external surface of the droplet for spherical beads and lc is the shortest distance between two adjacent growing-nucleolus fronts, for example, the distance between the fronts when the growth fronts collide. The NG picture shows that eq 1 should be applied for the gels with a wide range of radii, R, for R . a, where the length a denotes the size of Curdlan molecules or their aggregates. This also asserts that eq 1 including the proportional coefficient, C, is validly satisfied in both twoand three-dimensional geometrical boundary conditions, since the values of l and lc should be constant irrespective of the geometrical structure of dialysis. The proportional relationship in Figure 2 and the inset of this figure indicates that eq 1 holds at least down to R ∼ 330 µm, irrespective of two-dimensional and three-dimensional LCG/AGs. This experimental result supports the universality of eq 1. From the observed proportional coefficient, C ) 0.37, we obtain lc/l ) 0.63 for both two-dimensional and three-dimensional LCGs. The mechanism of this unique phenomenon is considered as follows. Curdlan takes two conformations, random coil at high sodium hydroxide and triple helix at low sodium hydroxide.5 When the Curdlan in aqueous 0.3 M sodium hydroxide is made in contact with aqueous calcium chloride at the droplet surface, the Curdlan molecules on the surface change the conformation from random coil to triple helix and they are cross-linked by calcium cations6 to form a thin gel membrane. Through the membrane, hydroxide ions are transferred to the dispersing medium and calcium ions are transferred into the droplet simultaneously by diffusion. Since the Curdlan molecules have a lower affinity to the outside calcium chloride solution than the inside sodium hydroxide solution, the molecules 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 Curdlan molecules are radially arranged. 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 increasing angle, the orderedness (5) Harada, T. ACS Symp. Ser. 1977, 45, 265-283. (6) Konno, A.; Kimura, H. Kinran Tanki Daigaku Kenkyushi 1992, 23, 173-182.
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Figure 3. Proportional coefficient, C, between the thickness of the LCG layer and the diameter of the gel beads as a function of the calcium chloride concentration in the dispersing medium. The arrow indicates the estimated calcium concentration in the gel.
of the radial arrangement structure lessens as the position moves closer to the center. The proportional coefficient, C, in eq 1 does not depend on the geometrical structure of dialysis but depends on the chemical properties of the dispersing medium. Figure 3 shows the calcium concentration dependence of C. The coefficient C increases with increasing calcium concentration and saturates at a high calcium concentration limit. The calcium concentration [CaCl2]G in the outermost LCG layer in the gel prepared with the same condition was 0.66 mol/g, or 7 g/dL, which is indicated by the arrow in Figure 3. The molar ratio of [CaCl2]G and Curdlan is close to that determined in the previous paper 2.12 under a different condition of 0.4 M NaOH, 7 wt % Curdlan, and 10 g/dL dispersing [CaCl2], that suggests a quantitative reaction of calcium cations and hydroxide groups of Curdlan, or two calcium cations corresponds to one glucose unit. Here, we note the value of [CaCl2]G was mistaken to be described as 0.66 mol/g, not the correct value 0.97 mol/g in the previous paper.1 Curdlan gels are induced by calcium ion binding (calcium gel) and also induced by hydrogen bonding7 (hydrogen bonding gel), and a competition of the two gelation mechanisms results in a variety
Letters
of structures of Curdlan gel. Even when the dispersing medium contains no calcium or the calcium concentration is very low, the inner Curdlan solution is gelled (hydrogen bonding gel) but has no liquid crystal structure. This state corresponds to the small value of C in Figure 3 at low calcium concentration. As the calcium concentration is increased, the calcium-induced gelation become predominant. Thus, in the medium concentration range, the liquid crystalline layer thickness increases with calcium concentration. At a high enough calcium concentration larger than ∼7 g/dL, all the calcium binding sites of Curdlan bind with calcium ions and the excess amount of calcium ion cannot stay at the LCG phase. We note a significant increase of the birefringence of LCG at the external spherical surface was observed with increasing calcium concentration of the medium. The calcium increases the orderedness of the liquid crystalline layer. For the higher order structure to be formed, the ordered structure should be grown from fewer nucleation points. Therefore, the nucleus point distance at the surface, l, is expected to depend on the calcium concentration. However, there are no reasons to suppose the calcium concentration dependence of lc. Therefore, the calcium concentration dependence of lc could be attributed to the change of l. In conclusion, spherical beads with alternating LCG/AG structures were prepared by the insolubilization reaction and it was confirmed that eq 1 holds in a wide range of radii, irrespective of the geometrical boundary conditions of dialysis. The calcium concentration in the dispersing medium determines the liquid crystalline layer/gel radius ratio and the orderedness. These characteristics are useful for controlling the structure of Curdlan LCG/AG and its drug release characteristics. Acknowledgment. This work was partly supported by Grant-in-Aid for Science Research from The Ministry of Education, Culture, Sports, Science and Technology in Japan (Grant No. 16540366). LA047901W (7) Harada, T.; Fujimori, K.; Hirose, S.; Maeda, M. Agric. Biol. Chem. 1967, 31, 1184.
