Notes
Characteristics of Drug Release from Gel Beads Formed by Hydrolysis of Alginic Acid into Guluronic Acid Blocks
2013 Volume 61 Issue 9 Pages 967-970
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2013 Volume 61 Issue 9 Pages 967-970
Alginic acid (Alg) is a natural anionic polysaccharide, which consists of α-L-guluronic acid (G) and β-D-mannuronic acid (M). G-G sequence-rich chain regions, known as G-blocks (GB), are important regions for gelation of Alg using divalent cations. In this study, calcium-induced GB gel beads were prepared, and drug release profiles and degradation properties of the GB gel beads were investigated in aqueous media. The GB gel beads swelled slightly in JP XVI 1st fluid (pH 1.2), and only slight release of sodium diclofenac (DF) from the GB gel beads was observed. Disintegration of the GB gel beads was not observed in the 1st fluid. On the other hand, the GB gel beads disintegrated in JP XVI 2nd fluid (pH 6.8), and the rate of disintegration depended on the concentration of calcium chloride used to prepare the GB gel beads. The DF release profiles of the GB gel beads in the 2nd fluid could be controlled by the concentration of CaCl2 used to prepare the GB gel beads. The initial release profile of DF from GB gel beads was not consistent with the profile of disintegration. According to the Higuchi-plot of the percentage of drug content released against the square root of time, gel disintegration did not affect the release of DF from GB gel beads. It appears that a diffusion-type mechanism was responsible for DF release. We propose that the GB gel bead gel matrix is an effective medium by which to control the release of drug within the gastrointestinal tract.
Alginic acid (Alg) is a natural anionic polysaccharide extracted from brown algae. It has been used as a treatment for gastric ulcers, and also as a food additive. Alg consists of α-L-guluronic acid (G) and β-D-mannuronic acid (M). The molecular weight of Alg, the ratio of the two uronic acids (M/G ratio), and the sequence referred to as “block structure,” are dominant factors in the characterization of the polymer.1) The solution of sodium alginate immediately forms a cured gel matrix in the presence of divalent cations such as Ca2+; this characteristic has led to the practical use of Alg as a bioreactor.2–4) In addition, calcium-induced alginate gel beads have been utilized as a vehicle for drug delivery.5–7) This gelation is known to arise mainly at G-G sequence-rich chain regions, or G-blocks (GB). GB are particularly liable to form a divalent cation chelate, forming characteristic egg-box junctions. Each alginate chain dimerizes in GB to form junctions with many other chains, and as a result, gel networks are formed.
G Alg and its sodium and calcium salts are regarded as generally non-toxic and biocompatible. Thus, the calcium-induced Alg gel has been reported as a vehicle for drug delivery and as a material in biomedical engineering.8–10) Alg with a high content of GB gives gels of considerably higher strength compared to Alg rich in M-blocks because G residues exhibit a stronger affinity for divalent ions than M residues.11,12) Thus, other researchers have examined Alg gels prepared from G-rich Alg and the role of the Ca2+–G interaction on Alg.13,14) However, few studies have investigated the use of GB as a component of the materials of the vehicle for drug delivery. The drug release profile from the calcium-induced Alg gel is reportedly affected by the addition of GB to the gel matrix and by coating the surface of the gel matrix with GB.15,16) Thus, orally administered calcium-induced GB gel beads are hypothesized to enable controlled drug release in the gastrointestinal tract. However, to the best of our knowledge, no previous studies have prepared calcium-induced gel beads from only GB and investigated their water-soluble drug release profiles. In the present study, we prepared sphere gel matrixes with GB, and examined the degradation profiles of the GB gel beads under conditions mimicking those of the gastrointestinal tract. Finally, the relationship between drug release and erosion of the GB gel beads was investigated.
Sodium alginate (300) (Alg) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Sodium diclofenac (DF) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were of analytical grade.
Preparation of GBAlg was partially hydrolyzed (0.2 M HCl, 2 h, 100°C), and GB was separated using the method of Haug et al.17) GB was washed twice with ethanol, and subsequently washed twice with diethyl ether. GB was dried under reduced pressure in a desiccator in the presence of P2O5.
Preparation of the GB Gel BeadsThe GB gel beads were prepared as follows: 0.1 g of GB and 0.01 g of a model drug, DF, was added to 0.89 g of distilled and demineralized water and mixed to homogeneity. Then, 0.1 g of the suspension was added drop-wise into 10 mL of calcium chloride solution, and left to stand at room temperature for 1 h. The hydrogel beads were then washed twice with 25 mL distilled water and dried at 37°C for 8 h in a culture dish, after which they were dried under reduced pressure in a desiccator in the presence of P2O5.
Gel Disintegration TestJP XVI 1st fluid (pH 1.2) or JP XVI 2nd fluid (pH 6.8) was used as test media. Dried GB gel beads prepared from 0.1 g of hydrogel were added to 20 mL of test medium maintained at 37°C in a glass vial. The vial was shaken at 250 rpm in a shaker incubator at 37°C. A 0.3-mL aliquot of the solution was periodically removed for analysis and replaced with 0.3 mL of fresh test solution to maintain a constant volume. The sample was mixed with 0.3 mL of 50 mM sodium phosphate buffer (pH 7.0) and centrifuged at 10000 rpm for 5 min. The supernatant was filtered (Millex-HV, 0.45 µm; Merck Millipore, Billerica, MA, U.S.A.) and subjected to HPLC analysis. The amount of GB in the solution was determined by the method of Matsubara et al.,18) with some modifications. A 50-µL aliquot of the diluted sample was loaded onto a high-performance gel permeation column (Superdex 75 10/300 GL, 10 mm×300 mm; GE Healthcare Bio-Sciences AB) and eluted with 50 mM sodium phosphate buffer (pH 7.0) as the mobile phase, using a flow rate of 0.5 mL/min (L-2130; Hitachi, Tokyo, Japan). The GB in the effluent from the column was detected at 210 nm using a UV spectrophotometer (L-2400; Hitachi).
Drug Release TestThe release of DF from GB gel beads into JP XVI 1st fluid or JP XVI 2nd fluid was determined. Dried GB gel beads, corresponding to 0.1 g of hydrogel, were added to 20 mL of test medium maintained at 37°C in a glass vial. The vial was shaken at 250 rpm in a shaker incubator at 37°C. A 0.7-mL aliquot of the solution was removed periodically for analysis and replaced with 0.7 mL of fresh test medium (pre-warmed to 37°C) to maintain a constant volume. The sample was centrifuged at 10000 rpm for 5 min, and a 0.6-mL aliquot of the supernatant was mixed with 0.6 mL of fresh test medium. The absorbance of each diluted sample was determined with a spectrophotometer (UV-1200; Shimadzu, Kyoto, Japan) at 275 nm. All tests were performed in triplicate.
GB, which are sequences of G-G, were obtained by partial degradation with dilute HCl and separated by using the method of Haug et al.17) We previously described gel permeation chromatography of Alg and its hydrolysates. The molecular weight of GB was one-eighth that of Alg.16) The fluidity of the GB suspension influenced the formation of GB gel beads. In fact, spherical beads did not form when highly viscous solutions, such as 12% GB, were used. Spherical hydrogel beads formed immediately after a 10% GB suspension was added drop-wise into 0.02–2.0 M CaCl2. The diameter of the dried GB gel beads is shown in Table 1. As the CaCl2 concentration used to prepare the GB gel beads was increased from 0.2 to 2.0 M, the diameter of the dried GB gel beads increased. The GB gel beads incorporated drug into their gel matrix. DF was detected within the dried GB gel beads at 84.3% of its theoretical yield. Although a 10% suspension of GB also resulted in formation of spherical hydrogel beads in 0.01 M CaCl2, the GB gel beads were very soft, rendering them unable to be washed with distilled water.
| CaCl2 concentration (M) | Diameter (mm)a) | Loading capacity (%)b) |
|---|---|---|
| 0.02 | 0.93±0.09 | 77.9±3.1 |
| 0.2 | 0.94±0.08 | 84.3±3.2 |
| 2.0 | 1.23±0.12 | 94.6±4.7 |
a) Diameter data represent the mean±S.D. (n=20). b) Loading capacity data represent the mean±S.D. (n=3).
When a dried GB gel bead was soaked in aqueous solution, it either swelled, disintegrated, or both. The GB gel beads swelled slightly in the 1st fluid (pH 1.2), but did not disintegrate within a 3-h incubation period. The total amount of GB released from the GB gel beads into the 1st medium is shown in Fig. 1A. Release of GB from the GB gel beads was not observed by HPLC analysis after 3 h, and this result was consistent with visual observations.
The concentrations of CaCl2 used to prepare the GB gel beads were as follows: open circles, 0.02 M; closed triangles, 0.2 M; and closed squares, 2.0 M. (A) Release of GB in the 1st fluid. (B) Release of GB in the 2nd fluid. Data represent the mean±S.D. (n=3).
When a GB gel bead prepared with 0.02–2.0 M CaCl2 was soaked in the 2nd fluid (pH 6.8), the gel was visibly eroded. As shown in Fig. 1B, after 3 h, 73.7% of the components of the gel matrix were released from the GB gel beads prepared with 0.02 M CaCl2. The GB release rates decreased with exposure to increasing concentrations of CaCl2 during preparation.
Drug Release from the GB Gel BeadsDrug could be readily incorporated within the matrixes of the GB gel beads. In all cases, the drug loading capacity of the GB gel beads exceeded 77%, as shown in Table 1. As the CaCl2 concentration used to prepare the GB gel beads was increased from 0.02 to 2.0 M, the drug loading capacity of the GB gel beads increased. It was reported that the DF loading capacity of Alg gel beads prepared with 0.2 M CaCl2 is 67.2%.19) The GB gel beads prepared with 0.2 M CaCl2 concentration incorporated 84.3% of DF into their gel matrix. Thus, the ability of GB gel beads to incorporate drugs is superior to that of Alg gel beads.
The release profiles of DF from the GB gel beads prepared with 0.02–2.0 M CaCl2 in the 1st fluid are shown in Fig. 2A. Only slight release of DF from the GB gel beads was observed, and the release rates were not affected by the CaCl2 concentrations used to prepare the GB gel beads. The release profiles of drug from the GB gel beads in the 1st fluid were similar to that of Alg gel beads.13)
The concentrations of CaCl2 used to prepare the GB gel beads were as follows: open circles, 0.02 M; closed triangles, 0.2 M; and closed squares, 2.0 M. (A) Release of GB in the 1st fluid. (B) Release of GB in the 2nd fluid. Data represent the mean±S.D. (n=3).
Release of DF from the GB gel beads increased dramatically when disintegration of the gel matrixes occurred in the 2nd fluid, as shown in Fig. 2B. For example, after 3 h, all of the incorporated DF was released into the 2nd fluid from GB gel beads prepared with 0.02 M CaCl2. The release rates were affected by the CaCl2 concentrations used to prepare the GB gel beads, since the gel matrixes were composed of GB–Ca2+ complexes. The release rates of DF from the GB gel beads decreased as the concentration of CaCl2 used to prepare the GB gel beads increased from 0.02 to 2.0 M. However, the initial release profile of DF from GB gel beads prepared with 2.0 M CaCl2 was not consistent with the initial release profile of GB from the GB gel beads prepared with 2.0 M CaCl2. In order to understand mechanism of drug release, the profiles of release of DF from GB gel beads were used to plot the percentage release of drug against the square root of time (Fig. 3). The profiles of release of DF from the GB gel beads were linear on the Higuchi plot in the range of about 5% to 90%, and the slope decreased as the concentrations of CaCl2 used to prepare the GB gel beads increased. The release rate constants in the 2nd fluid from the beads prepared with 0.02 M, 0.2 M, and 2.0 M CaCl2 were 11.7%/min1/2 (r2=0.989), 8.9%/min1/2 (r2=0.994), and 3.1%/min1/2 (r2=0.997), respectively. According to these observations, the release of DF from GB gel beads was not affected by gel disintegration, and it appears that the mechanism of DF release was diffusion type. The controlled release of a water-soluble drug from Alg gel beads is difficult because the drug immediately diffuses through the matrix to the gel surface. However, the release profiles of a water-soluble drug, DF, from the GB gel beads could be controlled by the concentration of CaCl2 used to prepare the GB gel beads.
The concentrations of CaCl2 used to prepare the GB gel beads were as follows: open circles, 0.02 M; closed triangles, 0.2 M; and closed squares, 2.0 M. Data represent the mean±S.D. (n=3). There was a significant difference between the release rate constant of the GB gel beads prepared using 0.02 M CaCl2 and that of the GB gel beads prepared using 0.2 M CaCl2 (p<0.05). Furthermore, there was a significant difference between the release rate constant of the GB gel beads prepared using 0.2 M CaCl2 and that of the GB gel beads prepared using 2.0 M CaCl2 (p<0.05).
In this study, we prepared calcium-induced GB gel beads and investigated their drug release profiles. We demonstrate that the release of DF from the GB gel beads in the 1st fluid was slight and that release rates were not affected by the CaCl2 concentrations used to prepare the GB gel beads. On the other hand, the DF release profiles of the GB gel beads in the 2nd fluid could be controlled by the concentration of CaCl2 used to prepare the GB gel beads, although controlled release of a water-soluble drug, DF, from Alg gel beads in the 2nd fluid was difficult. The GB gel beads suppressed the release of DF in the 1st fluid and controlled the release of DF in the 2nd fluid. Therefore, the use of GB gel beads is feasible for controlled release in the small intestine of drugs that have the side effect of gastric ulcer. We propose that GB gel beads show promise with regard to the development of controlled release drug formulations.
