Abstract
The purpose of this work was to develop and optimize gliclazide-loaded alginate–methyl cellulose mucoadhesive microcapsules by ionotropic gelation using central composite design. The effect of formulation parameters like polymer blend ratio and cross-linker (CaCl2) concentration on properties of gliclazide-loaded alginate–methyl cellulose microcapsules like drug encapsulation efficiency and drug release were optimized. The optimized microcapsules were subjected to swelling, mucoadhesive, and in vivo studies. The observed responses coincided well with the predicted values from the optimization technique. The optimized microcapsules showed high drug encapsulation efficiency (83.57 ± 2.59% to 85.52 ± 3.07%) with low T50% (time for 50% drug release, 5.68 ± 0.09 to 5.83 ± 0.11 h). The in vitro drug release pattern from optimized microcapsules was found to be controlled-release pattern (zero order) with case II transport release mechanism. Particle sizes of these optimized microcapsules were 0.767 ± 0.085 to 0.937 ± 0.086 mm. These microcapsules also exhibited good mucoadhesive properties. The in vivo studies on alloxan-induced diabetic rats indicated the significant hypoglycemic effect that was observed 12 h after oral administration of optimized mucoadhesive microcapsules. The developed and optimized alginate–methyl cellulose microcapsules are suitable for prolonged systemic absorption of gliclazide to maintain lower blood glucose level and improved patient compliance.
Key words: alginate–methyl cellulose, anti-diabetic activity, gliclazide, microcapsules, mucoadhesive
INTRODUCTION
Gliclazide, 1-(3-azabicyclo-[3, 3, 0]-oct-3-yl)-3-(p-tolyl sulfonyl) urea, is one of the second generation sulfonylureas used as oral hypoglycemic agent in the treatment of non-insulin-dependent diabetes mellitus (1). Previous reports showed that gliclazide possesses good general tolerability and lower rate of secondary failure (2,3). However, the gliclazide absorption rate from gastrointestinal tract is slow (4). Slower absorption of gliclazide has been suggested which may be due to either its poor dissolution rate owing to its hydrophobic nature or poor permeability across the gastrointestinal membrane (5). Therefore, the incorporation of gliclazide in controlled-release dosage forms such as microcapsules can control its absorption from gastrointestinal tract and thus overcomes variability problems.
Microencapsulation is one of the processes to prolong the drug release and reduce the adverse effects (6). However, the success of microcapsules for controlled drug delivery is limited due to their short residence time at the site of absorption. Therefore, it would be advantageous to have means by providing an intimate contact of the drug delivery systems with the absorbing surface of mucous membranes, i.e., mucoadhesion (7,8). It is mostly achieved by the use of mucoadhesive polymers. The mucoadhesive polymer containing oral drug delivery systems have the capacity to prolong the gastric residence time of drugs at the site of absorption and facilitate intimate contact with underlying absorptive surface to enhance the bioavailability of drugs (9–12).
Over the past few years, pharmaceutical formulators and scientists have shown an increased interest in using alginates as biopolymer in the development of drug delivery systems, due to its hydrogel-forming properties (13,14). These are abundant in nature and found as structural components of brown marine algae (15). Alginate, the monovalent form of alginic acid, belongs to a family of linear co-polymers composed of β-d-mannuronic acid monomers, regions of ∞-l-guluronic acid residues, and regions of interspersed both the residues (16). Alginates undergo ionotropic gelation in aqueous solution in the presence of divalent cations like Ca2+, Ba2+, etc. and trivalent cation like Al3+, due to an ionic interaction between the carboxylic acid groups located on the polymer backbone and these cations (17,18). Alginates have mucoadhesive property, but the cross-linked alginates are usually fragile (19,20). Therefore, to formulate various cross-linked alginate mucoadhesive microcapsules for controlled drug delivery, blending with mucoadhesive polymers is one of the most popular approaches. Again, blending with suitable polymers can improve the drug encapsulation and stability (21), which is found lower in alginate microcapsules, prepared by ionotropic gelation. A few investigations have been carried out to formulate alginate-based mucoadhesive microcapsules or beads for controlled gliclazide delivery. Al-Kassas et al. prepared alginate beads of gliclazide by ionotropic gelation (5). In another investigation, various mucoadhesive microcapsules of gliclazide using sodium alginate and mucoadhesive polymers such as sodium carboxymethyl cellulose, carbopol 934 P, and hydroxyl propyl methyl cellulose by ionotropic gelation was formulated by Prajapati et al. (22). Nevertheless, it is found that no attempt has been taken to formulate gliclazide-loaded alginate-based microcapsule or bead system using methyl cellulose as a mucoadhesive polymer. Therefore, in the present investigation, an attempt was made to develop and evaluate gliclazide-loaded alginate–methyl cellulose mucoadhesive microcapsules with special reference to anti-diabetic activity.
Designing controlled-release formulations with the minimum number of trials is very crucial for pharmaceutical scientists (23). Central composite design, a response surface design, has been widely used for formulation and process optimization (24). Therefore, the objectives of the present investigation were (a) to evaluate the effect of two process variables like polymer blend ratio and cross-linker concentration on the properties of gliclazide-loaded alginate–methyl cellulose microcapsules like drug encapsulation efficiency and drug release from these new microcapsules; (b) to optimize these process variables, which powerfully influence the properties and performances of gliclazide-loaded alginate–methyl cellulose microcapsules by central composite design; and (c) to evaluate the optimized gliclazide-loaded alginate–methyl cellulose microcapsules in vitro and in vivo.
MATERIALS AND METHODS
Materials
Gliclazide (Lupin Ltd., India), sodium alginate (CDH Laboratories, India), methyl cellulose (Loba Chemie, India), and calcium chloride (Merck Ltd., India) were used for the present investigation. All other chemicals and reagents used were of analytical grade.
Methods
Preparation of Microcapsules
The microcapsules were prepared by ionotropic gelation technique. Briefly, sodium alginate and methyl cellulose solutions were prepared separately using deionized water and well mixing together. Then, gliclazide was added to the polymeric mixture. The ratio of drug to polymer was maintained 1:1 in all formulations. The final mixture containing gliclazide was homogenized for 10 min at 1,000 rpm using homogenizer (Remi Motors, India), and the resulting mixture was dropped in calcium chloride (CaCl2) solution via 26 gauge needles. After 15 min, the microcapsules were collected by decantation, washed repeatedly using deionized water, and dried at 45°C for 12 h.
Experimental Design
To reduce the number of trials necessary to attain maximum numbers of information on product properties, the screening was performed applying a circumscribed central composite design. The polymer blend ratio (sodium alginate to methyl cellulose, 1:9) and cross-linker concentration (CaCl2, 5:10%, w/v) were defined as factors, while drug encapsulation efficiency (DEE; in percent) and time for 50% drug release (T50%, in hours) were used as responses. The process variables (factors) and levels with experimental values are reported in Table I. Design-Expert® Software (V.7.0, Stat-Ease Inc., USA) was used for generation and evaluation of experimental design.
Table I.
Factors and Levels of the Circumscribed Central Composite Design
| Normalized levels | Experimental settings | |
|---|---|---|
| SA/MC (X 1) | CaCl2 (%, w/v) (X 2) | |
| −1.414 | 1.00 | 5.00 |
| −1 | 2.20 | 5.70 |
| 0 | 5.00 | 7.50 |
| 1 | 7.80 | 9.30 |
| 1.414 | 9.00 | 10.00 |
SA/MC sodium alginate-to-methyl cellulose ratio
Drug Encapsulation Efficiency (in Percent) Estimation
One hundred milligrams of microcapsules was taken and crushed using pestle and mortar. The crushed powders were placed in 500 ml of phosphate buffer (pH 7.4) and kept for 48 h with occasionally shaking at 37 ± 0.5°C. The polymer debris formed after disintegration of microcapsules was removed by filtering through Whatman® filter paper (no. 40). The drug content in the filtrate was determined quantitatively by UV–VIS spectrophotometer (Shimadzu, Japan) at 226.5 nm wavelength. The DEE (in percent) was calculated using the following formula:
 |
1 |
Particle Size Measurement
Average particle size of 100 microcapsules from each batch was measured by optical microscope (Olympus Co., Japan). The ocular micrometer was previously calibrated by stage micrometer.
Morphology Analysis
Microcapsules were gold-coated in an ion sputter (Hitachi E1010, Japan), and morphology was examined by scanning electron microscopy (SEM) (Hitachi S3400, Japan).
In Vitro Drug Release Study
The in vitro gliclazide release from microcapsules was tested in 900 ml of phosphate buffer (pH 7.4) using dissolution test apparatus (paddle type) (Campbell Electronics, India) at 37 ± 1°C under 50 rpm speed (25). A sample of microcapsules equivalent to 100 mg gliclazide was used in each test. Five-milliliter aliquot was collected at regular time intervals, and same amount of fresh medium was replaced into dissolution vessel to maintain sink condition throughout the experiment. The collected aliquots were filtered and estimated quantitatively for gliclazide content using UV–VIS spectrophotometer (Shimadzu, Japan) at 226.5 nm wavelength.
Swelling Behavior Evaluation
One hundred milligrams of microcapsules was soaked in phosphate buffer (pH 7.4) and 0.1 N HCl (pH 1.2). The swelled microcapsules were removed at predetermined time interval and weighed after drying their surfaces using tissue paper. Swelling index was determined by using the following formula:
 |
2 |
Mucoadhesion Testing
The mucoadhesive properties of microcapsules were evaluated by in vitro wash-off method (24). Freshly excised pieces of goat intestinal mucosa (2 × 2 cm) (collected from slaughterhouse) were mounted on glass slide (7.5 × 2.5 cm) using thread. About 50 microcapsules were spread onto the wet, ringed tissue specimen, and the prepared slide was hung onto a groove of disintegration test apparatus. The tissue specimen was given a regular up and down movement in a vessel containing 900 ml of phosphate buffer (pH 7.4) and 0.1 N HCl (pH 1.2), separately, at 37 ± 0.5°C. After regular time intervals, the machine was stopped and the number of microcapsules still adhering to the tissue was counted.
In Vivo Studies
In vivo studies were performed in alloxan-induced diabetic albino rats of either sex (weighing 275–338 g) (22,26). The acclimatized rats were kept fasting for 24 h with water ad libitum. All experiments were performed between 8 am to 12 pm to minimize circadian influences.
The animal experimental protocol was approved by the Institutional Animal Ethical Committee and was cleared before starting. The experimental design was subjected to the scrutiny of IFTM University Ethical Committee (reg. no. IFTM/837ac/0159). The animals were handled as per the guidance of the Committee for the Purpose of Control and supervision on Experimental animals (CPCSEA), New Delhi, India. All efforts were made to minimize both the suffering and number of animals used. The rats were made diabetic by intraperitoneal administration of freshly prepared alloxan solution at a dose of 150 mg/kg dissolved in 2 mM citrate buffer (pH 3.0). After 1 week of alloxan administration, alloxanized rats with fasting blood glucose of 300 mg/dl or more were considered diabetic and were employed in the study for 12 h. The alloxan-induced diabetic rats were divided randomly into four groups of three rats each and treated as below.
Group A was administered with pure gliclazide in suspension form. Group B (O-1), C (O-2), and D (O-3) were administered with optimized gliclazide-loaded alginate–methyl cellulose microcapsules, both at a dose equivalent to 2 mg/kg of gliclazide by using oral feeding needle. Blood samples were withdrawn (0.1 ml) from tail tip of each rat at regular time intervals for 12 h under mild ether anesthesia and were analyzed for blood glucose by oxidase peroxidase method using commercial glucose kit. Comparative in vivo blood glucose level in alloxan-induced diabetic rats after oral administration of pure gliclazide and optimized alginate–methyl cellulose mucoadhesive microcapsules containing gliclazide were evaluated.
Statistical Analysis
For optimization, polynomial equations involving individual factors and interaction factors were selected based on model analysis, lack of fit and R2 analysis, and predicted residual sum of squares (PRESS) for measured responses. The quadratic mathematical model generated by circumscribed central composite design is in the following (24):
 |
3 |
where Y is the response; b0 is the intercept; and b1, b2, b3, b4, b5 are regression coefficients. X1 and X2 are individual effects; X21 and X22 are quadratic effects; X1X2 is the interaction effect. One-way ANOVA was applied to estimate the significance of the model (p < 0.05).
All measured data are expressed as mean ± standard deviation (SD). Each measurement was done in triplicate (n = 3).
RESULTS
Optimization
In the central composite design, total 13 experimental formulations of alginate–methyl cellulose microcapsules containing gliclazide were prepared by ionotropic gelation taking two process variable factors like polymer blend ratio (sodium alginate/methyl cellulose) and cross-linker (CaCl2) concentration (Table I). Overview of the experimental plan and observed response values are presented in Table II. The outcome of model analysis, lack of fit and R2 analysis, and PRESS value for measured responses are presented in Table III. The model was evaluated statistically applying one-way ANOVA (p < 0.05), which is shown in Table IV. The model equations were generated to fit the data from the experimental design.
Table II.
Experimental Plan and Observed Response Values from Randomized Run in Central Composite Design
| Experimental formulations | Normalized levels of factors | Responsesa | ||
|---|---|---|---|---|
| SA/MC (X 1) | CaCl2 (%, w/v) (X 2) | DEE (%) | T 50% (h) | |
| F-1 | −1 | −1 | 75.55 ± 2.26 | 4.63 ± 0.05 |
| F-2 | −1 | 1 | 82.72 ± 2.58 | 5.57 ± 0.12 |
| F-3 | 1 | −1 | 63.84 ± 2.04 | 3.67 ± 0.05 |
| F-4 | 1 | 1 | 68.38 ± 2.12 | 4.33 ± 0.08 |
| F-5 | −1.414 | 0 | 83.76 ± 2.66 | 5.78 ± 0.10 |
| F-6 | 1.414 | 0 | 64.08 ± 2.27 | 3.83 ± 0.06 |
| F-7 | 0 | −1.414 | 64.63 ± 2.12 | 3.97 ± 0.08 |
| F-8 | 0 | 1.414 | 73.60 ± 2.38 | 4.98 ± 0.08 |
| F-9 | 0 | 0 | 69.68 ± 2.07 | 4.64 ± 0.08 |
| F-10 | 0 | 0 | 70.39 ± 2.85 | 4.62 ± 0.09 |
| F-11 | 0 | 0 | 69.95 ± 2.46 | 4.66 ± 0.08 |
| F-12 | 0 | 0 | 70.07 ± 2.82 | 4.63 ± 0.10 |
| F-13 | 0 | 0 | 69.31 ± 2.23 | 4.75 ± 0.07 |
SA/MC sodium alginate-to-methyl cellulose ratio, DEE (%) drug encapsulation efficiency (in percent), T 50%, (h) time for 50% drug release from microcapsules
aObserved response values: mean ± SD (n = 3)
Table III.
Summary of Results of Model Analysis, Lack of Fit, and R 2 Analysis for Measured Responses
| Source | DEE (%) | T 50% (h) | ||||||
|---|---|---|---|---|---|---|---|---|
| Sum of squares | p value | Sum of squares | p value | |||||
| Model analysis | ||||||||
| Mean vs total | 65,957.99 | 277.48 | ||||||
| Linear vs mean | 437.35 | <0.0001 | 4.22 | <0.0001 | ||||
| 2FI vs linear | 1.73 | 0.5551 | 0.02 | 0.3570 | ||||
| Quadratic vs 2FI | 37.62 | 0.0002 | 0.12 | 0.0227 | ||||
| Cubic vs quadratic | 0.46 | 0.7220 | 0.04 | 0.1011 | ||||
| Residual | 3.33 | 0.03 | ||||||
| Total | 64434.49 | 281.91 | ||||||
| Lack of fit | ||||||||
| Linear | 42.48 | 0.0014 | 0.20 | 0.0158 | ||||
| 2FI | 40.75 | 0.0011 | 0.18 | 0.0142 | ||||
| Quadratic | 3.13 | 0.0543 | 0.05 | 0.0530 | ||||
| Cubic | 2.67 | 0.0161 | 0.01 | 0.0842 | ||||
| Pure error | 0.67 | 0.01 | ||||||
| R 2 analysis | Adjusted predicted | Adjusted predicted | ||||||
| R 2 | R 2 | R 2 | PRESS | R 2 | R 2 | R 2 | PRESS | |
| Linear | 0.9102 | 0.8922 | 0.8227 | 85.21 | 0.9533 | 0.9440 | 0.9031 | 0.44 |
| 2FI | 0.9138 | 0.8851 | 0.7739 | 108.62 | 0.9577 | 0.9437 | 0.8813 | 0.53 |
| Quadratic | 0.9921 | 0.9865 | 0.9515 | 23.31 | 0.9857 | 0.9754 | 0.9105 | 0.40 |
| Cubic | 0.9931 | 0.9834 | 0.6423 | 171.90 | 0.9943 | 0.9863 | 0.7881 | 0.94 |
DEE (%) drug encapsulation efficiency (in percent), T 50% (h) time for 50% drug release from microcapsules, 2FI two-factor interaction, PRESS predicted residual sum of squares
Table IV.
Summary of ANOVA for the Response Parameters
| Source | Sum of squares | df | Mean square | F value | p value Prob > F |
|---|---|---|---|---|---|
| For DEE (%) | |||||
| Model | 476.70 | 5 | 95.34 | 175.87 | <0.0001 |
| X 1 | 363.02 | 1 | 363.02 | 669.64 | <0.0001 |
| X 2 | 74.33 | 1 | 74.33 | 137.12 | <0.0001 |
| X 1 X 2 | 1.73 | 1 | 1.73 | 3.19 | 0.1173 |
| X 21 | 36.63 | 1 | 36.63 | 67.58 | <0.0001 |
| X 22 | 0.05 | 1 | 0.05 | 0.09 | 0.7738 |
| For T 50% (h) | |||||
| Model | 5.95 | 5 | 1.19 | 4,577.93 | <0.0001 |
| X 1 | 4.18 | 1 | 4.18 | 16,076.90 | <0.0001 |
| X 2 | 1.61 | 1 | 1.61 | 6,191.17 | <0.0001 |
| X 1 X 2 | 0.14 | 1 | 0.14 | 526.67 | <0.0001 |
| X 21 | 0.02 | 1 | 0.02 | 72.48 | <0.0001 |
| X 22 | 0.01 | 1 | 0.01 | 33.33 | 0.0007 |
X 1 and X 2 represent the main effects (factors); X 21 and X 22 are the quadratic effect; X 1 X 2 is the interaction effect
DEE (%) drug encapsulation efficiency (in percent), T 50% (h) time for 50% drug release from microcapsules
The model equation relating DEE T50% (in hours) (in percent) as response is shown in Eq. 4:
 |
4 |
It can be noted that the coefficient b3 and b5 of Eq. 4 had no statistic significance (p > 0.05) for response Y1 (DEE, in percent), since the statistic p value of b3 and b5 were 0.1173 and 0.7738, respectively.
The model equation relating T50% (h) as response is shown in Eq. 5:
 |
5 |
In Eq. 5, the coefficient b3 and b4 had no statistic significance (p > 0.05) for response Y2 (T50%, in hours), since the statistic p value of b3 and b4 were 0.1849 and 0.1968, respectively.
The three-dimensional response surface plots (Figs. 1 and 2) and corresponding contour plots (Figs. 3 and 4) are presented to reveal the effects of the independent variables on each response.
Fig. 1.
Effect of main factors on DEE (in percent) presented by response surface plot
Fig. 2.
Effect of main factors on T 50% (in hours) presented by response surface plot
Fig. 3.
Effect of main factors on DEE (in percent) presented by contour plot
Fig. 4.
Effect of main factors on T 50% (in hours) presented by contour plot
After generating the polynomial equations relating the responses, alginate–methyl cellulose containing gliclazide were optimized for both responses, Y1 (DEE, in percent) and Y2 (T50%, in hours). The desirable ranges of factors were restricted as sodium alginate-to-methyl cellulose ratio within 1:5 and CaCl2 concentration within 5:10% (w/v). In addition, the responses, DEE (in percent) and T50% (in hours) were restricted to 85% ≤ Y1 ≤ 100% and 5 h ≤ Y2 ≤ 6 h, respectively. The optimal values of responses were obtained by numerical analysis using the Design-Expert® software (V.7.0, Stat-Ease Inc., USA) based on the criterion of desirability. In order to evaluate the optimization capability of these models generated according to the optimal process variable settings given by the circumscribed central composite design, three formulations of gliclazide-loaded alginate–methyl cellulose microcapsules were selected and formulated. The optimized microcapsules (O-1, O-2, and O-3) were evaluated also for DEE (in percent) and T50% (in hours). Table V lists the results of experiments with predicted responses by the mathematical model and those observed.
Table V.
Results of Experiments for Confirming Optimization Capability
| Code | Factors | Responses | ||||||
|---|---|---|---|---|---|---|---|---|
| SA/MC | CaCl2 (%, w/v) | DEE (%) | T 50% (h) | |||||
| Predicted | Observeda | Error (%)b | Predicted | Observeda | Error (%)b | |||
| O-1 | 1.00 | 9.00 | 87.24 | 85.52 ± 3.07 | 1.97 | 5.96 | 5.83 ± 0.11 | 2.18 |
| O-2 | 1.60 | 9.50 | 85.57 | 83.82 ± 2.77 | 2.04 | 5.86 | 5.78 ± 0.08 | 1.37 |
| O-3 | 1.30 | 8.70 | 85.23 | 83.57 ± 2.59 | 1.95 | 5.83 | 5.68 ± 0.09 | 2.57 |
SA/MC sodium alginate-to-methyl cellulose ratio, DEE (%) drug encapsulation efficiency (in percent), T 50% (h) time for 50% drug release from microcapsules
aObserved response values: mean ± SD (n = 3)
bError (%) = [Difference between predicted value and observed value/Predicted value] × 100
Particle Size and Morphology
Particle size of gliclazide-loaded various alginate–methyl cellulose microcapsules was measured by optical microscopic method applied for each formulation. The mean diameters of all these microcapsules are shown in Table VI. The morphological analysis of microcapsules was done by SEM and presented in Fig. 5.
Table VI.
Mean Diameter of Alginate–Methyl Cellulose Microcapsules Containing Gliclazide, Measured by Optical Microscopic Method
| Formulation codesa | Mean diameterb (mm) |
|---|---|
| F-1 | 0.904 ± 0.097 |
| F-2 | 0.845 ± 0.084 |
| F-3 | 0.937 ± 0.086 |
| F-4 | 0.778 ± 0.068 |
| F-5 | 0.962 ± 0.092 |
| F-6 | 0.767 ± 0.085 |
| F-7 | 0.926 ± 0.087 |
| F-8 | 0.803 ± 0.078 |
| F-9 | 0.854 ± 0.080 |
| F-10 | 0.833 ± 0.091 |
| F-11 | 0.851 ± 0.068 |
| F-12 | 0.847 ± 0.068 |
| F-13 | 0.850 ± 0.092 |
| O-1 | 0.859 ± 0.084 |
| O-2 | 0.848 ± 0.079 |
| O-3 | 0.853 ± 0.090 |
aF-1 to O-3 were alginate–methyl cellulose microcapsules containing gliclazide. Among them, O-1, O-2, and O-3 were optimized formulations
bMean ± SD
Fig. 5.
SEM photograph of gliclazide-loaded alginate–methyl cellulose microcapsules (O-1)
In Vitro Drug Release Studies
The in vitro drug release studies were carried out for gliclazide-loaded alginate–methyl cellulose microcapsules in phosphate buffer (pH, 7.4). Various microcapsules (SP-1 to SP-13 and O-1 to O-3) showed prolonged release of gliclazide over 8 h (Figs. 6 and 7). The in vitro drug release data of optimized microcapsules were evaluated kinetically using various mathematical models (27–30):
- Zero-order kinetics
F = k0 t, where F represents the fraction of drug released in time t and k0 is the zero-order release constant
- First-order kinetics
ln (1 − F) = −k1 t, where F represents the fraction of drug released in time t and k1 is the first-order release constant
- Higuchi model
F = kHt½, where F represents the fraction of drug released in time t and kH is the Higuchi dissolution constant
- Korsmeyer–Peppas model
F = kPtn, where F represents the fraction of drug released in time t, kP is the rate constant, and n is the diffusion exponent; this indicates the drug release mechanism
The results of the curve fitting into these above-mentioned mathematical models are presented in Table VII.
Fig. 6.
In vitro drug release from alginate–methyl cellulose microcapsules containing gliclazide (F-1 to F-13) (mean ± SD, n = 3)
Fig. 7.
In vitro drug release from optimized alginate–methyl cellulose microcapsules containing gliclazide (O-1 to O-3) (mean ± SD, n = 3)
Table VII.
Results of Curve Fitting of the In Vitro Gliclazide Release Data from Different Optimized Alginate–Methyl Cellulose Microcapsules
| Formulation codes | Correlation coefficient (R 2) values | ||
|---|---|---|---|
| O-1 | O-2 | O-3 | |
| Zero-order model | 0.9945 | 0.9939 | 0.9924 |
| First-order model | 0.9849 | 0.9816 | 0.9842 |
| Higuchi model | 0.9794 | 0.9777 | 0.9792 |
| Korsmeyer–Peppas Model | 0.9872 | 0.9860 | 0.9761 |
| Diffusion coefficient (n) | 0.8697 | 0.9225 | 0.8743 |
Swelling Behavior
The swelling behavior of optimized alginate–methylcellulose microcapsules containing gliclazide was evaluated in gastric pH (0.1 N HCl, pH 1.2) and intestinal pH (phosphate buffer, pH 7.4). The swelling index of these microcapsules in both the medium is measured at various time intervals and shown in Table VIII.
Table VIII.
Results of the Swelling Behavior of Gliclazide-Loaded Alginate–Methyl Cellulose Microcapsules in pH 1.2 and pH 7.4
| Time (h) | Swelling ratio (%)a | |||||
|---|---|---|---|---|---|---|
| O-1 (pH 1.2) | O-2 (pH 1.2) | O-3 (pH 1.2) | O-1 (pH 7.4) | O-2 (pH 7.4) | O-3 (pH 7.4) | |
| 0.5 | 111.74 ± 1.79 | 114.63 ± 1.86 | 110.42 ± 2.04 | 118.83 ± 2.06 | 117.98 ± 1.98 | 113.84 ± 2.33 |
| 1 | 122.67 ± 2.52 | 108.64 ± 1.44 | 116.72 ± 2.02 | 348.49 ± 3.88 | 350.12 ± 3.73 | 344.66 ± 3.76 |
| 2 | 122.06 ± 2.26 | 124.64 ± 2.06 | 120.06 ± 3.03 | 716.43 ± 6.06 | 682.06 ± 6.34 | 695.75 ± 6.85 |
| 3 | 144.02 ± 2.62 | 128.24 ± 3.33 | 137.00 ± 3.17 | 923.56 ± 6.87 | 931.34 ± 7.73 | 924.90 ± 7.22 |
| 4 | 152.36 ± 3.88 | 154.55 ± 2.05 | 148.98 ± 3.13 | 665.33 ± 8.76 | 660.85 ± 7.05 | 662.43 ± 7.98 |
| 6 | 156.58 ± 3.05 | 147.09 ± 2.77 | 150.06 ± 3.37 | 190.74 ± 4.45 | 187.83 ± 4.56 | 185.07 ± 4.63 |
| 8 | 160.59 ± 3.85 | 160.06 ± 3.65 | 158.56 ± 3.56 | 2.11 ± 0.21 | 2.02 ± 0.25 | 2.29 ± 0.15 |
aMean ± SD, n = 3
Mucoadhesivity
The in vitro wash-off test for assessing mucoadhesivity of these optimized alginate–methyl cellulose microcapsules containing gliclazide was performed using goat intestinal mucosa at both gastric pH (0.1 N HCl, pH 1.2) and intestinal pH (phosphate buffer, pH 7.4) for 8 h. The result of in vitro wash-off test is presented in Fig. 8.
Fig. 8.
Results of in vitro wash-off test to assess mucoadhesive properties of the optimized alginate–methyl cellulose microcapsules containing gliclazide (mean ± SD, n = 3)
In Vivo Blood Glucose Evaluation
In vivo efficiencies of optimized mucoadhesive alginate–methyl cellulose microcapsules containing gliclazide (O-1 to O-3) were performed in alloxan-induced diabetic rats and estimated by measuring the blood glucose level. The comparative in vivo blood glucose level and the mean percentage reduction in blood glucose level in alloxan-induced diabetic rats after oral administration of pure gliclazide and optimized alginate–methyl cellulose mucoadhesive microcapsules containing gliclazide is presented in Figs. 9 and 10.
Fig. 9.
Comparative in vivo blood glucose level in alloxan-induced diabetic rats after oral administration of pure gliclazide and optimized alginate–methyl cellulose mucoadhesive microcapsules containing gliclazide (O-1 to O-3) (mean ± SD, n = 3)
Fig. 10.
Comparative in vivo mean percentage reductions in blood glucose level in alloxan-induced diabetic rats after oral administration of pure gliclazide and optimized alginate–methyl cellulose mucoadhesive microcapsules containing gliclazide (O-1 to O-3)
DISCUSSION
Gliclazide-loaded alginate–methyl cellulose microcapsules were prepared by ionotropic gelation technique according to the circumscribed central composite design (Table I). The result of experimental run by the central composite design (Table II) noticed that DEE (in percent) was increased with decreasing of sodium alginate-to-methyl cellulose ratio and increasing CaCl2 concentration. This may be due to higher degree of cross-linking by CaCl2 and increased viscosity of polymeric solution with methyl cellulose addition. This might have been prevented drug leaching to the cross-linking solution. The microcapsules prepared using lower CaCl2 concentration might have larger pores, due to insufficient cross-linking and resulted lower drug encapsulation (31). However, T50% (in hours) was decreased with decreasing of sodium alginate-to-methyl cellulose ratio and increasing CaCl2 concentration.
For optimization, the quadratic model was selected based on statistically insignificant lack of fit and smallest values of PRESS for both responses (DEE, in percent and T50%, in hours) (Table III). The smaller the PRESS statistic, the better for the model fitting to data points (32). These models were also evaluated statistically by ANOVA (p < 0.05) (Table IV), and the result indicated that these models were significant for the responses, studied in this investigation.
The influence of main effects on responses was further elucidated by response surface methodology. The response surface methodology has been widely used for optimization (33,34). The three-dimensional response surface plots (Figs. 1 and 2) and contour plots (Figs. 3 and 4) demonstrate changes in DEE (in percent) and T50% (in hours) influenced by process variable factors, studied in this investigation.
The optimized microcapsules (O-1, O-2, and O-3) were formulated using selected process variable settings by numerical analysis according to the circumscribed central composite design and evaluated for DEE (in percent) and T50% (in hours) (Table V). All these optimized microcapsules showed maximum DEE (83.57 ± 2.59% to 85.52 ± 3.07%) with low T50% (5.68 ± 0.09 to 5.83 ± 0.11 h) with small error values. This reveals that mathematical models obtained by the central composite design were well fitted.
The particle size range of these alginate–methyl cellulose microcapsules were 0.767 ± 0.085 to 0.937 ± 0.086 mm (Table VI). Increasing particle size of microcapsules was found with increasing methyl cellulose incorporation into formulations. This could be attributed due to increase in viscosity of polymer solution with methyl cellulose incorporation in increasing ratio, which increased droplet sizes during addition of polymer solution to cross-linking solution. Again, the particle size of microcapsules was decreased due to shrinkage of polymeric gel by higher degree of cross-linking; when more concentrated CaCl2 solution was used.
Rigid microcapsules were obtained, when polymer (sodium alginate and methyl cellulose)–gliclazide mixture was dropped into CaCl2 solution. The SEM photograph indicated that microcapsules were spherical with rough surfaces and completely covered with the coat polymer (Fig. 5).
Various alginate–methyl cellulose microcapsules (SP-1 to SP-13 and O-1 to O-3) showed prolonged in vitro gliclazide release in phosphate buffer (pH, 7.4) over 8 h (Figs. 6 and 7). In case of microcapsules containing higher methyl cellulose amount, the more hydrophilic property of methyl cellulose may bind better with water to form viscous gel structure, which may block the pores on microcapsule surfaces and sustain drug release. The high degree of cross-linking by higher CaCl2 concentration may slower the drug release from highly cross-linked microcapsules. Optimized microcapsules (O-1 to O-3) showed only 61.06 ± 2.02 to 64.12 ± 2.16% of gliclazide release in 8 h (Fig. 6). The gliclazide release from optimized microcapsules was found to follow zero-order kinetics (R2 = 0.9924 to 0.9939) over a period of 8 h (Table VII), indicating the controlled drug release from these microcapsules. The Korsmeyer–Peppas model was also employed to distinguish two competing release mechanisms, Fickian diffusional release (n ≤ 0.43) and case II transport (n ≥ 0.85) (27). The values of diffusion coefficient (n) ranged 0.8697 to 0.9225 (Table VII), indicating the drug release followed the case II transport mechanism controlled by swelling and relaxation of polymeric matrix.
The swelling index of optimized alginate–methylcellulose microcapsules was lower in acidic pH (1.2) in comparison with that of in alkaline pH (7.4) (Table VIII). Maximum swelling was observed at 2–3 h in alkaline pH; after which, erosion and dissolution took place. The swelling behavior of optimized microcapsules in alkaline pH could be explained by the ion exchange phenomenon between the calcium ion of cross-linked alginate–methyl cellulose microcapsules and the sodium ions present in phosphate buffer, with the influence of calcium sequestrate phosphate ions, which resulted in disaggregation of alginate–methyl cellulose matrix structure leading to matrix erosion and dissolution of swollen microcapsules (35).
In gastric pH, microcapsules adhering to goat intestinal mucosa varied from 55.50 ± 3.26% to 70.67 ± 4.05%, whereas this was from 4.50 ± 0.08% to 6.67 ± 0.15% in intestinal pH (Fig. 7). The rapid wash-off observed at intestinal pH could be due to ionization of carboxyl and other functional groups of polymers, which increased their solubility with reduced adhesive strength (35). The results of wash-off test indicated that these optimized microcapsules had fairly good mucoadhesivity.
A rapid reduction of blood glucose level in alloxan-induced diabetic rats was observed for a period of 2 h after oral administration of pure gliclazide (group A). After that, blood sugar level was recovered toward the normal (Figs. 9 and 10). However, the reductions in blood glucose level of groups treated with optimized microcapsules (groups B, C, and D) were slower than that of the group treated with pure gliclazide (group A). In case of groups treated with optimized microcapsules, the reduction in blood glucose level reached a maximum within 3 to 4 h and was sustained over 12 h after oral administration of optimized mucoadhesive alginate–methyl cellulose microcapsules containing gliclazide, which was almost similar with the previously reported gliclazide-loaded microcapsules by Prajapati et al. (22) in alloxan-induced diabetic rat model. A reduction of 25% in blood glucose level is considered a significant hypoglycemic effect (22,25). In the previous report by Prajapati et al. (22), it was found that the reduction on blood glucose level was slow and reached maximum reduction within 3 h of oral administration of alginate–methyl cellulose mucoadhesive microcapsules of gliclazide in rat model. So, it can be concluded from the present investigation that the drug release pattern from optimized alginate–methyl cellulose microcapsules was much sustained in comparison to the previously reported mucoadhesive microcapsules containing gliclazide. Therefore, the sustained anti-diabetic effect by optimized microcapsules was observed over a longer period. The above studies also indicated that these mucoadhesive microcapsules swelled slowly in stomach and accordingly adhered to the stomach mucosa allowing more gliclazide to be absorbed by prolonging gastric residence and then subsequently moved to upper intestine, where they swelled more and released drug through the polymeric gel layer, formed at matrices periphery.
CONCLUSION
The optimized alginate–methyl cellulose mucoadhesive microcapsules containing gliclazide by ionotropic gelation was developed based on central composite design. The drug encapsulation efficiency of these optimized microcapsules was found to be maximum (83.57 ± 2.59% to 85.52 ± 3.07%) with a controlled drug release pattern (zero order) and the drug release mechanism followed the case II transport. All of these optimized microcapsules exhibited good mucoadhesive behavior. The in vivo study demonstrated that the significant hypoglycemic effect was observed after oral administration of optimized mucoadhesive microcapsules containing gliclazide. Therefore, the developed and optimized alginate–methyl cellulose microcapsules are suitable for prolonged systemic absorption of gliclazide through controlled drug release and mucoadhesive properties after oral administration in the treatment of non-insulin-dependent diabetes mellitus with maintaining lower blood glucose level and improved patient compliance.
ACKNOWLEDGEMENTS
One of the authors is thankful to Dr. R M Dubey, Vice Chancellor, IFTM University for providing necessary facilities for animal experiments.
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