Development, optimization, and evaluation of emulsion-gelled floating beads using natural polysaccharide-blend for controlled drug release.
Oral controlled release drug delivery systems have been developed over the past few years due to their considerable therapeutic benefits such as ease of administration, patient compliance, and flexibility in formulation. However, this approach is suffered with several draw-backs such as inability to retain and locate the controlled release dosage forms within the gastrointestinal tract (GIT) due to variable gastric emptying and motility. Furthermore, the relatively brief gastric emptying time in humans, which normally averages 2-3 h through the major absorption zone, i.e., stomach and upper intestinal part, can result in incomplete drug release from dosage forms leading to insufficient efficacy of the administered dose (1). This consideration has led to the development of oral controlled dosage forms with sufficient gastroreten-tive properties. After oral administration, such dosage forms could lengthen the gastric residence time of dosage forms for prolonged time in the upper GIT and gradually released incorporated drug molecules in sustained manner to obtain optimal bioavailability (2), (3). Several gastrore-tentive drug delivery approaches have been reported in the literature such as floatation (4), (5), mucoadhesion (6), sedimentation (7), unfoldable, expandable, or swellable systems (8), superporous hydrogel systems (9), and magnetic systems (10). Low-density floating systems are designed to be remained buoyant on the gastric contents because of their lower bulk density compared to that of aqueous medium, thus retained in the stomach for several hours allowing slower drug release at a desired rate. Multiple-unit floating dosage forms show several advantages over single-unit ones, in terms of uniform distribution along the GIT, absence of impairing performance due to failure of a few units, more predictable drug release kinetics, and lowering dose-dumping chances.
Among various multiple-unit systems, alginate beads have been developed in recent years as drug delivery vehicle due to its biocompatibility, biodegradability, reproducibility, simple method of preparation, abundant sources, low cost, and minimal processing requirements (11), (12). Alginate is a linear anionic polysaccharide and is found as structural components of brown marine algae. It is a copolymer of [alpha]-L-guluronic acid (G) and [beta]-D-mannuronic acid (M), having 1, 4-glycosidic linkages between them. Alginate undergoes ionotropic gelation in aqueous solution in presence of divalent and trivalent cations such as [Ca.sup.2+], [Pb.sup.2+], [Cu.sup.2+], [Zn.sup.2+], [Cd.sup.2+], and [Al.sup.3+], due to iono-tropically cross-linking interaction with intermolecular bonding between the carboxylic acid groups located on the polymer back-bone and these cations (13). However, various floating alginate beads suffered from low drug entrapment, rapid burst drug-release, less buoyant duration with long buoyant lag-time, etc. These drawbacks can be overcome by the addition of further additives such as suitable polymer-blends, incorporation of low-density oils and effervescent agents, etc. Again, incorporation of various low-density oils such as mineral oils, olive oil, sunflower oil, castor oil, mentha oil, and linseed oil, imparts the buoyancy of the alginate beads (14-16).
Tamarind seed polysaccharide (TSP) is a natural polysaccharide. obtained from the seed kernel of Tarnarindus indica L. TSP is composed of (1 [right arrow] 4)-[beta]-D-glucan backbone substituted with side chains of [alpha]-D-xylopyranose and [beta]-a-galactopyranosyl (1 [right arrow] 2)--D-xylopyranose linked (1 [right arrow] 6) to glucose residues (17). TSP has high stability and biocompatibility (18). It is used as binder, gelling agent, thickening agent, emulsifying agent, and suspending agent in pharmaceutical formulations. Recently, our research group has reported the development of pH-sensitive TSP-alginate beads for controlled drug release and these beads showed improved drug entrapment with sustained drug release profile (19). In this regard, the present work deals with the development of groundnut oil-entrapped alginate-based floating beads for controlled gastroretentive drug delivery using TSP-alginate blend by ionotropically emulsion-gelation technique. Again, the hydrophilic and high viscous nature of TSP can help the beads for mucoadhesion on the gastric mucosa. The current investigation aims at developing, optimizing and evaluating groundnut oil-entrapped TSP-alginate beads using ionotropically emulsion-gelation method as multiple-unit floating drug delivery system for controlled drug release.
Diclofenac sodium, a nonsteroidal anti-inflammatory drug, which is widely used clinically as strong analgesic was used as model drug in the present investigation. It is also used in the treatment of rheumatoid arthritis, and osteoarthritis (20). The biological half-life of diclofenac sodium is about 1-2 h (21); therefore, it requires multiple dosing to maintain therapeutic drug-blood level. Hence, the attempt to formulate multiple-unit gastroretentive floating systems for controlled diclofenac sodium release can eliminate the need for multiple dosing with improved patient compliance and decreased side effects. Computer aided optimization technique using three-factor and two-level ([2.sup.3]) factorial design was used to investigate the effects of three independent process variables (factors), i.e., polymer to drug ratio, groundnut oil to water ratio and sodium alginate to TSP ratio on the properties of groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium such as drug entrapment, density, and drug release.
MATERIALS AND METHODS
Diclofenac sodium was purchased from B. S. Trader, India. Sodium alginate (Central Drug House, India), calcium chloride (Merck, India), groundnut oil (relative density = 0.92, BD Pharmaceutical Works, India) were used. Tamarind seed was purchased from Jharpokharia market. All chemicals and reagents used were of analytical grade.
Isolation of TSP from Tamarind Seed
Raw seeds of tamarind (Tamarindus indica L.) were cleaned with distilled water to remove any extra pulp. Two-hundred fifty gram of cleaned seeds were broken into small pieces and ground into fine powder. The powder was added in 500 ml water and then, boiled on water-bath at 80-100[degrees]C with constant stirring until a viscous solution was obtained. The solution was filtered using muslin cloth to throw away the undissolved fraction, and the supernatant was dried at 40[degrees]C for overnight. Then the dried material is called tamarind kernel powder. TSP was prepared from this tamarind kernel powder. In brief, 20 g of tamarind kernel powder was added in 200 ml of distilled water, and slurry was prepared. The slurry was poured into 800 ml of distilled water. The solution was boiled for 20 min under stirring condition in a water bath. The resulting clear solution was kept overnight so that most of the proteins and fibers settled out. The solution was then centrifuged at 5000 rpm for 20 min. The supernatant was separated and poured into twice the volume of absolute ethanol with continuous stirring. The precipitate was dried at 40[degrees]C. The dried film obtained was crushed to fine powder through sieve no. 12 and kept in airtight desiccators.
Three-factor and two-level factorial design was used for optimization. Polymer to drug ratio by weight ([X.sub.1]), groundnut oil to water ratio by volume ([X.sub.2]) and sodium alginate to TSP ratio by weight ([X.sub.3]) as the prime selected independent variables (factors), which were varied at two levels (low and high). The drug entrapment efficiency (DEE), density, and cumulative drug release after 8 h ([R.sub.8h]) in simulated gastric fluid, pH 1.2 used as dependent variable (responses). Design-Expert 220.127.116.11 software (Stat-Ease) was used for generation and evaluation of the statistical experimental design. The matrix of the design including investigated factors and responses are shown in Table 1.
TABLE I. [2.sup.3] full factorial design (coded values in bracket) with observed response values for different groundnut oil-entrapped tamarind seed polysaccharide-alginate beads containing diclofenac sodium. Normalized Respones levels of independent variables (factors,) used Code Polymer to Oil to water SA to TSP DEE (%) drug ratio (by ratio (by ratio (a) (b), (c) weight) volume) (by weight) [X.sub.1] [X.sub.2] [X.sub.3] F-1 3(+1) 5(+1) 4(+1) 82.32 [+ or -] 0.92 F-2 3(+1) 5(+1) 2(-1) 84.60 [+ or -] 0.80 F-3 3(+1) 3(+1) 4(+1) 68.24 [+ or -] 0.86 F-4 3(+1) 3(-1) 2(-1) 74.13 [+ or -] 1.12 F-5 2(-1) 5(+1) 4(+1) 81.87 [+ or -] 0.93 F-6 2(-1) 5(+1) 2(-1) 84.14 [+ or -] 0.82 F-7 2(-1) 3(-1) 4 (+1) 64.05 [+ or -] 0.72 F-8 2(-1) 3(-1) 2(-1) 70.44 [+ or -] 0.96 Code Density [R.sub.8h] (g/[cm.sup.3]) (%) (c), (d) (d) F-1 0.81 [+ or -] 33.00 [+ or 0.07 -] 1.52 F-2 0.82 [+ or -] 38.42 [+ or 0.06 -] 1.02 F-3 0.83 [+ or -] 35.66 [+ or 0.09 -] 0.67 F-4 0.86 [+ or -] 44.98 [+ or 0.08 -] 1.22 F-5 0.67 [+ or -] 33.20 [+ or 0.04 -] 1.45 F-6 0.68 [+ or -] 36.25 [+ or 0.06 -] 1.32 F-7 0.77 [+ or -] 36.40 [+ or 0.08 -] 0.52 F-8 0.79 [+ or -] 43.48 [+ or 0.05 -] 0.86 (+1) = higher values and (-1) = lower values. (a.) SA to TSP ratio = sodium alginate to tamarind seed polysaccharide ratio. (b.) DEE (%) = drug entrapment efficiency (%). (c.) (Mean [+ or -] S.D.; n = 3). (d.) [R.sub.8h] (%) = cumulative drug release after 8 h.
For optimization, the effects of independent variables upon the responses were modelled using following first-order polynomial equations involving independent variables and their interactions for various measured responses, studied in this investigation. The effects of independent variables upon the DEE (%), density (g/[cm.sup.3]), and [R.sub.8h] (%) were modelled using following mathematical model generated by [2.sup.3] factorial design is following:
Y = [b.sub.0] + [b.sub.1][X.sub.1] + [b.sub.2][X.sub.2] + [b.sub.3][X.sub.3] + [b.sub.4][X.sub.1][X.sub.2] + [b.sub.5][X.sub.1][X.sub.3] + [b.sub.6][X.sub.2][X.sub.3]
where Y is the dependent variable, while [b.sub.0] is the intercept, [b.sub.1], [b.sub.2], [b.sub.3], [b.sub.4], [b.sub.5], [b.sub.6] and [b.sub.7] are regression coefficients; [X.sub.1], [X.sub.2], and [X.sub.3] are independable variables; [X.sub.1][X.sub.2], [X.sub.2][X.sub.3], and [X.sub.1][X.sub.3] are interaction between variables. One-way ANOVA was applied to estimate the significance of the model (p < 0.05) and individual response parameters.
Preparation of Beads Containing Diclofenac Sodium
Groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium were prepared by ionotropically emulsion-gelation technique. Briefly, required amount of polymers (sodium alginate and TSP) was dissolved in 100 ml demineralized water with constant stirring. Diclofenac sodium and groundnut oil were added to polymer solution. The final mixture of sodium alginate, TSP, groundnut oil and diclofenac sodium was homogenized for 10 min at 1000 rpm using a homogenizer (BL 232, BIO-LAB Instruments Mfg., India) and stirring at 5000 rpm continuously for 30 mins until the stable emulsion is formed. This resultant emulsion was dropped through 21G needles into 10% w/v calcium chloride solution (100 ml) and the added droplets were retained for 15 min in the calcium chloride solution to complete the curing reaction. Then the prepared beads were filtered and washed twice with petroleum ether and kept the beads after drying at room temperature for 24 h. The dried groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium were stored in desiccators until used.
Determination of DEE
Accurately weighed 100 mg of prepared beads from each batch were taken separately and were crushed using pestle and mortar. The crushed powders were placed in 500 ml of phosphate buffer, pH 7.4, and kept for 24 h with occasionally shaking at 37[degrees]C [+ or -] 0.5[degrees]C. After the stipulated time, the mixture was stirred at 500 rpm for 15 min on a magnetic stirrer. The polymer debris formed after disintegration of bead was removed filtering through Whatman filter paper (No. 40). The drug content in the filtrate was determined spectrophotometrically using a UV--visible spectrophotometer (Shimadzu, Japan) at 276 nm. The DEE of beads was calculated by using this following formula:
DEE, % = Actual drug content in beads/Theoretical drug content in beads x 100 (2)
Determination of Bead Size
The diameters of dried beads were measured using digital slide calipers (CD-6" CS, Mitutoyo, Japan) by inserting the beads in between the space of two metallic plates and diameter of resultant beads were displayed in the digital screen of the previously calibrated equipment. The average size was then calculated by measuring the diameter of three sets of 20 beads from each batch.
Sin face Morphology Analysis
Beads were gold coated by mounted on a brass stub using double-sided adhesive tape and under vacuum in an ion sputter (Hitachi E1010, Japan) with a thin layer of gold (3-5 nm) for 75 sec and at 40 W to make them electrically conductive and their morphology was examined by scanning electron microscopy (SEM) (Hitachi S3400, Japan).
The mean weights and diameters of the beads were measured and used to calculate densities of beads using the following equations:
[rho] = M/V, and V = 4/3 [pi][r.sup.3] (3)
where [rho], M, V, and r are the density (g/[cm.sup.3]), weight (g), volume ([cm.sup.3]), radius (cm) of the beads, respectively.
In Vitro Floatation Study
The floatation ability of beads was determined using dissolution apparatus type-II (Campbell Electronics, India). Fifty beads were placed in the dissolution vessel containing 500 ml of simulated gastric fluid (pH 1.2) maintained at 37[degrees]C [+ or -] 0.5[degrees]C for 8 h and the paddles were rotated at 50 rpm. The floating ability of beads was measured by visual observation. The time taken to float at the surface of dissolution medium (known as floating lag-time) and duration of floating were noted.
Fourier Transform-Infrared (FTIR) Spectroscopy
Samples were reduced to powder and analyzed as KBr pellets by using a FTIR spectroscope (Perkin--Elmer Spectrum RX I). The pellet was placed in the sample holder. Spectral scanning was taken in wavelength region between 4000 and 400 [cm.sup.-1] at a resolution of 4 [cm.sup.-1] with 1 cm/sec scan speed.
Nuclear Magnetic Resonance (NMR) Spectroscopy
[.sup.1]H NMR (600 MHz, 25[degrees]C) spectra of solution of various samples in dimethyl sulfoxide were recorded on a Bruker Avance[TM] III 500 spectrometer (Bruker Biospin Gmbh, Germany) operating at 500.13 MHz using a 4-mm CP-MAS probe head.
In Vitro Drug Release Studies
The in vitro release of diclofenac sodium from groundnut oil-entrapped TSP-alginate beads was tested using dissolution apparatus type-II. An equivalent weight of beads containing 100 mg diclofenac sodium was placed into 900 ml of simulated gastric fluid (pH 1.2), maintained at 37[degrees]C -[+ or -] 0.5[degrees]C and 50 rpm paddle speed. Ten milliliter of aliquots was collected at regular time intervals, and same amount of fresh dissolution medium was replaced into dissolution vessel to maintain the sink condition throughout the experiment. The collected aliquots were filtered, and further diluted suitably to analyze using UV--visible spectrophotometer (Shimadzu, Japan) at 276 nm.
Analysis of In Vitro Drug Release Kinetics and Mechanism
To predict and correlate the release behavior of diclofenac sodium from formulated beads in simulated gastric fluid (pH 1.2), it is necessary to fit into a suitable mathematical model. The in vitro drug release data were evaluated kinetically in important mathematical models (22):
Zero - order model: Q = kt + [Q.sub.0] (4)
where Q represents the drug released amount in time t, and [Q.sub.0] is the start value of Q; k is the rate constant.
First--order model: Q = [Q.sub.0][e.sup.kt] (5)
where Q represents the drug released amount in time t. and [Q.sub.0] is the start value of Q; k is the rate constant.
Hixson - Crowell model: [Q.sup.1/3] = kt + [Q.sub.0.sup.1/3] (6)
where Q represents the drug released amount in time t, and [Q.sub.0] is the start value of Q; k is the rate constant.
Weibull model: m = 1 - exp[-[(t).sup.b]/a] (7)
where in represents the drug released amount in time 1. a is the time constant and h is the shape parameter.
Baker - Lonsdale model: 3/2 [1 - [(1 - Q).sup.2/3]] - Q = kt (8)
where Q represents the drug released amount in time t, and k is the rate constant.
Higuchi model: Q = [kt.sup.0.5] (9)
where Q represents the drug released amount in time r, and k is the rate constant.
Korsmeyer -- Peppas model: Q = [kt.sup.n] (10)
where Q represents the drug released amount in time t, k is the rate constant and n is the diffusional exponent, indicative of drug release mechanism.
The accuracy and prediction ability of these models were compared by calculation of squared correlation coefficient ([R.sup.2]) and root mean squared error (RMSE) using KinetDS 3.0 Rev. 2010 software.
The Korsmeyer-Peppas model was used in in vitro drug release behavior analysis of beads to distinguish competing mechanisms: Fickian (diffusion-controlled) release, non-Fickian (anomalous) release, and case-II transport (relaxation-controlled release) (17). When n is [less than or equal to] 0.43, it is Fickian release. The n value between 0.43 and 0.85 is defined as non-Fickian release. When, n = 0.85, it is case-II transport.
Anti-Inflammatory Activity Evaluation
The carrageenan-induced rat-paw oedema model (23) was performed to assess anti-inflammatory activity evaluation of the optimized groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium (F-0) on male albino rats of either sex (weighing 266-340 g). The acclimatized rats were kept fasting for 24 h with water ad libitum. The experimental protocol was subjected to the scrutiny of the Institutional Animal Ethical Committee and was cleared before starting. The animals were handled as per guidelines of committee for the purpose of control and supervision on experimental animals (CPCSEA), New Delhi.
The fasted animals were divided into 3 groups (n = 6) and treated as follows:
Group A (control): administered with 2.5 ml sodium carboxymethyl cellulose (Na CMC) aqueous solution (0.5% w/v);
Group B (standard): administered with diclofenac sodium (10 mg/kg body weight) in 2.5 ml Na CMC aqueous solution (0.5% w/v); and
Group C (test): administered with optimized groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium (F-0) (equivalent to 10 mg diclofenac sodium/kg body weight) in 2.5 ml Na CMC aqueous solution (0.5% w/v).
After oral administration of samples using feeding needles, rats of all groups were challenged by subcutaneous injection of 0.05 ml of 1% w/v solution of carrageenean in saline into plantar site of the left hind paw. Paw volumes were measured before and after carrageenan administration at different time intervals with a plethysmometer (INCO, India). Mean increase in paw volume and % inhibition was calculated for all time intervals by using following formula:
% Inhibition = (1 - [D.sub.t]/[D.sub.c]) x 100 (11)
where, [D.sub.t] = Difference in paw volume in drug treated group; [D.sub.c] = Difference in paw volume in control animals.
Statistical optimization was performed using Design-Expert 18.104.22.168 software (Stat-Ease). The in vivo data was tested for significant differences (p < 0.05) by paired samples t-test. All other data was analyzed with simple statistics. The simple statistical analysis and paired samples t-test were conducted using MedCale software version 22.214.171.124.
RESULTS AND DISCUSSION
Isolation of TSP and Preparation of Beads Containing Diclofenac Sodium
TSP was isolated from tamarind (Tarnarinclus indica L.) seeds, and the average yield of dried TSP was found 47% w/w. The groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium was prepared by ion-otropically emulsion-gelation technique using calcium chloride solution as cross-linker. When the emulsions of sodium alginate, TSP, groundnut oil, and diclofenac sodium were dropped into the solutions containing calcium ions, gelled spherical groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium were formed instantaneously due to electrostatic interaction between negatively charged sodium alginate and positively charged calcium ion.
Traditionally, pharmaceutical formulators develop formulations by changing one variable at a time and the method is time consuming. It is therefore important to understand the influence of formulation variables on the formulation quality with a minimal number of trials and subsequent selection of formulation variables to develop optimized formulation using established statistical tools such as factorial design (24).
For the [2.sup.3] factorial design, a total 8 trial formulations were proposed by Design-Expert 126.96.36.199 software (Stat-Ease) for three independent variables: polymer to drug ratio by weight ([X.sub.1]), groundnut oil to water by volume ([X.sub.2]), and sodium alginate to TSP ratio by weight ([X.sub.3]), which were varied at two different levels (high and low). The effects of these independent variables on DEE (%), density (g/[cm.sup.3]), and [R.sub.8h] (%) in simulated gastric fluid (pH 1.2) were investigated as optimization response parameters in the present study. According to this trial proposal, various groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium were prepared by ionotropically emulsion-gelation technique. Overview of the experimental trial and observed responses were presented in Table 1.
The Design-Expert 188.8.131.52 software provided suitable polynomial model equations involving individual main factors and interaction factors. The model equation relating DEE (%) as response became:
DEE (%) = +44.10 + 8.80 X1 + 8.47 [X.sub.2] - 6.28 [X.sub.3] - 1.74 [X.sub.1][X.sub.2]+ 0.12 [X.sub.1][X.sub.3] + 0.97 [X.sub.2][X.sub.3]
[[R.sup.2] = 0.9999; F - value = 2315.09; p <0.05] (12)
The model equation relating density (g/[cm.sup.3]) as response became:
Density (g/[cm.sup.3]) = 1.09 - 0.04 [X.sub.1] - 0.14 [X.sub.2] - 0.02 [X.sub.3] + 0.04 [X.sub.1][X.sub.2] - 0.00 [X.sub.1][X.sub.3] + 0.00 [X.sub.2][X.sub.3]
[[R.sup.2] = 0.9996; F - value = 449.00; p < 0.05] (13)
The model equation relating [R.sub.8h] (%) as response became:
[R.sub.8h] (%) = 60.95 + 2.93 [X.sub.1] - 6.22 [X.sub.2] - 3.89 [X.sub.3] + 0.38 [X.sub.1][X.sub.2] - 1.23 [X.sub.1][X.sub.3] + 0.95 [X.sub.2][X.sub.3]
[[R.sup.2] = 0.9998; F - value = 993.09; p <0.05] (14)
The results of ANOVA, as shown in Table 2, indicated that all models were significant (p < 0.05) for all response parameters investigated. Model simplification was carried out by eliminating nonsignificant terms (p > 0.05) in polynomial equations (25), giving:
DEE (%) = +44.10 + 8.80 [X.sub.1] + 8.47 [X.sub.2] - 6.28 [X.sub.3] - 1.74 [X.sub.1][X.sub.2] + 0.97 [X.sub.2][X.sub.3] (15)
Density (g/[cm.sup.3]) = 1.09 - 0.04 [X.sub.1] - 0.14 [X.sub.2] + 0.04 [X.sub.1][X.sub.2], (16)
[R.sub.8h] (%) = 60.95 - 6.22 [X.sub.2] - 3.8 + 0.95 [X.sub.2][X.sub.3] (17)
TABLE 2. Summary of ANOVA for the response parameters. Source Sum of d. f. Mean F value p-value squares (a) square prob > F (a) For DEE (%) (h) M0del 451.62 6 75.27 2315.09 0.0159 (S) [X.sub.1] 9.66 1 9.66 297.06 0.0369 (S) [X.sub.2] 392.98 1 392.98 12087.06 0.0058 (S) [X.sub.3] 35.41 1 35,41 1089.00 0.0193 (S) [X.sub.1] 6.07 1 6.07 186.78 0.0465 [X.sub.2] (S) [X.sub.1] 0.03 1 0.03 0.92 0.5127 [X.sub.3] (NS) [X.SUB.2] 7.4s7 1 7.47 229.73 0.0419 [X.SUB.3] (S) (b) For density (g/[cm.sub.3]) Model 0.03 6 0.01 449.00 0.0361 (S) [X.sub.1] 0.02 1 0.02 1681.00 0.0155 (S) [X.sub.2] 0.01 1 0.01 729.00 0.0236 (S) [X.sub.3] 0.00 1 0.00 49.00 0.0903 (NS) [X.sub.1] 0.00 1 0.00 225.00 0.0425 [X.sub.2] (S) [X.sub.1] 0.00 1 0.00 1.00 0.5000 [X.sub.3] (NS) [X.sub.2] 0.00 1 0.00 9.00 0.2048 [X.sub.3] (NS) (C) For [R.sub.8h] Model 137.72 6 22.95 993.09 0.0243 (S) [X.sub.1] 1.15 1 1.15 49.65 0.0897 (NS) [X.sub.2] 46.80 1 46.80 2025.00 0.0141 (S) [X.sub.3] 79.19 1 79.19 3426.33 0.0109 (S) [X.sub.1] 0.29 1 0.29 12.33 0.1766 [X.sub.2] (NS) [X.sub.1] 3.01 1 3.01 130.38 0.0556 [X.SUB.3] (NS) [X.sub.2] 7.28 1 7.28 314.86 0.0358 [X.sub.3] (S) (a.) d. f. indicates degree of freedom. (b.) DEE (%) = drug entrapment efficiency (%). (c.) [R.sub.8h] (%) = cumulative drug release after 8 h. [X.sub.1], [X.sub.2], and [X.sub.3] represent polymer to drug ratio by weight, sodium alginate to TSP ratio by weight, and water to groundnut oil ratio by volume, respectively; [X.sub.1][X.sub.2], [X.sub.1][X.sub.3], and [X.sub.2][X.sub.3] are the interaction effects. S and NS indicate significant and not significant, respectively.
In addition, Design-Expert 184.108.40.206 software generated three-dimensional response surface plots and corresponding two-dimensional contour plots relating various measured responses. These plots were presented to estimate the effects of the independent variables on each response (Figs. 1-3). A numerical optimization technique using the desirability approach was used to develop new formulations with desired response. The desirable ranges of independent variables were restricted to: [X.sub.1] = 3.50, [X.sub.2] = 5.00, and [X.sub.3] = 1.50; whereas the desirable ranges of responses were restricted to 80 [less than or equal to] DEE [less than or equal to] 100%, 0.50 [less than or equal to] density [less than or equal to] 0.90 g/[cm.sup.3], and 40 [less than or equal to] [R.sub.8h] [less than or equal to] 60%. The optimal values of responses were obtained by numerical analysis using the Design-Expert 220.127.116.11 software based on the criterion of desirability. To evaluate optimization capability of models generated according to the results of [2.sup.3] factorial design, optimized groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium was prepared using the optimal process variable settings. The optimized groundnut. oil-entrapped TSP-alginate beads containing diclofenac sodium (F-0) was evaluated for DEE (%), density (g/[cm.sup.3]), and [R.sup.8h] (470). Table 3 lists the results of experiments with predicted responses by the mathematical model and those actually observed. The optimized groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium (F-0) showed DEE of 82.48% [+ or -] 2.34%, density of 0.88 [+ or -] 0.07 g/[cm.sup.3], and [R.sup.8h] of 41.02% [+ or -] 0.82% with small error-values (-3.20, -1.12, and -1.28, respectively). This reveals that mathematical models obtained from the [2.sup.3] factorial design were well fitted.
TABLE 3. Results of experiments for confirming optimization capability. Code Polymer lo Oil to water SA lo TSP Responses drug ratio ratio (by ratio (by (by weight) volume) weight) [X.sub.1] [X.sub.2] [X.sub.3] DEE (%) (a) F-0 3.50 5.00 1.50 82.48 [+ or -] 2.34 85.21 % Error -3.20 (d) Code Density [R.sub.8h] (g/[cm.sup.3]) (%) (b) F-0 Actual values (c) 0.88 [+ or -] 41.02 [+ or 0.07 -] 0.82 Predicted values 0.89 41.55 % Error -1.12 -1.28 (d) (a.) DEE (%) = drug entrapment efficiency (%). (b.) [R.sub.8h] (%) = cumulative drug release after 8 h. (c.) Mean [+ or -] S.D.; n = 3. (d.) Percentage of error (%) = (Actual value--Predicted value)/ Predicted value x 100.
The DEE of formulated groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium ranged from 64.05 [+ or -] 0.72 to 84.60% [+ or -] 0.80% (Tables 1 and 3). It was found that the drug entrapment in groundnut oil-entrapped TSP-alginate beads was increased with decreasing values of sodium alginate to TSP ratio and increasing values of both polymer to drug ratio and oil to water ratio. In general, increase in sodium alginate content in the used polymer-blend increases the cross-linking by interacting with the calcium ion present in the cross-linking solution, which should increase the DEE (%) of the TSP-alginate beads. Oppositely, it was found that decreasing sodium alginate to TSP ratio resulted increase in DEE (%) in these formulated TSP-alginate beads. The increased DEE (%) with decreasing sodium alginate to TSP ratio may be due to increase in viscosity of the polymeric solution by the TSP addition as polymeric blend with sodium alginate, so that, it might have been prevented drug leaching to the cross-linking solution. The phenomenon of increasing drug entrapment in the groundnut oil-entrapped TSP-alginate beads with increasing oil to water ratio may be due to either partitioning of some amount of drug (diclofenac sodium) in the oil (groundnut oil) phase or formation of oil barrier, which might have been obstructed the passage of drug molecules to external media during preparation. Moreover, increased DEE (%) with an increase in the polymer to drug ratio in these beads may be due to entanglement of higher drug amount inside intricate cross-linked alginate-TSP gel network.
The average size of these dried beads ranged from 1.07 [+ or -] 0.03 to 1.94 [+ or -] 0.09 mm (Table 4). Increasing bead size was found with increasing of both polymer to drug ratio and decreasing of sodium alginate to TSP ratio. This could be attributed due to the increase in viscosity of emulsion with the increasing proportion of total polymers as well as TSP amount in polymer blend with sodium alginate in increasing ratio that in turn increased the droplet size during addition of the groundnut oil-TSP-alginate containing diclofenac sodium emulsion to the crosslinking solution.
TABLE 4. Bead size, floatation, and floating lag-time of various groundnut oil-entrapped TSP-aiginate beads containing diclofenac sodium. Batch code Diameter (mm) Floatation (h) Floating lag-lime (a) (min) F-1 1.52 [+ or -] >8 7.40 0.06 F-2 1.86 [+ or -] >8 7.45 0.09 F-3 1.25 [+ or -] >8 8.05 0.07 F-4 1.67 [+ or -] >8 8.10 0.08 F-5 1.43 [+ or -] >8 5.30 0.04 F-6 1.70 [+ or -] >8 5.45 0.05 F-7 1.07 [+ or -] >8 6.30 0.03 F-8 1.38 [+ or -] >8 6.45 0.05 F-O 1.94 [+ or -] >8 8.25 0.09 (a.) Mean [+ or -] S.D.; n = 3.
Density values of various groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium ranged from 0.67 [+ or -] 0.04 to 0.89 [+ or -] 0.07 g/[cm.sup.3] (Tables 1 and 3). All these beads showed the density < 1. The decreasing densities of various groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium with the increasing of groundnut oil to water ratio. Results of lower density values obtained in the presence of higher oil concentrations entrapped in polymeric systems.
In Vitro Floating Lag-Time and Floatation
The in vitro floatation results of groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium in simulated gastric fluid (pH 1.2) is presented in Table 4. The density values of all formulated beads (F-1 to F-8, and F0) were less than the density of in simulated gastric fluid (pH 1.2) (i.e., 1.004 g/[cm.sup.3]) imparting their flotation (buoyancy) and these beads were floated well over 8 h. These beads were floated within 10 min after being placed in simulated gastric fluid, pH 1.2. The entrapped oil (here groundnut oil) in these beads was responsible for floating. Actually, entrapped low-density oils can lower the density of the polymeric systems. Thus, a clear correlation was evidenced between the density values of these groundnut oil-entrapped TSP-alginate beads containing diclofenac sodium with their floatation and floating lag-time.
Scanning electron microphotograph of diclofenac sodium-loaded groundnut oil-entrapped TSP-alginate bead surface showed a rough surface with small pores or channels (Fig. 4). The surface had an "orange peel" appearance with corrugations. No large crystals of drug were found on the surface of bead, indicating the presence of drug particles as finely dispersed state in the polymeric matrix.
The FTIR spectra of sodium alginate, TSP, diclofenac sodium, and groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium are shown in Fig. 5 (A-D, respectively). The FTIR spectrum of diclofenac sodium showed that the principal IR peaks at 1283.39 and 1305.57 [cm.sup.-1] resulted from C--N stretching; whereas at 1507.10 [cm.sup.-1] and 1575.56 cm-1 resulted from C=C stretching and C=0 stretching of carboxyl group, respectively. In the FTIR spectrum of TSP, the characteristic peaks showed principal peaks at 1043.06, 1650.37, 2925.55, and 3403.48 [cm.sup.-1] for --HC=0 stretching vibration,--CH--OH stretching vibration, aliphatic C--H stretching, and for the--OH groups, respectively. In the FTIR spectrum of sodium alginate, the characteristic peaks were appeared 1420.70, 1620.25, and 3442.50 [cm.sup.-1], for symmetric --[COO.sup.-] stretching vibration, asymmetric --[COO.sup.-] stretching vibration and for the --OH groups, respectively. In the FTIR spectrum of groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium (F-0), various characteristic peaks of sodium alginate, TSP, and diclofenac sodium were appeared without any significant shifting of these peaks. In short, the groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium had significant characters of diclofenac sodium in the FTIR spectrum, suggesting, there were no interaction between the diclofenac sodium and the polymers (TSP and sodium alginate) used in the bead formulation. The FTIR analysis confirmed the compatibility of the diclofenac sodium with TSP and sodium alginate used to prepare the groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium.
[.sup.1]H NMR Spectroscopy
The [.sup.1]H NMR spectra of blank groundnut oil-entrapped TSP-alginate beads, groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium and diclofenac sodium are shown in Figs. 6-8, respectively. In the [.sup,1]H NMR spectra of blank TSP-alginate beads and diclofenac sodium loaded TSP-alginate beads, typical characteristic signals of polysaccharides were crowded in a narrow region between 3 and 5 ppm indicating the presence of many similar sugar residues (26). Characteristic signals present in NMR spectra of
diclofenac sodium were also emerged in that of groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium indicating no interaction between the diclofenac sodium and the polymers (TSP and sodium alginate) used.
In Vitro Drug Release
The in vitro drug release studies were carried out for all formulated groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium in simulated gastric fluid (pH 1.2). All beads showed prolonged sustained release of diclofenac sodium over 8 h (Fig. 9). The cumulative drug released from these floating beads containing diclofenac sodium in 8 h ([R.sub.8h], %) was within the range of 33.00% [+ or -] 1.52% to 44.98% [+ or -] 1.22%. From the result of optimization, it was found that a significant increase in drug release from these TSP-alginate floating beads containing cliclofenac sodium was observed with the increasing of oil to water ratio, and decreasing of sodium alginate to TSP ratio in the formulated beads. The most reasonable explanation of the slow sustained drug release from highly oil-entrapped TSP-alginate floating beads was that the most of the drugs remained saturated and dispersed in the oil pockets of the beads to form a drug-oil dispersed matrix. Actually, drug transportation from beads to the dissolution medium may undergo two steps. Firstly, the drug may diffuse out of oil pockets into bead-matrix. Secondly, it may diffuse out of the matrix in to the dissolution medium. In case of comparatively higher TSP containing beads, the more hydrophilic property of TSP binds better with aqueous medium to form viscous gel structure, which may blockade pores on the surface of beads and thus, sustain release profile of the diclofenac sodium from these floating beads.
The in vitro drug release data from various groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium were evaluated kinetically using various mathematical models such as zero-order, first-order, Hixson-Crowell, Weibull, Baker-Lonsdale, Higuchi, and Korsmeyer-Peppas models. The [R.sup.2] and RMSE values of these models were determined for evaluation of accuracy and prediction ability of these models using KinetDS 3.0 Rev. 2010 software. The result of the curve fitting into various mathematical models is given in Table 5. When the respective [R.sup.2] of groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium were compared, it was found to follow the zero-order model ([R.sup.2] = 0.9842-0.9947) over a period of 8 h with minimum RMSE values (0.77-1.69). This indicates that the drug release from these floating beads in simulated gastric fluid (pH 1.2) was in controlled-release manner throughout the in vitro dissolution study. The values of diffusional exponent (n) determined from Korsmeyer-Peppas model of in vitro drug release data of these floating bead matrices ranged from 0.93 to 1.03, indicating the drug release from these beads followed the super case-II transport mechanism controlled by swelling and relaxation of polymeric-blend (TSP-alginate) matrix. This could be attributed due to polymer dissolution and polymeric chain enlargement or relaxation.
TABLE 5. Results of curve fitting of the in vitro diclofenac sodium release data from groundnut oil-entrapped TSP-alginate floating beads in simulated gastric fluid (pH 1.2). Formulation code Models F-l F-2 F-3 F-4 F-5 Zero order [R.sup.2] 0.9900 0.9876 0.9904 0.9902 0.9897 RMSE 1.01 1.32 1.32 1.32 1.02 First order [R.sup.1] 0.9510 0.9507 0.9606 0.9589 0.9465 RMSE 3.68 4.32 3.32 4.17 3.73 Hixson -Crowell [R.sup.2] 0.9783 0.9743 0.9815 0.9812 0.9728 RMSE 2.07 2.56 1.92 2.30 2.22 Weibull [R.sup.2] 0.9816 0.9691 0.9709 0.9680 0.9793 RMSE 1.25 1.65 1.54 2.00 1.21 Baker-Lonsdale [R.sup.2] 0.9357 0.9372 0.9387 0.9235 0.9488 RMSE 21.07 24.89 22.75 22.91 21.46 Higuchi [R.sup.2] 0.6143 0.6310 0.6661 0.6326 0.6501 RMSE 6.31 7.18 6.09 8.06 7.24 Korsmeyer-Peppas [R.sup.2] 0.9867 0.9739 0.9762 0.9750 0.9828 RMSE 1.14 1.53 1.37 1.70 1.14 n 1.03 1.00 0.95 0.99 0.99 Models F-6 F-7 F-8 F-O Zero order [R.sup.2] 0.9947 0.9916 0.9842 0.9930 RMSE 0.77 0.97 1.69 1.02 First order [R.sup.1] 0.9513 0.9462 0.9592 0.9514 RMSE 3.46 3.71 4.68 4.22 Hixson -Crowell [R.sup.2] 0.9781 0.9730 0.9783 0.9776 RMSE 1.97 2.26 2.76 2.41 Weibull [R.sup.2] 0.9811 0.9822 0.9614 0.9781 RMSE 1.20 1.23 2.23 1.46 Baker-Lonsdale [R.sup.2] 0.9435 0.9515 0.9311 0.9408 RMSE 23.20 23.80 27.78 26.19 Higuchi [R.sup.2] 0.6820 0.7022 0.6130 0.6540 RMSE 5.97 3.48 8.35 7.01 Korsmeyer-Peppas [R.sup.2] 0.9852 0.9862 0.9685 0.9831 RMSE 1.01 1.11 2.03 1.25 n 0.95 0.93 1.03 0.98 R2, squared correlation coefficients; RMSE, root mean squared error; n, diffusional exponent. All results were obtained using KinetDS 3.0 Rev. 2010 software.
Anti-inflammatory effect of the optimized groundnut oil-entrapped TSP-alginate floating beads containing diclofenac sodium (F-0) was evaluated using carrageenan-induced rat-paw oedema model. After oral administration of samples use in the study, rats of all groups (control, standard and test) were challenged by a subcutaneous injection of 0.05 ml of 1% w/v solution of carrageenan in saline into the plantar site of left hind paw. The mean increase in paw volumes (ml) and inhibition of paw oedema (%) for all samples at various time intervals were calculated and presented in Figs. 10 and 11, respectively. The mean increase in paw volumes after oral administration of standard (pure diclofenac sodium) and test (F-0) was significantly (p < 0.05) lower than that of pure diclofenac sodium (standard) after 2 h of oral administration. The pure diclofenac sodium exhibited comparatively rapid inhibition of paw oedema than that of the optimized floating beads tested indicating rapid anti-inflammatory activity up to 3rd h and after that, this was decreased. The optimized floating beads showed slower inhibition of paw oedema and maintained increasing inhibition of paw oedema over 8 h after oral administration indicating sustained action of diclofenac sodium.
Emulsion-gelled floating beads containing diclofenac sodium using TSP-alginate blend for controlled gastroretentive delivery was successfully developed using 23 factorial design. These developed TSP-alginate floating beads combining excellent DEE, appreciate floating ability in gastric fluid with a minimum floating lag-time, suitable controlled drug release pattern and excellent anti-inflammatory activity over prolonged period, which could possibly be advantageous in terms of increased bioavailability of diclofenac sodium. The ionotropically emulsion-gelation technique for the preparation of these newly developed floating beads containing diclofenac sodium for controlled gastroretentive delivery were found to be simple, reproducible, easily controllable, economical and consistent process. In addition, the excipients used for the formulation of these new floating beads were cheap and easily available. This type of floating beads can also be developed for other drugs used for the treatment of diseases associated with GIT to improve their bioavailability and therapeutic efficacy.
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Amit Kumar Nayak, (1) Dilipkumar Pa1, (2) Jadupati Malakar (3)
(1.) Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj, Orissa 757086, India
(2.) Department of Pharmaceutical Sciences, Guru Ghasidas Viswavidyalay, Koni, Bilaspur, Chhattisgarh 495 009, India
(3.) Department of Pharmaceutics, Bengal College of Pharmaceutical Sciences and Research, Durgapur, West Bengal 713212, India
Correspondence to: Dilipkumar Pal; e-mail: firstname.lastname@example.org
Published online in Wiley Online Library (wileyonlinelibrary.com).
[C] 2012 Society of Plastics Engineers
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|Author:||Nayak, Amit Kumar; Pal, Dilipkumar; Malakar, Jadupati|
|Publication:||Polymer Engineering and Science|
|Date:||Feb 1, 2013|
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