INTRODUCTION:

Symptomatic relief from musculo-skeletal disorders like rheumatoid arthritis, osteoarthritis and ankylosing spondylitis is achieved with non-steroidal anti inflammatory drugs (NSAIDs) ^1,2. However, long term therapy with these drugs may induce minor gastric irritation to severe bleeding and ulceration of gastric mucosa due to both inhibition of synthesis of prostaglandins and direct contact of the drug with mucosa ³. A rational dosage form for NSAIDs should be designed to prevent or minimize the drug release in stomach. Enteric coated tablets of NSAIDs are prescribed to achieve this objective. However, due to all or none hypothesis and considerable delay in gastric emptying, dose–dumping as well as intra- and inter-subject variation in the onset of therapeutic action may be exhibited by drugs administered as enteric coated tablets⁴.

Multiple unit drug delivery systems like microcapsules, pellets offer several advantages over single unit dosage form like more predicable gastric emptying, release drugs in a predictable manner and prevent dose dumping ^5,6. Recently much research efforts have been directed to the use of natural polymers to prepare such dosage forms.

Among the natural polymers, polysaccharides which are obtained from algae, plant, microorganism and animal sources, are abundant in nature, less expensive, non toxic, safe and do not require organic solvents for processing. Moreover, various functional groups present on the molecular chain of the polysaccharides are amenable to chemical and biochemical modifications ⁷ and this offers many important functional properties to the formulations ⁸. In addition, hydrophilic nature of the polysaccharides makes them to absorb water and swell. These properties have been used to prepare hydrogels which are three-dimensional hydrophilic networks capable of absorbing water without dissolving ⁹. The water absorption and swelling properties of the hydrogels can be controlled by physical or chemical cross-linking methods ¹⁰. That is why hydrogels are becoming an important device in drug delivery in addition to biochemical, biomedical and sensing fields ^11-14.

Quite often, a single polymer may not provide desired properties and performances with respect to formulation aspects. Therefore, polymer blend technology has been successfully used to overcome such drawbacks. Introduction of another polymer may give a new character of such polymer combination. However, success of such technology requires adequate knowledge on polymer structure, properties, characteristics and selection of ideal combination of polymer and their appropriate ratio.

Xanthan gum is a polysaccharide obtained from Xanthomonas campestris. Though xanthan gum is unable to form gel beads through ionotropic gelation with Al³⁺ ions, sodium carboxymethyl xanthan (SCMX), obtained through o-carboxymethylation of xanthan gum, can form gel beads ¹⁵. Carboxymethyl xanthan has been used to prepare gel beads of diltiazem ¹⁶ and albumin ¹⁷.

Cellulose is another most abundant biopolymer in nature ¹⁸. Sodium carboxymethyl cellulose (SCMC), a semi synthetic derivative of cellulose, is widely used in ranging from flocculation, drag reduction, oil drilling operation ¹⁹ to food industries, cosmetics and pharmaceuticals ²⁰. Recently SCMC has been used for IPN beads with gelatin for drug delivery system ²¹.

In this present work, aceclofenac (ACF) loaded aluminium carboxmethyl xanthan (Al-CMX), aluminium carboxmethyl cellulose (Al-CMC) and new bipolymeric hydrogel beads composed of SCMX and SCMC were prepared by ionotropic gelation with Al³⁺ ions with an aim that the formulation will minimize the drug release in stomach to avoid the harsh effects of NSAIDs and can provide controlled release in small intestine. Most of the reports described the effect of various formulation parameters on the physical parameters of the beads. It, however, appears that the viscosity of the polymer or combination of polymers may have an impact on the formation of hydrogel beads and hence, on the physical properties of the beads. An attempt was also made to correlate the formation and physical characteristics of the beads with the viscosity of the polymer solution in addition to other formulation variables. The compatibility of the drug in the hydrogel beads was assessed by FTIR, XRD and DSC analyses. Bipolymeric hydrogel beads were subjected to in-vitro drug release study in acid solution and in phosphate buffer solution and their performance was compared with the drug release behavior of single polymeric beads. ACF was selected as a model NSAID.

EXPERIMENTAL:

Materials:

Aceclofenac (Indian Pharmacopoeia) was obtained as gift sample from Torrent Pharmaceuticals, (Himachal Pradesh, India). Sodium carboxymethyl cellulose (viscosity 500-600cps of 2% aqueous solution) and monochloro acetic acid (Loba Chemie Pvt. Ltd, Mumbai, India), xanthan gum (viscosity 600 cps of 1 % aqueous solution) and AlCl₃.6H₂O (SD Fine Chem Pvt. Ltd, Mumbai, India), and all other analytical grade reagents were obtained commercially and used as received.

Preparation of Sodium Carboxymethyl Xanthan (SCMX):

SCMX having O-carboxymethyl substitution of 0.7 was prepared from xanthan gum following the method reported previously (15). In brief, required amount of xanthan gum was dispersed in ice cold solution of 45% w/v sodium hydroxide. The dispersion was kept at 5-8°C with continuous stirring for 1h. Monochloroacetic acid solution (75% w/v) was added with stirring in the reaction mixture and the temperature was raised slowly to 15-18°C. After 30 min, the temperature was raised to 75°C and maintained for additional 30 min. The reaction mixture was cooled to room temperature and dried at 50°C. The dried product was milled, washed with 80% v/v methanol and dried.

Preparation of hydrogel bead:

SCMX and SCMC were dissolved in deionized water with constant stirring until a homogeneous solution was formed. Required amount of ACF was added to the polymer solution and dispersed homogenously. The resulting dispersion was extruded through 21 G flat-tip hypodermic needle into AlCl₃ solution and gelled for different periods of time. The beads were then collected by filtration, washed with deionized water, dried at 45°C into a hot air oven to constant weight, and kept in a desiccator until used. The beads were prepared using the following variables:

1) Keeping the drug load constant at 20% w/w of total polymer, AlCl₃ concentration at 2% w/v, gelation time at 0.5 h and total polymer concentration at 2.5% w/v, the weight ratio of SCMX: SCMC was varied from 0%-100%.

2) Keeping the drug load constant at 20% w/w of total polymer, the gelation time at 0.5 h , and weight ratio of SCMX:SCMC at 50:50 (total polymer concentration 2.5% w/v), concentration of AlCl₃ was varied from 2-8% w/v.

3) Keeping the weight ratio of SCMX:SCMC constant at 50:50 (total polymer concentration 2.5% w/v), gelation time at 0.5 h and AlCl₃ concentration at 2% w/v, drug load was varied from 20-60% w/w of total polymer.

4) Keeping the drug load constant at 20% w/w of total polymer, AlCl₃ concentration at 2% w/v, gelation time at 0.5 h and weight ratio of SCMX:SCMC at 50:50, the total polymer concentration was varied from 1.5-3.5% w/v.

The composition of beads is shown in Table 1. Each formulation was prepared in triplicate.

Fourier Transform Infrared (FTIR) Analysis:

FTIR spectra of pure ACF and drug loaded bipolymeric bead were recorded in a FTIR spectrophotometer (Spectrum RX-1, Perkin- Elmer, UK). Each sample was mixed with KBr and converted into disc at 100 kg pressure using a hydraulic press. The spectra were recorded within 4000-400 cm-1 wave numbers.

Table1: Effect of formulation variables

Formulation Code	Total polymer Concentration (%w/v)	SCMX: SCMC (%ratio of total polymer)	Drug Load (% w/w of total polymer)	Concentration of AlCl₃ (% w/v)	Gelation time (h)	DEE Mean±SD (n=3)	Diameter (mm) Mean±SD (n=50)
F1	2.5	100:0	20	2	0.5	99.53±2.34	0.93 ±0.07
F2	2.5	75:25	20	2	0.5	99.17±0.83	1.05 ±0.10
F3	2.5	50:50	20	2	0.5	95.83±0.45	1.14 ±0.89
F4	2.5	25:75	20	2	0.5	85.83±1.38	1.18 ±0.13
F5	2.5	0:100	20	2	0.5	71.67±1.16	1.23 ±0.10
F6	2.5	50:50	20	4	0.5	75.83±1.37	1.08 ±0.10
F7	2.5	50:50	20	8	0.5	64.17±2.77	1.02 ±0.09
F8	2.5	50:50	40	2	0.5	96.25±1.59	1.30 ±0.08
F9	2.5	50:50	60	2	0.5	97.22±0.97	1.44 ±0.07
F10	3.5	50:50	20	2	0.5	93.33±0.81	1.42 ±0.09
F11	1.5	50:50	20	2	0.5	94.06±1.20	0.87 ±0.05

Powder X-Ray Diffraction (XRD) Study:

Qualitative XRD studies were performed using an X-ray diffractometer (Miniflex XRD, Rigaku Corporation, Japan). Pure ACF and powdered drug loaded bipolymeric beads were scanned from 5° to 50° diffraction angle (2θ) range under the following measurement conditions: Source, Ni-filtered Cu-Kα (λ = 1.54) radiation; voltage, 40 kV; current, 40 mA; scan speed, 1°/min.

Differential Scanning Calorimetry (DSC) Study:

DSC thermograms of pure ACF and powdered drug loaded bipolymeric beads were obtained in the following way: A weighed amount (about 6 mg) of sample was kept in a hermetically sealed aluminium pan and heated at a scan speed of 10°C/min over a temperature range of 35°C- 300°C in a differential scanning calorimeter (Model Pyris Diamond TG/DTA, Perkin-Elmer, UK) which was calibrated against indium. A nitrogen purge (20 ml/min) was used throughout the runs.

Scanning Electron Microscopic (SEM) Study:

The drug-loaded beads were mounted onto stubs using double-sided adhesive tape and sputter coated with gold using a sputter coater (S150, Edward, UK). The coated beads were observed under scanning electron microscope (SEM) (JSM-5200, Jeol, Japan) at the required magnification at room temperature. The acceleration voltage used was 10 kV.

Drug Entrapment Efficiency (DEE):

Drug loaded hydrogel beads (50 mg) were accurately weighed in an electronic balance (Precisa XB 600 MC, Precisa Instrument Ltd, Switzerland), immersed in 250 ml USP phosphate buffer (PB) solution (pH 6.8), and shaken for 24 h on a mechanical shaker. The beads were crushed and further shaken for 1h. The solution was filtered and an aliquot, following suitable dilution, was analyzed at 272 nm in a UV-Visible spectrophotometer (UV-2450 Shimadzu, Japan). The content of the beads was determined using a calibration curve constructed using PB solution of pH 6.8. The reliability of the above analytical method was judged by conducting recovery analysis at three levels of spiked drug solution in the presence or absence of the polymers for three consecutive days. The recovery averaged 98.45 ± 2.68%.

DEE was determined using the following relation:

DEE (%) =

(Determined drug content/Theoretical drug content) × 100

In-Vitro Drug Release Study:

In-vitro drug release study was carried out in acidic solution (USP HCl buffer, pH 1.2) and in USP PB solution (pH 6.8) using USP-II dissolution rate test apparatus (TDP-06P Electro Lab, India). 50 mg drug loaded beads were placed in 500 ml acidic solution or 500 ml PB solution (37 ± 0.5°C) and rotated with paddle at 75 rpm. At different time interval, aliquot was withdrawn and replenished immediately with the same volume of fresh solution. Undiluted or suitably diluted withdrawn samples were analyzed spectrophotometrically at 272 nm for acidic solution and PB solution. The amount of drug released in acidic solution and PB solution were calculated from the calibration curves drawn respectively, in HCl buffer (pH 1.2) and PB solution (pH 6.8). Each release study was conducted three times.

Swelling Study:

Dried drug-free hydrogel beads (50 mg) were immersed in 50 ml acidic solution (pH 1.2) at 37ºC. The beads were removed at different time intervals by filtration using a stainless steel grid and blotted carefully to remove excess surface water. The swollen beads were weighed. The swelling ratio of the beads was determined using the following formula:

Swelling ratio = (weight of swollen beads-weight of dry beads)/weight of dry beads

Swelling ratio of the drug-free beads in PB solution (pH 6.8) was determined in a similar way.

Viscosity Measurement:

Viscosity of 1% w/v aqueous solution single and blended polymers was measured in Brookfield viscometer (TokiSangyo viscometer, model no. TV-10, Japan) using spindle no. M1.

Measurement of Bead Diameter:

The diameter of the drug loaded beads was measured using a digimatic caliper (model CD-6”CS, Mitutoyo, Japan) having an accuracy of 0.01 mm. The average diameter of the 50 particles per batch was calculated.

RESULTS AND DISCUSSION:

Morphology of beads:

Scanning electron micrographs demonstrated that Al-CMX beads were discrete and spherical with a few depressions on the surface (Fig. 1a). On the other hand, Al-CMC beads appeared to be slightly elongated, and the surface was almost devoid of any depression (Fig. 1e). The bipolymeric beads were also discrete and spherical; however, the number of depression on the surface decreased as the amount of SCMC in the beads was increased (Fig. 1b-1d). At higher magnification under SEM (Fig. 2a-2e), the surface of Al-CMX beads appeared smooth and as the amount of SCMC was increased, the surface of the bipolymeric beads tended to become denser and the surface of Al-CMC beads was rough and folded. The change in gross and surface morphology of the beads could be related to the viscosity of the polymer solutions and the degree of cross linking with Al³⁺ ions.

Low viscosity of SCMX (113.6 cp of 1% w/v solution) provided sufficient fluidity to the beads to acquire spherical shape. In addition to the low viscosity, less number of O-carboxymethyl groups (degree of substitution 0.7) provided lower degree of cross linking with Al³⁺ ions that in turn, produced less rigidity to the matrix leading to the formation of depression on the surface of the beads when dried. Substitution of SCMX with increasing amount of SCMC not only increased the viscosity of the solutions of the blended polymers (SCMX:SCMC=75:25, 149.43 cp; SCMX:SCMC=50:50, 194.04 cp ; SCMX:SCMC=25:75, 254.87cp) but also increased the number of carboxymethyl groups as the degree of substitution of SCMC was found to be 1.1. This provided greater rigidity to the matrix leading to decrease in the number of depressions on the surface of beads. The viscosity of SCMC (283.47 cp of 1% w/v solution) was too high and extrusion through the needle produced elongated beads. Moreover, larger number of carboxymethyl groups produced higher cross linking. This resulted in an increase in the rigidity of the matrix with rough and folded surface.

Figure 1a

Figure 1b

Figure 1c

Figure 1d

Figure 1e

Figure 1: Scanning electron micrographs of various homopolymeric and IPN hydrogel beads (at low magnification, 35 X). Key: a) Al-CMX:Al-CMC=100:0 b) Al-CMX:Al-CMC=75:25 c) Al-CMX:Al-CMC=50:50 d) Al-CMX:Al-CMC=25:75 e) Al-CMX:Al-CMC=0:100 .

Figure 2a

Figure 2b

Figure 2c

Figure 2d

Figure 2e

Figure 2: Scanning electron micrographs of various homopolymeric and IPN hydrogel beads (at high magnification, 150 X). Key: a) Al-CMX:Al-CMC=100:0 b) Al-CMX:Al-CMC=75:25 c) Al-CMX:Al-CMC=50:50 d) Al-CMX:Al-CMC=25:75 e) Al-CMX:Al-CMC=0:100.

Diameter of beads:

The size of the prepared hydrogel beads appeared to be dependent on the formulation factors. Keeping the total polymer concentration (2.5% w/v), drug load (20 % w/w), concentration of AlCl₃ solution (2% w/v) and gelation time (0.5 h) constant, substitution of SCMX with increasing amount of SCMC increased the average diameter of beads (Table 1). Similarly, keeping all other variables constant, increase in total polymer concentration from 1.5 % to 3.5% w/v increased the size of the beads. As stated earlier, the viscosity of the SCMX solution was the lowest; substitution of SCMX with increasing amount of SCMC increased the viscosity of the polymer solutions, and the viscosity of SCMC solution was the highest. High viscosity of polymer solution produced bigger droplets during extrusion through the needle, and resulted in the formation of bigger particles. This is in agreement with the results relating to the formation of semi IPN microspheres of gelatin and SCMC ²¹ chitosan poly(ethylene oxide-g-acrylamide) microspheres ²² and polyacrylamide grfted xanthan-CMC IPN beads²³. Increase in drug load from 20-60 % w/w in the polymer solution having a definite composition also increased the viscosity of the solution because the drug might have occupied the interstitial spaces between the polymer segments and thus led to the formation of beads of larger diameter ²⁴. On the other hand, increase in the concentration of AlCl₃ in gelation medium tended to reduce the size of the beads due to squeezing of water out of the beads in hyperosmotic gelation medium. Many research reports also stated that higher concentration of glutaraldehyde or AlCl₃ used as cross-linking agent in gelation medium causes rapid shrinkage of the beads leading to the formation of smaller sized beads ^21,25.

Compatibility of Drug in Bead:

FTIR analysis:

Interaction of drug with polymer in the bipolymeric beads were studied through FTIR analysis of pure ACF and drug loaded bipolymeric beads. FTIR spectrum of ACF (Fig. 3) showed characteristics peaks at 3319 cm^-1, 2936 cm^-1, 1715 cm^-1, 1508 cm^-1, 1254 cm^-1, 666 cm^-1 representing respectively OH stretching vibration, CH stretching vibration, COO- asymmetric stretching and C=0 stretching, C=C aromatic ring stretching, C-O-C stretching and C-Cl stretching. Drug loaded bipolymeric bead generated the respective peaks at 3319 cm^-1, 2936 cm^-1, 1716 cm^-1, 1506 cm^-1, 1256 cm^-1, 665 cm^-1 that were almost at the same wave numbers as those of the pure drug. This indicates the absence of any interaction of the drug with the polymer.

Figure 3: FTIR spectra of: a) aceclofenac b) aceclofenac loaded IPN bead (Al-CMX:Al-CMC=50:50)

XRD study:

The diffractograms obtained from XRD study of pure ACF and drug loaded bipolymeric beads are shown in Fig. 4. XRD trace of ACF showed reflection to the interplanner distance of 5.01, 4.76, 3.96 and 3.41 Å respectively at 17.66, 18.62, 22.4 and 26.09 ° 2θ. The drug loaded bipolymeric bead showed reflection to the interplanner distance of 5.02, 4.75, 3.97 and 3.41Å respectively at 17.63, 18.65, 22.37and 26.06 ° 2θ. The results demonstrated that the characteristic peaks of the drug appeared almost at the same 2θ values in the X-ray diffractograms of drug loaded bipolymeric beads although intensity of the peaks was considerably reduced. It indicates that the crystalline nature of the drug was retained in the beads.

Figure 4: X-ray diffractograms of: a) aceclofenac b) aceclofenac loaded IPN bead (Al-CMX:Al-CMC=50:50)

DSC study:

The DSC thermograms of pure ACF and drug loaded bipolymeric beads are shown in Fig. 5. The pure ACF showed a sharp endothermic peak at 152.49˚C whereas drug loaded bipolymeric beads showed an endothermic peak at 158.26˚C. Since the endothermic peak of the drug in pure state and in bipolymeric bead were almost at the same temperature, it may be considered that the crystalline nature of the drug was retained in the bipolymeric bead and no molecular dispersion or amorphization of the drug took place during the preparation of the beads.

Figure 5: DSC thermograms of a) aceclofenac b) aceclofenac loaded IPN bead (Al-CMX:Al-CMC=50:50)

DEE of beads:

DEE of various beads has been shown in Table 1 which indicates that DEE of Al-CMX beads were the highest. Substitution of SCMX with SCMC decreased the DEE of bipolymeric beads, and DEE of Al-CMC beads was the lowest. A recent research report shows that entrapment efficiency of ketorolac in semi IPN microspheres composed of gelatin and SCMC decreased to some extent with increase in the amount of SCMC and the lower entrapment efficiency was accounted for leaching of drug ²¹. Though leaching phenomenon is indeed responsible for low entrapment efficiency of many drugs, it may not be completely applicable for poorly water soluble drugs like ACF. High viscosity of the polymer solution together with low aqueous solubility of ACF should decrease the leaching of drug, and thus Al-CMC beads are expected to have higher DEE. A possible reason for such low DEE may be nonuniform drug dispersion, even after homogenization in 2.5% w/v SCMC solution. To ascertain this apprehension, beads were prepared using 1% w/v and 2 % w/v SCMC solution. Although the resulting beads were flattened instead of being spherical in shape, DEE of the beads prepared with 1% (293.47 cp) and 2% (550cp) SCMC solution were respectively 93.35% and 74.16%. The result clearly indicates that increase in viscosity led to nonuniform drug dispersion and hence produced lower DEE of Al-CMC beads. It has been reported that use of highly viscous alginate solution may lead to improper mixing of drug ²⁶.

The effect of drug load on DEE of the beads was examined by preparing beads using SCMX and SCMC in ratio of 50:50, gelation time 0.5 h and 2% w/v AlCl₃ solution. DEE was found to vary within 95.83 to 97.22% indicating that initial drug loading did not produce any significant change ( p>0.05 ) in DEE. Similar observations have been reported by other workers^21,27.The entrapment efficiency of ACF was found to decrease significantly when the concentration of cross-linking agent (AlCl₃) was increased from 2 to 8 % w/v. This could be due to increase in gel porosity with increase in the concentration of AlCl₃. Small angle X-ray scattering analysis revealed that increase in the ratio of calcium and alginate concentration increases the cross sectional radius of gyration of the junction zones leading to increase in porosity of Ca-alginate gel ^28,29 also reported that increase in the concentration of Ca²⁺ ion up to a certain value increased the porosity of calcium alginate gel as evident from blue dextran release experiment.

Increase in total polymer concentration from 1.5 to 3.5% w/v did not produce any appreciable change in DEE.

In-Vitro Drug Release:

The effect of amounts of the two polymers on drug release in acid solution was examined for a period of 3 h using the beads prepared with 20 % w/w drug and gelling for 0.5 h in 2% w/v AlCl₃ solution, and the results are presented in Fig. 6. Drug release from Al-CMX beads was rapid, releasing 14.20 % of loaded drug in 3 h. Substitution of SCMX with increasing amount of SCMC in the beads decreased the drug release, and the release of the drug from Al-CMC was the lowest, releasing 7.98 % drug in 3 h (Fig. 6). This decrease in drug release was also verified from the area under the curves (AUCs) which were measured from the cumulative percent drug release versus time curves using trapezoidal rule. One way ANOVA revealed that AUC values decreased significantly (p< 0.05) from 27.98 to 14.48 (%mg. h/ml) as SCMX was substituted with increasing amount of SCMC. Both SCMX and SCMC possess COOH groups which can bind with Al³⁺ ion through ionotropic gelation process to form water insoluble gel beads of Al-CMX and Al-CMC. The release of a drug from such an ionically cross linked polymer depends on the extent of swelling of the polymer that in turn, is controlled by the pH of the aqueous medium. Similar to calcium alginate, which becomes converted in to alginic acid and swells to a very less extent in acid solution of low pH ³⁰, both Al-CMX and Al-CMC may be converted in to their corresponding acid forms in solution of low pH. The unioniozed carboxyl groups of the polymers exert insignificant electrostatic repulsive forces and hence, relaxation of the macromolecular chain does not take place ³¹. As a result, the beads do not swell in acidic solution to a great extent and drug release takes place slowly. Comparison of the swelling behavior of the beads made of various amounts of two polymers demonstrated that the swelling ratio of Al-CMX beads in acid solution was high. Substitution of SCMX with increasing amount of SCMC decreased the swelling of the beads and swelling ratio of Al-CMC was the lowest (Fig. 7). The swelling capacity of beads made of hydrophilic polymer also depends on the number of –OH groups present in the polymer. As the number of –OH groups per unit residue of SCMX in greater than that of SCMC, Al-CMX beads swell to a greater extent and release the drug faster than the beads containing increasing amount of SCMC.

Figure 6: Release of aceclofenac from various beads in HCl buffer (pH 1.2) (dotted line) and in phosphate buffer solution (pH 6.8) (Firm line). Key: ∆=F1(Al-CMX:Al-CMC=100:0), ●=F2(Al-CMX:Al-CMC=75:25), ▲=F3(Al-CMX:Al-CMC=50:50), ■=F4(Al-CMX:Al-CMC=25:75) , ○=F5(Al-CMX:Al-CMC=0:100).

Figure 7: Swelling ratio of blank beads in HCl buffer (pH 1.2). Key: ○=F1(Al-CMX:Al-CMC=100:0) , ■=F2(Al-CMX:Al-CMC=75:25), ♦=F3(Al-CMX:Al-CMC=50:50), ▲=F4(Al-CMX:Al-CMC=25:75) , ●=F5(Al-CMX:Al-CMC=0:100).

Figure 8: Swelling ratio of blank beads in phosphate buffer (pH 6.8). Key: ○=F1(Al-CMX:Al-CMC=100:0) , ■=F2(Al-CMX:Al-CMC=75:25), ♦=F3( Al-CMX:Al-CMC=50:50), ▲=F4(Al-CMX:Al-CMC=25:75) , ●=F5(Al-CMX:Al-CMC=0:100).

Table 2: Derived release parameters and release kinetics of drug from various hydrogel beads.

Formulation Code	t_50%in PB solution (min) Mean ± SD (n=3)	t_80%in PB solution (min) Mean ± SD (n=3)	Release Kinetics
			In acidic solution		In PB Solution
			n	r²	n	r²
F1	144.9±1.36	190.7±1.66	0.50	0.99	0.82	0.86
F2	147.6±0.85	202.6±2.68	0.44	0.98	0.79	0.92
F3	184.7±6.58	233.5±3.17	0.41	0.99	0.78	0.87
F4	242.2±5.12	317.1±5.90	0.34	0.98	0.74	0.89
F5	197.1±1.88	274.8±3.16	0.25	0.92	0.69	0.92
F6	135.4±3.36	192.9±2.83	0.59	0.99	0.54	0.82
F7	126.7±6.19	182.1±9.76	0.65	0.99	0.48	0.83
F8	146.4±4.31	206.0±3.72	0.31	0.98	1.45	0.99
F9	123.4±0.50	182.0±3.96	0.34	0.99	1.07	0.97
F10	171.6±3.10	225.2±1.35	0.49	0.99	0.96	0.86
F11	67.0±4.76	102.5±4.92	0.38	0.98	0.77	0.97

Except the release of the drug from Al-CMC beads, the drug release in PB solution from Al-CMX and bipolymeric beads followed the same order as that found in acid solution. The release of the drug from Al-CMC beads was higher than that from the bipolymeric beads made of 25% SCMX and 75% SCMC (Fig. 6). The time required for 50% (t_50%) and 80% (t_80%) drug release gradually increased with increase in substitution of SCMX with SCMC. However, the same parameters made of SCMC alone decreased (Table 2). Al-CMX, Al-CMC and bipolymeric beads may swell in PB solution following the same mechanisms responsible for swelling of calcium alginate beads. In PB solution, the ionotropically gelled structure of the beads breaks down due to ion exchange between the gel forming Al³⁺ ions and PO₄^3- ions of the PB solution. The ionized carboxylate ions create strong repulsive forces ³² and results in relaxation of the macromolecular chain³¹ which accelerate the swelling and erosion of the gel structure. All these factors make the beads to swell to a great extent and results in higher drug release. However, the reswelling of ionotropically gelled beads also depends on the rigidity or cross linked density of the matrix formed through interaction between the COOH groups of the polymers and Al³⁺ ions. The gel strength of Al-CMX beads may be expected to be less due to presence of smaller number of COOH (degree of substitution 0.7) in SCMX than in SCMC (degree of substitution 1.1). Thus Al-CMX beads swell faster in PB solution (Fig. 8) and release the embedded drug rapidly (Fig. 6). Substitution of SCMX with increasing amount of SCMC increased the gel strength and thereby, decreased the swelling of the beads. As a result, drug release decreased gradually. Theoretically, the drug release from Al-CMC beads should have been the slowest. Release studies, however, demonstrated that the drug release from Al-CMC beads was faster than the beads containing 25% SCMX and 75% SCMC (Fig. 6). This anomalous release result may be related to the high viscosity of SCMC solution that retarded the influx of Al³⁺ ion into the beads producing nonhomogeneous or lower extent of cross linking resulting faster release of drug. Comparison of the swelling behavior also demonstrated that swelling of SCMC beads was only marginally less than that of the bipolymeric beads composed of 25% SCMX and 75% SCMC. This means that the degree of crosslinking of Al-CMC beads did not increased considerably due to resistance in the influx of Al³⁺ ions offered by the higher viscosity of SCMC solution.

The effect of the concentration (2-8%) of AlCl₃ used as a cross linking agent, on the release of ACF was studied with the beads containing 20% drug load and prepared using SCMX and SCMC in ratios of 50:50 and gelling for 0.5 h. Increase in the concentration of AlCl₃ increased the release of the drug in both the dissolution media (Fig. 9). The results are not in harmony with the general trends. Because, increase in AlCl₃ concentration would increase the extent of cross-linking and forming a dense and rigid matrix which would produce slower drug release. This anomalous result may be explained in the following way. During the preparation of the beads in water containing higher concentration of AlCl₃, a thick gel layer might have been formed along the periphery of the beads that provided diffusional resistance to further influx of Al³⁺ions. As a result inhomogenous gel beads having less densely cross-linked matrix in the core are formed. Once the outer gel layer breaks down in the dissolution media, rapid drug release takes place. On the contrary, preparation of beads at low AlCl₃ concentration provides uniform diffusion of Al³⁺ions into the beads and results in the formation of homogenously cross-linked gel beads which provides slow release of the drug.

Figure 9: Effect of AlCl₃ concentration on release profiles of aceclofenac in HCl buffer (pH 1.2) (dotted line) and in phosphate buffer solution (pH 6.8) (Firm line) from IPN beads composed of Al-CMX:Al-CMC=50:50 and 20% w/w drug. Key: ●=F3(2%w/vAlCl₃), ■=F6(4% w/v AlCl₃), ▲=F7(8% w/v AlCl₃).

The effect of drug loading on the release of ACF was studied with beads prepared using 50% SCMX and 50% SCMC and gelling for 0.5 h in 2% AlCl₃ solution. Increase in drug load increased the release of the drug (Fig. 10).Since the drug was loaded on weight basis, increase in the amount of drug decreases the amount of polymer per unit weight and this makes the gel network structure weak. In addition, higher drug loading may increase the free volume within the network and create more tortuous pathway for water penetration in to beads. As a result, increase in drug load increased the release of the drug. Similar observations were reported by other researchers for different types of drug loaded beads ^21-23.

Figure 10: Effect of drug load on release profiles of aceclofenac in HCl buffer (pH 1.2) (dotted line) and in phosphate buffer solution (pH 6.8) (Firm line) from IPN beads composed of Al-CMX:Al-CMC=50:50 . Key: ●=F3(20% w/w drug load), ■=F8(40% w/w drug load), ▲= F9(60 % w/w drug load).

The effect of total polymer concentration of the bipolymeric beads on drug release was studied with the beads prepared using SCMX and SCMC in a ratio of 50%:50%, 20 % w/v drug load and gelling for 0.5 h in 2% w/v AlCl₃ solution and the results are shown in Fig. 11. Increase in total polymer concentration from 1.5% w/v to 2.5% w/v decreased the drug release in acidic dissolution medium. Although further increase in total polymer concentration to 3.5% w/v marginally decreased the drug release, comparison of AUC values indicated significant difference (p< 0.05). In PB solution drug release was found to be considerably decreased when the total polymer concentration was increased from 1.5% to 2.5 % w/v. The values of t_50%and t_80%(Table 2) increased respectively from 67 min to 184.7 min and 184.7 min to 233.5 min. Further increase in total polymer concentration to 3.5% tended to increase the drug release as the t_50%and t_80% values decreased to 171.9 min and 225.2 min respectively (Table 2). However, total drug release from the beads appeared to be almost same, as one way analysis of variance revealed no significant difference (p> 0.05) in AUCs of the release profiles of the beads prepared with 2.5% and 3.5% w/v total polymer concentration. This means that increase in total polymer concentration up to a certain value decreases the drug release and further increase do not produce any significant change in drug release. This observation again confirms the effect of viscosity of polymer solution on cross-linking density of the matrix. The high viscosity of 3.5% solution of the blended polymer did not increase the cross link density of the matrix due to hindrance in influx of Al³⁺ ions into the beads. The structure of nonhomogeneously gelled beads breaks down quickly and produces faster release.

Figure 11: Effect of total polymer concentration on release profiles of aceclofenac in HCl buffer (pH 1.2) (dotted line) and in phosphate buffer solution (pH 6.8) (Firm line) from IPN beads composed of Al-CMX:Al-CMC=50:50 . Key: ●=F3(2.5 w/v total polymer), ■=F10(3.5% w/v total polymer), ▲=F11(1.5 % w/v total polymer).

Figure 12: Dynamic drug release from IPN beads composed of Al-CMX:Al-CMC=50:50. Key: ●=F3(20% w/w drug load), ■= F8(40% w/w drug load), ▲= F9(60 %w/w drug load).

Dynamic drug release:

Following oral administration, a dosage form encounters various pH conditions in the gastrointestinal tract. To simulate the changing environment, at least to some extent, the beads, prepared using SCMX and SCMC in a ratio of 50:50, were transferred in PB solution (pH 6.8) following the dissolution study in acid solution (pH 1.2) for 2 h and the dissolution was carried out till complete drug release was achieved. Fig. 12 shows that only 10% drug was released in 2 h in acidic solution and 80-90% drug release was achieved in PB solution within 4 h depending on the drug load. As usual, the higher the drug load, the faster was the drug release. It can, therefore, be expected from the above results that the beads may be capable of minimizing the release of ACF in the stomach and then provide controlled release in the intestine. Such release behavior is desirable to reduce the gastric irritation of NSAID and provide controlled release in the intestine.

Release Kinetics:

Drug release from a swellable matrix is a complex process; it depends on the degree of gelation, hydration, chain relaxation, and erosion of polymer. In order to understand the mode of drug transport through the bipolymeric beads, the release data were fitted to the classical power law expression³³

M_t/M_α = Ktⁿ

Where M_t and M_α are, respectively, the amount of drug released at time t and at infinite time, K represents a constant that incorporates structural and geometrical characteristics of the dosage forms, n denotes the diffusion exponent indicative of the mechanism of drug release. For spheres values of n within 0.5indicates Fickian or diffusion controlled release, values of n ranging from 0.5 to 0.89 indicate non-Fickian or anomalous release, and above 0.89 indicates Case-II transport mechanism. Values of n have been calculated from the above equation by fitting the drug release data up to 60% and shown in Table 2 along with the correlation co-efficient (r²). The results indicate that drug release in acidic medium followed Fickian diffusion controlled model. However, increase in AlCl₃ concentration tended to shift the release mechanism from Fickian to non-Fickian mechanism. In PB solution drug release followed non Fickian mechanism. However, increase in drug load and total polymer concentration in the beads tended to shift the drug release mechanism from non-Fickian to case II transport model. When the swelling data of drug-free beads were fitted to the above power law expression, it was found that swelling in acidic medium took place following the diffusion controlled mechanism and that in PB solution followed non-Fickian mechanism (Table 3).

Table 3: Swelling behavior of drug-free homopolymeric and IPN hydrogel beads in various media.

Formulation Code	Swelling Kinetics
	In acidic solution		In PB Solution
	n	r²	n	r²
F1	0.36	0.97	0.87	0.86
F2	0.34	0.97	0.88	0.88
F3	0.30	0.98	0.87	0.86
F4	0.33	0.99	0.91	0.92
F5	0.35	0.97	0.95	0.91
F6	0.32	0.96	0.93	0.91
F7	0.35	0.96	0.73	0.93

CONCLUSION:

ACF loaded single polymeric and bipolymeric beads were prepared by ionotropic gelation method using AlCl₃ as a common ionic cross-linking agent for both polymers. FTIR, XRD and DSC analyses apparently did not indicate any interaction of the drug with the polymers. However, the morphology and DEE of the beads were found to be influenced by the viscosity of polymer dispersion in addition to the ratios of the two polymers and the concentration of the total polymer and AlCl₃. In-vitro drug release study demonstrated that substitution of SCMX with increasing amount of SCMC decreased the drug release from the hydrogel beads with the exception of SCMC beads which released the drug slightly faster. While, increase in the concentration of AlCl₃and drug load increased the drug release, increase in total polymer concentration tended to decrease the same. The viscosity which was related to the polymer combination and total polymer concentration affected all the physical characteristics of the beads. This study further revealed that the single polymeric and bipolymeric beads released very less amount of drug in acid solution and provided complete release in PB solution in a controlled fashion.

ACKNOWLEDGEMENTS:

We greatly acknowledge Torrent pharmaceuticals (Himachal Pradesh, India) for providing Aceclofenac as a gift sample.

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Received on 22.10.2011 Modified on 02.11.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 103-113