INTRODUCTION:

Multiparticulate systems attracted much attention since several years in controlling and sustaining of release rate of many active pharmaceutical ingredients. The use of natural biodegradable polymers as rate controlling agents also has been tremendously increased¹. Microbeads are small, solid, free-flowing particulate carriers that contain distributed drug particles in solution or crystalline form, allowing for continuous or multiple release profiles of therapy with a variety of active agents without incurring side effects.Beads have been attracted a significant attention as potential drug carriers to obtain a controlled release, site-specific delivery and to increase drug bioavailability^2-5.

Biodegradability and biocompatibility made alginate and chitosan the most popularly used hydrogels in the formulation of such systems.

Sodium alginatewhich is a hydrophilic biopolymer has a unique property of gel formation in the presence of multivalent cations such as calcium ions in aqueous media, Chitosan, on the other hand, is a polysaccharide made up of glucosamine and N-acetylglucosamine that has distinctive polycation properties. Studies reported some problems during alginate beads preparation. Drug loss and shrinkage of the alginate polymer take place when subjected to an acidic medium, while it dissolves at higher pH values. On the contrary, chitosan beads dissolve at low pH and are insoluble in alkaline media. Due to its hydrophilicity, chitosan alone, according to certain research, cannot control medication release. Therefore, the alginate-chitosan polyelectrolyte complex was a promising medium to reduce the porosity of alginate beads and decrease the leakage of the encapsulated drug. Because of its nontoxic, odourless nature, biodegradable qualities, and biocompatibility in animal tissues, chitosan has sparked a strong interest in medical and pharmaceutical applications. Blending of chitosan with other polymers is a convenient and effective method of improving its physical and mechanical properties for practical applications^6-16.

In the present study, Rifaximin (RFX) was used as a model drug. Rifaximin a semi-synthetic antibiotic, is related to rifamycin and is not absorbed systemically. Rifaximin has been authorized for the treatment of gram-positive and gram-negative bacteria. This broad-spectrum oral antibioticis used clinically to treat a variety of gastrointestinal disorders, including traveller’s diarrhoea, hepatic encephalopathy, irritable bowel syndrome, and the inflammatory bowel diseases^17,18. The aim of the present study was to develop a controlled release delivery system of Rifaximin using alginate-chitosan beads.

MATERIALS AND METHODS:

Materials:

Rifaximin was received as a gift sample from Lupin Pharmaceuticals Ltd, Aurangabad, India. Sodium alginate and Chitosan were purchased from Research lab, fine chem Industries, Mumbai, India. All the other substances were analytical grade and were utilised just as they were.

Method:

Calibration curve of Rifaximin:

Accurately weighed RFX was dissolved and diluted with PBS pH 6.8 to make stock solution (100μg/ml). The solution was diluted up to 20μg/ ml, which was scanned spectrophotometrically in the range of 200nm – 400nm for determination of λmax.The stock solution (100μg/ ml) was diluted with PBS pH 6.8 to make solutions of concentration of 2 to 20μg/ml.The UV absorbances were recorded at λmax and equation for regression was obtained by plotting the graph of concentrations on X – axis vs. absorbances on Y – axis.

Preparation of chitosan beads:

The chitosan beads were prepared by ionotropic gelation process, with Sodium tri poly phosphate (TPP) as an ionic agent¹⁹. Briefly, required quantity of drug was added to chitosansolution in 2% v/v acetic acid with gentle stirring. The solution was kept aside for the removal of air bubbles. The beads were formed by injecting chitosan solution drop wise into the curing solution (4%w/v sodium TPP) under gentle agitation on a magnetic bead stirrer. The beads formed instantaneously and were kept for half an hour to cure. The solidified beads, rinsed with distilled water, was then added in the flask containing 20mL 1% glutaraldehyde solution for the hardening of beads and then dried at room temperature for 24 hrs.

Preparation of alginate beads:

The microbeads (Table 1) were prepared with different concentrations of Na alginate as polymer and calcium chloride as counter ion. The drug polymer suspension was dropped through a syringe into calcium chloride solution with stirring. The gelled beads were separated after 30minutes by filtration, washed with distilled water, and dried at room temperature. Prepared beads were stored in airtight container before further use in their characterization.

Preparation of chitosan-alginate beads^20-27:

Drug alginate mixture was added to the chitosan-calcium chloride solution dropwise through a 21 G syringe at a constant rate with gentle stirring over thermal controlled magnetic stirrer. The formed microbeads were filtered, washed with acetone, and dried in an oven at 35°C. The dried formulations were stored in an amber-coloured bottle and kept in a desiccator until used.

Evaluation of hydrogel beads^24-28:

Morphological characterization:

For visualizing surface morphology of beads,scanning electron microscopy SEM is promising technique. Morphology and surface topography of the hydrogel beads were studied by SEM(FEI Nova NanoSEM 450 Bruker X Flash 6130).

Table 1: Composition of hydrogel beads

Formulation Batch code	Drug (mg)	Chitosan (%w/v)	Sodium alginate (%w/v)	Sodium tri polyphosphate (%w/v)	Calcium chloride (%w/v)
CR1	200	0.25	-	4
CR2	200	0.5	-	4
CR3	200	0.75	-	4
CR4	200	1	-	4
CR5	200	1.25	-	4
SA-1	200	-	0.5	-	3
SA-2	200	-	1	-	3
SA-3	200	-	2	-	3
SA-4	200	-	3	-	3
SA-5	200	-	4	-	3
SCR4	200	1	1	-	3
SCR5	200	1	1.5	-	3
SCR6	200	1	2	-	3

Micromeritic properties:

The beads were evaluated for its flow property by calculating the angle of repose, carr’s index and hausner’s ratio.

Particle size analysis:

An optical microscope with a calibrated eyepiece was used to measure the particle size of prepared beads. Particle size was determined by taking the average of 50 beads.

Percentage yield:

The prepared microbeads were collected and weighted. The actual weight of obtained microbeads divided by the total amount of all material that was used for the preparation of the microbeads.

Actual weight of product

Percentage yield = ------------------------------------ × 100

Total weight of excipients

Determination of % drug content:

For determination of drug content, 10mg of beads were placed in 100ml of phosphate buffer pH 6.8. The filtered solution was measured for Rifaximin content using a UV spectrophotometer at 445nm. The drug loading capacity of the beads was calculated using the following equation:

Total amount of drug in particies

Drug loading (%) = ------------------------------------- ×100

Weight of particies taken

Percentage entrapment efficiency:

In a glass mortar, about 50mg of precisely weighed drug-loaded microbeads were crushed and combined with 100 ml phosphate buffer (pH 6.8) and kept for 24 hours. The solution was stirred on a magnetic stirrer for 15 min, filtered and 1ml of the filtrate was diluted using phosphate buffer (pH 6.8) and analysed spectrophotometrically at 445nm. The following formula was used to compute the drug entrapment efficiency:

Actual drug content

Entrapment efficiency (%) = ------------------------- × 100

Theoretical drug content

Cumulative invitro drug release:

The in vitro dissolution study of hydrogel beads was done in USP type-I apparatus using pH 6.8 phosphate buffer at 37⁰C±0.5⁰C and 100rpm. The measurement of % drug release was carried out by UV-Visible spectroscopy method at 445nm.

Stability studies:

Selected batches of beads were subjected to stability studies under accelerated storage conditions according to the International Conference of Harmonization (ICH) guidelines. The stability study [ICH Q1A (R2)] for Rifaximin hydrogel beads were carried out by placing them in stability chamber. Two different storage conditions viz. long-term study (25 ± 2⁰C/60% RH) for 6 monthsand accelerated study (40 ± 2⁰C/75%RH) for three months were used for the study. During the stability study the formulations were evaluated for physical appearance, drug content and drug dissolution after 3 and 6 months.

RESULTS AND DISCUSSION:

The aim of this study was to evaluatethe applicability of sodium alginate and chitosan as the carrier for oral delivery of RFX hydrogel beads. The hydrogel beads were prepared by ionotropic gelation method and evaluated for the percentage yield, drug content, and invitro cumulative percentage drug release.

Figure1: Calibration curve of Rifaximin in phosphate buffer pH 6.8

Morphological characterization:

The morphological characterization of the hydrogel beads was performed by optical microscope and scanning electron microscopy (SEM). The beads were found to be spherical in shape with a diameter of around 2mm.

Figure 2: Microscopic picture of Rifaximin loaded alginate-chitosan beads.

Figure3:SEM of Chitosan(A), Sodium alginate(B) and Chitosan-Alginate(C) beads at 500x magnification and Chitosan(D), Sodium alginate(E) and Chitosan-Alginate(F) beads at 200x magnification.

Micromeritic properties:

Table 2: Micromeritic properties ofhydrogel beads

Formulation code	Carr’s index (%)	Hausner’s Ratio	Angle of repose (⁰)
CR1	2.9	1.03	25.56±0.03
CR2	11.05	1.12	30.75±0.04
CR3	6.66	1.07	24.48±0.08
CR4	4.76	1.04	23.46±0.01
CR5	16.68	1.20	32.75±0.02
SA-1	5.68	1.06	34.9±0.003
SA-2	4.88	1.05	35.3±0.002
SA-3	5.00	1.05	34.8±0.008
SA-4	5.81	1.06	34.6±0.001
SA-5	4.59	1.04	35.7±0.005
SCR4	6.05	1.06	25.43±0.009
SCR5	6.45	1.06	28.31±0.007
SCR6	10.79	1.12	27.98±0.001

All formulations showed good flow property that is within the range of good to excellent flowability. This could be attributed to better uniformity in shape and size.

Characterization of the beads:

The percentage yield of the formulation was within the range of 66.1 to 103.44%.The method followed gave good encapsulation efficiency i.e., between 84% - 96% The size of beads from different batches exhibited diameter ranging between 610-1014µm. The drug polymer ratio plays an important role in formation of beads; the sizes of beads were increased in formulation with increasing polymer concentration. Low concentration of chitosan produced a less viscous solution leading to smaller particle size. Whereas higher chitosan concentration resulted in highly viscous solution, thus difficulty in extrusion through the needle and bigger bead size.Similarly higherbead size wasalso obtained when the concentration of sodium alginate was increased.

In vitro drug release:

Invitro drug release study of different batches of chitosan beads showed that percentage of drug released from batch CR3 after 2hrs was the highest i.e.,71.64%. The percentage drug release (80.88%) from batch SA3 was found to be highest in comparison with other alginate batches. The release profiles of RFX from Sodium alginate- chitosan (SCR) hydrogel beads at pH 6.8 are illustrated in figure. A higher drug release was observed when a combination of polymerwas used when compared to the release profile of beads with sodium alginate and chitosan alone.

Table 3: Characterization of hydrogel beads

Formulation code	Particle diameter (µm)	*Percentage yield (%)**	*Drug content (%)**	*Entrapment efficiency (%)**
CR1	894	99.30±0.10	69.18±1.72	92.35±1.58
CR2	918	90.28±0.13	66.91±1.23	94.31±2.01
CR3	930	80.00±0.21	78.45±1.51	96.47±2.84
CR4	969	103.44±0.11	62.18±1.25	93.72±2.31
CR5	1014	66.1±0.19	52.12±0.99	90.10±1.25
SA-1	850	96.8±0.97	78.56±1.15	84.2±1.38
SA-2	863	96.0±1.58	82.34±1.23	88.9±1.54
SA-3	880	95.3±1.27	81.60±0.99	90.7±1.62
SA-4	891	95.1±0.94	85.87±2.12	93.1±0.78
SA-5	901	92.3±1.49	90.13±1.89	95.6±0.91
SCR4	807	96.6±0.45	91.47±2.13	86.63±1.14
SCR5	610	95.54±0.53	91.48±1.44	90.28±1.23
SCR6	659	97.42±0.27	91.09±1.69	91.30±1.74

* (n=3, mean ± S.D.)

In vitro drug release:

Figure 4: Cumulative % drug release

Stability studies:

Since SCR6 formulation showed better results from characterization and invitro drug release of beads, it was selected for further studies.The stability studies of the SCR6 formulation revealed that no significant changes in the physical parameters when stored at humidity conditions of 40⁰C/75% RH and at room temperature. The active drug content of the hydrogel beads was not reduced significantly over a period of six months as well as drug release studies shows no significant change in drug release as shown below.

Figure 5: Stability studies of selected formulation

CONCLUSION:

Chitosan alginate beads were prepared successfully for oral delivery of Rifaximin. The results of micromeritic studies showed goodflowability of Rifaximin beads. From the experimental results it can be concluded that batch SCR6 is most suitable with regard to percentage yield, drug content, and encapsulation efficiency. Invitro drug release study of alginate-chitosan complex formulation indicated that Rifaximin was released in desirable manner. In conclusion the proposed dual cross-linked beads can serve as a convenient carrier for oral controlled release of Rifaximin.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors thank Savitribai Phule Pune University for their help in SEM studies.

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Received on 02.03.2022 Modified on 05.07.2022

Research J. Pharm. and Tech 2023; 16(6):2985-2990.

DOI: 10.52711/0974-360X.2023.00493