Research paper
Reversed chitosan–alginate polyelectrolyte complex for stability improvement of alpha-amylase: Optimization and physicochemical characterization

https://doi.org/10.1016/j.ejpb.2006.07.014Get rights and content

Abstract

The present work explores, using response surface methodology, the main and interaction effects of some process variables on the preparation of a reversed chitosan–alginate polyelectrolyte complex (PEC) with entrapped α-amylase for stability improvement. A 33 full factorial design was used to investigate the effect of the chitosan and alginate concentrations and hardening time on the percent entrapment, time required for 50% (T50) and 90% (T90) enzyme release, and particle size. The beads were prepared by dropping chitosan containing α-amylase into a sodium alginate solution without any salt. The in vitro enzyme release profile of the beads was fitted to various release kinetics models to study the release mechanism. A topographical characterization was carried out using scanning electron microscopy (SEM), and the entrapment was confirmed using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zones III and IV. Beads prepared using 2.5% w/v chitosan and 3% w/v sodium alginate with a hardening time of 60 min had more than 90% entrapment and a T90 value greater than 48 min. Moreover, the shelf-life of the enzyme-loaded beads was found to increase to 3.68 years, compared with 0.99 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare a reversed PEC of chitosan and alginate with good mechanical strength, provided both the reactants are in a completely ionized form at the time of the reaction. Proper selection of the reaction pH, polymer concentration and hence charge density, and hardening time is important and determines the characteristics of the PEC.

Introduction

α-Amylase, a major enzyme used for replacement of pancreatic enzymes [1], is not reabsorbed in the intestine like other proteins used for systemic therapy. Fungal α-amylases (EC 3.2.1.1; CAS 9000-90-2) are obtained from various strains of Aspergillus species (mainly A. niger, A. awamori, and A. usamii). α-Amylases catalyze the hydrolysis of α-1,4 glycosidic linkages in starch and other related carbohydrates. The active site has a large number of charged groups, among which are three acids (Asp231, Glu261, and Asp328, numbered according to the BLA sequence) essential to catalytic activity. α-Amylases are used in several industrial processes such as starch liquefaction, laundering, dye removal, and feed preprocessing, but the largest volume is sold to the starch industry for the production of high-fructose syrups and ethanol [2].

Pharmaceutical formulations containing α-amylase and other digestive enzymes need to be stored under cold (2–8 °C) or cool (8–25 °C) conditions and have a shelf-life of up to 1 year. Entrapment of the α-amylase in a biodegradable polyelectrolyte complex (PEC) may improve the stability of the parent enzyme [3], [4] and make it less prone to interference from the various excipients of the formulation. Immobilized enzymes are stable at higher temperatures and may be stored at room temperature with an extended shelf-life [5]. These advantages are of great interest from a commercial viewpoint for the pharmaceutical industry. Hence, it was the objective of this research to develop an extended shelf-life formulation of α-amylase by entrapping it in biodegradable chitosan–alginate PEC beads, with a resultant improved and efficient utilization of the enzyme. This paper also deals with in vitro dissolution studies and physicochemical characterization carried out to evaluate the beads and their release behavior.

Chitin (β(1–4)-N-acetyl-d-glucosamine), the second most abundant naturally occurring biopolymer after cellulose, is the major structural component of the invertebrate exoskeleton and the fungal cell wall [6]. Waste produced in the processing of seafood, mainly crab, shellfish, lobster, and shrimp, is an abundant source of chitin. Chitosan, obtained by partial alkaline deacetylation of chitin, is a polycationic polysaccharide consisting of β-[1  4]-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucopyranose (GlcN; D-unit) (Fig. 1A) [7] and has a macro pKa value in the range of 6.3–6.5. Chitosan and chitin are commercially interesting compounds because of their high nitrogen content (6.89%; the repeating unit contains the –NH2 group at the C-2 position) [8] compared with synthetically substituted cellulose (1.25%). This makes chitosan a useful chelating agent [9]. This polysaccharide becomes water-soluble under acidic conditions (pH < 6), allowing the preparation of biocompatible and often biodegradable polymer solutions [10]. Also, it has excellent cell-adhesive properties [11], promotes wound–healing [12], and has bacteriostatic effects [13]. Moreover, chitosan is metabolized by certain human enzymes, e.g., lysozyme [14], α-amylase [14], and hyaluronidase, and can be considered biodegradable [14]. Finally, chitosan is abundant in nature, and it is cheap to produce, apart from being ecologically interesting [15].

Alginate, a high-molecular-mass hyaluronic acid-like biodegradable polymer, is a naturally occurring copolymer of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) (Fig. 1B) extracted from brown seaweeds (Phaeophyceae, mainly Laminaria). The pKa values of M- and G- residues are 3.38 and 3.65, respectively. By virtue of the carboxyl groups on the constituent uronic acid residues, the pKa value of alginic acid ranges between 3.4 and 4.4, depending on the type of alginate and the salts present in the mixture [16]. It has been shown that the G and M units are joined together in blocks, and as such, three types of block may be found: homopolymeric G blocks (GG), homopolymeric M blocks (MM), and heteropolymeric sequentially alternating blocks (MG). Alginate is chemically very stable at pH values between 5 and 10. High acid concentrations cause decarboxylation of alginate [17]. Alginate beads have the advantages of being non-toxic orally and having high biocompatibility [18]. Alginate is used as an entrapment matrix for cells and enzymes and as a pharmaceutical and food adjuvant.

The formation of polyanion–polycation (polyelectrolyte) complexes is mainly driven by an electrostatic mechanism where charge neutralization and possible local overcompensation or bridging (such as hydrogen bonding, Coulomb forces, van der Waals forces, and transfer forces) mediated by a multivalent counterion induces attraction between topologically separated segments of the polyelectrolytes [3]. The lower the charge density of the polymer, the higher is the polymer proportion in the PEC, since more polymeric chains are required to react with the other polymer, leading to ‘bulky’ PEC [19]. Since chitosan has a rigid, stereo-regular structure containing bulky pyranose rings, the formation of PEC can induce a conformational change of the other polyelectrolyte if the latter has a non-rigid structure [20]. PECs of different characteristics can be obtained by changing the chemical characteristics of the component polymers, such as the molecular weight, flexibility, functional group structure, charge density, hydrophilicity and hydrophobicity balance, and stereo-regularity and compatibility, as well as the reaction conditions: the pH, ionic strength, concentration, mixing ratio, and temperature [3].

Beads produced from sodium alginate with calcium chloride present in the chitosan solution bind ∼100 times more chitosan than do capsules produced by dropping the alginate solution in a chitosan solution in the absence of salt, and most beads, micro- and macrocapsules, and microspheres are produced by these methods. Reversed chitosan–alginate complex coacervate capsules, formed by dropwise addition of chitosan solution into alginate solution, were reported to be fragile even after hardening for 3 h [21]. To avoid the limitation of this reverse coacervation, the present study employs a novel approach, wherein chitosan and alginate are reacted in their completely ionized states to increase the counterion charge density of the polymers, which increases the overall interaction and hence the mechanical strength of the PEC membrane. This was achieved by maintaining the pH values of the chitosan and alginate solutions at 2 and 6.5, respectively. Further, the pH value of 2 of the chitosan solution suppresses the chitolytic activity of the added α-amylase, which is active in a pH range of 4–5.

Section snippets

Materials

Potassium dihydrogen phosphate, sodium hydroxide (NaOH), hydrochloric acid (HCl) (Qualigens Fine Chemicals, Mumbai, India), and soluble starch (Himedia Laboratories Pvt. Ltd., Mumbai, India) were used as received. Fungal α-amylase, sodium alginate, iodine, and potassium iodide were purchased from S. D. Fine-Chem Ltd., Mumbai, India. Chitosan (Chito Clear®) with a reported degree of deacetylation of 89% (titration method) was a kind gift from Primex ehf, Ireland. The average molecular weight of

Principle of PEC formation

The electrostatic attraction between the cationic amino groups of chitosan (the macro pKa value is about 6.5) [38] and the anionic carboxyl groups of the alginate is the main interaction leading to the formation of the PEC. It is stronger than most secondary binding interactions [38]. For preparation of beads with reverse engineering, the formation of a membrane of good mechanical strength (precipitate) at the interface of the polymers is essential. This is required to avoid homogeneous mixing

Conclusions

Reversed chitosan–alginate PEC beads exhibited a promising improvement in stability of entrapped α-amylase and can find a place in the design of multiparticulate drug delivery systems. Optimization of the process using response surfaces resulted in >90% entrapment and a >48 min T90 value (experiment 12). The T50 and T90 values were increased with an increase in alginate concentration and hardening time, while they decreased with an increase in chitosan concentration. The percentage of entrapment

Acknowledgments

We acknowledge the University Grants Commission (New Delhi, India) for availing Senior Research Fellowship to Mr. Mayur G. Sankalia. We greatly appreciate the Relax Pharmaceuticals (Vadodara, India) for skillful assistance and providing FTIR testing facility in this study.

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