Stability improvement of alpha-amylase entrapped in kappa-carrageenan beads: physicochemical characterization and optimization using composite index

PURPOSE: This work examines the influence of various process parameters on alpha-amylase entrapped in crosslinked kappa-carrageenan beads for stability improvement. A three level full factorial design was employed to investigate the effect of three process variables namely kappa-carrageenan concentration, potassium chloride concentration and hardening time on % entrapment, time required for 50% (T50) and 90% (T90) of enzyme release and particle size. METHODS: The beads were prepared by dropping the kappa-carrageenan-containing alpha-amylase to magnetically stirred potassium chloride solution. The composite index was applied to optimize the process under study. 'In vitro' enzyme release profile of the beads was fitted to various release kinetics models to understand the release mechanism. Topographical characterization was carried out by SEM and entrapment was confirmed by FTIR and DSC. Stability testing according to the ICH guidelines for zone III and IV was carried out. RESULTS: With the use of ionotropic gelation method, a polymeric matrix prepared by 3.5% (w/v) kappa-carrageenan, 0.7 M potassium chloride and hardening time of 30 min resulted in the production of beads characterized by disc shaped with collapsed center, absence of aggregates, % entrapment of 73.79, T90 of 74.4 min, and composite index of 83.01. Moreover, shelf-life of the enzyme loaded beads was found to increase up to 3.53 years compared to 0.99 year of the conventional formulation. CONCLUSIONS: It can be inferred that the proposed method can be used to prepare alpha-amylase loaded kappa-carrageenan beads for stability improvement. Also the proper selection of rate-controlling carrageenan concentration and its interactive potential for crosslinking is important and will determine the overall size and shape of beads, the duration and pattern of dissolution profiles and enzyme loading capacity.     

Microencapsulation of a recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in chitosan-coated alginate beads

A recombinant aminopeptidase (90 kDa) of Lactobacillus rhamnosus S93 produced by E. coli was encapsulated in alginate or chitosan-treated alginate beads prepared by an extrusion method. This study investigated the effects of alginate, CaCl2, chitosan concentrations, hardening time, pH and alginate/enzyme ratios on the encapsulation efficiency (EE) and the enzyme release (ER). Chitosan in the gelling solution significantly increased the EE from 30.2% (control) to 88.6% (coated). This polycationic polymer retarded the ER from beads during their dissolution in release buffer. An increase in alginate and chitosan concentrations led to greater EE and lesser ER from the beads. The greatest EE was observed in a pH 5.4 solution (chitosan-CaCl2) during bead formation. Increasing the CaCl2 concentration over 0.1 M neither affected the EE nor the ER. Increasing hardening time beyond 10 min led to a decrease in EE and the alginate:enzyme ratio (3 : 1) was optimal to prevent the ER.     

Physicochemical characterization of papain entrapped in ionotropically cross-linked kappa-carrageenan gel beads for stability improvement using Doehlert shell design

This work examines the influence of various process parameters on papain entrapped in cross-linked kappa-carrageenan beads for improvement of its stability. A Doehlert shell design (DSD) was employed to investigate the effect of three process variables, namely kappa-carrageenan concentration, KCl concentration, and hardening time, on the entrapment, time required for 50% enzyme release (T50), time required for 90% enzyme release (T90), and particle size. The beads were prepared by dropping the kappa-carrageenan containing papain into a magnetically stirred KCl solution. Topographical characterization was carried out by scanning electron microscopy and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zone III and IV. A polymeric matrix was prepared with kappa-carrageenan (3.5% w/v) and potassium chloride (0.5 M) using the ionotropic gelation method, with a hardening time of 20 min. Beads characterized by a spherical disc shape with a collapsed center, an absence of aggregates, an entrapment of 82.75%, a T90 value of 55.36 min, and a composite index of 88.55 were produced. The shelf-life of the enzyme-loaded beads was found to increase to 3.63 years compared with 1.01 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare papain-loaded kappa-carrageenan beads for stability improvement.     

Microencapsulation of a recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in chitosan-coated alginate beads

A recombinant aminopeptidase (90 kDa) of Lactobacillus rhamnosus S93 produced by E. coli was encapsulated in alginate or chitosan-treated alginate beads prepared by an extrusion method. This study investigated the effects of alginate, CaCl₂, chitosan concentrations, hardening time, pH and alginate/enzyme ratios on the encapsulation efficiency (EE) and the enzyme release (ER). Chitosan in the gelling solution significantly increased the EE from 30.2% (control) to 88.6% (coated). This polycationic polymer retarded the ER from beads during their dissolution in release buffer. An increase in alginate and chitosan concentrations led to greater EE and lesser ER from the beads. The greatest EE was observed in a pH 5.4 solution (chitosan-CaCl₂) during bead formation. Increasing the CaCl₂ concentration over 0.1 M neither affected the EE nor the ER. Increasing hardening time beyond 10 min led to a decrease in EE and the alginate:enzyme ratio (3 : 1) was optimal to prevent the ER.     

Characterization of microcrystalline cellulose loaded diclofenac calcium alginate gel beads in vitro

Diclofenac calcium alginate (DCA) beads containing microcrystalline cellulose (MCC) were prepared using ionotropic gelation method. The effect of MCC amounts on physicochemical characteristics of the DCA beads was examined. The particle size and entrapment efficiency of diclofenac sodium (DS) of the DCA beads increased with increasing amount of MCC. MCC could be involved in the calcium alginate formation to create a complex matrix in the DCA beads, which was revealed using FTIR spectroscopy. The MCC-DCA beads provided greater water uptake in distilled water, but retarded swelling rate in pH 6.8 phosphate buffer. A longer lag time and a similar drug release rate of the MCC-DCA beads in pH 6.8 phosphate buffer were found. The MCC-DCA beads also gave higher drug release rates in distilled water when compared with the DCA beads. However, the increase of MCC content over 0.5% in the DCA beads did not affect the drug release in distilled water. In conclusion, MCC could improve drug entrapment efficiency and modify drug release from the DCA beads.     

In vitro evaluation of chitosan coated- and uncoated-calcium alginate beads containing methyl salicylate-lactose physical mixture

Context: Methyl salicylate–lactose physical mixture (1:1 and 1:1.5 ratios) was incorporated into calcium alginate beads by a coacervation method involving an ionotropic gelation/polyelectrolyte complexation approach.

Objectives: This study aims to determine the influence of chitosan coating over the beads on drug entrapment efficiency (DEE) and release characteristics in artificial saliva compared to that of the uncoated beads.

Results and discussion: Changes in formulation parameters (gelation time, concentrations of Ca²⁺ and alginate) resulted in decrease in DEE of chitosan-uncoated beads (p?<?0.05). This is due to the combined effects of drug leach-out from the physical mixture by Ca²⁺ ions, alginate gel matrix cross-linking and free drug diffusion from chitosan-uncoated beads. However, an increment in the DEE was seen for chitosan-coated beads. A rapid drug release profile was noted for uncoated beads, but for chitosan-coated beads a sustained release profile was depicted depending upon the coating conditions. Chitosan-coated beads had reduced swelling and erosion properties and thus behaved as a physical barrier to drug release. Shifting from anomalous transport type to Fickian transport confirmed the formation of physical barrier onto chitosan-coated beads.

Conclusion: Calcium alginate beads could be used as a controlled-release system for methyl salicylate–lactose physical mixture.     

Release characteristics of chitosan treated alginate beads: II. Sustained release of a low molecular drug from chitosan treated alginate beads

The aim of this paper was to investigate the possible applicability of chitosan treated alginate beads as a controlled release system of small molecular drugs with high solubility. Timolol maleate (mw 432.49) was used as a model drug. The beads were prepared by the ionotropic gelation method and the effect of various factors (alginate, chitosan, drug and calcium chloride concentrations, the volume of external and internal phases and drying methods) on bead properties were also investigated. Spherical beads with 0.78-1.16mm diameter range and 10.8-66.5% encapsulation efficiencies were produced. Higher encapsulation efficiencies and retarded drug release were obtained with chitosan treated alginatebeads. Amongthedifferentfactors investigatedsuchas alginate, drug, chitosan and CaCl₂ concentrations, the volumes of the external and internal phases affected bead properties. The drying technique has an importance on the bead properties also. The release data was kinetically evaluated. It appeared that chitosan treated alginate beads may be used for a potential controlled release system of small molecular drugs with high solubility, instead of alginate beads.     

Swelling studies and in vitro release of verapamil from calcium alginate and calcium alginate-chitosan beads

The aim of the present work was to investigate the swelling behavior and the in vitro release of the antihypertensive drug verapamil hydrochloride from calcium alginate and chitosan treated calcium alginate beads. Calcium-alginate beads, chitosan-coated alginate beads and alginate-chitosan mixed beads were synthesized and their morphology was investigated by scanning electron microscopy. The swelling ability of the beads in different media was found to be dependent on the presence of the polyelectrolyte complex between alginate and chitosan, the pH of the aqueous media and the initial physical state of the beads. The results revealed that the encapsulation of verapamil in both calcium-alginate and calcium alginate-chitosan mixed beads exceeded 80%. Considering the in vitro stability of verapamil encapsulating beads, 70% of the drug released from wet and dry plain calcium alginate beads within 1 and 3h, respectively. The presence of chitosan was found to retard significantly the release from wet beads. However, in the case of dry beads the presence of chitosan had no significant effect on the initial release stage and significantly increased the release on the later stage. The results were analyzed by using a semi-empirical equation and it was found that the drug release mechanisms were either "anomalous transport" or "case-II transport".     

Release characteristics of chitosan treated alginate beads: I. Sustained release of a macromolecular drug from chitosan treated alginate beads

Alginate and chitosan treated alginate beads were prepared and compared as an oral controlled release system for macromolecular drugs. Dextran (M.W. 70,000) was used as a model substance. The beads were prepared by the ionotropic gelation method and the effect of various factors (alginate, chitosan, drug and calcium chloride concentrations, the volume of external and internal phases and drying methods) on bead properties were investigated. The addition of chitosan increased the drug loading capacity of the beads, and larger beads were obtained in the presence of chitosan. On the other hand, addition of chitosan in the gel structure reduced the drug release from beads. The erosion of the beads was suppressed by chitosan treatment. The drying method was important to the properties of the chitosan-alginate beads. It is proposed that chitosan treated alginate beads may be used as a potential controlled release system of such macromolecules.     

Release characteristics of chitosan treated alginate beads: II. Sustained release of a low molecular drug from chitosan treated alginate beads

The aim of this paper was to investigate the possible applicability of chitosan treated alginate beads as a controlled release system of small molecular drugs with high solubility. Timolol maleate (mw 432.49) was used as a model drug. The beads were prepared by the ionotropic gelation method and the effect of various factors (alginate, chitosan, drug and calcium chloride concentrations, the volume of external and internal phases and drying methods) on bead properties were also investigated. Spherical beads with 0.78-1.16 mm diameter range and 10.8-66.5% encapsulation efficiencies were produced. Higher encapsulation efficiencies and retarded drug release were obtained with chitosan treated alginate beads. Among the different factors investigated such as alginate, drug, chitosan and CaCl2 concentrations, the volumes of the external and internal phases affected bead properties. The drying technique has an importance on the bead properties also. The release data was kinetically evaluated. It appeared that chitosan treated alginate beads may be used for a potential controlled release system of small molecular drugs with high solubility, instead of alginate beads.     

Bile salt-reinforced alginate-chitosan beads

A polymeric delayed release protein delivery system was investigated with albumin as the model drug. The polysaccharide chitosan was reacted with sodium alginate in the presence of calcium chloride to form beads with a polyelectrolyte. In this study, attempts were made to extend albumin release in the phosphate buffer at pH 6.8 from the alginate-chitosan beads by reinforcing the matrix with bile salts. Sodium taurocholate was able to prevent albumin release at pH 1.2, protecting the protein from the acidic environment and extending the total albumin release at pH 6.8. This effect was explained by an interaction between the permanent negatively charged sulfonic acid of sodium taurocholate with the amino groups of chitosan. Mild formulation conditions, high bovine serum albumin (BSA) entrapment efficiency, and resistance to gastrointestinal release seem to be synergic and promising factors toward the development of an oral protein delivery form.     

Alternate polyelectrolyte coating of chitosan beads for extending drug release

In the present study, we addressed the factors modifying ciprofloxacin release from multiple coated beads. Beads were prepared by simple ionic cross-linking with sodium tripolyphoshate and coated with alginate and/or chitosan to prepare single, double, or multilayered beads. The water uptake capacity depended on the nature of beads (coated or uncoated) and pH of test medium. The number of coatings given to the beads influenced ciprofloxacin release rate. The coating significantly decreased the drug release from the beads in comparison to uncoated beads (p < 0.001). When the beads were given three coatings, viz., alginate, chitosan, and again alginate, the drug release appeared to follow the pattern exhibited by colon-targeted drug delivery systems with time dependent release behavior. The increase in coating formed a barrier for easy ingress of dissolution medium into the bead matrix, reducing the diffusion of drug.     

A novel approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery

A novel approach was developed to improve the mechanical strength of tripolyphosphate (TPP)/chitosan beads prepared under coagulation condition at 4 degrees C in the presence of gelatin. Cross-sectional analysis indicated that the beads had a homogeneous crosslinked structure, as a result the beads were strengthened greatly (the mechanical strength increased more than ten times). Furthermore sodium alginate (a polyanion) can interact with cationic chitosan on the surface of these TPP/chitosan beads to form polyelectrolyte complex film for the improvement of the drug sustained release performances. The loading efficiency of model drugs (brilliant blue and FITC-dextran) in these beads was very high (more than 90%). Crosslinking time, TPP solution pH and other preparation factors had an effect on the drug release performance of beads. The release period of brilliant blue (a poor water soluble dye) was more than 2-months at a fairly constant rate in 0.9% NaCl, 10 mM PBS pH 7.4. However, for FITC-dextran (a water soluble polysaccharide) only 1-2 days in the same conditions. It seems that TPP/chitosan bead prepared by the novel method is a promising formulation for drug delivery.     

Calcium pectinate gel beads for controlled release drug delivery: II. Effect of formulation and processing variables on drug release.

The effect of four formulation and processing variables, calcium concentration, drying condition, concentration of hardening agent and hardening time on the bead properties and the release characteristics of a model drug from calcium pectinate gel (CPG) beads were studied. A poorly soluble compound, indomethacin, was used as the model drug. The investigated variables affected the bead size, the entrapment efficiency and the release of indomethacin from CPG beads. Drug release was found to be a function of the formulation and processing variables. The slower drug release was achieved from the formulations with higher calcium concentration, higher concentration of hardening agent and longer hardening time. The drying condition, however, did not influence the drug release. The mechanism of indomethacin release from CPG beads followed the diffusion controlled model for an inert porous matrix. All drug release data fitted well to the Higuchi square root time expression.     

酮洛芬果胶钙凝胶小球和海藻酸钙凝胶小球的制备及性能比较

目的酮洛芬果胶钙凝胶小球和酮洛芬海藻酸钙凝胶小球的制备及性能比较。方法利用果胶、海藻酸钠及二者不同比例,以酮洛芬为模型药物采用滴制法制备凝胶小球,考察2种多糖物质对药物包封率和释放行为的影响。利用大鼠肠囊外翻实验对凝胶小球的生物黏附性能进行比较,通过对释放机理的探讨和凝胶小球溶胀性的测定进一步证明2种凝胶小球释药行为的不同。结果酮洛芬果胶钙凝胶小球和酮洛芬海藻酸钙凝胶小球均具有良好的生物黏附性能,果胶钙凝胶小球主要通过溶胀作用缓慢释药,而海藻酸钙凝胶小球的释药与凝胶小球慢慢吸水后骨架溶蚀有关。结论酮洛芬果胶钙凝胶小球和酮洛芬海藻酸钙凝胶小球通过与生物黏膜的紧密结合缓慢释药,而二者的释放行为有所不同。     

The release behavior of brilliant blue from calcium-alginate gel beads coated by chitosan: the preparation method effect.

The aim of this study is to reveal how the release behavior of a model drug (brilliant blue, BB) from chitosan coating calcium-alginate gel beads (CCAGB) was influenced by the preparation methods. The CCAGB were prepared by dropping alginate solution into CaCl(2)/chitosan solution (method 1(a)), or into chitosan solution then gelled by CaCl(2) (method 1(b)), or into CaCl(2) solution then coated by chitosan (method 2). Scanning electron microscopy was used for morphology observation, and elemental analysis was applied to determine the chitosan content bound on calcium-alginate gel beads (CAGB). Compared to CAGB, the dried CCAGB had poorer shape and rougher surface morphology especially in methods 1(a) and (b); moreover, CCAGB was found to be more instable in 0.9% NaCl and serious burst of beads occurred when high concentration of alginate (3.0 and 5.0% w/v) was used. The influence on BB release from the beads by chitosan coating was not only related to the chitosan density on bead surface, but also preparation method and other factors. Under un-dried bead state in method 1(a), the increase of chitosan content prolonged BB release in 0.9% (w/v) NaCl; while in method 2, the increase of chitosan concentration over 0.1% (w/v) (3.0% (w/v) alginate concentration was used) resulted in more serious burst of beads and hence facilitated BB release. Furthermore, in both methods 1(a) and 2, the increase of alginate from 1.5 to 3.0 or 5.0% (w/v) usually resulted in the significant burst of beads and accelerated BB release when 0.3 or 0.5% (w/v) chitosan was used for coating. Drying process greatly influenced BB release profile due to the destroying of alginate-chitosan film. The acceleration of BB release from CCAGB by drying process was more significant in the case of method 1 than of method 2.     

Chitosan-alginate multilayer beads for controlled release of ampicillin

The aim of this study is to develop multilayer beads with improved properties for controlled delivery of the antibiotic ampicillin. Ionotropic gelation was applied to prepare single and multilayer beads using various combinations of chitosan and Ca²⁺ as cationic components and alginate and polyphosphate as anions. Beads prepared with higher concentrations of chitosan entrapped more ampicillin. During incubation in simulated gastric fluid, the beads swelled and started to float but did not show any sign of erosion. Single layer chitosan–alginate beads released 70% of the drug within 4 h. Multilayer beads released only 20–30% in the same period of time. During subsequent incubation in simulated intestinal fluid, both single and multilayer beads continued to release drug. At least part of this release is due to disintegration of the beads. The rate of release both in gastric and intestinal fluid and the kinetics of disintegration in intestinal fluid can be controlled by changing the chitosan concentration in the coagulation fluid. The release of the drug can also be controlled by the degree of cross-linking using polyphosphate. Cross-linked multilayer beads were prepared that released only 40% of the entrapped drug during 24 h. It is concluded that chitosan–alginate multilayer beads, cross-linked with polyphosphate offer an opportunity for controlled gastrointestinal passage of compounds with low molecular weight like ampicillin.     

Calcium pectinate gel beads for controlled release drug delivery: II. Effect of form ulation and processing variables on drug release

The effect of four formulation and processing variables, calcium concentration, drying condition, concentration of hardening agent and hardening time on the bead properties and the release characteristics of a model drug from calcium pectinate gel (CPG) beads were studied. A poorly soluble compound, indomethacin, was used as the model drug. The investigated variables affected the bead size, the entrapment efficiency and the release of indomethacin from CPG beads. Drug release was found to be a function of the formulation and processing variables. The slower drug release was achieved from the formulations with higher calcium concentration, higher concentration of hardening agent and longer hardening time. The drying condition, however, did not influence the drug release. The mechanism of indomethacin release from CPG beads followed the diffusion controlled model for an inert porous matrix. All drug release data fitted well to the Higuchi square root time expression.     

Application of mathematical modelling to alginate chitosan polyelectrolyte complexes for the prediction of system behavior with Venlafaxine HCl as a model charged drug

PurposeThis work aimed to develop and analyze the performance of chitosan/alginate polyelectrolyte complex (PEC). Multiple regression and Lab fit curve fitting were applied to derive empirical models for the prediction of zeta potential of plain systems as a function of alginate chitosan ratio. Venlafaxine-HCl was loaded as a model charged drug and empirical models for prediction of its release as a function of time were also derived.MethodsCoacervation method was used for the preparation of green PECs. Preliminary studies were conducted to optimize the preparation method. Pre-adjustment of the pH of alginate and chitosan sols enabled the formation of PECs at alginate/chitosan ratios starting from 1:9 to 9:1. On mixing of alginate and chitosan sols, equal volume dilution method produced spherical particles, while direct mixing method gave fibrous particles. Twenty-seven PECs nanoparticle formulae were prepared using nine alginate/chitosan ratios and three levels of total polymer concentrations.ResultsStatistical analysis showed that Zeta potential of the nanoparticle was significantly dependent on alginate/chitosan ratio, while particle size was a function of total polymer concentration. Nine fiber formulae were prepared and evaluated for their appearance and zeta potential. Venlafaxine-HCl release followed anomalous transport mechanism. FT-IR and DSC studies confirmed complexation at the carboxylate and amine site at alginate and chitosan respectively.ConclusionChitosan/alginate PECs were successfully obtained without a cross-linker and empirical equations were obtained to help finding the best composition for loading charged drugs and to predict their release profiles.     

Xanthan-alginate composite gel beads: molecular interaction and in vitro characterization

Xanthan gum (XG), a trisaccharide branched polymer, was applied to reinforce calcium alginate beads in this study. Composite beads consisting of XG and sodium alginate (SA) were prepared using ionotropic gelation method. Diclofenac calcium-alginate (DCA) beads incorporated with different amounts of XG were produced as well. Molecular interaction between SA and XG in the composite beads and the XG-DCA beads was investigated using FTIR spectroscopy. Physical properties of the XG-DCA beads such as entrapment efficiency of diclofenac sodium (DS), thermal property, water uptake, swelling and DS release in various media were examined. XG could form intermolecular hydrogen bonding with SA in the composite beads with or without DS. Differential scanning calorimetric study indicated that XG did not affect thermal property of the DCA beads. The DS entrapment efficiency of the DCA beads increased with increasing amount of XG added. The XG-DCA beads showed higher water uptake and swelling in pH 6.8 phosphate buffer and distilled water than the DCA beads. A longer lag time and a higher DS release rate of the XG-DCA beads in pH 6.8 phosphate buffer were found. In contrast, the 0.3%XG-DCA beads could retard the drug release in distilled water because interaction between XG and SA gave higher tortuosity of the bead matrix. However, higher content of XG in the DCA beads increased the release rate of DS. This can be attributed to erosion of small aggregates of XG on the surface of the DCA beads. This finding suggested that XG could modulate physicochemical properties and drug release of the DCA beads, which based on the existence of molecular interaction between XG and SA.