Formation of alginate microspheres produced using emulsification technique

This study investigated the formative process of alginate microspheres produced using an emulsification technique. The alginate microspheres were produced by cross-linking alginate globules dispersed in a continuous organic phase using various calcium salts: calcium chloride, calcium acetate, calcium lactate and calcium gluconate. The size, shape, drug content and Ca²⁺ content of the microspheres were evaluated. The tack, viscosity and pH of the calcium salt solution and percentage of Ca²⁺ partitioned into the organic phase were determined. Microscopic examination of the test emulsion at various stages of the emulsification process was also carried out. The propensity of cross-linking reaction was found to be dependent on successful collision between alginate and calcium salt globules. Examination of the characteristics of microspheres indicated that the formed microsphere was a resultant product of alginate globule clustering. The growth propensity of microspheres was promoted by the higher rate and extent of cross-linkage which was governed by the pH, tack and/or Ca²⁺ content of the cross-linking solution, as well as the dissociation constant and diffusivity of the calcium salt. Overall, the amount of free Ca²⁺ cross-linked with alginate in the formed microspheres was in the following order: calcium acetate > calcium chloride + calcium acetate > calcium chloride + calcium gluconate; calcium chloride + calcium lactate > calcium chloride. In microencapsulation by emulsification, the mean size of the microspheres produced can be modified by varying the tack, pH and Ca²⁺ content of the cross-linking solution and through the use of a combination of calcium salts. The shape of the microspheres produced was, nonetheless, unaffected by the physicochemical properties of the cross-linking solution.     

Production of alginate microspheres by internal gelation using an emulsification method

Alginate is a natural polysaccharide found in brown algae. Alginates are widely used in the food and pharmaceutical industries and have been employed as a matrix for the entrapment of drugs, macromolecules and biological cells. Alginate microspheres can be produced by the external or internal gelation method using calcium salts. The addition of calcium chloride solution in the final phase of production of microspheres by external gelation method using an emulsification technique causes the disruption of the equilibrium of the system being stirred, resulting in a significant degree of clumping of microspheres. Therefore, in this study, production of alginate microspheres by the internal gelation method using a modified emulsification technique was explored. The influence of calcium salt, added in varying amounts and at different stages, on the morphology of the microspheres was investigated. The effects of other hardening agents and different drying methods were also studied.     

Chitosan-reinforced alginate microspheres obtained through the emulsification/internal gelation technique.

Alginate microspheres prepared by emulsification/internal gelation were chosen as carriers for a model protein, hemoglobin (Hb). Reinforced chitosan-coated microspheres were obtained by an uninterrupted method, in order to simplify the coating process, minimize protein losses during production and to avoid Hb escape under acidic conditions. Microspheres recovery was evaluated as well as its morphology by determination of Hb encapsulation efficiency and microscopic observation, respectively. The formation of chitosan membrane made of it interaction with alginate was assessed by DSC (differential scanning calorimetry) and FT-IR (Fourier-transform infrared spectrometry) studies. Spherical uncoated microspheres with a mean diameter of 20 microm and encapsulation efficiency above 89% were obtained. Coated microspheres provided similar encapsulation efficiency but a higher mean diameter was obtained due to microspheres clumping during the coating step. Protein loss occurred mainly during emulsification rather than recovery. FT-IR and DSC together indicated electrostatic interactions between alginate carboxylate and chitosan ammonium groups as the main forces for complex formation. Hb release from microspheres showed a pH-dependent profile and was affected by chitosan coating. Under simulated gastric conditions, a total Hb burst release from uncoated microspheres was decreased with one-stage and two-stage chitosan coatings (68% and 28%, respectively). At pH 6.8, the Hb release from coated microspheres was fast but incomplete. These results suggest an optimization of the coating method to protect Hb under acidic conditions and to permit a complete but sustained release of Hb.     

Drug encapsulation in alginate microspheres by emulsification.

A method based on an emulsification process was developed for the production of calcium alginate microspheres. Isopropyl alcohol and acetone, which are strong dehydrating agents, were used to aid in the hardening and drying of the microspheres. However, the amount of drug encapsulated was very low. This was due to the drug being soluble in the dehydrating solvents. In the absence of the solvents a high percentage of drug was encapsulated, and this was further increased by forming the microspheres by phase inversion. It was also found that a suspension of the drug particles was required for effective microencapsulation. The efficiency of drug encapsulation generally increased with the ratio of drug to encapsulating material. The microspheres produced were free-flowing and most of them were smaller than 150 microns.     

Influence of partially cross-linked alginate used in the production of alginate microspheres by emulsification

Spherical and discrete calcium alginate microspheres had been produced by the emulsification technique. The microencapsulation process was highly efficient, but drug release from microspheres was rapid. A more orderly chain arrangement of the polymeric chains would give rise to a stronger and less permeable matrix capable of sustaining drug release. Therefore, the potential of using partially cross-linked alginate in the production of microspheres by emulsification was explored. The size and roundness of the microspheres, its drug content and drug release property were determined. The more viscous alginate solutions when reacted with more calcium salt added resulted in larger microspheres produced. Microspheres made from partially cross-linked alginate exhibited lower drug content and higher T75% values in drug release studies. This was due to decreased flexibility of the polymer chains which were partially held together by calcium ions, reducing subsequent interaction with the calcium ions resulting in lower drug entrapment efficiency and a more permeable microsphere matrix.     

Influence of viscosity and uronic acid composition of alginates on the properties of alginate films and microspheres produced by emulsification

This study investigated the influence of viscosity and uronic acid composition of alginates on the properties of alginate films and microspheres produced by emulsification. Tensile properties of films were determined while the yield, size, drug contents and release characteristics of the microspheres were examined. Tensile properties of calcium alginate matrix were significantly affected by the orientation and arrangement of the polymer chains. High viscosity alginates gave rise to higher yields and bigger microspheres. Generally, microspheres with high drug content and slower rate of drug release had high Ca²⁺ contents and were produced from alginates of higher viscosity. Within an alginate microsphere batch, small sized microsphere fractions had higher drug contents but showed faster drug release rates. Microspheres having a defined size range revealed great dependence of encapsulation efficiency and drug release rates on viscosity and extent of Ca²⁺-alginate interaction. Viscosity appeared to exert a predominant influence on the microsphere properties.     

Influence of viscosity and uronic acid composition of alginates on the properties of alginate films and microspheres produced by emulsification

This study investigated the influence of viscosity and uronic acid composition of alginates on the properties of alginate films and microspheres produced by emulsification. Tensile properties of films were determined while the yield, size, drug contents and release characteristics of the microspheres were examined. Tensile properties of calcium alginate matrix were significantly affected by the orientation and arrangement of the polymer chains. High viscosity alginates gave rise to higher yields and bigger microspheres. Generally, microspheres with high drug content and slower rate of drug release had high Ca2+ contents and were produced from alginates of higher viscosity. Within an alginate microsphere batch, small sized microsphere fractions had higher drug contents but showed faster drug release rates. Microspheres having a defined size range revealed great dependence of encapsulation efficiency and drug release rates on viscosity and extent of Ca2+-alginate interaction. Viscosity appeared to exert a predominant influence on the microsphere properties.     

Microencapsulation of hemoglobin in chitosan-coated alginate microspheres prepared by emulsification/internal gelation

Chitosan-coated alginate microspheres prepared by emulsification/internal gelation were chosen as carriers for a model protein, hemoglobin (Hb), owing to nontoxicity of the polymers and mild conditions of the method. The influence of process variables related to the emulsification step and microsphere recovering and formulation variables, such as alginate gelation and chitosan coating, on the size distribution and encapsulation efficiency was studied. The effect of microsphere coating as well its drying procedure on the Hb release profile was also evaluated. Chitosan coating was applied by either a continuous microencapsulation procedure or a 2-stage coating process. Microspheres with a mean diameter of less than 30 microm and an encapsulation efficiency above 90% were obtained. Calcium alginate cross-linking was optimized by using an acid/CaCO(3) molar ratio of 2.5, and microsphere-recovery with acetate buffer led to higher encapsulation efficiency. Hb release in gastric fluid was minimal for air-dried microspheres. Coating effect revealed a total release of 27% for 2-stage coated wet microspheres, while other formulations showed an Hb release above 50%. Lyophilized microspheres behaved similar to wet microspheres, although a higher total protein release was obtained with 2-stage coating. At pH 6.8, uncoated microspheres dissolved in less than 1 hour; however, Hb release from air-dried microspheres was incomplete. Chitosan coating decreased the release rate of Hb, but an incomplete release was obtained. The 2-stage coated microspheres showed no burst effect, whereas the 1-stage coated microspheres permitted a higher protein release.     

Release characteristics of pectin microspheres prepared by an emulsification technique

The potential application of pectin as a matrix polymer for making microspheres by an emulsification technique was explored, and the drug release property of these pectinate microspheres containing drug cores of varying aqueous solubilities: sulphanilamide, sulphaguanidine and sulphathiazole, was investigated using different dissolution media. The size and size distribution, specific surface area, drug content and drug release property of the pectinate microspheres were determined. The solubility and solution pH of drugs and their propensity to interact with pectin were characterized. Pectinate microspheres were successfully prepared by external gelation, using a modified emulsification technique. The kinetics of drug release from the microspheres best fitted Higuchi's model. Interestingly, the lowest percentage of drug released was produced by microspheres which were smallest in size and, therefore, largest in specific surface area, and containing sulphanilamide, the most aqueous soluble and the lowest molecular weight drug. Mathematical correlation study indicated that the drug release profile of pectinate microspheres was notably affected by the drug content and the extent of drug-pectin interaction in the microspheres. Generally, a higher percentage of drug was released from the microspheres with a higher drug content and/or lower extent of drug-pectin interaction. The extent of drug-pectin interaction was highest in microspheres containing sulphanilamide, followed by sulphaguanidine and sulphathiazole, opposite to that of drug content.     

Release characteristics of pectin microspheres prepared by an emulsification technique

The potential application of pectin as a matrix polymer for making microspheres by an emulsification technique was explored, and the drug release property of these pectinate microspheres containing drug cores of varying aqueous solubilities: sulphanilamide, sulphaguanidine and sulphathiazole, was investigated using different dissolution media. The size and size distribution, specific surface area, drug content and drug release property of the pectinate microspheres were determined. The solubility and solution pH of drugs and their propensity to interact with pectin were characterized. Pectinate microspheres were successfully prepared by external gelation, using a modified emulsification technique. The kinetics of drug release from the microspheres best fitted Higuchi's model. Interestingly, the lowest percentage of drug released was produced by microspheres which were smallest in size and, therefore, largest in specific surface area, and containing sulphanilamide, the most aqueous soluble and the lowest molecular weight drug. Mathematical correlation study indicated that the drug release profile of pectinate microspheres was notably affected by the drug content and the extent of drug-pectin interaction in the microspheres. Generally, a higher percentage of drug was released from the microspheres with a higher drug content and/or lower extent of drug-pectin interaction. The extent of drug-pectin interaction was highest in microspheres containing sulphanilamide, followed by sulphaguanidine and sulphathiazole, opposite to that of drug content.     

Preparation of regular sized Ca-alginate microspheres using membrane emulsification method

Abstract

Terbutaline sulphate microcapsules were prepared by coacervation-phase separation induced by solvent evaporation technique. The cellulose acetate phthalate was employed as coating material alone and in combination with ethyl cellulose. The prepared microcapsules were evaluated for their drug content, particle size distribution (microscopic method), flow properties, bulk density and in vitro dissolution.     

Preparation of regular sized Ca-alginate microspheres using membrane emulsification method.

Monodisperse Ca-alginate microspheres were prepared using the membrane emulsification method. Three ionic types of drugs (anionic, cationic and non-ionic) were incorporated into the microspheres, and the effects of sodium alginate concentration and the pressure applied during the dispersing process on the properties of the microspheres were examined. Monodisperse microspheres were obtained when the concentration of alginate solution was 2 wt% and the pressure applied was 0.4 x 10(5) Pa. The mean size of microspheres was approximately 4 microm. Lidocaine x HCl (cationic), sodium salicylate (anionic) and 4-acetamidophenol (non-ionic) were selected as ionic model drugs and included in the alginate microspheres. Lidocaine x HCl (cationic drug) release was more retarded than that of the anionic drug, because of the electrostatic attraction between the negative charge of the ionized carboxyl group in the alginate chain and the positive charge of the cationic drug. In acidic release medium, a slow release was observed due to the low swelling characteristic and the increased viscosity of alginate, regardless of ionic type of drug.     

Evaluation of furosemide-loaded alginate microspheres prepared by ionotropic external gelation technique

Alginate microspheres containing furosemide were prepared by the ionotropic external gelation technique using Ca2+, Al3+ and Ba2+ ions. The incorporation efficiency of the prepared microspheres ranged between 65% and 93%. The effect of sodium alginate concentration, cross-linking ions and drying conditions was evaluated with respect to entrapment efficiency, particle size, surface characteristics and in vitro release behavior. Infrared spectroscopic study confirmed the drug-polymer compatibility. Differential scanning calorimetric analysis revealed that the drug was molecularly dispersed in the alginate microsphere matrices. Scanning electron microscopic study of microspheres showed the rough surface due to higher concentration of drug molecules dispersed at molecular level in the alginate matrices. The mean particle size and entrapment efficiency were found to be varied by changing various formulation parameters. The in vitro release profile could be altered significantly by changing various formulation parameters to give a sustained release of drug from the microspheres. The kinetic modeling of the release data indicate that furosemide release from the alginate microspheres follow anomalous transport mechanism after an initial lag period when the drug release mechanism was found to be Fickian diffusion controlled.     

Preparation of chitosan microspheres using membrane emulsification and its size modelling

The chitosan microspheres were prepared by a membrane emulsification method with variations of the N₂ gas pressure and the chitosan concentration. The pressure of N₂ gas was varied within the range from 0.2?×?10⁵ to 0.8?×?10⁵?Pa at chitosan concentration 1.5?wt%. In addition, the concentration of chitosan was varied between 0.5?～?2.0?wt% at 0.4?×?10⁵?Pa of N₂ gas pressure. Using this method, it is possible to prepare stable emulsions with a very narrow droplet size distribution in comparison with conventional methods. The average size of the microspheres was dependent on the N₂ gas pressure and the concentration, that is it was increased with the pressure and the concentration. The modelling of the size for the microspheres according to the concentration was carried out using Macleod's relation and Parkins & Brown equation. The former shows the relationship between density and surface tension and the latter demonstrates the correlation between the volume of the microspheres and the interfacial tension. The modelling results were in good agreement with the experimental data to predict the microspheres size with the variation of concentration.     

Preparation of chitosan microspheres using membrane emulsification and its size modelling

The chitosan microspheres were prepared by a membrane emulsification method with variations of the N2 gas pressure and the chitosan concentration. The pressure of N2 gas was varied within the range from 0.2 x 10(5) to 0.8 x 10(5) Pa at chitosan concentration 1.5 wt%. In addition, the concentration of chitosan was varied between 0.5 approximately 2.0 wt% at 0.4 x 10(5) Pa of N2 gas pressure. Using this method, it is possible to prepare stable emulsions with a very narrow droplet size distribution in comparison with conventional methods. The average size of the microspheres was dependent on the N2 gas pressure and the concentration, that is it was increased with the pressure and the concentration. The modelling of the size for the microspheres according to the concentration was carried out using Macleod's relation and Parkins & Brown equation. The former shows the relationship between density and surface tension and the latter demonstrates the correlation between the volume of the microspheres and the interfacial tension. The modelling results were in good agreement with the experimental data to predict the microspheres size with the variation of concentration.     

Novel alginate gel microspheres produced by impinging aerosols for oral delivery of proteins

Lysozyme and insulin were encapsulated in alginate gel microspheres using impinging aerosols method. High loadings of around 50% weight/dry microspheres weight were obtained with encapsulation efficiencies of at least 48%. Environmental scanning electron microscopy revealed smooth spherical hydrated microspheres (30–60?µm) in diameter. No lysozyme or insulin release was measured in simulated gastric fluid (HCl, pH 1.2, 37°C). Total insulin release occurred in simulated intestinal fluid (SIF; phosphate buffer saline, pH 7.4, 37°C) in 8?h following 2?h incubation in SGF and was found to retain 75% activity using the ARCHITECT® assay. Lysozyme was released completely in SIF in 10?h following 2?h incubation in SGF and was found to exhibit at least 80% bioactivity using the Micrococcus lysodeikticus assay. The absence of protein release in HCl and the retention of high levels of biological activity demonstrate the potential of alginate gel microspheres, for improving oral delivery of biopharmaceuticals.     

Novel alginate gel microspheres produced by impinging aerosols for oral delivery of proteins

Lysozyme and insulin were encapsulated in alginate gel microspheres using impinging aerosols method. High loadings of around 50% weight/dry microspheres weight were obtained with encapsulation efficiencies of at least 48%. Environmental scanning electron microscopy revealed smooth spherical hydrated microspheres (30-60 μm) in diameter. No lysozyme or insulin release was measured in simulated gastric fluid (HCl, pH 1.2, 37°C). Total insulin release occurred in simulated intestinal fluid (SIF; phosphate buffer saline, pH 7.4, 37°C) in 8 h following 2 h incubation in SGF and was found to retain 75% activity using the ARCHITECT? assay. Lysozyme was released completely in SIF in 10 h following 2 h incubation in SGF and was found to exhibit at least 80% bioactivity using the Micrococcus lysodeikticus assay. The absence of protein release in HCl and the retention of high levels of biological activity demonstrate the potential of alginate gel microspheres, for improving oral delivery of biopharmaceuticals.     

Effect of alginate composition and purity on alginate microspheres

BACKGROUND: Alginate is commonly used to microencapsulate islets in experiments with islet allografts and xenografts for the treatment of Type I diabetes. The purpose of the present study is to determine the effects of alginate composition and purity on the morphology and size of microspheres. METHODS: Microcapsules produced with the impure alginate types, medium-viscosity high-guluronic acid (IMVG), low-viscosity high-G (ILVG), low-viscosity high-mannuronic acid (ILVM) and medium-viscosity high-M (IMVM) were compared with one another and others generated with a highly purified LVM (HPLVM) alginate. Droplets of 1.5% alginate from an air-syringe pump were gelled in 1.1% CaCl2 solution. While leaving the alginate pressure and needle recess constant, the air-jacket pressure was varied between 9.5-10.5 PPSI to enhance stable microcapsule generation and different batches of microbeads were made from each alginate type. RESULTS: The sizes of the high-guluronic acid alginate microbeads were consistently bigger than those of the corresponding high-mannuronic acid alginate beads at all air-jacket settings. At the optimal air-jacket pressure of 9.0 PPSI, the mean+SD diameter of the IMVG microbeads was 780 + 20 microm, while that of IMVM was 607 + 44 microm (p < 0.0001, n=30). Similarly, the mean ILVG microbead diameter was 816+28 microm compared to 656+26 microm for ILVM capsules (p<0.0001, n=30). Less polymorphism was found with the HPLVM microspheres than with the ILVM microbeads. CONCLUSION: Highly purified high-mannuronic acid alginate will provide smaller, spherical microcapsules suitable for islet cell transplantation.     

Effect of alginate composition and purity on alginate microspheres

Background: Alginate is commonly used to microencapsulate islets in experiments with islet allografts and xenografts for the treatment of Type I diabetes. The purpose of the present study is to determine the effects of alginate composition and purity on the morphology and size of microspheres. Methods: Microcapsules produced with the impure alginate types, medium-viscosity high-guluronic acid (IMVG), low-viscosity high-G (ILVG), low-viscosity high-mannuronic acid (ILVM) and medium-viscosity high-M (IMVM) were compared with one another and others generated with a highly purified LVM (HPLVM) alginate. Droplets of 1.5% alginate from an air-syringe pump were gelled in 1.1% CaCl₂ solution. While leaving the alginate pressure and needle recess constant, the air-jacket pressure was varied between 9.5–10.5 PPSI to enhance stable microcapsule generation and different batches of microbeads were made from each alginate type. Results: The sizes of the high-guluronic acid alginate microbeads were consistently bigger than those of the corresponding high-mannuronic acid alginate beads at all air-jacket settings. At the optimal air-jacket pressure of 9.0 PPSI, the mean+SD diameter of the IMVG microbeads was 780+20?µm, while that of IMVM was 607+44?µm (p<0.0001, n?=?30). Similarly, the mean ILVG microbead diameter was 816+28 µm compared to 656+26?µm for ILVM capsules (p<0.0001, n?=?30). Less polymorphism was found with the HPLVM microspheres than with the ILVM microbeads. Conclusion: Highly purified high-mannuronic acid alginate will provide smaller, spherical microcapsules suitable for islet cell transplantation.     

Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction

An important application of hepatocyte cultures is identification of drugs acting as inducers of biotransformation enzymes that alter metabolic clearance of other therapeutic agents. In the present study we optimized an in vitro system with hepatocytes cultured in alginate microspheres that allow studies of enzyme induction with excellent sensitivity. Induction factors obtained with standard inducers, such as 3-methylcholanthrene or phenobarbital, were higher compared to those with conventional hepatocyte co-cultures on collagen coated dishes. This is illustrated by activities of 7-ethoxyresorufin-O-deethylase (EROD) after incubation with 5 microM 3-methylcholanthrene (3-MC), a standard inducer for cytochrome P4501A1 and 1A2. Mean activities for solvent controls and 3-MC exposed cells were 2.99 and 449 pmol/min/mg protein (induction factor: 150) for hepatocytes cultured in microspheres compared to 2.72 and 80.6 pmol/min/mg (induction factor: 29.6) for hepatocytes on collagen coated dishes. To compare these in vitro data to the in vivo situation male Sprague Dawley rats, the same strain that was used also for the in vitro studies, were exposed to 3-MC in vivo using a protocol that guarantees maximal induction. Activities were 29.2 and 1656 pmol/min/mg in liver homogenate of solvent and 3-MC treated animals (induction factor: 56.7). Thus, the absolute activities of 3-MC exposed hepatocytes in microspheres are lower compared to the in vivo situation. However, the induction factor in vitro was even higher compared to the in vivo situation (150-fold versus 56.7-fold). A similar scenario was observed using phenobarbital (0.75 mM) for induction of CYP2B and 3A isoenzymes: induction factors for testosterone hydroxylation in position 16beta were 127.5- and 50.4-fold for hepatocytes in microspheres and conventionally cultured hepatocytes, respectively. The new in vitro system with hepatocytes embedded in solid alginate microspheres offers several technical advantages: (i) the solid alginate microspheres can be liquefied within 60s, allowing a fast and complete harvest of hepatocytes; (ii) alginate capsules are stable allowing transport and mechanical stress; (iii) high numbers of hepatocytes can be encapsulated in short periods; (iv) defined cell numbers between 600 hepatocytes, the approximate number of cells in one capsule, and 18 x 10(6) hepatocytes, the number of hepatocytes in 6 ml alginate, can be transferred to a culture dish or flask. Thus, encapsulated hepatocytes allow a flexible organization of experiments with respect to cell number. In conclusion, we optimized a technique for encapsulation of hepatocytes in alginate microspheres that allows identification of enzyme induction with an improved sensitivity compared to existing systems.