Stabilizing effects of calcium alginate microspheres on Mycobacterium bovis BCG intended for oral vaccination

In this study, alginate microspheres containing BCG were prepared at a diameter of approximately 10 microm by emulsification-internal gelation of an alginate-BCG solution dispersed in olive oil using a high rate speed stirrer. The stability of BCG was assayed at 4 degrees C showing that the encapsulated BCG was more stable than free BCG at least for 5 weeks; however, BCG in sodium alginate solution was not stable at all. On the other hand, the studies using media with different pH (1.2, 4.4, 6.2, 6.8 and 7.5) have demonstrated that the alginate microspheres are stable in acidic medium for upto 1.5 h without any sign of disintegration. Moreover, BCG incorporated in alginate microspheres demonstrated an almost 9-fold increase in viable bacilli in simulated gastric fluid (SGF) after 1.5 h in comparison with free BCG.     

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?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.     

5-Fluorouracil-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(lactide-co-glycolide) polymers: Characterization and drug release

5-Fluorouracil (5-FU), a hydrosoluble anti-neoplastic drug, was encapsulated in microspheres of poly(D,L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) polymers using the spray-drying technique, in order to obtain small size microspheres with a significant drug entrapment efficiency. Drug-loaded microspheres included between 47?±?11 and 67?±?12?µg 5-FU?mg^?1 microspheres and the percentage of entrapment efficiency was between 52?±?12 and 74?±?13. Microspheres were of small size (average diameter: 0.9?±?0.4–1.4?±?0.8?µm microspheres without drug; 1.1?±?0.5–1.7?±?0.9?µm 5-FU-loaded microspheres) and their surface was smooth and slightly porous, some hollows or deformations were observed in microspheres prepared from polymers with larger T_g. A fractionation process of the raw polymer during the formation of microspheres was observed as an increase of the average molecular weight and also of T_g of the polymer of the microspheres. The presence of 5-FU did not modify the T_g values of the microspheres. Significant interactions between the drug and each one of the polymers did not take place and total release of the included drug was observed in all cases. The time needed for the total drug release (28–129?h) was in the order PLA?>?PLGA 75/25?>?PLGA 50/50. A burst effect (17–20%) was observed during the first hour and then a period of constant release rate (3.52?±?0.82–1.46?±?0.26?µg 5-FU?h^?1 per milligram of microspheres) up to 8 or 13?h, depending on the polymer, was obtained.     

Preparation and characterization of Mitomycin-C loaded chitosan-coated alginate microspheres for chemoembolization

Mitomycin-C loaded and chitosan-coated alginate microspheres were prepared for use in chemoembolization studies. In this respect, first alginate microspheres were prepared by using a spraying method using an extrusion device with a small orifice and following suspension cross-linking in an oil phase. Chitosan-coating onto the alginate microspheres was achieved by polyionic complex formation between alginate and chitosan. CaCl₂ was used as a cross-linker for alginate microspheres. The obtained chitosan-coated alginate microspheres were spherical shaped and ～100–400?µm average size. The microspheres were evaluated based on their swellability and the swelling ratio was changed between 50–280%. CaCl₂ concentration, stirring rate, chitosan molecular weight, chitosan concentration and time for coating with chitosan were selected as the effective parameters on microsphere size and swelling ratio. Equilibrium swellings were achieved in ～30?min. On the other hand, chitosan molecular weight, chitosan concentration and time for coating with chitosan were found as the most effective parameters on both drug loading ratio and release studies. Maximum drug loading ratio of 65% was achieved with high molecular weight (HMW) chitosan, highest chitosan concentration (i.e. 1.0% v/v) and shortest time for coating with chitosan (i.e. 1?h) values.     

Effects of alginate coated on PLGA microspheres for delivery tetracycline hydrochloride to periodontal pockets

The effects of alginate coated on tetracycline (Tc) loaded poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres fabricated by double emulsion solvent evaporation technique for local delivery to periodontal pocket were investigated. Alginate coated PLGA microspheres showed smoother surface but enlarged their particle sizes compared with those of uncoated ones. In addition, alginate coated microspheres enhanced Tc encapsulation efficiency (E.E.) from 11.5?±?0.5% of uncoated ones to 17.9?±?0.5%. Moreover, all of the coated PLGA microspheres even fabricated at different conditions could prolong Tc release from 9–12 days with 50% or higher in cumulative release of Tc compared with those of uncoated ones. The swelling ratios of PLGA microspheres for alginate coated or uncoated ones, one of the possible mechanisms for enhancing Tc release for the coated ones, were measured. The results showed that 20% or higher in swelling ratio for the coated microspheres at the earlier stage of hydration (e.g.?≤?24?h) could be an important factor to result in high Tc release compared to the uncoated ones. In conclusion, alginate coated Tc loaded PLGA microspheres could enhance Tc delivery to periodontal pocket by enhancing drug encapsulated efficiency, released quantities and sustained release period compared with uncoated ones.     

A novel impinging aerosols method for production of propranolol hydrochloride-loaded alginate gel microspheres for oral delivery

Propranolol hydrochloride was directly encapsulated in alginate gel microspheres (40-50?μm in diameter) using a novel method involving impinging aerosols of CaCl(2) cross-linking solution and sodium alginate solution containing the drug. Microspheres formulated using 0.1?M CaCl(2) exhibited the highest drug loading (14%, w/w of dry microspheres) with 66.5% encapsulation efficiency. Less than 4% and 35% propranolol release occurred from hydrated and dried microspheres, respectively, in 2?h in simulated gastric fluid (SGF). The majority of the drug load (90%) was released in 5 and 7?h from hydrated and dried microspheres, respectively, in simulated intestinal fluid (SIF). Prior incubation of hydrated microspheres (cross-linked using 0.5?M CaCl(2)) in SGF prolonged the time of release in SIF to 10?h, which has implications for the design of protocols and correlation with in?vivo release behaviour. Restricted propranolol release in SGF and complete extraction in SIF demonstrate the potential of alginate gel microspheres for oral delivery of pharmaceuticals.     

Chitosan-coated alginate microspheres for embolization and/or chemoembolization: In vivo studies

In this study, chitosan-coated alginate microspheres were prepared by the ionic complexation of alginate and chitosan biopolymers to use in embolization and/or chemoembolization studies. Biopolymeric microspheres were prepared by the ionic gelation technique of alginate with a suitable divalent cation (i.e. CaCl₂) in a suspension medium composed of mineral oil and petroleum ether including emulsifier (i.e. Tween-80) and then obtained microspheres were coated with chitosan in an aqueous chitosan solution while the medium was magnetically stirred. The obtained microspheres are in the size range of 100–400?µm and they can be prepared as required by changing the preparation conditions (i.e. stirring rate, concentration of biopolymers, molecular weight and concentration of chitosan, etc.). In the in vivo studies, New Zealand rabbits were used as the test animals. Both complete and partial embolization of the kidney were achieved by using the microspheres. The renal angiograms obtained before/after embolization and the histopathological observations showed the feasibility of the chitosan-coated alginate microspheres as an alternative embolization and/or chemoembolization agent.     

Sophoridine-loaded PLGA microspheres for lung targeting: preparation,in vitro,and in vivo evaluation

Lung-targeting sophoridine-loaded poly(lactide-co-glycolide) (PLGA) microspheres were constructed by a simple oil-in-oil emulsion-solvent evaporation method. The obtained microspheres were systematically studied on their morphology, size distribution, drug loading, encapsulation efficiency, in vitro release profile, and biodistribution in rats. The drug-loaded microparticles showed as tiny spheres under SEM and had an average size of 17?μm with 90% of the microspheres ranging from 12 to 24?μm. The drug loading and encapsulation efficiency were 65% and 6.5%, respectively. The in vitro drug release behavior of microspheres exhibited an initial burst of 16.6% at 4?h and a sustained-release period of 14 days. Drug concentration in lung tissue of rats was 220.10?μg/g for microspheres and 6.77?μg/g for solution after intraveneous injection for 30?min, respectively. And the microsphere formulation showed a significantly higher drug level in lung tissue than in other major organs and blood samples for 12 days. These results demonstrated that the obtained PLGA microspheres could potentially improve the treatment efficacy of sophoridine against lung cancer.     

Preparation and characterization of 5‐fluorouracil‐loaded poly(ϵ‐caprolactone) microspheres for drug administration

Microspheres of 5‐fluorouracil‐loaded poly(?‐caprolactone) (PCL) were prepared by spray‐drying procedure. The degradation characteristics and 5‐fluorouracil release in vitro as well as in vivo were investigated. The average molecular weight, weight loss, crystallinity, and morphology of microspheres were determined using GPC, DSC, and SEM, at different times during the in vitro degradation process. The size distribution of the microspheres indicated that most of the particles were smaller than 3 µm. A 30% weight loss as well as an increase of crystallinity were observed on day 330 of incubation. The percentage of entrapment efficiency of 5‐FU was 49% (44 µg of drug/mg of microspheres). The in vitro total release of 5‐FU took place in 8 days. Determination of plasma 5‐FU concentration in vivo using s.c. injection of 5‐FU‐loaded microspheres in Wistar rats by HPLC with analysis of data using a non‐compartmental model showed drug in plasma 18 days after administration with a maximum drug concentration of 1.5 µg/ml at 96 h. Pharmacokineticallly, a significant increase of AUC and MRT of 5‐FU with regard to the administration of the drug in solution. Scanning electron microscopy and histological studies indicated that the microspheres were surrounded by connective tissue and inflammatory processes were not evident. As a result of these characteristics, the 5‐FU‐loaded PCL microspheres could be used for drug delivery. Drug Dev Res 63:41–53, 2004. © 2004 Wiley‐Liss, Inc.     

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.     

Application of self-assembled ultra-thin film coatings to stabilize macromolecule encapsulation in alginate microspheres

Alginate-based hydrogels have several unique properties that have enabled them to be used as a matrix for the entrapment of a variety of enzymes, proteins and cells for applications in bioprocessing, drug delivery and chemical sensing. However, control over release rates or, in some cases, stable encapsulation remains a difficult goal, especially for small particles with high surface-area-to-volume ratios. In this work, the potential to limit diffusion of macromolecules embedded in alginate spheres with nanofilm coatings was assessed. Alginate microspheres were fabricated using an emulsification process with high surfactant concentration to form beads in the size range of 2-10 microm. Using calcium chloride for ionotropic gelation, dextran was encapsulated in the gel phase by mixing with the alginate in solution. The exterior surface was then modified with polyelectrolyte coatings using the layer-by-layer self assembly technique. Leaching studies to assess retention of dextran with varying molecular weights confirmed that the application of multi-layer thin films to the alginate microspheres was effective in reducing leaching rate and total loss of the encapsulated material from the microspheres. For the best case, the rate of release for dextran of 2,000,000 Dalton molecular weight decreased from 1% h(-1) in bare microspheres to 0.1% h(-1) in polyelectrolyte-coated microspheres. The effectiveness of nanofilms reducing loss of the encapsulated macromolecules was found to vary between different polycation materials used. These studies support the feasibility of using these microsystems for development of long-term stable encapsulated systems, such as implantable biosensors.     

Polyethyleneglycol diacrylate microspheres: a novel carrier for laccase immobilisation

Abstract

Laccase was immobilised on polyethyleneglycol diacrylate (PEGDA) microspheres. The optimal preparation conditions of PEGDA microspheres were as follows: 3.0% (w/v) 2,2-azobisisobutyro-nitrite (AIBN), 4.0–5.0% (w/v) polyvinylpyrrolidone (PVP), 5.0–8.0% (w/v) glucose and 4.0% (w/v) PEGDA in glucose solution. The volume ratio of PEGDA solution, glucose/PVP solution and AIBN solution was 25: 100: 1. Microspheres obtained exhibited good characteristics with small sizes (1–4?µm). The immobilised laccase showed a higher stability in a wide pH range. Thermal stability and storage stability of immobilised laccase were enhanced. The activity of immobilised laccase was 45.0% after six cycles uses. Only 62.7% of the activity remained for free laccase while there was a 60.4% increased for immobilised laccase with storage at 4?°C for 25?d. The K_m value of laccase increased from 21.9 to 114.0?µmol/l after immobilisation.     

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.     

Insulin delivery through nasal route using thiolated microspheres

Abstract

The aim of the present study was to investigate the potential of developed thiolated microspheres for insulin delivery through nasal route. In the present study, cysteine was immobilized on carbopol using EDAC. A total of 269.93?µmol free thiol groups per gram polymer were determined. The prepared nonthiolated and thiolated microspheres were studied for particle shape, size, drug content, swellability, mucoadhesion and in vitro insulin release. The thiolated microspheres exhibited higher mucoadhesion due to formation of covalent bonds via disulfide bridges with the mucus gel layer. Drug permeation through goat nasal mucosa of nonthiolated and thiolated microspheres were found as 52.62?±?2.4% and 78.85?±?3.1% in 6?h, respectively. Thiolated microspheres bearing insulin showed better reduction in blood glucose level (BGL) in comparison to nonthiolated microspheres as 31.23?±?2.12% and 75.25?±?0.93% blood glucose of initial BGL were observed at 6?h after nasal delivery of thiolated and nonthiolated microspheres in streptozotocin-induced diabetic rabbits.     

Microspheres of essential oil in polylactic acid and poly(methyl methacrylate) matrices and their blends

This study is focussed on micro-encapsulation of essential oils in polylactic acid (PLA) and a poly(methyl methacrylate) (PMMA) matrix as well as blends of the same. Microspheres were prepared by the solvent evaporation technique and characterised by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier transform infra-red spectroscopy (FTIR). The encapsulation efficiencies and release profiles of the essential oils were studied by gas chromatography mass spectrometry (GC-MS) and head-space solid-phase microextraction GC-MS, respectively. Furthermore, the microspheres were tested for antibacterial activity against both Gram-negative and Gram-positive bacterial strains.

The results showed that the microspheres compositions (PLA/PMMA ratio) have significant effect on their characteristics. The process adopted for preparing the microspheres promoted formation of spherical particles at the sizes of 1.5–9.5?µm. The highest encapsulation efficiency of the prepared microspheres was observed in systems consisting of linalool (81.10?±?10.0?wt. % for PLA system and 76.0?±?3.3?wt. % for PMMA system). Confirmation was also made that the release rate of the microspheres was affected by the size of the same.     

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.     

Encapsulation of vitamin C in tripolyphosphate cross-linked chitosan microspheres by spray drying

This paper describes vitamin C-encapsulated chitosan microspheres cross-linked with tripolyphosphate (TPP) using a new process prepared by spray drying intended for oral delivery of vitamin C. Thus, prepared microspheres were evaluated by loading efficiency, particles size analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), zeta potential and in vitro release studies. The microspheres so prepared had a good sphericity and shape but varied with the volume of cross-linking agent solution added. They were positively charged. The mean particle size ranged from 6.1–9.0?µm. The size, shape, encapsulation efficiency, zeta potential and release rate were influenced by the volume of cross-linking agent. With the increasing amount of cross-linking agent, both the particle size and release rate were increased. Encapsulation efficiency decreased from 45.05–58.30% with the increasing amount of TPP solution from 10–30?ml. FTIR spectroscopy study showed that the vitamin C was found to be stable after encapsulation. XRD studies revealed that vitamin C is dispersed at the molecular level in the TPP-chitosan matrix. Well-defined change in the surface morphology was observed with the varying volume of TPP. The sphericity of chitosan microspheres was lost at higher volume of cross-linking agent. The release of vitamin C from these microspheres was sustained and affected by the volume of cross-linking agent added. The release of vitamin C from TPP-chitosan microspheres followed Fick's law of diffusion.     

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.     

Microencapsulation of ovalbumin in poly(lactide-co-glycolide) by an oil-in-oil (o/o) solvent evaporation method

Abstract

The objective of this study was to produce biodegradable poly(lactide-co-glycolide) (PLGA; 50/50) microspheres by an oil-in-oil (o/o) solvent evaporation method to prolong the in vitro release of ovalbumin (OVA) as a model protein. The effects, on loading efficiency, microsphere yield, morphology and drug release, of two dispersing agents, aluminum tristearate and Span 80, in mineral oil were examined. PLGA 50/50 microspheres containing OVA powder (sieved through a 53 μm mesh) were prepared using an o/o solvent evaporation method. When aluminum tristearate was employed as a dispersing agent, the loading efficiency and yield of OVA had maximum values of 89 and 72% at 0·15% (w/v) aluminum tristearate, respectively. Morphology studies suggested that the obtained microspheres were spherical, and had a smooth surface. The diameters of the microspheres ranged between 100 and 200 μm. The loading efficiency, or yield, for microspheres decreased significantly above or below 0·15% (w/v) aluminum tristearate, and microspheres wkh irregular shapes were observed. The minimum sedimentation volume ratio (F) was obtained at a dispersity of carbon black particles in ethanol containing 0·15% (w/v) aluminum tristearate by a sedimentation study, and the cloudy supernatant suggested a defiocculated suspension. However, on the contrary, when Span 80 was added into the mineral oil as a dispersing agent, the concentration of Span 80 had little or no effect on the characteristics of the prepared microspheres. Drug loadings (60–70%) were obtained within the Span 80 concentrations employed in the present study (0·05–1·0% (w/v)). The yields were also in the same levels. The microspheres prepared in mineral oil containing Span 80 had an average diameter less than 50 μm in all cases. Sustained-release characteristics were demonstrated for PLGA microspheres prepared in mineral oil containing aluminum tristearate as a dispersing agent, even though a burst release at the initial phase was observed. This initial burst release from PLGA microspheres was reduced to some extent by micronization of the OVA powder using a planetary-type ball mill. However, PLGA microspheres prepared in mineral oil containing Span 80 as a dispersing agent, exhibited a large initial burst release. This burst release seems to be due to the smaller size of microspheres and the OVA powder adhering to the surface of PLGA microspheres (confirmed by scanning electron microscope (SEM) study).