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Polyelectrolyte nanocomposites rarely reach a stable state and aggregation often occurs. Here, we report the synthesis of nanocomposites for the oral delivery of insulin composed of alginate, dextran sulfate, poly-(ethylene glycol) 4000, poloxamer 188, chitosan, and bovine serum albumin. The nanocomposites were obtained by Ca 2+ -induced gelation of alginate followed by an electrostatic-interaction process among the polyelectrolytes. Chitosan seemed to be essential for the final size of the nanocomposites and there was an optimal content that led to the synthesis of nanocomposites of 400-600 nm hydrodynamic size. The enhanced stability of the synthesized nanocomposites was assessed with LUMiSizer after synthesis. Nanocomposite stability over time and under variations of ionic strength and pH were assessed with dynamic light scattering. The rounded shapes of nanocomposites were confirmed by scanning electron microscopy. After loading with insulin, analysis by HPLC revealed complete drug release under physiologically simulated conditions., Competing Interests: The authors declare no conflict of interest.

Cashew gum (CG) is a biopolymer that presents a favorable chemical environment for structural modifications, which leads to more stable and resistant colloidal systems. The gum was subjected to an acetylation reaction using a fast, simple, solvent-free and low cost methodology. The derivative was characterized by infrared and NMR spectroscopy, elemental analysis, coefficient of solubility and zeta potential. The modified biopolymer was used as a platform for drug delivery systems using insulin as a model drug. Nanoparticles were developed through the technique of polyelectrolytic complexation and were characterized by size, surface charge, entrapment efficiency and gastrointestinal release profile. The nanoparticles presented size of 460 nm with a 52.5% efficiency of entrapment of insulin and the electrostatic stabilization was suggested by the zeta potential of + 30.6 mV. Sustained release of insulin was observed for up to 24 h. The results showed that acetylated cashew gum (ACG) presented potential as a vehicle for sustained oral insulin release., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The potential of nanoparticles (NPs) to overcome the barriers for oral delivery of protein drugs have led to the development of platforms capable of improving their bioavailability. However, despite the progresses in drug delivery technologies, the success of oral delivery of insulin remains elusive and the disclosure of insulin mechanisms of absorption remains to be clarified. To overcome multiple barriers faced by oral insulin and to enhance the insulin permeability across the intestinal epithelium, here insulin-loaded alginate/dextran sulfate (ADS)-NPs were formulated and dual-coated with chitosan (CS) and albumin (ALB). The nanosystem was characterized by its pH-sensitivity and mucoadhesivity, which enabled to prevent 70% of in vitro insulin release in simulated gastric conditions and allowed a sustained insulin release following the passage to simulated intestinal conditions. The pH and time-dependent morphology of the NPs was correlated to the release and permeation profile of insulin. Dual CS/ALB coating of the ADS-NPs demonstrated augmented intestinal interactions with the intestinal cells in comparison to the uncoated-NPs, resulting in a higher permeability of insulin across Caco-2/HT29-MTX/Raji B cell monolayers. The permeability of the insulin-loaded ALB-NPs was reduced after the temperature was decreased and after co-incubation with chlorpromazine, suggesting an active insulin transport by clathrin-mediated endocytosis. Moreover, the permeability inhibition with the pre-treatment with sodium chlorate suggested that the interaction between glycocalix and the NPs was critical for insulin permeation. Overall, the developed nanosystem has clinical potential for the oral delivery of insulin and therapy of type 1 diabetes mellitus., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Alginate-dextran sulfate-based particles obtained by emulsification/internal gelation technology can be considered suitable carriers for oral insulin delivery. A rational study focused on the emulsification and particle recovery steps was developed in order to reduce particles to the nanosize range while keeping insulin bioactivity. There was a decrease in size when ultrasonication was used during emulsification, which was more pronounced when a cosurfactant was added. Ultrasonication add-on after particle recovery decreased aggregation and led to a narrower nanoscale particle-size distribution. Insulin encapsulation efficiency was 99.3%±0.5%, attributed to the strong pH-stabilizing electrostatic effect between insulin and nanoparticle matrix polymers. Interactions between these polymers and insulin were predicted using molecular modeling studies through quantum mechanics calculations that allowed for prediction of the interaction model. In vitro release studies indicated well-preserved integrity of nanoparticles in simulated gastric fluid. Circular dichroism spectroscopy proved conformational stability of insulin and Fourier transform infrared spectroscopy technique showed rearrangements of insulin structure during processing. Moreover, in vivo biological activity in diabetic rats revealed no statistical difference when compared to nonencapsulated insulin, demonstrating retention of insulin activity. Our results demonstrate that alginate-dextran sulfate-based nanoparticles efficiently stabilize the loaded protein structure, presenting good physical properties for oral delivery of insulin.

Oral insulin able to induce an efficient antihyperglycemic effect either to replace or complement diabetes mellitus therapy is the major goal of health providers, governments and diabetic patients. Oral therapy is associated not only with the desire to exclude needles from the daily routine of diabetic patient but also with the physiological provision of insulin they would get. Despite numerous efforts over the past few decades to develop insulin delivery systems, there is still no commercially available oral insulin. The reasons why the formulations developed to administer insulin orally fail to reach clinical trials are critically discussed in this review. The principal features of nanoformulations used so far are also addressed as well as the undergoing clinical trials.

The oral route is the most suitable and physiological delivery route. Oral insulin delivery would minimize the health hazard implied in repeated injection, surpass complications arising from the need for sterile techniques associated with parenteral formulations and provide better glucose homeostasis. However, it is limited by various physiological barriers and still remains a scientific challenge. The desire to deliver insulin by the oral route in a conveniently and effectively way has led to the intense investigation of new delivery systems. Nanodelivery systems have been proposed to enhance the bioavailability of insulin after oral administration. This review article describes the gastrointestinal barriers to oral insulin delivery, including chemical, enzymatic and absorption barriers. The potential transport mechanisms of insulin delivered by nanoparticles across the intestinal epithelium are also addressed. Finally, how nanoparticles characteristics affect insulin pharmacological activity and bioavailability is discussed.

Alginate-dextran sulfate (ADS) microgel has been used to protect insulin from gastrointestinal attack and as a carrier to promote insulin permeation through intestinal epithelium. The throughput of ADS submicron particles generation by emulsification/internal gelation is limited by its wide size distribution. The aim of this work was to study the recovery protocol influence on ADS particles through the determination of its impact on particles' size distribution and bioactivity. ADS particles showed a wide and multimodal distribution, characterized by a high aggregation phenomenon. In an attempt to reverse particles' tendency to aggregate and to homogenize particle size ADS populations were submitted to ultrasonication, while particle size distribution, physical and chemical stability, and the bioactivity of entrapped insulin were investigated. After ultrasonication a narrower particle population shifted to the nanoscale, with higher physical stability and significant insulin bioactivity was obtained. Emulsification internal/gelation followed by ultrasonication constituted a valid strategy to obtain ADS particles at the submicron range, with high stability and without significantly compromising insulin bioactivity, so offering promises, under previously well established conditions, to evaluate impact of ADS particle's size on biopharmaceutical and pharmacokinetics phases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The purpose of this study was to evaluate hepatic glucose metabolism of diabetic induced rats after a daily oral load of insulin nanoparticles over 2 weeks. After the 2-week treatment, an oral glucose tolerance test was performed with [U-¹³C] glucose and ²H₂O. Plasma glucose ²H and ¹³C enrichments were quantified and the contribution of glycogenolysis and gluconeogenesis to overall glucose production were estimated. Animals with the insulin nanoparticles displayed the lowest glycemia before the oral glucose tolerance test. In all animals, 75% of the total glucose production was from gluconeogenesis and glycogen synthesis was only detected in some animals. Gluconeogenic pathway was an active contributor to hepatic glucose production and the treatment with oral delivered insulin nanoparticles did not alter this contribution, suggesting that under this treatment, protocol hepatic glucose metabolism is not the most relevant target of insulin action but instead a more generalised effect in peripheral tissues.

The effect of nanoparticulate delivery system on enhancing insulin permeation through intestinal membrane was evaluated in different intestinal epithelial models using cell cultures and excised intestinal tissues. Multilayered nanoparticles were formulated by encapsulating insulin within a core consisting of alginate and dextran sulfate nucleating around calcium and binding to poloxamer, stabilized by chitosan, and subsequently coated with albumin. Insulin permeation through Caco-2 cell monolayer was enhanced 2.1-fold, facilitated by the nanoparticles compared with insulin alone, 3.7-fold through a mucus-secreting Caco-2/HT29 co-culture, and 3.9-fold through excised intestinal mucosa of Wistar rats. Correlation of Caco-2/HT29 co-culture cells with the animal-model intestinal membrane demonstrates that the mucus layer plays a significant role in determining the effectiveness of oral nanoformulations in delivering poorly absorbed drugs. Albumin was applied to the nanoparticles as outermost coat to protect insulin through shielding from proteolytic degradation. The effect of the albumin layering on insulin permeation was compared with albumin-free nanoparticles that mimic the result of albumin being enzymatically removed during gastric and intestinal transport. Results showed that albumin layering is important toward improving insulin transport across the intestinal membrane, possibly by stabilizing insulin in the intestinal conditions. Transcellular permeation was evidenced by internalization of independently labeled insulin and nanoparticles into enterocytes, in which insulin appeared to remain associated with the nanoparticles. Transcellular transport of insulin through rat intestinal mucosa may represent the predominant mechanism by which nanoparticles facilitate insulin permeation. Nanoformulations demonstrated biocompatibility with rat intestinal mucosa through determination of cell viability via monitoring of mitochondrial dehydrogenases. Insulin permeation facilitated by the biocompatible nanoparticles suggests a potential carrier system in delivering protein-based drugs by the oral route., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Intestinal uptake, insulinemia and hypoglycemic effect of orally delivered insulin encapsulated in polyelectrolytically stable nanoparticles were evaluated in streptozotocin-induced Wistar diabetic rats. Nanoparticles with 396nm mean diameter were formed by alginate and dextran sulfate nucleating around calcium and binding to poloxamer, stabilized by chitosan, and subsequently coated with albumin. The resulting negatively charged nanoparticles retained insulin bioactivity and enhanced pharmacological availability by shielding insulin from enzymatic degradation and through chemical and physical facilitation of permeation through the intestinal membrane. Insulin nanoencapsulated through a simplified method avoiding harsh conditions and organic solvents, reduced plasma glucose levels to 40% of the basal values with a sustained hypoglycemic effect over 24h. Pharmacodynamic and pharmacokinetic parameters were evaluated at a dose of 50IU/kg nanoencapsulated insulin, and 13% oral bioavailability showed a threefold increase in comparison to free insulin. Confocal microscopy showed internalization of nanoencapsulated insulin in the small intestinal mucosa using independently labeled insulin-FITC and alginate-RBITC. Therefore the nanoformulation facilitated the oral delivery of insulin, and potentially that of other therapeutic proteins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Strategies to design effective and safe colloidal carriers for biopharmaceuticals have evolved through applying the knowledge gained in nanotechnology to medicine. Designing a colloidal carrier to serve as a protein delivery device requires an understanding of the effect of different materials on the physicochemical, physiological and toxicological parameters for clinical application. The purpose of this study was to evaluate the influence of formulation components on the physicochemical factors and biological function involved in the development and optimization of newly designed nanoparticles for orally dosed insulin. Biodegradable, biocompatible, mucoadhesive and protease-protective biomaterials were combined through ionotropic pre-gelation and polyelectrolyte complexation forming an alginate, dextran sulfate and poloxamer hydrogel containing insulin, stabilized in nanoparticles with chitosan and poly(ethyleneglycol) and coated with albumin. Nanoparticles ranged in size from 200 to 500nm with 70-90% insulin entrapment efficiency, and electrostatic stabilization was suggested by zeta potential values lower than -30mV. This combination of formulation components was selected for insulin protection against harsh gastric pH and proteolytic conditions, and to improve insulin absorption through intestinal mucosa by combining nanoparticle uptake and insulin release at the site of absorption. Insulin was shown to be bioactive after nanoparticle formulation and release in neutral pH conditions. Fourier transform infrared spectroscopy was used to confirm the presence of formulations components in the nanoparticle structure and to identify potential interactions between biomaterials.

Design of nanoparticles integrating biomaterials that govern the functional behavior of orally dosed insulin is focused on improving insulin stability and absorption by facilitating its uptake and translocation throughout the intestinal membrane, while providing protection from acidic and enzymatic degradation in the gastrointestinal tract. The purpose of the study was to optimize a nanoparticle formulation by investigating the relationship between design factors and experimental data by response surface methodology. Designed nanoparticles consisting of calcium crosslinked alginate, dextran sulfate, poloxamer 188 and chitosan followed by an outermost coating of albumin are described as multilayer complex retaining insulin within the nanoparticle. A 3-factor 3-level Box-Behnken design was used to optimize nanoparticle formulation. The screened independent variables were the concentration of calcium chloride, chitosan and albumin, and the dependent variables were particle size, polydispersity index, zeta potential, entrapment efficiency and insulin release in enzyme-free simulated digestive fluids. Experimental responses of a total of 15 formulations resulted in mean nanoparticle diameters ranging from 394 to 588 nm, with polydispersity index from 0.77 to 1.10, zeta potential values ranging from -36.6 to -44.5 mV, and entrapment efficiency of insulin was over 85%. Insulin release from nanoparticles in enzyme-free digestive fluids was prevented during 120 min in gastric conditions, and over 80% of insulin was released after 180 min in simulated intestinal fluid. Based on the experimental responses and the criteria of desirability defined by constraints, solutions of 0.20% calcium chloride, 0.04% chitosan and 0.47% albumin constitute the optimum formulation of nanoparticles for orally dosed insulin.

Nanospheres are being developed for the oral delivery of peptide-based drugs such as insulin. Mucoadhesive, biodegradable, biocompatible, and acid-protective biomaterials are described using a combination of natural polyelectrolytes, with particles formulated through nanoemulsion dispersion followed by triggered in situ gel complexation. Biomaterials meeting these criteria include alginate, dextran, chitosan, and albumin in which alginate/dextran forms the core matrix complexed with chitosan and albumin coat. Smaller size and higher albumin-based acid-protective formulation was orally administered to diabetic rats and glucose reduction and physiological response analyzed. Insulin encapsulation efficiency was 90, 82, and 66% for uncoated, chitosan-coated, and albumin-chitosan-coated alginate nanospheres, respectively. The choice of coating polymer seems to influence insulin release profile and to be crucial to prevent peptic digestion. Physiological response following oral delivery showed that insulin albumin-chitosan-coated alginate nanospheres reduced glycemia approximately 72% of basal values. Albumin serves as an important enteric coating providing acid- and protease protection enabling uptake of active drug following oral dosage.

The design of strategies that improve the absorption of insulin through the gastrointestinal tract is a considerable challenge in the pharmaceutical sciences and would significantly enhance the treatment of diabetes mellitus. Several strategies have been devised to overcome physiologic and morphologic barriers to insulin absorption, including the inhibition of acidic and enzymatic degradation, enhancement of membrane permeability or widening of tight junctions, chemical modification of insulin, and the formulation of carrier systems. In particular, the concept of nanoparticulate carriers for oral insulin delivery has evolved through remarkable advances in nanotechnology. Investigations focused on uptake and translocation via Peyer's patches have demonstrated high levels of nanoparticle absorption based on significant alterations in the glycemic response to various glucogenic sources. This paper reviews the mechanisms for insulin and particle uptake and translocation through the gastrointestinal tract, and the potential barriers to this, outlines the design of nanoparticulate carriers for the oral delivery of insulin, and presents prospects for its clinical application.

Purpose: To evaluate the pharmacological activity of insulin-loaded alginate/chitosan nanoparticles following oral dosage in diabetic rats., Methods: Nanoparticles were prepared by ionotropic pre-gelation of an alginate core followed by chitosan polyelectrolyte complexation. In vivo activity was evaluated by measuring the decrease in blood glucose concentrations in streptozotocin induced, diabetic rats after oral administration and flourescein (FITC)-labelled insulin tracked by confocal microscopy., Results: Nanoparticles were negatively charged and had a mean size of 750 nm, suitable for uptake within the gastrointestinal tract due to their nanosize range and mucoadhesive properties. The insulin association efficiency was over 70% and insulin was released in a pH-dependent manner under simulated gastrointestinal conditions. Orally delivered nanoparticles lowered basal serum glucose levels by more than 40% with 50 and 100 IU/kg doses sustaining hypoglycemia for over 18 h. Pharmacological availability was 6.8 and 3.4% for the 50 and 100 IU/kg doses respectively, a significant increase over 1.6%, determined for oral insulin alone in solution and over other related studies at the same dose levels. Confocal microscopic examinations of FITC-labelled insulin nanoparticles showed clear adhesion to rat intestinal epithelium, and internalization of insulin within the intestinal mucosa., Conclusion: The results indicate that the encapsulation of insulin into mucoadhesive nanoparticles was a key factor in the improvement of its oral absorption and oral bioactivity.

Alginate nanoparticles were prepared from dilute alginate sol by inducing a pre-gel with calcium counter ions, followed by polyelectrolyte complex coating with chitosan. Particles in the nanometer size range were obtained with 0.05% alginate and 0.9 mM Ca2+. The mean particle size was influenced by time and stirring speed of nanoparticle preparation, by alginate guluronic acid content and chitosan molecular weight and by the initial alginate:chitosan mass ratio. The association efficiency of insulin into alginate nanoparticles, as well as loading capacity were mainly influenced by the alginate:chitosan mass ratio. Under optimized size conditions, the association efficiency and loading capacities were as high as 92% and 14.3%, respectively. Approximately 50% of the protein was partially retained by the nanoparticles in gastric pH environment up to 24 hours while a more extensive release close to 75% was observed under intestinal pH conditions. Mild formulation conditions, optimum particle size range obtained, high insulin entrapment efficiency, and resistance to gastrointestinal release seem to be synergic and promising factors toward development of an oral insulin delivery form.

Insulin-loaded alginate-dextran nanospheres were prepared by nanoemulsion dispersion followed by triggered in situ gelation. Nanospheres were characterized for mean size and distribution by laser diffraction spectroscopy and for shape by transmission electron microscopy. Insulin encapsulation efficiency and in vitro release were determined by Bradford protein assay and bioactivity determined in vitro using a newly developed Western blot immunoassay and in vivo using Wistar diabetic rats. Nanospheres ranged from 267 nm to 2.76 microm in diameter and demonstrated a unimodal size distribution. Insulin encapsulation efficiency was 82.5%. Alginate-dextran particles suppressed insulin release in acidic media and promoted a sustained release at near neutral conditions. Nanoencapsulated insulin was bioactive, demonstrated through both in vivo and in vitro bioassays.

Alginate microparticles produced by emulsification/internal gelation were investigated as a promising carrier for insulin delivery. The procedure involves the dispersion of alginate solution containing insulin protein, into a water immiscible phase. Gelation is triggered in situ by instantaneous release of ionic calcium from carbonate complex via gentle pH adjustment. Particle size is controlled through the emulsification parameters, yielding insulin-loaded microparticles. Particle recovery was compared using several washing protocols. Recovery strategies are proposed and the influence on particle mean size, morphology, recovery yield (RY), encapsulation efficiency, insulin release profile, and structural integrity of released insulin were evaluated. Spherical micron-sized particles loaded with insulin were produced. The recovery process was optimized, improving yield, and ensuring removal of residual oil from the particle surface. The optimum recovery strategy consisted in successive washing with a mixture of acetone/hexane/isopropanol coupled with centrifugation. This strategy led to small spherical particles with an encapsulation efficiency of 80% and a RY around 70%. In vitro release studies showed that alginate itself was not able to suppress insulin release in acidic media; however, this strategy preserves the secondary structure of insulin. Particles had a mean size lower than the critical diameter necessary to be orally absorbed through the intestinal mucosa followed by their passage to systemic circulation and thus can be considered as a promising technology for insulin delivery., ((c) 2006 Wiley Periodicals, Inc.)

A nanoparticle insulin delivery system was prepared by complexation of dextran sulfate and chitosan in aqueous solution. Parameters of the formulation such as the final mass of polysaccharides, the mass ratio of the two polysaccharides, pH of polysaccharides solution, and insulin theorical loading were identified as the modulating factors of nanoparticle physical properties. Particles with a mean diameter of 500 nm and a zeta potential of approximately -15 mV were produced under optimal conditions of DS:chitosan mass ratio of 1.5:1 at pH 4.8. Nanoparticles showed spherical shape, uniform size and good shelf-life stability. Polysaccharides complexation was confirmed by differential scanning calorimetry and Fourier transformed infra-red spectroscopy. An association efficiency of 85% was obtained. Insulin release at pH below 5.2 was almost prevented up to 24h and at pH 6.8 the release was characterized by a controlled profile. This suggests that release of insulin is ruled by a dissociation mechanism and DS/chitosan nanoparticles are pH-sensitive delivery systems. Furthermore, the released insulin entirely maintained its immunogenic bioactivity evaluated by ELISA, confirming that this new formulation shows promising properties towards the development of an oral delivery system for insulin.

Insulin-loaded alginate microspheres prepared by emulsification/internal gelation were reinforced by blending with polyanionic additive polymers and/or chitosan-coating in order to increase the protection of insulin at simulated gastric pH and obtain a sustained release at simulated intestinal pH. Polyanionic additive polymers blended with alginate were cellulose acetate phtalate (CAP), Eudragit L100 (EL100), sodium carboxymethylcellulose (CMC), polyphosphate (PP), dextran sulfate (DS) and cellulose sulfate (CS). Chitosan-coating was applied by using a one-stage procedure. The influence of additive polymers and chitosan-coating on the size distribution of microspheres, encapsulation efficiency and release profile of insulin in simulated gastrointestinal pH conditions was studied. The mean diameter of blended microspheres ranged from 65 to 106 microm and encapsulation efficiency of insulin varied from 14 to 100%, reaching a maximum value when CS and DS were incorporated in the alginate matrix. Insulin release, at pH 1.2, was almost prevented by the incorporation of PP, DS and CS. When uncoated microspheres were transferred to pH 6.8, a fast dissolution occurred, independently of the additive polymer blended with alginate, and insulin was completely released. Increasing the additive polymer concentration in the alginate matrix and/or chitosan-coating the blended alginate microspheres did not promote a sustained release of insulin from microspheres at pH 6.8.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate., (Copyright 2006 John Wiley & Sons, Ltd.)

Recombinant human insulin was encapsulated within alginate microspheres by the emulsification/internal gelation technique with the objective of preserving protein stability during encapsulation procedure. The influence of process and formulation parameters was evaluated on the morphology and encapsulation efficiency of insulin. The in vitro release of insulin from microspheres was studied under simulated gastrointestinal conditions and the in vivo activity of protein after processing was assessed by subcutaneous administration of extracted insulin from microspheres to streptozotocin-induced diabetic rats. Microspheres mean diameter, ranging from 21 to 287 microm, decreased with the internal phase ratio, emulsifier concentration, mixer rotational speed and increased with alginate concentration. Insulin encapsulation efficiency, near 75%, was not affected by emulsifier concentration, mixer rotational speed and zinc/insulin hexamer molar ratio but decreased either by increasing internal phase ratio and calcium/alginate mass ratio or by decreasing acid/calcium molar ratio and alginate concentration. A high insulin release, above 75%, was obtained at pH 1.2 and under simulated intestinal pH a complete dissolution of microspheres occurred. Extracted insulin from microspheres decreased hyperglycemia of diabetic rats proving to be bioactive and showing that encapsulation in alginate microspheres using the emulsification/internal gelation is an appropriate method for protein encapsulation.