Insulin Encapsulation Efficiency Research Articles

The Effect of Insulin-Loaded Chitosan Particle–Aggregated Scaffolds in Chondrogenic Differentiation

Osteochondral defect repair requires a tissue engineering approach that aims at mimicking the physiological properties and structure of two different tissues (cartilage and bone) using a scaffold-cell construct. One ideal approach would be to engineer in vitro a hybrid material using a single-cell source. For that purpose, the scaffold should be able to provide the adequate biochemical cues to promote the selective but simultaneous differentiation of both tissues. In this work, attention was paid primarily to the chondrogenic differentiation by focusing on the development of polymeric systems that provide biomolecules release to induce chondrogenic differentiation. For that, different formulations of insulin-loaded chitosan particle-aggregated scaffolds were developed as a potential model system for cartilage and osteochondral tissue engineering applications using insulin as a potent bioactive substance known to induce chondrogenic differentiation. The insulin encapsulation efficiency was shown to be high with values of 70.37 +/- 0.8%, 84.26 +/- 1.76%, and 87.23 +/- 1.58% for loadings of 0.05%, 0.5%, and 5%, respectively. The in vitro release profiles were assessed in physiological conditions mimicking the cell culture procedures and quantified by Micro-BCA protein assay. Different release profiles were obtained that showed to be dependent on the initial insulin-loading percentage. Further, the effect on prechondrogenic ATDC5 cells was investigated for periods up to 4 weeks by studying the influence of these release systems on cell morphology, DNA and glycosaminoglycan content, histology, and gene expression of collagen types I and II, Sox-9, and aggrecan assessed by real-time polymerase chain reaction. When compared with control conditions (unloaded scaffolds cultured with the standard chondrogenic-inducing medium), insulin-loaded scaffolds upregulated the Sox-9 and aggrecan expression after 4 weeks of culture. From the overall results, it is reasonable to conclude that the developed loaded scaffolds when seeded with ATDC5 can provide biochemical cues for chondrogenic differentiation. Among the tested formulations, the higher insulin-loaded system (5%) was the most effective in promoting chondrogenic differentiation.

Insulin-S.O (sodium oleate) complex-loaded PLGA nanoparticles: Formulation, characterization and in vivo evaluation

S.O (sodium oleate) is an anionic surfactant, which is able to forman ionic complex with positively charged insulin at suitable pH. In a previous study, the insulin-S.O (Ins-S.O) complex was prepared by a hydrophobic ion pairing (HIP) method to improve the apparent liposolubility of insulin. The formation of the complex was further confirmed by Zeta potential and X-ray method. Based on the preliminary study, poly(lactide-co-glycolide) (PLGA) nanoparticles harbouring Ins-S.O complex was prepared via an emulsion solvent diffusion method. The effects of key parameters such as concentration of PVA, concentration of PLGA and initial-loaded drug on the properties of the nanoparticles were investigated. The insulin encapsulation efficiency (EE(%)) reached up to 91.2% and mean diameter of the nanoparticles was sized ∼160 nm under optimal conditions. The pharmacological effects of the nanoparticles made of PLGA (75/25, Av Mw 15 000) were further evaluated to confirm their potential suitability for oral delivery. In order to evaluate hyperglycaemic effect of the nanoparticles for oral administration, Ins-S.O complex-loaded PLGA nanoparticles (20 IU/Kg) were administered orally by force-feeding to diabetic rats. In the case of the nanoparticles, the plasma glucose level reduced to 23.85% from the initial one 12 h post-administration and this continued for 24 h. The results showed that the use of Ins-S.O complex-loaded PLGA nanoparticles is an effective method of reducing plasma glucose levels. The insulin nanoparticles also improved the glycaemic response to an oral glucose challenge.

Assessment of Insulin Stability Inside Diblock Copolymer PEG-PLA Microspheres

Insulin-loaded PEG2-PLA40 and PEG5-PLA20 microspheres containing 5% bovine insulin were manufactured using single emulsion and w/o/w multiple emulsion-solvent evaporation techniques. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 °C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the loading efficiencies were higher in case of insulin loaded PEG2-PLA40 and PEG5-PLA20 microspheres prepared by single emulsion emulsion-solvent evaporation technique. Insulin release was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 6, 12 and 18 days of PEG2-PLA40 microspheres erosion showed that insulin was intact inside the eroding microspheres. In addition, only small amounts of protein undergo degradation under these conditions (only 11.69% ± 1.13 of the initially loaded insulin loading were detected as degradation products after 18 days. Mass spectra recorded at these retention times confirmed the presence of insulin with a molar mass of 5734 Da and other two products of molar masses of 5587 Da and 5487 Da.

Evaluation of polyanhydride microspheres for basal insulin delivery: Effect of copolymer composition and zinc salt on encapsulation, in vitro release, stability, in vivo absorption and bioactivity in diabetic rats

The potential of poly 1,3-bis-(p-carboxyphenoxy) propane-co-sebacic acid (p(CPP:SA)) microspheres was investigated for controlled delivery of basal insulin. CPP:SA copolymers with molar compositions of 20:80, 40:60, and 50:50 were synthesized, characterized, and used in the fabrication of microspheres by water-in-oil-in-water double emulsion solvent evaporation technique. Insulin stability was assessed using various analytical methods and in vivo insulin absorption and bioactivity were studied in diabetic rats. Microspheres exhibited smooth surfaces and mean particle size ranged from 41.5 to 49.8 µm. Insulin encapsulation efficiency (EE) and in vitro release kinetics were influenced by the molar ratios of CPP:SA copolymer. Increasing CPP content and addition of zinc oxide increased EE, reduced burst release, and prolonged insulin in vitro release over a month. Dimer aggregates were observed for insulin encapsulated in CPP:SA 50:50 microspheres and addition of zinc oxide prevented dimer formation. Subcutaneous administration of CPP:SA 50:50 microspheres in diabetic rats controlled insulin release over a month, and the released insulin was bioactive as determined by lowering blood glucose levels. The results indicate that CPP:SA microspheres controlled insulin release in vitro and in vivo over a month and the released insulin was conformationally and chemically stable, and bioactive. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4237–4250, 2009

The effect of formulation variables on the characteristics of insulin-loaded poly(lactic-co-glycolic acid) microspheres prepared by a single phase oil in oil solvent evaporation method

Biodegradable polymeric microspheres are ideal vehicles for controlled delivery applications of drugs, peptides and proteins. Amongst them, poly(lactic-co-glycolic acid) (PLGA) has generated enormous interest due to their favorable properties and also has been approved by FDA for drug delivery. Insulin-loaded PLGA microparticles were prepared by our developed single phase oil in oil (o/o) emulsion solvent evaporation technique. Insulin, a model protein, was successfully loaded into microparticles by changing experimental variables such as polymer molecular weight, polymer concentration, surfactant concentration and stirring speed in order to optimize process variables on drug encapsulation efficiency, release rates, size and size distribution. A 2 4 full factorial design was employed to evaluate systematically the combined effect of variables on responses. Scanning electron microscope (SEM) confirmed spherical shapes, smooth surface morphology and microsphere structure without aggregation. FTIR and DSC results showed drug–polymer interaction. The encapsulation efficiency of insulin was mainly influenced by surfactant concentration. Moreover, polymer concentration and polymer molecular weight affected burst release of drug and size characteristics of microspheres, respectively. It was concluded that using PLGA with higher molecular weight, high surfactant and polymer concentrations led to a more appropriate encapsulation efficiency of insulin with low burst effect and desirable release pattern.

Insulin Loaded PLGA Microspheres: Effect of Zinc Salts on Encapsulation, Release, and Stability

The purpose of this study was to investigate the effect of three zinc salts (i.e., zinc oxide, zinc carbonate, and zinc acetate) on insulin encapsulation efficiency (EE), stability, and in vitro release kinetics from poly(lactic‐co‐glycolic acid) (PLGA) microspheres. Microspheres were prepared by water‐in‐oil‐in‐water (w/o/w) double emulsion solvent evaporation technique and characterized. Integrity of the encapsulated insulin and stability of the released insulin was assessed using a wide range of comprehensive analytical techniques. The EE of the formulation prepared without the addition of a zinc salt was 69%, the secondary structure of the encapsulated insulin in this formulation was found to be altered. Further, desamido insulin and aggregates were observed during in vitro release. When insulin was encapsulated with a zinc salt, EE increased significantly, secondary structure was unaltered, and no degradation or aggregation products were found. Initial burst release and release kinetics were markedly changed with the addition of zinc salts. More than 87% of the encapsulated insulin was released over a 2‐week period with the addition of a zinc salt. In conclusion, zinc salts can be useful to increase the EE and stability of insulin in PLGA microspheres prepared by w/o/w technique. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:529–542, 2009

Evaluation of hydrophobic nanoparticulate delivery system for insulin

Insulin loaded hydrophobic nanoparticles were prepared by solvent diffusion followed by lyophilization. Nanoparticles were characterized for mean size by dynamic laser scattering and for shape by scanning electron microscopy. Insulin encapsulation efficiency, in vitro stability of nanoparticles in presence of proteolytic enzymes and in vitro release were determined by high pressure liquid chromatography analysis. The biological activity insulin from the nanopraticles was estimated by enzyme-linked immunosorbant assay and in vivo using Wister diabetic rats. Nanoparticles ranged 0.526±0.071 μm in diameter. Insulin encapsulation efficiency was 95.7±1.2%. Insulin hydrophobic nanoparticles suppressed insulin release promoted sustained release in pH 7.4 phosphate buffer and shown to protect insulin from enzymatic degradation in vitro in presence of chymotripsin. Nanoencapsulated insulin was bioactive, demonstrated through both in vivo and in vitro.

Insulin-egg yolk dispersions in self microemulsifying system

Formulation of insulin into a microemulsion very often presents a physicochemical instability during their preparation and storage. In order to overcome this lack of stability and facilitate the handling of these colloidal systems, stabilization of insulin in presence of hydrophobic components of a microemulsion appears as the most promising strategy. The present paper reports the use of egg yolk for stabilization of insulin in self microemulsifying dispersions. Insulin loaded egg yolk self microemulsifying dispersions were prepared by lyophilization followed by dispersion into self microemulsifying vehicle. The physicochemical characterization of selfmicroemulsifying dispersions includes such as insulin encapsulation efficiency, in vitro stability of insulin in presence of proteolytic enzymes and in vitro release. The biological activity of insulin from the dispersion was estimated by enzyme-linked immunosorbant assay and in vivo using Wistar diabetic rats. The particle size ranged 1.023±0.316 μm in diameter and insulin encapsulation efficiency was 98.2±0.9 %. Insulin hydrophobic self microemulsifying dispersions suppressed insulin release in pH 7.4 phosphate buffer and shown to protect insulin from enzymatic degradation in vitro in presence of chymotripsin. Egg yolk encapsulated insulin was bioactive, demonstrated through both in vivo and in vitro.

Polyelectrolyte Biomaterial Interactions Provide Nanoparticulate Carrier for Oral Insulin Delivery

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 ∼ 72% of basal values. Albumin serves as an important enteric coating providing acid- and protease protection enabling uptake of active drug following oral dosage.

Insulin-loaded alginate microspheres for oral delivery – Effect of polysaccharide reinforcement on physicochemical properties and release profile

Oral administration of insulin requires protein protection from degradation in the gastric environment and its absorption improvement in the intestinal tract. To achieve this objective several types of microspheres composed of alginate, chitosan and dextran sulphate have been prepared by ionotropic gelation. Parameters such as the mean particle size, swelling behaviour, insulin encapsulation efficiency, loading capacity and release profiles in simulated gastric and intestinal fluids have been compared for the systems developed. In this study, attempts have been made to increase insulin protection and to improve its release from microspheres by reinforcing the alginate matrix with chitosan and/or dextran sulphate. Dextran sulphate was able to avoid insulin release at pH 1.2, protecting the protein from the acidic environment and reducing the total insulin released at pH 6.8. This effect was explained by an interaction between the permanent negatively charged groups of dextran sulphate and insulin molecules.

Nanoparticulate delivery system for insulin: Design, characterization and in vitro/ in vivo bioactivity

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 μm 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

In vitro and in vivo evaluation of a novel oral insulin formulation

To develop a stable self-emulsifying formulation for oral delivery of insulin. Caco-2 cell line and diabetic beagles were used as in vitro and in vivo models to study the absorption mechanism and the hypoglycemic efficacy of the formulation. In addition, various physicochemical parameters of the formulation such as droplet size, insulin encapsulation efficiency and stability were evaluated. This formulation enabled changes in barrier properties of Caco-2 monolayers, as referred by transepithelial electrical resistance (TEER) and apparent permeability coefficients (P(app)) of the paracellular marker ranitidine (20-fold greater than control) but not transcellular marker propranolol, suggesting that the opening of tight junctions was involved. In diabetic beagle dogs, the bioavailability of this formulation was up to 15.2% at a dose of 2.5 IU/kg in comparison with the hypoglycemic effect of native insulin (0.5 IU/kg) delivered by subcutaneous injection. This formulation, recently approved by the China State Food and Drug Administration to enter clinical trials, was stable, degradation-protected and absorption-enhanced, and provided a promising formulation for oral insulin delivery.

Insulin encapsulation in reinforced alginate microspheres prepared by internal gelation

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 μm 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.

Cyclodextrin–insulin complex encapsulated polymethacrylic acid based nanoparticles for oral insulin delivery

Present investigation was aimed at developing an oral insulin delivery system based on hydroxypropyl β cyclodextrin–insulin (HPβCD–I) complex encapsulated polymethacrylic acid-chitosan-polyether (polyethylene glycol-polypropylene glycol copolymer) (PMCP) nanoparticles. Nanoparticles were prepared by the free radical polymerization of methacrylic acid in presence of chitosan and polyether in a solvent/surfactant free medium. Dynamic light scattering (DLS) experiment was conducted with particles dispersed in phosphate buffer (pH 7.4) and size distribution curve was observed in the range of 500–800 nm. HPβCD was used to prepare non-covalent inclusion complex with insulin and complex was analyzed by Fourier transform infrared (FTIR) and fluorescence spectroscopic studies. HPβCD complexed insulin was encapsulated into PMCP nanoparticles by diffusion filling method and their in vitro release profile was evaluated at acidic/alkaline pH. PMCP nanoparticles displayed good insulin encapsulation efficiency and release profile was largely dependent on the pH of the medium. Enzyme linked immuno sorbent assay (ELISA) study demonstrated that insulin encapsulated inside the particles was biologically active. Trypsin inhibitory effect of PMCP nanoparticles was evaluated using N-α-benzoyl- l-arginine ethyl ester (BAEE) and casein as substrates. Mucoadhesive studies of PMCP nanoparticles were conducted using freshly excised rat intestinal mucosa and the particles were found fairly adhesive. From the preliminary studies, cyclodextrin complexed insulin encapsulated mucoadhesive nanoparticles appear to be a good candidate for oral insulin delivery.

Alginate microspheres prepared by internal gelation: Development and effect on insulin stability

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 μm, 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.

Design of insulin-loaded alginate nanoparticles: Influence of the calcium ion on polymer gel matrix properties

Alginate-based nanoparticles were produced by dispersing alginate aqueous solution containing an insoluble calcium salt within mineral oil forming a water-in-oil emulsion. Subsequently, alginate gelled upon contact with the calcium ions due to the physical cross-linking between the carboxylate anions of the alginate and the calcium ions. The influence of the calcium salt, added in varying amounts, on gel integrity and on particle size was investigated. The efficiency of encapsulating active biological compounds by nanoparticles was also assayed. The calcium concentration was seen to be a crucial parameter in particle production, influencing the particle size, the viscosity of the solutions at different stages of the emulsification/gelation process and, finally, the encapsulation efficiency. The most appropriate mass relation between calcium and alginate was 7% (w/w). Under this condition, the smallest mean diameter obtained was 2.604 ? 2.141 ?m combined with the narrowest range of particle sizes. The encapsulation efficiency of insulin was over 71 %. These previous characteristics appear to be best suited for producing small, well-dispersed and stable nanoparticles with high encapsulation of insulin. This particulate system may be considered as a promising carrier for the oral delivery of insulin.

The effect of coencapsulation of bovine insulin with cyclodextrins in ethylcellulose microcapsules

Polymeric microcapsules have been widely investigated for protein delivery. Common problems include: low stability, low encapsulation efficiency, lack of uniformity, and burst release. Cyclodextrins (CDs) are known to enhance stability and solubility of proteins in solution. This research examines the effect of α-, β-, and γ-CDs on: (1) stability, (2) encapsulation, and (3) release of insulin from ethylcellulose microcapsules. All CDs improved thermal stability of insulin by lowering the enthalpy of unfolding by 16–52%. α- and γ-CDs also increased the encapsulation efficiency of insulin and improved uniformity of the microcapsule formulations. Two mathematical models were proposed to account for insulin release and consisted of multiple zero order and first order input processes, and a single first order output process. All CDs decreased the initial burst release of insulin by up to 30%. This research demonstrates the potential for CDs to improve stability, uniformity, and encapsulation of proteins in microcapsule formulations.

Stability of insulin during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres

In recent years, the acylation of peptides during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres has been described in the literature. To investigate whether insulin is prone to the covalent attachment of lactic or glycolic acid, insulin-loaded PLA and PLGA microspheres containing 5% bovine insulin were manufactured using a w/o/w multiple emulsion-solvent evaporation technique. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 °C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the insulin loading efficiencies of PLA and PLGA microspheres were 75.18% and 79.63%, respectively. The microspheres were spherical with relatively porous surfaces with an average diameter of 40 and 53 μm, respectively. Insulin release from the microspheres was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 18 days of erosion in phosphate buffer pH 7.4 at 37 °C revealed that deamidation was the major mechanism of instability. Surprisingly, no acylation products were found. Control experiments in concentrated lactic acid solutions confirmed a minimal reactivity of the peptide under these conditions.

Solid lipid micro-particles carrying insulin formed by solvent-in-water emulsion–diffusion technique

The study aimed to produce solid lipid insulin-loaded micro-particles by the solvent-in-water emulsion–diffusion technique, using isobutyric acid as solvent phase, glyceryl monostearate or cetyl palmitate as lipid, soya lecithin and taurodeoxycholate as emulsifiers. Isobutyric acid, a partially water-miscible solvent with low toxicity, was used due to its high insulin-solubilization capacity. Solid lipid micro-particles of spherical shape were prepared by simple dilution of the emulsion with water. To increase the lipid load the process was conducted at 50 °C, and in order to reach sub-micron size, a high-shear homogeniser was used. Insulin encapsulation efficiency was about 80%. Analysis of microsphere content after processing showed that insulin did not undergo any chemical modification within the micro-particles. The in vitro release of insulin from the micro-particles was very low, and an initial burst effect of 20% of the dose was observed. After treatment of the solid lipid micro-particles with pepsin solution, an insulin loss of about 24% of the total englobed insulin was observed. The solid lipid micro-particles appear to have interesting possibilities as delivery systems for oral administration of insulin.

How cyclodextrin incorporation affects the properties of protein-loaded PLGA-based microspheres: the case of insulin/hydroxypropyl-β-cyclodextrin system

The aim of this work was to study the influence of cyclodextrin (CD) incorporation on the properties of protein-loaded poly(lactide-co-glycolide) (PLGA) microspheres, with particular regards to protein release kinetics. To this purpose, insulin-loaded microspheres were prepared by spray-drying emulsion or solution formulations, with or without hydroxypropyl-β-cyclodextrin (HPβCD), and fully characterized for encapsulation efficiency and release kinetics of both insulin and cyclodextrin. Homogeneous populations of spherical microparticles entrapping both insulin and HPβCD were obtained. In order to get an insight into insulin/HPβCD interactions occurring inside microspheres, Fourier transform infrared (FTIR) analysis in the Amide I region was performed. FTIR spectra of dried microspheres containing HPβCD showed a change in insulin secondary structure, attributed to the presence of insulin/HPβCD complexes within microspheres. Insulin release was affected by the presence of HPβCD depending on the initial formulation conditions. In the case of microspheres prepared from emulsion, cyclodextrin reduced only insulin burst, whereas in the case of microspheres obtained from solution, the overall insulin release rate was slowed down. Combining the release kinetics of HPβCD with the FTIR results on hydrated microspheres, it was concluded that the formation of insulin/HPβCD complexes inside microspheres is critical to decrease protein diffusivity in the polymer matrix and achieve an effective modulation of protein release rate.