Influence of chitosan coating on the liposomal surface on physicochemical properties and the release profile of nanocarrier systems

Chitosan-coated nanoliposomes containing etofenprox or alpha-cypermethrin prepared by ultrasonic homogenization maintained a size distribution in the nanometre range. Nanoliposomes were constructed using different types and concentrations of chitosan to regulate the mean size and surface charge. As the chitosan concentration (0.1-0.5%, w/v) and the degree of deacetylation increased, surface charge also increased. The encapsulation efficiency and release profile were measured by gas chromatography. Encapsulation efficiency decreased slightly as chitosan concentration increased (0.1-0.5%, w/v). As the intrinsic surface charge or concentration of the coating material increased, the release period of the entrapped core material was extended (chitosans A and B; 0.1 and 0.3%, w/v). The results indicate that diverse preparation conditions could affect the physicochemical properties and release profile of the resulting nanocarrier systems.     

Application of the combined use of ultrasonic homogenization and electro-spraying in the formation of nano carrier systems

Chitosan-coated nano-liposomes containing etofenprox were prepared by ultrasonic homogenization (UH) and a combined use of UH and electro-spraying. The physicochemical properties of the resulting samples were examined and compared. The two methods yielded similar values and tendencies, except for encapsulation efficiency that differed by an average of 15%. In the coating process, as the chitosan concentration increased (0.1–0.5%, w/v) and the degree of deacetylation increased (chitosans A, B and C), the surface charge of the nano carrier likewise increased and carrier size distribution was altered. The encapsulation efficiency as measured by gas chromatography decreased slightly with the increasing chitosan concentration (0.1–0.5%, w/v). The results indicate that diverse preparation conditions could affect the physicochemical properties of the resulting nano carrier systems.     

Application of the combined use of ultrasonic homogenization and electro-spraying in the formation of nano carrier systems

Chitosan-coated nano-liposomes containing etofenprox were prepared by ultrasonic homogenization (UH) and a combined use of UH and electro-spraying. The physicochemical properties of the resulting samples were examined and compared. The two methods yielded similar values and tendencies, except for encapsulation efficiency that differed by an average of 15%. In the coating process, as the chitosan concentration increased (0.1-0.5%, w/v) and the degree of deacetylation increased (chitosans A, B and C), the surface charge of the nano carrier likewise increased and carrier size distribution was altered. The encapsulation efficiency as measured by gas chromatography decreased slightly with the increasing chitosan concentration (0.1-0.5%, w/v). The results indicate that diverse preparation conditions could affect the physicochemical properties of the resulting nano carrier systems.     

Effect of manufacturing parameters on the characteristics of vitamin C encapsulated tripolyphosphate-chitosan microspheres prepared by spray-drying

To prepare the sustained release vitamin C carriers, vitamin C was successfully encapsulated in tripolyphosphate (TPP) cross-linked chitosan (TPP-chitosan) microspheres by the spray-drying method at different manufacturing conditions. Manufacturing parameters (inlet temperature, liquid flow rate, chitosan concentration and volume of 1% w/v TPP solution) had a significant influence on the characteristics of thus prepared microspheres. The optimum spray-drying conditions such as inlet temperature, liquid flow rate and compressed air flow rate for the encapsulation of vitamin C in TPP-chitosan microspheres was found to be 170 degrees C, 2 ml min(-1) and 101 min(-1), respectively. The size and yield of the TPP-chitosan microspheres ranged from 3.9-7.3 microm and 54.5-67.5%, respectively. The encapsulation efficiency of TPP-chitosan microspheres ranged from 45.72-68.7% and it decreased with the increasing volume of 1% w/v TPP solution. At the same cross-linking extent, the encapsulation efficiency of TPP-chitosan microspheres increased when the concentration of chitosan was increased from 0.5-1% w/v. Effect of volume of 1% w/v TPP solution on the surface morphology of chitosan microspheres was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These studies revealed that chitosan solution (250 ml) cross-linked with 15 ml 1% w/v TPP solution produced more porous microspheres than that cross-linked with 5 and 10 ml TPP solution. The release rate of vitamin C from TPP-chitosan microspheres decreased when the concentration of chitosan was increased from 0.5-1.0% w/v. Vitamin C release rate was also modulated by varying the volume of 1% w/v TPP solution. The release rate of vitamin C from TPP-chitosan microspheres decreased with increasing volume (5-15 ml) of 1% w/v TPP solution.     

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

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

Formulation and evaluation of carnosic acid nanoparticulate system for upregulation of neurotrophins in the brain upon intranasal administration

To develop formulations of carnosic acid nanoparticles and to assess their in vivo efficacy to enhance the expression of neurotrophins in rat model. Carnosic acid loaded chitosan nanoparticles were prepared by ionotropic gelation technique using central composite design. Response surface methodology was used to assess the effect of three factors namely chitosan concentration (0.1–1% w/v), tri-poly phosphate concentration (0.1–1% w/v) and sonication time (2–10?min) on the response variables such as particle size, zeta potential, drug encapsulation efficiency and drug release. The neurotrophins level in the rat brain upon intranasal administration of optimized batch of carnosic acid nanoparticles was determined. The experimental values for the formulation were in good agreement with those predicted by the mathematical models. A single intranasal administration of the optimized formulation of carnosic acid nanoparticles was sufficient to result in comparable levels of endogenous neurotrophins level in the brain that was almost on par with four, once a day intranasal administration of solution in rats. The results clearly demonstrated the fact that nanoparticulate drug delivery system for intranasal administration of carnosic acid would require less number of administrations to elicit the required pharmacological activity owing to its ability to localize on the olfactory mucosal region and provide controlled delivery of carnosic acid for prolonged time periods.     

The characteristics of betamethasone-loaded chitosan microparticles by spray-drying method

Betamethasone (BTM)-loaded microparticles prepared by a spray drying method using chitosan (CTS) as raw material, type-A gelatin and ethylene oxide-propylene oxide block copolymer (Pluronic F68) as modifiers. The BTM-loaded in varied chitosan/Pluronic F68/gelatin microparticle formulations was investigated. By properly choosing excipient type and concentration a high degree of control was achieved over the physical properties of the BTM-loaded microparticles. Microparticle characteristics (zeta potential, tap density, particle size and yield), loading efficiencies, microparticle morphology and in-vitro release properties were examined. Surface morphological characteristics and surface charges of prepared microparticles were observed by using scanning electron microscopy (SEM) and microelectrophoresis. A SEM micrograph shows that the particle sizes of the varied chitosan composed microparticles ranged from 1.1-4.7 microm and the external surfaces appear smooth. The BTM-loaded microparticles entrapped in the chitosan/Pluronic F68/gelatin microparticles with trapping efficiencies up to 93%, collected yield rate 44%, and mean particle size varied between 1-3 microm, positive surface charge (20-40 mv), and tap densities (0.04-0.40 g/cm3) were obtained. The collected BTM yield and size of particle was increased with increasing BTM-loaded amount but both zeta potential and tap density of the particles decreased with increasing BTM-loaded amount. The in vitro release of BTM showed a dose-dependent burst followed by a slower release phase that was proportional to the drug concentration in the concentration range between 5-30%w/w. The in vitro drug release from the chitosan/Pluronic F68/gelatin 1/0.1/0.4 microspheres had a prolong release pattern. These formulation factors were correlated to particulate characteristics for optimizing BTM microspheres in pulmonary delivery.     

Cytotoxicity of 5-fluorouracil entrapped in gelatin microspheres

Gelatin microspheres were prepared by water/oil emulsion polymerization and by cross-linking with glutaraldehyde. For the microsphere preparation procedure, two different gelatin (5 or 10% w/v) and three different glutaraldehyde (5, 0.5 or 0.1% v/v) concentrations were used. The influence of preparation compositions on microsphere recovery, particle size and morphology, swelling and degradation, 5-fluorouracil loading and release, and cytotoxicity were investigated. The concentrations of gelatin and glutaraldehyde influenced the size and surface properties of microspheres. The decrease in gelatin concentration and the increase in glutaraldehyde concentration resulted in the formation of smaller (140.82-71.47 microm for gelatin microspheres with a 5% gelatin content; 297.67-97.44 microm for gelatin microspheres with a 10% gelatin content) microspheres with smoother surface properties. Swelling values were decreased as the amount of glutaraldehyde was increased. In particular, for microspheres with a high glutaraldehyde content (5% v/v), only about 15% were degraded in 12 days, whereas for those with 0.5% (v/v) glutaraldehyde, almost 97% degradation occurred in the same period. The most rapid 5-fluorouracil release was observed from uncross-linked microspheres (about 88% in 4 h), whereas a particular slower release (about 36% in 4 h) profile was obtained for the highly cross-linked ones. Cytotoxicity tests of free and entrapped 5-fluorouracil were carried out with MCF-7 breast cancer cell line. Free 5-fluorouracil produced an immediate effect, whereas entrapped 5-fluorouracil showed a prolonged cytotoxic effect.     

Preparation and in vitro evaluation of mucoadhesive properties of alginate/chitosan microparticles containing prednisolone

This study describes the preparation of mucoadhesive alginate/chitosan microparticles containing prednisolone intended for colon-specific delivery. Two methods have been used for the preparation of the particles: the one-step method is the method in which prednisolone was dispersed within sodium alginate solution and this dispersion was then dropped in a solution containing both calcium chloride and chitosan. The two-step method consisted also of the dispersion of prednisolone in alginate solution and then dropping this dispersion into a solution containing calcium chloride, the particles were then transferred to a chitosan solution. The concentration of sodium alginate solution at 2% (w/v), various concentrations of calcium chloride solution (0.5-1.0%, w/v), chitosan solutions (0.5, 1.0 and 1.5%, w/v) and prednisolone drug load (2, 5, 10 and 15%, w/v) have been used. The results for both preparation methods show that the particle size and drug content were mainly depending on the amount of the drug concentration and not the amount of chitosan and calcium chloride. The in vitro mucoadhesive tests for particles prepared from both methods were carried out using the freshly excised gut of pigs. The particles prepared by the one-step method exhibited excellent mucoadhesive properties after 1h test. Increased chitosan concentrations from 0, 0.5, 1.0 to 1.5% (w/v) resulted in 43, 55, 82 and 88% of the particle remaining attached on the gut surface after 1 h, respectively. However, the particles prepared by the two-step method showed significant less mucoadhesion under the same experimental conditions. At chitosan concentrations of 0, 0.5, 1.0 and 1.5% (w/v) the amount of particles remaining attached to the mucosal surface of the pig gut after 1 h was 43, 3, 11 and 11%, respectively. The prednisolone release at a pH of 6.8 after 4 h was between 63 and 79% for the particles prepared by the one-step method and between 57 and 88% for the particles prepared by the two-step method with a prednisolone drug load of 5 and 10% (w/v), respectively. The results show that depending on the preparation method these chitosan coated alginate particles show different mucoadhesiveness whereas their other properties are not statistically significant different.     

Poloxamer/Cyclodextrin/Chitosan-Based Thermoreversible Gel for Intranasal Delivery of Fexofenadine Hydrochloride

To enhance permeation and solubility of an intranasal delivery system of fexofenadine hydrochloride (FXD HCl), a new formulation using poloxamer 407 (P407)/hydroxypropyl-β-cyclodextrin (HP-β-CD)-based thermoreversible gels with chitosan, was developed. Prepared gels were characterized by gelation temperature, viscosity, viscoelasticity, and drug release profile. The in vitro permeation study was performed in primary human nasal epithelial cell monolayers cultured by air–liquid interface method. The addition of chitosan caused the slight elevation of gelation temperature and viscosity-enhancing effect. Viscosity enhancement by the incorporation of chitosan caused the retardation of drug release from P407 gels in in vitro release test. The in vitro permeation profile showed that the increase in chitosan content (0.1% and 0.3%, w/v) significantly enhanced the permeation of FXD HCl. After intranasal administration of P407/HP-β-CD–based thermoreversible gels containing 0.1% and 0.3% of chitosan in rabbits at 0.5 mg/kg dose, plasma concentrations of FXD HCl were significantly higher than those of nasal solutions (p < 0.05). In particular, the bioavailability of the optimized thermoreversible gel containing 0.3% chitosan was about 18-fold higher than that of the solution type. These results suggested the feasibility that thermosensitive gels could be used as an effective dosage form to enhance the nasal absorption of FXD HCl.     

Release characteristics of brilliant blue from calcium-alginate beads coated with quaternized chitosan

Calcium-alginate beads coated with quaternized chitosan were prepared in a neutral environment, and morphologies were observed by SEM. Optimum conditions for the encapsulation and retention of a model drug (brilliant blue, BB) in acid were obtained from studies of preparation conditions, including alginate and quaternized chitosan concentration, calcium chloride (CaCl2) concentration in the gelling medium and by comparing one-step and two-step preparation methods. Results showed that very high BB encapsulation efficiency (99%, w/w) and low leakage in acid (8%, w/w) was achieved from dry beads when 2.0% (w/v) alginate was dropped into 1.0% (w/v) CaCl2 containing 0.3% (w/v) quaternized chitosan by a one-step method. The release of BB in 0.9% (w/v) NaCl was modulated by coating calcium-alginate with different weight average molecule weight (Mw) and degree of substitution (DS) of quaternized chitosan. A decreased of Mw accelerated the release of BB and a high DS value significantly decreased the release in 0.9% (w/v) NaCl.     

Encapsulation and controlled release of recombinant human erythropoietin from chitosan-carrageenan nanoparticles

Novel chitosan-carrageenan nanoparticles were produced through the process of ionotropic gelation for the encapsulation and controlled release of recombinant human erythropoietin (rHu-EPO). The effects of chitosan concentration, chitosan to carrageenan mass ratio and solution pH on the nanoparticle diameter, polydispersity and surface charge were explored through both screening and response surface modeling (RSM) methods. The chitosan-carrageenan nanoparticles created had particle diameters between 200 and 1000nm, surfaces charges between 40 and 55mV, and polydispersity between 0.2 and 0.35. RSM optimized chitosan-carrageenan nanoparticles demonstrated an increased rHu-EPO encapsulation efficiency of 47.97?4.10% and a more sustained in vitro release of ~50% over a 2 week period when compared to previous nano/microparticle delivery systems. Studies on the effect of surface charge and chitosan molecular weight on the encapsulation and controlled release of rHu-EPO revealed that increasing either led to improved encapsulation efficiency and reduced release rate.     

Nasal delivery of insulin using chitosan microspheres

Nasal delivery of insulin is an alternative route for administration of this drug. The objective of this study was preparation of chitosan microspheres for insulin nasal delivery. After preparation of insulin chitosan microspheres by emulsification-cross linking process, the effect of chitosan quantity (200–400?mg), cross-linker type (ascorbic acid or ascorbyl palmitate) and amount (70–140?mg) were studied on the morphology, particle size, loading efficiency, flow and release of insulin from the microspheres by a factorial design. Optimized formulation was administered nasally in four groups of diabetic rats and their serum insulin levels were analysed by the insulin enzyme immunoassay kit and the serum glucose by the glucose oxidase kits. Insulin loading in microspheres was between 4.7–6.4% w/w, preparation efficiency more than 65% and mean particle size was 20–45?µm. In most cases, drug released followed a Higuchi model. Ascorbic acid caused an increase in stability, particle size and T_50% while decreased the loading efficiency and production efficiency. Increasing the chitosan content, increased particle size, flow and insulin release rate form the microspheres. The increase of cross-linking percentage decreased the flow and size of the microspheres while increase of cross-linking percentage promoted the stability and decreased DE₈% of insulin. Microspheres containing 400?mg of chitosan and 70?mg ascorbyl palmitate caused a 67% reduction of blood glucose compared to i.v. route and absolute bioavaliability of insulin was 44%. The results showed that chitosan microspheres of insulin are absorbable from nasal route.     

Formation of size-controlled nano carrier systems by self-assembly

Nano carrier systems were prepared by forming self-assembled liposomes having a size distribution in the nano range through use of an ultrasonic homogenizer. Phosphatidylcholine and cholesterol were utilized as an amphiphilic compound and a shape stabilizer, respectively. The size of prepared samples was decreased (up to 150 nm) by elevating ratio of lecithin and extending homogenization time (2 ～ 6 min). After secondary coating with alginic acid (0.1, 0.3 and 0.5%, W/V), size was remarkably changed in the range of ±30 nm and zeta-potential was altered (only chitosan coating (molecular weight: 30 000 Da, 0.2%, W/V): 10.3 mV, Alginic acid coating (0.5%, W/V) after the chitosan coating: ?21.8 mV). The low molecular weight chitosan (0.1%, W/V)-coated nano-liposomes had a lower absolute value of zeta-potential than the high molecular weight chitosan (0.1%, W/V)-coated nano-liposomes. The encapsulation efficiency was measured by gas chromatography. The efficiency was decreased slightly by elevating chitosan concentration (0.1 ～ 0.5%, W/V).     

Hydrogels based on interpenetrating network of chitosan and polyvinyl pyrrolidone for pH-sensitive delivery of repaglinide

The aim of this study was to develop a pH-sensitive chitosan/polyvinyl pyrrolidone (PVP) based controlled drug release system for repaglinide. The hydrogels were synthesised by crosslinking chitosan and PVP blend with glutaraldehyde to form a semi-interpenetrating polymer network (semi-IPN). These semi-IPNs were studied for their content uniformity, swelling index (SI), mucoadhesion, wettability, in vitro release and their release kinetics. The hydrogels showed more than 95% loading of repaglinide. These hydrogels showed high swelling and mucoadhesion under acidic conditions. The swelling was found due to the protonation of a primary amino group on chitosan. In acidic condition chitosan was ionized, and adhesion occurred between the positively charged chitosan and the negatively charged mucus. In the physiological condition less swelling was noticed. In vitro release study revealed that formulation containing chitosan (2% w/v) and PVP (4% w/v) in the ratio of 14:6 w/w showed complete drug release after 12h. Release profile showed that all the formulations followed non-fickian diffusion mechanism (diffusion coupled with swelling). Fourier transform infrared (FTIR) spectroscopic analysis revealed proper crosslinking of polymer and formation of semi-IPN as well as presence of drug in the formulation. Differential scanning calorimetry (DSC) and powder x-ray diffraction (p-XRD) study revealed the presence of repaglinide in crystalline form in the formulations. The surface morphology of semi-IPN was studied before and after dissolution in simulated gastric fluid (SGF, pH 1.2) which indicated generation of open channel-like structure in hydrogel after dissolution. The results of study suggest that semi-IPNs of chitosan/PVP are potent candidates for delivery of repaglinide in acidic environment.     

Preparation and characterization of drug‐loaded chitosan–tripolyphosphate microspheres by spray drying

The present study reports on the preparation of chitosan–tripolyphosphate (TPP) microspheres by the spray‐drying method using acetaminophen as a model drug substance. Chitosan–TPP microspheres were spherical and had a smooth surface. Perfectly spherical chitosan–TPP microparticles loaded with acetaminophen were obtained in the size range of 3.1–10.1 µm. Spray‐dried chitosan–TPP microspheres were positively charged (zeta potential ranged from +18.4 to +31.8). The encapsulation efficiency of these microspheres was in the range of 48.9–99.5%. The swelling capacity of chitosan–TPP microspheres increased with increases in the molecular weight of chitosan and decreases with increasing volume of 1% wt/vol TPP solution used for the cross‐linking reaction. The effect of chitosan concentration, drug loading, volume of TPP solution used for cross‐linking, and chitosan molecular weight on surface morphology and drug release rate was extensively investigated. Microparticles with spherical shape and slower release rates were obtained from chitosan–TPP microspheres prepared using a higher concentration of chitosan, higher volume of TPP solution, a higher molecular weight chitosan and/or a higher drug loading. Most importantly, the drug release rate was mainly controlled by the chitosan–TPP matrix density and, thus, by the degree of swelling of the hydrogel matrix. Drug release from chitosan–TPP microspheres occurred via diffusion as the best fit for drug release was obtained using the Higuchi equation. Drug Dev. Res. 64:114–128, 2005. © 2005 Wiley‐Liss, Inc.     

Chitosan oligomers as potential and safe absorption enhancers for improving the pulmonary absorption of interferon-alpha in rats

Effects of chitosan oligomers on pulmonary absorption of interferon-alpha (IFN) were examined by means of an in vivo pulmonary absorption experiment. Chitosan oligomers used in this study were chitosan dimer, tetramer, hexamer, and water-soluble (WS) chitosan. A significant increase in serum IFN concentrations was observed after intratracheal administration of IFN with these oligomers. Of these chitosan oligomers, 0.5% w/v chitosan hexamer appeared to be more effective in enhancing the pulmonary absorption of IFN than other oligomers at the same concentration, and the AUC value of IFN with chitosan hexamer increased 2.6-fold as compared with the control. On the other hand, chitosan polymers, which have relatively high molecular weights (22-96 kDa), were not effective in enhancing the pulmonary absorption of IFN due to their low solubility in water. Additionally, the effect of different concentrations (0.1%-1% w/v) of chitosan hexamer on the pulmonary absorption of IFN was studied. Of these different concentrations of chitosan hexamers, the highest AUC value of IFN was obtained in the presence of 0.5% w/v chitosan hexamer. Furthermore, chitosan oligomers did not cause any membrane damage to the rat pulmonary tissues, as determined by leakage of protein and lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid. Therefore, these findings indicated that the use of chitosan oligomers would be a promising approach for improving of the pulmonary absorption of biologically active peptides including IFN.     

Engineering and Development of Chitosan-Based Nanocoatings for Ocular Contact Lenses

This article reports on electrohydrodynamic atomization to engineer on-demand novel coatings for ocular contact lenses. A formulation approach was adopted to modulate the release of timolol maleate (TM) using chitosan and borneol. Polymers polyvinylpyrrolidone and poly (N-isopropylacrylamide) were utilized to encapsulate TM and were electrically atomized to produce optimized, stationary contact lens coatings. The particle and fiber diameter, thermal stability, material compatibility of the formed coatings, their in vitro release-modulating effect, and ocular tolerability were investigated. Results demonstrated highly stable nanomatrices with advantageous morphology and size. All formulations yielded coatings with high TM encapsulation (>88%) and excellent ocular biocompatibility. Coatings yielded biphasic and triphasic release, depending on composition. Kinetic modeling revealed a noticeable effect of chitosan; the higher the concentration, the more the release of TM because of chitosan swelling, with the mechanism changing from Fickian diffusion (1% w/v; n = 0.5) to non-Fickian (5% w/v, 0.45 < n < 0.89). The use of electrohydrodynamic atomization has not yet been explored in depth within the ocular research remit, engineering on-demand lens coatings capable of sustaining TM release. This is likely to offer an alternative dosage form for management of glaucoma with particular emphasis on improving poor patient compliance.     

Encapsulation of olive leaf antioxidants in microbeads: Application of alginate and chitosan as wall materials

Edible microcapsule technology has been declared as a newly developed technology in 21st century by some certain authorities in order to preserve food products. Encapsulation of the bioactive materials in edible coatings is a blessing that can eliminate many undesirable situations that might arise when it is used as additive. In this study, olive leaf extract has been evaluated as active material to prepare microcapsules by using alginate as coating. Ionic gelation was used to produce microbeads. The experimental design of the encapsulation system, the effects of the process parameters, the modeling of the experimental data and the optimization of the conditions were carried out with Box-Behnken design of response surface method (Box-Behnken-RSM). Box-Behnken-RSM produced 17 experimental runs. Calcium chloride (2–15%, w/v) and sodium alginate concentrations (1–2%, w/v), and hardening time (15–45 min) were selected as independent variables, while encapsulation efficiency (EE) of the capsules in terms of total phenolic content (TPC) and oleuropein concentration were responses. Impact of chitosan as coating layer was also investigated with three different ratios of chitosan (0.4%, 0.7%, 1% w/v). Accelerated oxidation test was employed to measure the stability of the microcapsules against oxidation by means of Rancimat method. Encapsulation of the olive leaf extract in alginate microbeads was satisfying with >70% and >90% efficiencies with respect to TPC and oleuropein under optimum conditions (2.34% calcium chloride concentration and 2% sodium alginate for 26 min of hardening time).     

The effects of experimental parameters and calibration on MTDSC data

Numerous studies have shown that chitosan, a mucoadhesive polymer, is a potential enhancer for transmucosal drug delivery. To further understand the mechanisms involved in chitosan action on the mucosal barrier, the activity of chitosan on the function and structure of monolayers of intestinal epithelial cells was investigated. In Caco-2 cells, chitosan caused a reversible, time and dose-dependent decrease in transepithelial electrical resistance. The effect of chitosan on tight junctions was confirmed by an increased permeability coefficient for mannitol transport when cells were treated with 0.1-0.5% w/v chitosan solution for 60 min compared to control cells. Involvement of tight junctions was visualized by confocal scanning microscopy using occludin and ZO-1, tight junctional proteins. Following an incubation with 0.01 or 0.1% w/v chitosan, labeling of both proteins varied in localization and decreased in fluorescent intensity at the cell periphery. In addition, a focal condensation of actin was observed preferentially at areas of cell-to-cell contacts. However, after 24-h recovery, the cell structure resembled untreated control cells. Simultaneous addition of cycloheximide, a protein synthesis inhibitor, prevented full recovery. This implied that protein synthesis was required for the cells to return to baseline levels. Chitosan treatment appeared to slightly perturb the plasma membrane as assessed by an increased release of lactate dehydrogenase. However, addition of 0.5% chitosan for 60 min did not affect cell viability as shown by Trypan blue dye exclusion. These data suggest that chitosan increases cell permeability by affecting paracellular and intracellular pathways of epithelial cells, in a reversible manner. Copyright