Formulation, in vitro evaluation and kinetic analysis of chitosan-gelatin bilayer muco-adhesive buccal patches of insulin nanoparticles.

The present study was performed to optimise the formulation of a muco-adhesive buccal patch for insulin nanoparticles (NPs) delivery. Insulin NPs were synthesised by an ionic gelation technique using N-di methyl ethyl chitosan cysteine (DMEC-Cys) as permeation enhancer biopolymer, tripolyphosphate (TPP) and insulin. Buccal patches were developed by solvent-casting technique using chitosan and gelatine as muco-adhesive polymers. Optimised patches were embedded with 3 mg of insulin-loaded NPs with a homogeneous distribution of NPs in the muco-adhesive matrix, which displayed adequate physico-mechanical properties. The drug release characteristics, release mechanism and kinetics were investigated. Data fitting to Peppas equation with a correlation coefficient indicated that the mechanism of drug release followed an anomalous transport that means drug release was afforded through drug diffusion along with polymer erosion. In vitro drug release, release kinetics, physical and mechanical studies for all patch formulations reflected the ideal characteristics of this buccal patch for the delivery of insulin NPs.

Application of artificial neural networks for optimization of preparation of insulin nanoparticles composed of quaternized aromatic derivatives of chitosan.

The aim of this research was to develop an artificial neural network (ANN) in order to design a nanoparticulate oral drug delivery system for insulin. The pH of polymer solution (X1), concentration ratio of polymer/insulin (X2) and polymer type (X3) in 3 level including methylated N-(4-N,N- dimethyl aminobenzyl) chitosan, methylated N-(4-pyridinyl) chitosan, and methylated N-(benzyl) chitosan are considered as the input values and the particle size, zeta potential, PdI, and entrapment efficiency (EE %) as output data. ANNs are employed to generate the best model to determining the relationships between input and response values. In this research, a multi-layer percepteron with different topologies has been tested in order to define the one with the best accuracy and performance. The optimization was used by minimizing the error between the predicted and observed values. Three training algorithms (Levenberg-Marquardt (LM), Bayesian-Regularization (BR), and Gradient Descent (GD)) were employed to train ANNs with various numbers of nodes, hidden layers and transfer functions by random selection. The accuracy of prediction data were assayed by the mean squared error (MSE).The ability of all algorithms was in the order: BR>LM>GD. Thus, BR was selected as the best algorithm.

Evaluation and optimization of chitosan derivatives-based gene delivery system via kidney epithelial cells.

PURPOSE: Non-viral vectors have been widely proposed as safer alternatives to viral vectors, and cationic polymers have gained increasing attention because they can form self-assembly with DNA. Chitosan is also considered to be a good candidate for gene delivery systems, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential. However, low solubility and transfection efficiency need to be overcome prior to clinical trial. In this work, we focus on alkyl modified chitosan which might be useful in DNA condensing and efficient gene delivery. METHODS: N, N- Diethyl N- Methyl (DEMC) and N- Triethyl Chitosan (TEC) were synthesized from chitosan polymer. In order to optimize the polymers for gene delivery, we used FITC-dextran (FD). Then the optimized polymer concentrations were used for gene delivery. Fluorescent microscope was used, in order to evaluate the polymers' efficiency for gene delivery to human embryonic kidney epithelial cells (HEK 293T). RESULTS: This modification increased chitosan's positive charge, thus these chitosan derivatives spontaneously formed complexes with FD, green fluorescence protein plasmid DNA (pEGFP), red fluorescence protein plasmid DNA (pJred) and fluorescent labeled miRNA .RESULTS gained from fluorescent microscope showed that TEC and DEMC were able to transfer FD, DNA and miRNA (micro RNA) to HEK cell line. CONCLUSION: We conclude that these chitosan derivatives present suitable characteristics to be used as non-viral gene delivery vectors to epithelial cells.

Ex vivo evaluation of insulin nanoparticles using chitosan and arabic gum.

Polymeric delivery systems based on nanoparticles have emerged as a promising approach for peroral insulin delivery. The aim of the present study was to investigate the release of insulin nanoparticulate systems and ex vivo studies. The nanoparticles were prepared by the ion gelation method. Particle size distribution, zeta potential, and polydispersity index of the nanoparticles were determined. It was found that the nanoparticles carried positive charges and showed a size distribution in the range of 170-200 nm. The electrostatic interactions between the positively charged group of chitosan and negatively charged groups of Arabic gum play an important role in the association efficiency of insulin in nanoparticles. In vitro insulin release studies showed an initial burst followed by a slow release of insulin. The mucoadhesion of the nanosystem was evaluated using excised rat jejunum. Ex vivo studies have shown a significant increase in absorption of insulin in the presence of chitosan nanoparticles in comparison with free insulin.

Development of a Gas Empowered Drug Delivery system for peptide delivery in the small intestine.

The aim of this investigation was to design a novel Gas Empowered Drug Delivery (GEDD) system for CO(2) forced transport of peptide drugs together with mucoadhesive polymers to the surface of the small intestine. The GEDD effect of the core tablet was achieved using CO(2) gas to push insulin together with the mucoadhesive excipients poly(ethyleneoxide) (PEO) and the permeation enhancer trimethyl chitosan (TMC) to the surface of the small intestine. The in-vitro insulin release showed that almost 100% of the insulin was released from enterically coated tablets within 30 min at pH 6.8. The designed GEDD system was shown to increase the insulin transport by approximately 7 times in comparison with the free insulin across sheep's intestine ex-vivo. Three different peroral formulations were administered to male rabbits: F1 containing no TMC or PEO, F2 containing PEO but no TMC and F3 containing both PEO and TMC. The administrations of insulin using the formulation F1 resulted in a low FR value of 0.2%+/-0.1%, while the formulations F2 and F3 resulted in a much higher FR values of 0.6+/-0.2% and 1.1%+/-0.4%, respectively. Hence, the insulin permeation achieved by the GEDD system is primarily due to the enhancing effect of TMC and the mucoadhesive properties of PEO both of which synergistically increase the bioavailability of insulin.

Permeation enhancer effect of chitosan and chitosan derivatives: comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells.

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.

Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods.

TMC and DEMC, quaternized derivatives of chitosan, have been shown to have penetration enhancement properties and able to open the tight junctions of the intestinal epithelia at neutral and alkaline pH environments. The use of the nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Hence, the aim of this study was to synthesize and characterize TMC and DEMC, both with quaternization degrees of 50+/-5%, which were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and the polyelectrolyte complexation (PEC) techniques. The obtained nanoparticles were then characterized for size, zeta potential, insulin loading and release as well as antibacterial activities. The results showed that nanoparticles prepared by the PEC method had higher insulin loading efficiency and zeta potential than those made by the ionotropic gelation method and may subsequently be used for further in vitro, ex vivo and in vivo studies. Moreover, the antibacterial studies suggest that the polymers in free form have higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form.

Performance comparison of neural network training algorithms in modeling of bimodal drug delivery.

The major aim of this study was to model the effect of two causal factors, i.e. coating weight gain and amount of pectin-chitosan in the coating solution on the in vitro release profile of theophylline for bimodal drug delivery. Artificial neural network (ANN) as a multilayer perceptron feedforward network was incorporated for developing a predictive model of the formulations. Five different training algorithms belonging to three classes: gradient descent, quasi-Newton (Levenberg-Marquardt, LM) and genetic algorithm (GA) were used to train ANN containing a single hidden layer of four nodes. The next objective of the current study was to compare the performance of aforementioned algorithms with regard to predicting ability. The ANNs were trained with those algorithms using the available experimental data as the training set. The divergence of the RMSE between the output and target values of test set was monitored and used as a criterion to stop training. Two versions of gradient descent backpropagation algorithms, i.e. incremental backpropagation (IBP) and batch backpropagation (BBP) outperformed the others. No significant differences were found between the predictive abilities of IBP and BBP, although, the convergence speed of BBP is three- to four-fold higher than IBP. Although, both gradient descent backpropagation and LM methodologies gave comparable results for the data modeling, training of ANNs with genetic algorithm was erratic. The precision of predictive ability was measured for each training algorithm and their performances were in the order of: IBP, BBP>LM>QP (quick propagation)>GA. According to BBP-ANN implementation, an increase in coating levels and a decrease in the amount of pectin-chitosan generally retarded the drug release. Moreover, the latter causal factor namely the amount of pectin-chitosan played slightly more dominant role in determination of the dissolution profiles.

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

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

Diethyl methyl chitosan as an intestinal paracellular enhancer: ex vivo and in vivo studies.

Chitosan exhibits favorable biological properties such as no toxicity, biocompatibility and biodegradability; therefore, it has attracted great attention in both pharmaceutical and biomedical fields. Chitosan exhibits poor solubility at pH values above 6 that prevents enhancing effects at the sites of absorption of drugs. In the present work, N-diethyl methyl chitosan (DEMC) was prepared and the enhancing effect of this polymer was investigated. Ex vivo studies have shown a significant increase in absorption of brilliant blue in the presence of diethyl methyl chitosan in comparison with chitosan. DEMC with positive charges is able to interact with tight junctions of colon epithelial cells and hence increases permeability of brilliant blue across the tight junctions. In vivo investigations have exhibited the absorption enhancer effects of DEMC on the colon absorption of insulin in normal and diabetic rats. The insulin absorption from the rat's colon was evaluated by its hypoglycemic effect. A significant decrease in blood glucose was observed, when mixture of insulin and DEMC was introduced in ascending colon of rats.

Feasibility study on the retention of superporous hydrogel composite polymer in the intestinal tract of man using scintigraphy.

In recent years, many complex oral drug delivery systems have been developed using various polymers in order to achieve better drug targeting and drug absorption in the intestinal tract. Superporous hydrogel (SPH) and SPH composite (SPHC)-based drug delivery systems were also developed for the targeted delivery of peptide drugs into the intestinal tract. In the present study, the retention time of SPHC polymer is studied in man using the scintigraphy technique. To that purpose, SPHC polymers were radiolabelled with Tc-99m and administered orally in an enteric-coated gelatin capsule. The location of the radiolabelled polymer was monitored in five healthy volunteers while the subjects were sitting in front of a large field of view gamma camera. The results showed that enteric-coated gelatin capsules remained in the stomach for 75 to 150 min after oral administration to fasted volunteers and that the SPHC polymers thereafter attached to the upper part of the small intestine for at least 45 to 60 min due to their mechanical fixation properties. No discomfort was observed in any of the volunteers after oral administration of these polymers, which indicates that they are safe to be applied for oral drug delivery systems in man.

Intestinal absorption of human insulin in pigs using delivery systems based on superporous hydrogel polymers.

In this in vivo study, novel delivery systems based on superporous hydrogel (SPH) and SPH composite (SPHC) polymers were used to improve the intestinal absorption of insulin in healthy pigs. Six female pigs of approximately 35 kg body weight were used. A cannula was inserted into the jugular vein for blood sampling and a silicone fistula in the duodenum for administration of gelatin capsules containing the delivery systems or insulin solutions. The delivery systems consisted of two components, (1) conveyor system made of SPH and SPHC; (2) core containing insulin. The core was inserted either into the conveyor system (core inside, c.i.) or attached to the surface of conveyor system (core outside, c.o.). The following intestinal formulations were investigated: c.i., c.o. and intraduodenal (i.d.) administration of insulin solutions. Subcutaneous (s.c.) injection of insulin was also investigated for reasons of comparison. Blood samples were taken and analyzed for insulin and glucose concentrations. Relative bioavalibility values of 1.3+/-0.4 and 1.9+/-0.7% were achieved for c.o. and c.i. administrations, respectively. The bioavalibility for i.d. administration of insulin solution was 0.5+/-0.2%. These results indicate that the absorption of insulin was slightly increased using SPH/SPHC-based delivery systems. Furthermore, a large variability was observed, probably due to physiological and metabolic changes during the experiments. Blood glucose levels were slightly decreased after the c.o. and c.i administrations, whereas these levels did not decrease after i.d. administration of insulin solutions. In conclusion, SPH/SPHC-based delivery systems are able to enhance the intestinal absorption of insulin and are, therefore, considered as promising systems for peroral peptide drug delivery. However, insulin delivery from these delivery systems under in vivo have to be improved.

Acrylic resins as rate-controlling membranes in novel formulation of a nine-day 17beta-estradiol transdermal delivery system: in vitro and release modifier effect evaluation.

The feasibility of transdermal controlled delivery system of 17beta-estradiol was investigated by conducting in vitro release studies. Several new 17beta-estradiol unilaminate adhesive devices capable of releasing 17beta-estradiol in a controlled fashion over a 24-h, 36-h, 96-h, 104-h, 168-h, and 216-h period have been developed using acrylic resins (Eudragits E100, RSPO, and RLPO) as adhesive and rate-controlling polymers. The in vitro release profiles of 17beta-estradiol from various TDS unilaminate devices were characterized in a new developed dissolution tester vessel (total volume 200 ml), using a new paddle. The release of drug from different formulations was measured by a sensitive high-performance liquid chromatographic (HPLC) method. The release of drug from all prepared adhesive devices seems to obey zero-order kinetics (r > 0.98). The effect of two different plasticizers (acetyltriburyl citrate [ATBC] and triethyl citrate [TEC]) on the release patterns of 17beta-estradiol from TDS formulations was studied, and they were almost identical. The effect of two different release modifiers, propylene glycol (PG) and myristic acid (MA), on the release pattern of 17beta-estradiol from prepared unilaminate devices was evaluated. It was shown that the use of these release modifiers significantly increased the release of 17beta-estradiol from a TDS unilaminate patch. Furthermore, these data clearly demonstrated that the acrylic resins are suitable polymers for the preparation of 17beta-estradiol TDS adhesive devices.

Development and characterization of a novel peroral peptide drug delivery system.

Novel drug delivery systems were developed for peroral administration of peptide and protein drugs for site specific mechanical fixation at the gut wall and with specific release patterns. These so-called shuttle systems were designed by using superporous hydrogels (SPH) and SPH composite (SPHC) as the conveyor of a core which contained the model compound N-alpha-benzoyl-L-arginine ethylester (BAEE). Two different types of shuttle systems were evaluated: (a) core inside the shuttle system, and (b) core attached to the surface of shuttle system. Each of these systems was made of two parts: (1) the conveyor system made of SPHC which is used for keeping the dosage form at specific site(s) of the GI tract by mechanical interaction of the dosage form with the intestinal membranes, and (2) the core containing the active ingredient and incorporated in the conveyor system. The effect of formulation composition of the core on the release profile of BAEE was investigated by changing the type and amount of excipients in the formulations. In addition, the effect of various enteric-coat layers on the release profile and dissolving of the dosage form was investigated. The systems were also characterized for trypsin inactivation and Ca(2+) binding. The release profile of BAEE from the core formulation consisting of PEG 6000 microparticles or small tablets showed the desired burst release. When these core formulations were incorporated into the conveyor system made of SPH and SPHC, a suitable time-controlled release profile was obtained. Changing the type, concentration and thickness of the enteric-coat layer influenced the starting time of BAEE release from the dosage form, which indicates the necessary lag time for dissolving of the dosage form at any desired specific site of drug absorption in the intestine. Both SPH and SPHC were found to partly inhibit the activity of trypsin, due to two mechanisms: Ca(2+) binding and entrapment of the enzyme in these polymers. In conclusion, the presently developed delivery systems demonstrate suitable in vitro characteristics with an appropriate time-controlled release profile, making these systems very promising for effective peroral delivery of peptide and protein drugs.

In vitro release studies of piroxicam from oil-in-water creams and hydroalcoholic gel topical formulations.

The importance of piroxicam, a therapeutic anti-inflammatory drug, is well known. Because of gastrointestinal disorders, dermatological dosage forms are recommended most. In our first studies, oil-in-water (O/W) creams of piroxicam (1% concentration) were prepared using glyceryl monostearate (GMS), stearic acid, and triethanolamine as additive ingredients. In our second studies, hydroalcoholic transparent gel formulations of this drug in a 0.5% concentration were prepared using hydroxypropylcellulose (HPC) as the gelling agent. The release of piroxicam from all formulations via dialysis through a cellulose membrane into phosphate buffer pH 6.8 at 37 degrees C was studied. The effects of additives such as propylene glycol and 2-propanol on the drug release were also investigated. The release profiles from the standpoint of diffusion-controlled processes, as well as zero-order and first-order kinetics, were evaluated, and relevant parameters, such as diffusion coefficient, permeability coefficient, and partition coefficient, were calculated. The release obeys both the diffusion mechanism and first-order kinetics. The drug release from gel formulations containing 10%, 20%, and 30% propylene glycol was decreased due to the enhancement of viscosity. However, the limpidity of these formulations was improved. Moreover, the release of drug from gel formulations containing 15% and 20% of 2-propanol was increased. These results show that a hydroalcoholic gel formulation with HPC is a more suitable preparation of piroxicam when compared with an O/W cream formulation.

Permeability and adsorption capacity of dialysis membranes to lipid A.

Hemodialysis membranes were tested in vitro for possible penetration by low molecular weight endotoxins containing lipid A. Using lipid A from Escherichia coli as a model substance for this kind of pyrogen, different dialyzers (F4, E3. Acepal 1300, Altraflux, F 40, Polyflux 110, Filtral 12, F 60) were challenged by tangential filtration in aqueous medium. All membranes exhibited impermability to lipid A (as well as to LPS from Pseudomonas aeruginosa), which was proved by additional experiments using culture filtrates of Pseudomonas aeruginosa in bicarbonate dialysis fluid, as well as by employing miniaturized dialyzers with synthetic lipid A as a contaminant. Furthermore, the highest adsorption capacities were found for polysulfone and polyamide membranes.

Removal of lipid A and Pseudomonas aeruginosa endotoxin from dialysis fluids by high-flux polysulfone ultrafilter (dialyzer).

Ultrafilters (dialysis membranes) are generally considered to be impermeable to bacterial endotoxins (lipopolysaccharides) contaminating dialysates used for hemodialysis therapy, due to the self-aggregating properties of its molecules in aqueous media. The aim of the present investigation was to monitor the efficiency of high-flux polysulfone ultrafilters in removing lipid A and lipopolysaccharide (LPS) from dialysis fluids. To evaluate the safety of high-flux polysulfone membranes, an in-vitro circulation system (measuring lipopolysaccharide and lipid A penetrate from the ultrafilter to the other side and vice-versa), was utilized. Peritoneal dialysis solution was spiked with various concentration of Pseudomonas aeruginosa lipopolysaccharide (LPS) as well as lipid A diphosphoryl (E. coli F-583). Endotoxin and lipid A concentrations were detected by a chromogenic limulus amoebocyte lysate (LAL) assay. This investigation indicates that polysulfone ultrafiltration represents an efficient system to obtain an endotoxin and lipid A free dialysate when contaminated peritoneal solution was circulated through the dialysis membrane.