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.

Effect of zeolite nano-materials and artichoke (Cynara scolymus L.) leaf extract on increase in urinary clearance of systematically absorbed nicotine.

Nicotine, the main pharmacologically active component in tobacco and cigarette, has some toxic effects and also high potential for addiction. In this study, the effect of artichoke (Cynara scolymus L.) and zeolite nano-materials on urinary excretion of nicotine and consequently elimination of systematically absorbed nicotine was investigated. A simple, valid and highly sensitive high performance liquid chromatography method has been developed for determination of nicotine in rat urine according to guidelines for bioanalysis.It was found that nano-zeolites can cause increase in urinary concentration of nicotine due to its high surface adsorption. Artichoke leaf extract can cause increase in urinary excretion of nicotine in longer post administration times. It was observed that co-administration of nanozeolites and the leaf extract has the synergetic effect on increasing the urinary excretion of nicotine.

Mathematical modelling of the transport of hydroxypropyl-ß-cyclodextrin inclusion complexes of ranitidine hydrochloride and furosemide loaded chitosan nanoparticles across a Caco-2 cell monolayer.

Chitosan nanoparticles (CS-NPs) have been used to enhance the permeability of furosemide and ranitidine hydrochloride (ranitidine HCl) which were selected as candidates for two different biopharmaceutical drug classes having low permeability across Caco-2 cell monolayers. Drugs loaded CS-NPs were prepared by ionic gelation of CS and pentasodium tripolyphosphate (TPP) which added to the drugs inclusion complexes with hydroxypropyl-ß-cyclodextrin (HP-ßCD). The stability constants for furosemide/HP-ßCD and ranitidine HCl/HP-ßCD were calculated as 335 M(-1) and 410 M(-1), whereas the association efficiencies (AE%) of the drugs/HP-ßCD inclusion complexes with CS-NPs were determined to be 23.0 and 19.5%, respectively. Zetasizer and scanning electron microscopy (SEM) were used to characterise drugs/HP-ßCD-NPs size and morphology. Transport of both nano and non-nano formulations of drugs/HP-ßCD complexes across a Caco-2 cell monolayer was assessed and fitted to mathematical models. Furosemide/HP-ßCD-NPs demonstrated transport kinetics best suited for the Higuchi model, whereas other drug formulations demonstrated power law transportation behaviour. Permeability experiments revealed that furosemide/HP-ßCD and ranitidine HCl/HP-ßCD nano formulations greatly induce the opening of tight junctions and enhance drug transition through Caco-2 monolayers.

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.

In vivo transfection study of chitosan-DNA-FAP-B nanoparticles as a new non viral vector for gene delivery to the lung.

Gene therapy targeted at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. We recently reported that Chitosan-DNA-FAP-B nanoparticles are good candidates for targeted gene delivery to fibronectin molecules (FAP-B receptors) of lung epithelial cell membrane. In this study Chitosan-DNA-FAP-B nanoparticles were nebulized to mice using air jet nebulizer. The effect of nebulization on size, zeta potential and DNA binding ability of nanoparticles were studied. The level of gene expression in the mice lungs was evaluated. Nebulization did not affect the physicochemical properties of nanoparticles. Aerosol delivery of Chitosan-DNA-FAP-B nanoparticles resulted in 16-fold increase of gene expression in the mice lungs compared with Chitosan-DNA nanoparticles. This study suggested that Chitosan-FAP-B nanoparticle can be a promising carrier for targeted gene delivery to the lung.

N-Diethylmethyl chitosan for gene delivery to pancreatic cancer cells and the relation between charge ratio and biologic properties of polyplexes via interpolations polynomial.

In gene therapy of pancreatic cancer, non-viral vectors show an important role. These vectors are modified with the aim of improvement for pancreatic cancer gene therapy. For this aim, we used N,N-diethyl N-methyl chitosan (DEMC) for gene delivery to human pancreatic cancer cells (AsPC-1). pEGFP (Enhanced green fluorescent protein plasmid) was used as a model plasmid. In order to evaluate the efficiency of this polymer for gene delivery, the DEMC/pEGFP complexes are characterized via photon correlation spectroscopy, gel electrophoresis, fluorescence microscopy, flow cytometry and MTT assay. Also cancer cells' mean fluorescence intensity (MFI) and size changes after transfection are evaluated. The enhancement in polyplexes' charge ratios from 5 to 40, results in 16.70-fold increase in transfection efficiency. Higher MFI, cell size and cytotoxicity were observed as the N/P ratio increased. Considering that mathematical models can be used to understand and predict consequences associated with nanomedicine, the relation between DEMC/pDNA complexes charge ratio, cell transfection and toxicity was evaluated for the first time with Lagrange's interpolation polynomial method.

Preparation and evaluation of chitosan-DNA-FAP-B nanoparticles as a novel non-viral vector for gene delivery to the lung epithelial cells.

Gene delivery using cationic polymers such as chitosan shows good biocompatibility, but reveals low transfection efficiency. Fibronectin Attachment Protein of Mycobacterium bovis (FAP-B) which is responsible for the attachment of many Mycobacteria on the Fibronectin molecule of epithelial cell membrane can be considered as a new targeting ligand and can improve transfection rates in epithelial cells. In this study, chitosan-DNA nanoparticles were prepared using coacervation process. The effect of stirring speed and charge ratio (N/P) on the size and zeta potential of nanoparticles were evaluated. FAP-B ligand was added to nanoparticles at the specific condition to form chitosan-DNA-FAP-B nanoparticles via electrostatic attraction. Transfection efficiency of the final nanoparticles was investigated in A549 (alveolar epithelial cells). Cell viability was investigated using MTT assay. The optimum speed of stirring which was yielded the smallest chitosan-DNA nanoparticles with a narrow distribution (227±43 nm), was 500 rpm with the corresponding N/P ratio of 20. Chitosan-DNA-FAP-B nanoparticles presented the size of 279±27 nm with transfection efficiency about 10-fold higher than chitosan-DNA nanoparticles and resulted in 97.3% cell viability compared to 71.7% using Turbofect controls. Chitosan-DNA-FAP-B nanoparticles showed good transfection efficiency without cell toxicity. They have small particle size around 279 nm which make them a promising candidate as a novel non-viral gene vector for gene delivery to lung epithelial cells.

Development of novel budesonide pellets based on CODES(TM) technology: In vitro/in vivo evaluation in induced colitis in rats.

BACKGROUND AND THE PURPOSE OF THE STUDY: Budesonide is the drug of choice for treatment of active inflammatory bowel disease (IBD). The aim of this study was to develop budesonide pellets based on a novel colon drug delivery system (CODES). METHODS: Pellet cores containing lactulose or mannitol were prepared by extrusion/spheronization and coated with an acid soluble polymer (Eudragit E100), hydroxypropylmethyl cellulose (HPMC) and an enteric coat (Eudragit FS 30D) sequentially. In vitro drug release of coated pellets was studied using USP dissolution apparatus type II in buffers of pH 1.2 (2 hrs), pH of 7.4 (4 hrs) and pH of 6.8 containing 8% rat cecal contents (RCC) (18 hrs). The efficacy of the optimized formulation (containing 50% lactulose coated with Eudragit E (30% w/w) and Eudragit FS 30D (12% w/w)) was evaluated against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. RESULTS: The results of the kind of bacteria in vitro dissolution tests indicated absence of drug release in pHs of 1.2 and 7.4 and controlled release in buffer of pH 6.8 containing RCC. It was found that release rate was controlled by the type and amount of polysaccharide and the thickness of the acid soluble layer. The prepared formulation showed promising results in alleviating the conditions of experimental model of colitis. CONCLUSION: The results of this study suggest that pellets based on CODES technology could be useful for colonic delivery of budesonide.

Development and validation of a rapid HPLC method for simultaneous analysis of budesonide and its novel synthesized hemiesters in colon specific formulations.

A simple and reliable reversed-phase high performance liquid chromatographic (HPLC) method was developed, validated and applied for determination of budesonide and its novel synthesized hemiesters in colon specific formulations and dissolution media. The method was employed on a µ-Bondapak C(18) column (250 mm × 4.6 mm, 5 µm) at ambient temperature. The mobile phase consisted of acetonitrile: monobasic potassium phosphate containing orthophosphoric acid (55:45, pH 3.2) at a flow rate of 1 ml/min. The UV detection wavelength was set at 244 nm and 50 µL of sample was injected into the HPLC system. Dexamethasone was used as the internal standard. The retention times for internal standard and budesonide were 4.5 and 7.2 min, respectively. The method was linear in the concentration range of 1-20 µg/ml of budesonide (R(2)>0.999). Limit of detection and limit of quantitation were 0.05 and 0.5 µg/ml, respectively. The method presented the requisite accuracy, selectivity, sensitivity and precision and showed good resolution for separation of the drug and related derivatives in the presence of excipients. The proposed method was successfully used for analysis of the drug and its derivatives in dissolution media and oral colon specific formulations prepared in our laboratory with enough reproducibility.

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.

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.

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.