Within the past 20years, a considerable amount of work has been published on chitosan and its potential use in drug delivery systems. In contrast to all other polysaccharides having a monograph in a pharmacopeia, chitosan has a cationic character because of its primary amino groups. These primary amino groups are responsible for properties such as controlled drug release, mucoadhesion, in situ gellation, transfection, permeation enhancement, and efflux pump inhibitory properties. Due to chemical modifications, most of these properties can even be further improved. Within this review, an overview on the advantages of chitosan for various types of drug delivery systems is provided.

The aim of the present study was the development and evaluation in vitro as well as in vivo of an oral delivery system based on a novel type of thiolated chitosan, so-called S-protected thiolated chitosan, for the peptide drug antide. The sulfhydryl ligand thioglycolic acid (TGA) was covalently attached to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Absorptive transport studies of antide were evaluated ex vivo using rat intestinal mucosa. Matrix tablets of each polymer sample were prepared and their effect on the absorption of antide evaluated in vivo in male Sprague-Dawley rats. In addition, tablets were examined in terms of their disintegration, swelling and drug release behavior. The resulting S-protected thiomer (TGA-MNA) exhibited 840μmol of covalently linked 6-MNA per gram thiomer. Based on the implementation of this hydrophobic ligand on the thiolated backbone, the disintegration behavior was reduced greatly and a controlled release of the peptide could be achieved. Furthermore, permeation studies with TGA-MNA on rat intestine revealed a 4.5-fold enhanced absorptive transport of the peptide in comparison to antide in solution. Additional in vivo studies confirmed the potential of this novel conjugate. Oral administration of antide in solution led to only very small detectable quantities in plasma with an absolute and relative bioavailability (BA) of 0.003 and 0.03%, only. In contrast, with antide incorporated in TGA-MNA matrix tablets an absolute and relative BA of 1.4 and 10.9% could be reached, resulting in a 421-fold increased area under the plasma concentration time curve (AUC) compared to the antide solution. According to these results, S-protected thiolated chitosan as oral drug delivery system might be a valuable tool for improving the bioavailability of peptides.

Purpose: The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, so-named S-protected thiolated chitosan. Methods: Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD4 as model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (Papp) were calculated and compared to values obtained from each buffer control. Results: Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h permeation studies on excised rat intestine, Papp values of all S-protected chitosans were at least 1.3-fold higher compared to corresponding thiomers and more than twice as high as unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Due to the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. Conclusion: These features approve S-protected thiolated chitosan as promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

The purpose of this study was to develop thiolated nanoparticles to enhance the bioavailability for the nasal application of leuprolide. Thiolated chitosan-thioglycolic acid (chitosan-TGA) and unmodified chitosan nanoparticles (NPs) were developed via ionic gelation with tripolyphosphate (TPP). Leuprolide was incorporated during the formulation process of NPs. The thiolated (chitosan-TGA) NPs had a mean size of 252±82nm, a zeta potential of +10.9±4mV, and payload of leuprolide was 12±2.8. Sustained release of leuprolide from thiolated NPs was demonstrated over 6h, which might be attributed to inter- and/or intramolecular disulfide formation within the NPs network. Ciliary beat frequency (CBF) study demonstrated that thiolated NPs can be considered as suitable additives for nasal drug delivery systems. Compared to leuprolide solution, unmodified NPs and thiolated NPs provoked increased leuprolide transport through porcine nasal mucosa by 2.0 and 5.2 folds, respectively. The results of a pharmacokinetic study in male Sprague-Dawley rats showed improved transport of leuprolide from thiolated NPs as compared to leuprolide solution. Thiolated NPs had a 6.9-fold increase in area under the curve, more than 4-fold increase in elimination half-life, and a ∼3.8-fold increase in maximum plasma concentration compared to nasal solution alone. The relative nasal bioavailability (versus s.c. injection) of leuprolide thiolated NPs calculated on the basis of AUC((0-6)) was about 19.6% as compared to leuprolide solution 2.8%. The enhanced bioavailability of leuprolide is likely due to facilitated transport by thiolated NPs rather than improved release.

The aim of this study was to investigate in situ crosslinking systems of anionic thiolated polymers. In order to accelerate the increase in dynamic viscosity of thiolated polymers (thiomers), they were combined with hydrogen peroxide, carbamide peroxide and ammonium persulfate. Thiomers (pectin-cysteine (Pec-Cys), sodium carboxymethylcellulose-cysteine (NaCMC-Cys) and poly(acrylic acid)-cysteine (PAA-Cys)) were synthesized via amide bond formation between the carboxylic acid group of polymers and the primary amino group of l-cysteine. The rheological properties of 1%(m/v) thiomer solutions with oxidizing agents were compared by oscillatory measurements over time (120 min). Pec-Cys and NaCMC-Cys with hydrogen and carbamide peroxide showed a sol-gel phase transition within a few minutes and scored up to 13,000-fold increase in dynamic viscosity. Furthermore, only thiomers exhibiting a polysaccharide backbone (Pec-Cys and NaCMC-Cys) showed a significant increase in viscosity (p < 0.05). In contrast, measurements of carbohydrate thiomers in combination with ammonium persulfate showed an initial increase in viscosity. Afterwards, a decrease in viscosity was observed likely caused by chain scission. According to these results, carbohydrate thiomer/oxidizing agent systems might be useful for various pharmaceutical applications such as for in situ gelling liquid/semisolid formulations or in tissue engineering.

Nanoparticles generated by complex coacervation of plasmid DNA (pDNA) and modified chitosans namely chitosan-thioglycolic acid (TGA) conjugate and chitosan-HIV-1 Tat peptide conjugate were evaluated as gene delivery systems. In order to optimize transfection efficiency, chitosan-HIV-1 Tat peptide conjugate was combined with chitosan-TGA before its complexation with pDNA. Particle size and zeta potential measurements were performed to characterize the generated nanoparticles. The nanoparticles transfection efficiencies were assessed by exploitation of the green fluorescent protein (GFP) reporter gene. HEK293 cells were incubated for 24 h with the nanoparticles and the GFP positive cells were observed by fluorescence microscopy. The nanoparticles in the size range of 200-300 nm could transfect HEK293 cells as a model cell line with different transfection efficiencies. Unlike chitosan-TGA, chitosan-HIV-1 Tat peptide led to increased zeta potential of nanoparticles as compared to unmodified chitosan. The transfection efficiency of the nanoparticles generated by combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was comparatively higher than that of the nanoparticles generated by either chitosan-TGA or the combination of chitosan-HIV-1 Tat peptide with unmodified chitosan. After 72 h of incubation, the combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was found to be 7.12- and 67.37 times more efficient than unmodified chitosan and pDNA alone, respectively and showed a synergistic effect in transfection of pDNA into the cells. Moreover, none of the nanoparticles showed any severe cytotoxicity. Accordingly, this strategy might result in a potent carrier for gene delivery.

This study was aimed to synthesize polymeric excipients with improved mucoadhesive, cohesive and in situ-gelling properties to assure a prolonged retention time of dosage forms at a given target site, thereby achieving an increased uptake and improved oral bioavailability of certain challenging therapeutic agents such as peptides and proteins. Accordingly, poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-cys-2MNA) conjugates were synthesized by the oxidative S-S coupling of PAA-cys (100-, 250- and 450 kDa) with 2-mercaptonicotinic acid (2MNA). Unmodified PAAs, PAAs-cys (thiomers) and PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates were compressed into tablets to perform disintegration tests, mucoadhesion studies and rheological measurements. Moreover, cytotoxicty of the polymers was determined using Caco-2 cells. The resulting PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 113.5 ± 12.7, 122.7 ± 12.2 and 117.3 ± 4.6 μmol/g of 2-mercaptonicotinic acid, respectively. Due to the immobilization of 2MNA, the PAA-cys-2MNA (pre-activated thiomers) conjugates exhibit comparatively higher swelling properties and disintegration time to the corresponding unmodified and thiolated polymers. On the rotating cylinder, tablets based on PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 5.0-, 5.4- and 960-fold improved mucoadhesion time in comparison to the corresponding unmodified PAAs. Results achieved from tensile studies were found in good agreement with the results obtained by rotating cylinder method. The apparent viscosity of PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates was improved 1.6-, 2.5- and 206.2-fold, respectively, in comparison to the corresponding unmodified PAAs. Moreover, pre-activated thiomers/mucin mixtures showed a time dependent increase in viscosity up to 24 h, leading to 7.0-, 18.9- and 2678-fold increased viscosity in comparison to unmodified PAAs (100-, 250- and 450 kDa), respectively. All polymers were found non-toxic over Caco-2 cells. Thus, on the basis of achieved results the pre-activated thiomers seem to represent a promising generation of mucoadhesive polymers which are safe to use for prolonged residence time of drug delivery systems to target various mucosa.

The purpose of this study was the development of stable thiomer nanoparticles for mucosal drug delivery. Chitosan-thioglycolic acid (chitosan-TGA) nanoparticles (NP) were formed via ionic gelation with tripolyphosphate (TPP). In order to stabilize the NP inter- and intra-molecular disulfide bonds were formed via controlled oxidation with hydrogen peroxide (H(2)O(2)). Thereafter, stability was investigated in saline and simulated body fluids at pH 2 and pH 5.5 via optical density measurements. The mucoadhesive properties were evaluated in vitro on freshly excised porcine intestinal mucosa via the rotating cylinder method. Particles had a mean size of 158 ± 8 nm and a zeta potential of ~ + 16 mV. Three different degrees of oxidation were adjusted by the addition of H(2)O(2) in final concentrations of 10.60 µmol (chitosan-TGA (ox1)), 21.21 µmol (chitosan-TGA (ox2)), and 31.81 µmol (chitosan-TGA (ox3)) leading to 60%, 75%, and 83% of oxidized thiol groups, respectively. More than 99% of chitosan-TGA (ox3) NP, 70% of chitosan-TGA (ox2) NP, and 50% of chitosan-TGA (ox1) NP were stable over a 60-min period in simulated gastric fluid. In contrast, only 10% of unmodified chitosan and chitosan-TGA NP which were just ionically cross-linked remained stable in the same experiment. The adhesion times of covalently cross-linked chitosan-TGA (ox1), chitosan-TGA (ox2), and chitosan-TGA (ox3) were ~ 41-fold, 31-fold, and 25-fold longer in comparison to unmodified ionically cross-linked chitosan. The method described here might be useful for the preparation of stable nanoparticulate drug delivery systems.

The purpose of this study was to determine the concentration-dependent effect of selected solubilizers, used in common nasal drug formulations, on ciliary beat frequency (CBF) in human nasal epithelial cell cultures. CBF was measured by a high-speed digital imaging method. Excised ciliated human nasal epithelial cells were incubated for 60min with the solubilizers and determination of the half maximal inhibitory concentration (IC(50)), followed by a reversibility test. LDH test was performed on human nasal epithelial cells with the solubilizing agents. These were applied to nasal epithelial cells in IC(50) values. The following rank order in IC(50) values was obtained for the solubilizers: glycerol>propylene glycol>polyethylene glycol 300>N,N-dimethylacetamide>polyethylene glycol 400>ethanol>ethylendiamindihydrochloride>polyvinylpyrrolidon 25>polyvinylpyrrolidon 90. The highest reversibility of approximately 75% was shown by propylene glycol and polyethylene glycol 300 at a concentration of 30%(v/v). Results from the LDH test showed that N,N-dimethylacetamide displayed the highest cytotoxicity with 5.2% at a concentration of 14.5%(v/v). According to these results, several solubilizers can alter the CBF frequency and thus, have an impact on the nasal mucosa. Therefore, CBF studies with solubilizers used at a concentration relevant for nasal formulations are essential in the design of efficient and most notably safe nasal medicinal products.

It was the aim of this study to develop a sustained parenteral peptide (DALCE) delivery system by the immobilization of DALCE to thiolated carboxymethyl dextran-cysteine (CMD-Cys) via disulfide bond formation. The resulting CMD-Cys-DALCE conjugate displayed a 22.6±7.9%(m/m) of DALCE (mean±S.D.; n=3). The conjugation of DALCE with CMD-Cys was confirmed by FTIR-ATR spectroscopy. In vitro release studies of conjugate CMD-Cys-DALCE in the presence of 2μM/ml reduced glutathione (GSH) being also available in the plasma showed a sustained peptide release over a time period of 8h, because of thiol/disulfide exchange reactions. For in vivo pharmacokinetic study, DALCE and CMD-Cys-DALCE were administered intravenously to male Sprague-Dawley rats at a dose of 1mg/kg. The AUC(0-8)(ng.min/ml) was determined to be 268848±924 and 40019±495 for CMD-Cys-DALCE and DALCE, respectively. The mean residence time (MRT) was determined to be 256±8 and 53.1±9.5min for CMD-Cys-DALCE and for DALCE, respectively. CMD-Cys-DALCE showed a more than 5-fold increased elimination half-life (p<0.01), 3-fold decreased volume of distribution (p<0.01) and a 6.7-fold decreased plasma clearance rate (p<0.01) compared to DALCE. According to these findings, CMD-Cys-DALCE seems to act as prodrug by improving half-life and decreasing plasma clearance.