Deni Rahmat

Thiolated chitosan nanoparticles for the nasal administration of leuprolide: Bioavailability and pharmacokinetic characterization

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 ± 82 nm, a zeta potential of +10.9 ± 4 mV, 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.

S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties.

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. 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 FD(4) as the 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 (P(app)) were calculated and compared to values obtained from each buffer control. 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 of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of 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. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

Synthesis, characterization, mucoadhesion and biocompatibility of thiolated carboxymethyl dextran-cysteine conjugate.

This study was aimed at improving the mucoadhesive properties of carboxymethyl dextran by the covalent attachment of cysteine. Mediated by a carbodiimide, l-cysteine was covalently attached to the polymer. The resulting CMD-cysteine conjugate (CMD-(273) conjugate) displayed 273+/-20 micromol thiol groups per gram of polymer (mean+/-S.D.; n=3). Within 2h the viscosity of an aqueous mucus/CMD-(273) conjugate mixture pH 7.4 increased at 37 degrees C by more than 85% compared to a mucus/carboxymethyl dextran mixture indicating enlarged interactions between the mucus and the thiolated polymer. Due to the immobilization of cysteine, the swelling velocity of the polymer was significantly accelerated (p<0.05). In aqueous solutions the CMD-(273) conjugate was capable of forming inter- and/or intramolecular disulfide bonds. Because of this crosslinking process within the polymeric network, the cohesive properties of the conjugate were also improved. Tablets comprising the unmodified polymer disintegrated within 15 min, whereas tablets of the CMD-(273) conjugate remained stable for 160 min (means+/-S.D.; n=3). Results from LDH and MTT assays on Caco-2 cells revealed 4.96+/-0.98% cytotoxicity and 94.1+/-0.9% cell viability for the CMD-(273) conjugate, respectively. Controlled release of model compound from CMD-(273) conjugate tablets was observed over 6h. These findings suggest that CMD-(273) conjugate is a promising novel polymer for drug delivery systems providing improved mucoadhesive and cohesive properties, greater stability and biocompatibility.

Development and in vitro evaluation of slippery nanoparticles for enhanced diffusion through native mucus

AIM The aim of this study was to investigate the mucus-penetrating properties of neutral nanoparticles comprising poly(acrylic acid) (PAA) and poly(allylamine) (PAM). MATERIALS & METHODS PAA and PAM nanoparticles were prepared on the basis of ionic interactions between the two polymers. Nanoparticles were characterized by particle size as well as surface charge. The cytotoxicity was examined via resazurin and lactate dehydrogenase assays. Using a modified Ussing chamber with mucus, the diffusion properties of obtained neutral nanoparticles were compared with control particles. RESULTS The obtained PAA-PAM nanoparticles demonstrated no significant cytotoxicity and displayed smooth and spherical surfaces, a particle size range of 200 nm and ζ-potential of 0.9 mV. The diffusion efficiency of neutral nanoparticles was 2.5- and 1.8-fold higher than PAM and PAA nanoparticles, respectively. CONCLUSION Taking enhanced mucus-penetrating properties into account, neutral nanoparticles were shown to be very promising in drug delivery via mucus membranes of different cavities.

Design and synthesis of a novel cationic thiolated polymer

The purpose of this study was to design and characterize a novel cationic thiolated polymer. In this regard a hydroxyethylcellulose-cysteamine conjugate (HEC-cysteamine) was synthesized. Oxidative ring opening with periodate and reductive amination with cysteamine were performed in order to immobilize free thiol groups to HEC. The resulting HEC-cysteamine displayed 2035 ± 162 μmol immobilized free thiol groups and 185 ± 64 μmol disulfide bonds per gram of polymer being soluble in both acidic and basic conditions. Unlike the unmodified HEC, in case of HEC-cysteamine, a three-fold increase in the viscosity was observed when equal volumes of the polymer were mixed with mucin solution. Tablets based on HEC-cysteamine remained attached on freshly excised porcine mucosa for 8 0h and displayed increased disintegration time of 2h. Swelling behavior of HEC-cysteamine tablets in 0.1M phosphate buffer pH 6.8 indicated swelling ratio of 19 within 8h. In contrast, tablets comprising unmodified HEC detached from the mucosa within few seconds and immediately disintegrated. In addition, they did not exhibit swelling behavior. The transport of rhodamine 123 across freshly excised rat intestine enhanced by a value of approximately 1.6-fold (p-value = 0.0024) in the presence of 0.5% (m/v) HEC-cysteamine as compared to buffer control. Result from cytotoxicity test of HEC-cysteamine applied to Caco-2 cells in concentration of 0.5% (m/v) revealed 82.4 ± 4.60% cell viability. According to these results, HEC-cysteamine seems to be a promising polymer for various pharmaceutical applications especially for intestinal drug delivery.

HEC-cysteamine conjugates: influence of degree of thiolation on efflux pump inhibitory and permeation enhancing properties.

Within the present study hydroxyethyl cellulose-cysteamine conjugates are investigated regarding biocompatibility, in situ gelling, permeation enhancing and efflux pump inhibitory properties. For this purpose, a series of concentrations of sodium periodate was prepared to oxidize HEC leading to ring opening of glucose subunits. The resulting polymers showing varying degrees of oxidation (DO) were then conjugated with cysteamine stabilized via reductive amination. Consequently, HEC-cysteamine conjugates with increasing degree in thiolation were obtained. Since the conjugates are positively charged, potency of cytotoxicity was tested by resazurin assay. In situ gelling properties of the conjugates were studied to investigate change of their viscosity due to inter- and/or intramolecular crosslinking via disulfide bonds. The influence of the presence of the conjugates on transport of rhodamine 123 and fluoresceinisothiocyanate-dextran 4 (FD4) representing model compounds for P-glycoprotein (P-gp) inhibition and permeation enhancing studies, respectively, across Caco-2 cell monolayers was determined. The conjugates showed a degree of thiolation in the range of 316-2158 μmol/g. Within 30 min, dynamic viscosity of the conjugate with the lowest degree of thiolation 0.5% (m/v) increased up to 300-fold. The conjugates showed a degree of thiolation-dependent increase in cytotoxicity but they all were found comparatively low cytotoxic. The addition of the conjugate with thiol group content of 1670 μmol/g resulted in the highest improvement in the transport of both rhodamine 123 and FD4 as compared to buffer control. Accordingly, the degree of thiolation strongly influences the properties of the conjugates and the modulation of the degree of thiolation could be exploited for development of various drug delivery systems.

Enzymatic degradation of thiolated chitosan

The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100 mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate–plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9–26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4–50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan.

Preactivated thiomers: permeation enhancing properties.

Graphical abstract

Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency

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.

Thiolated hydroxyethyl cellulose: design and in vitro evaluation of mucoadhesive and permeation enhancing nanoparticles.

Within this study, HEC-cysteamine nanoparticles with free thiol groups in the range of 117-1548 μmol/g were designed and characterized. Nanoparticles were generated via ionic gelation of the cationic polymer with tripolyphosphate (TPP) followed by covalent crosslinking via disulfide bond formation using H2O2 as oxidant. The mean diameter of the particles was in the range of 270-360 nm, and zeta potential was determined to be +4 to +10 mV. Nanoparticles were evaluated in terms of mucoadhesive, permeation enhancing, and biocompatible properties as well as biodegradability. The particles remained attached to porcine intestinal mucosa up to 70% after 3h of incubation. The more nanoparticles were oxidized; however, the less were their mucoadhesive properties. Nanoparticles applied in a concentration of 0.5% (m/v) with the highest content of free thiol groups improved the transport of fluorescein isothiocyanate dextran 4 (FD4) across Caco-2 cell monolayer 3.94-fold in comparison with control (buffer). In addition, the transport of FD4 was even 1.84-fold enhanced in the presence of 0.5% (m/v) nanoparticles with the lowest free thiol group content. The higher the disulfide bond content within nanoparticles was, to a lower degree nanoparticles were hydrolyzed by cellulase. None of these nanoparticles showed pronounced cytotoxicity. Accordingly, HEC-cysteamine could be a promising excipient for nanoparticulate delivery systems for poorly absorbed drugs.