Duangkamon Sakloetsakun

Distribution of thiolated mucoadhesive nanoparticles on intestinal mucosa.

It was the aim of the present study to evaluate and compare the distribution of thiolated mucoadhesive anionic poly(acrylic acid) (PAA) and cationic chitosan (CS) nanoparticles on intestinal mucosa. Modifications of these polymers were achieved by conjugation with cysteine (PAA-Cys) and 2-iminothiolane (CS-TBA). Nanoparticles (NP) were prepared by ionic gelation and labelled with the strong hydrophilic fluorescent dye Alexa Fluor 488 (AF 488) and hydrophobic fluorescein diacetate (FDA). Unmodified and modified CS and PAA NP were examined in vitro in terms of their mucoadhesive and mucus penetrating properties on the mucosa of rat small intestine. To investigate the transport of NP across the mucus layer, their diffusion behaviour through natural porcine intestinal mucus was studied through a new diffusion method developed by our group. Lyophilised particles displayed 526 μmol/g (CS) and 513 μmol/g (PAA) of free thiol groups and a zeta potential of 20 mV (CS) and -14 mV for PAA NP. Nanoparticle distribution on rat intestine suggested that mucoadhesion of thiolated NP is higher than the diffusion into the intestinal mucosa. Modified particles displayed more than a 6-fold increase in mucoadhesion compared to unmodified ones. The rank order with regard to mucoadhesion of all particles was: CS-TBA>PAA-Cys>CS>PAA, whereas CS-TBA showed 2-fold higher mucoadhesive properties compared to PAA-Cys NP. Diffusion through intestinal mucus was much higher for unmodified than for thiolated as well as for anionic compared to cationic particles. Overall, it was shown that thiolated particles of both anionic and cationic polymers have improved mucoadhesive properties and could be promising carriers for mucosal drug delivery.

In situ gelling properties of chitosan-thioglycolic acid conjugate in the presence of oxidizing agents

The rheological behaviour of chitosan-thioglycolic acid conjugate (chitosan-TGA) in the presence of four oxidizing agents was investigated. Chitosan-TGA was synthesized via amide bond formation between the primary amino group of chitosan and the carboxylic acid group of thioglycolic acid. The sol-gel phase transition of the polymer was determined by rheological measurements. Moreover, cytotoxicity of the gel in combination with each oxidizing agent was evaluated utilizing LDH and MTT assay. The modified chitosan displayed 1053+/-44 micromol/g thiol groups. Results of rheological studies showed that 1% (m/v) chitosan-TGA without any oxidizing agents became gel within 40 min. In contrast, when the oxidizing agents hydrogen peroxide, sodium periodate, ammonium persulfate and sodium hypochlorite were added, respectively, gelation took place within a few minutes. Within 20 min, hydrogen peroxide having been added in a final concentration of 25.2 nmol/L increased dynamic viscosity of 1% (m/v) chitosan-TGA up to 16,500-fold. This can be explained by the formation of inter- and/or intramolecular disulfide bonds which were indirectly verified via the decrease in thiol groups. Additionally, evidence of an increase in cross-linking of thiolated chitosan as a function of time was provided by frequency sweep measurements. Furthermore, viability of Caco-2 cells having been incubated with chitosan-TGA/oxidizing agent systems assessed by MTT assay was 70-85% and the percentage of LDH release was only in case of the chitosan-TGA/ammonium persulfate system significantly (p<0.05) raising compared to the negative control. According to these results, chitosan-TGA/oxidizing agent combinations might be a promising novel in situ gelling system for various pharmaceutical applications such as a controlled drug release carrier or for tissue engineering.

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.

S-protected thiolated chitosan: Synthesis and in vitro characterization

Highlights ► Synthesis of a novel mucoadhesive thiolated chitosan with protected thiol groups. ► The novel conjugate exhibited promising mucoadhesive features. ► In vitro cytotoxicity of the new conjugate was evaluated and found to be non-toxic. ► Swelling behavior of the polymer decreased with the increase of protection. ► Enhanced cross linking within the novel conjugate resulted in improved stability.

Combining two technologies: multifunctional polymers and self-nanoemulsifying drug delivery system (SNEDDS) for oral insulin administration.

The aim of the study is to develop a self-nanoemulsifying drug delivery system (SNEDDS) based on thiolated chitosan for oral insulin administration. The preparations were characterized by particle size, entrapment efficiency, stability and drug release. Serum insulin concentrations were determined after oral administration of all formulations. Insulin SNEDDS formulation was served as control. The optimized SNEDDS consists of 65% (w/w) miglyol 840, 25% (w/w) cremophor EL, 10% (w/w) co-solvents (a mixture of DMSO and glycerol). The formulations in the presence or absence of insulin (5mg/mL) were spherical with the size range between 80 and 160 nm. Entrapment efficiency of insulin increased significantly when the thiolated chitosan was employed (95.14±2.96%), in comparison to the insulin SNEDDS (80.38±1.22%). After 30 min, the in vitro release profile of insulin from the nanoemulsions was markedly increased compared to the control. In vivo results showed that insulin/thiolated chitosan SNEDDS displayed a significant increase in serum insulin (p-value=0.02) compared to oral insulin solution. A new strategy to combine SNEDDS and thiolated chitosan described in the study would therefore be a promising and innovative approach to improve oral bioavailability of insulin.

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.

Thiolated hydroxyethylcellulose: Synthesis and in vitro evaluation

In recent years, thiomers have received considerable interest due to advantageous characteristics, such as improved mucoadhesive and permeation enhancing properties. Thiolated polymers, however, are characterized by an ionic charge which represents for various applications a great limitation. The aim of this study was therefore to synthesize a novel thiolated polymer not exhibiting ionizable groups. Hydroxyethylcellulose (HEC) was chosen as polymer backbone. The chemical modification was achieved by the replacement of hydroxyl groups on the carbohydrate structure with thiol moieties, using thiourea as thiolating reagent. The resulting thiolated hydroxyethylcellulose (HEC-SH) was characterized in vitro regarding its gelling properties, swelling behaviour, mucoadhesion on freshly excised porcine intestinal mucosa and permeation enhancing effect across rat intestinal mucosa. The new thiomer displayed up to 131.58 ± 11.17 μmol thiol groups per gram polymer, which are responsible for the observed in situ gelling capacity. The swelling behaviour and the mucoadhesive properties of tablets based on HEC-SH were 1.5-fold and 4-fold improved compared with unmodified HEC, respectively. The permeation enhancing effect of 0.5% (m/v) HEC-SH on rhodamine 123 (Rho-123) transport was 1.9-fold improved compared with buffer only. According to these results, HEC-SH seems to represent a promising tool for the development of in situ gelling, mucoadhesive delivery systems with permeation enhancing properties.

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 chitosans: influence of various sulfhydryl ligands on permeation-enhancing and P-gp inhibitory properties

Purpose: The influence of various sulfhydryl ligands on permeation-enhancing and P-glycoprotein (P-gp) inhibitory properties of the six established thiolated chitosan conjugates was investigated using Rhodamine-123 (Rho-123) and fluorescein isothiocyanate-dextran 4 (FD4) as model compounds. Methods: Permeation of these compounds was tested on freshly excised rat intestine in Ussing-type chambers. Apparent permeability coefficients (Papp) were calculated and compared to values obtained from the buffer only control. Results: The lyophilized polymers had a thiol group content in the range of 230–520 μmol/g. Results of this study led to the following rank order in permeation enhancement: chitosan-6-mercaptonicotinic acid (chitosan-6MNA) > chitosan-cysteine (chitosan-Cys) > chitosan-glutathione (chitosan-GSH) > chitosan-4-thiobutylamidine (chitosan-TBA) > chitosan-thioglycolic acid (chitosan-TGA) > chitosan-N-acetyl cysteine (chitosan-NAC). In P-gp inhibition studies, 0.5% (m/v) chitosan-NAC showed the highest inhibitory effect on P-gp, where the Papp was determined to be 3.78-fold increased compared with the buffer control. Among these thiolated chitosans, chitosan-NAC and chitosan-6MNA are the most effective polymers being responsible for P-gp inhibition and permeation enhancement, respectively. Conclusion: These thiolated chitosans would therefore be advantageous tools for enhancing the noninvasive bioavailability of active pharmaceutical ingredients.