Wongsakorn Suchaoin

Development and in vitro evaluation of zeta potential changing self-emulsifying drug delivery systems for enhanced mucus permeation.

The aim of this study was the development of zeta potential changing self-emulsifying drug delivery systems (SEDDS). Various cationic surfactants were incorporated into a formulation consisting of 30% Cremophor EL, 30% Capmul MCM, 30% Captex 355 and 10% propylene glycol (w/w). A substrate of intestinal alkaline phosphatase (IAP), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid sodium (PA), was thereafter incorporated into SEDDS. Size, zeta potential and polydispersity index were determined. Phosphate release studies were performed using three different models, namely, isolated IAP, Caco-2 cell monolayer and rat intestinal mucosa and the amount of released phosphate was quantified by malachite green assay. Interaction of SEDDS and mucus was investigated regarding surface charges and mucus diffusion studies were performed using rotating tube technique. SEDDS were diluted 1:100 in 100mM HEPES buffer and a negative zeta potential was obtained. By addition of isolated IAP, 15% to 20% phosphate was liberated from SEDDS within 3h and a shift of zeta potential from negative to positive was observed. On Caco-2 cell monolayer and rat intestinal mucosa, 12% and 23% phosphate were released, respectively, from SEDDS diluted 1:1000 in glucose-HEPES buffer. Positively charged droplets were bound to negatively charged mucus resulting in a decrease of zeta potential, whereas negatively charged SEDDS showed no interaction. Furthermore, negatively charged SEDDS diffused faster through mucus layer as higher extent of incorporated Lumogen was present in deeper mucus segments in comparison to positively charged ones. Accordingly, zeta potential changing SEDDS provide an effective mucus permeation combined with higher cellular uptake when droplets reach absorptive epithelium membrane.

Permeation Enhancement via Thiolation: In Vitro and Ex Vivo Evaluation of Hyaluronic Acid-Cysteine Ethyl Ester

It was the aim of this study to evaluate the permeation-enhancing effect of synthesized thiolated hyaluronic acid (HA). HA, a naturally found polysaccharide, was chemically modified with l-cysteine ethyl ether (C) via amide bond formation. In vitro permeation enhancement was tested on Caco-2 cells with two compounds, sulforhodamine (SR) and fluorescein isothiocyanate-dextran (FD4). Cytotoxicity assays as lactate dehydrogenase and thiazolyl blue tetrazolium bromide (MTT) were performed on colon carcinoma cell line. Transepithelial electrical resistance (TEER) measurements were conducted. Ex vivo evaluation was accomplished on rat intestinal mucosa in order to predict the permeation enhancing effect with SR, sodium fluorescein (SF), and FD4, respectively. The MTT as well as lactate dehydrogenase revealed no toxicity over time periods of 3 and 12 h, respectively. The bioconjugate is biocompatible and safe to use. Furthermore, TEER measurements showed the integrity of tight junctions. The in vitro permeation studies on cell studies exhibit 1.28-fold enhancement for SR and 1.47-fold enhancement for FD4 with hyaluronic acid-cysteine ethyl ester (HAC) in comparison to unmodified one. The ex vivo transport studies exhibit 1.9-fold enhancement for SF, 1.31-fold enhancement for Rhodamine123, and 1.3-fold enhancement for FD4 with HAC in comparison to unmodified one, respectively. Thus, the promising results encourage further investigations and exploitation of this versatile polysaccharide.

Novel bioadhesive polymers as intra-articular agents: Chondroitin sulfate-cysteine conjugates.

The aim of this study was to generate and characterize a chondroitin sulfate-cysteine conjugate (CS-cys) as a novel bioadhesive agent for intra-articular use. Mucoadhesive properties of synthesized CS-cys were investigated by rheological measurement of polymer-mucus mixture and rotating cylinder method, while bioadhesive features of CS-cys on porcine articular cartilage were evaluated via tensile studies. Thiolation was achieved by attachment of l-cysteine to CS via amide bond formation mediated by carbodiimide as a coupling reagent. The conjugate exhibited 421.17±35.14 μmol free thiol groups per gram polymer. The reduced CS-cys displayed 675.09±39.67 μmol free thiol groups per gram polymer after disulfide bonds reduction using tris(2-carboxyethyl)phosphine hydrochloride. The increase in dynamic viscosity of thiolated CS due to oxidative disulfide bond formation was demonstrated using capillary viscometer. The combination of CS-cys and mucus led to 4.57-fold increase in dynamic viscosity in comparison with mucus control. Furthermore, adhesion time to porcine mucosa of CS-cys-based test disk was enhanced by 2.48-fold compared to unmodified CS as measured by rotating cylinder method suggesting the interaction between thiomers and mucus gel layer via disulfide bonds formation. Tensile studies of thiolated CS on porcine articular cartilage showed 5.37- and 1.76-fold increase in the total work of adhesion and the maximum detachment force, respectively, in comparison with unmodified CS indicating bioadhesive features of CS-cys. Cytotoxicity of CS-cys was assessed in Caco-2 cells and rat primary articular chondrocytes using MTT and LDH release assay, thereby showing the safety of CS-cys at a concentration of 0.25% (w/v) in Caco-2 cells. Furthermore, 0.1% of CS-cys was found non-toxic to rat primary articular chondrocytes. According to these results, CS-cys provides improved bioadhesive properties that might be useful as an intra-articular agent for treatment of osteoarthritis.

Thiolated graphene oxide as promising mucoadhesive carrier for hydrophobic drugs

The aim of this study was to improve the mucoadhesive properties of graphene by conjugating thiol ligands, in order to formulate an oral delivery system for hydrophobic drugs showing long mucus residence time. Graphene oxide was obtained by oxidation of graphite and then was thiolated following two synthetic paths. On the one hand, the hydroxyl groups were conjugated with thiourea passing through the formation of a brominated intermediate. On the other hand, the carboxylic acid groups were conjugated with cysteamine via carbodiimide chemistry. The mucoadhesive properties of thiolated graphene were evaluated by rheological measurements and by residence time assay. Then, valsartan was loaded on thiolated graphene and the release profile was evaluated in simulated intestinal fluid. Following both synthetic paths it was possible to obtain thiolated graphene bearing 215-302μmol SH/g product. Both products induced after 1h incubation an increase of mucus viscosity of about 22-33-fold compared to unmodified graphite. The residence time assay confirmed that 60% of thiolated graphene could be retained on intestinal mucosa after 4h incubation, whereas just 20% of unmodified graphite could be retained. Valsartan could be loaded with a drug loading of about 31±0.3% and a sustained release profile was observed for both formulations. According to the presented data, the thiolation of graphene could improve its mucoadhesive properties. Therefore, thiolated graphene represents a promising platform for oral delivery of hydrophobic drugs, possessing a long residence time on intestinal mucosa which allows the release of the loaded drug close to the adsorptive epithelium.

Synthesis and In Vitro Evaluation of Thiolated Carrageenan

The aim of this study was to generate and characterize a thiolated carrageenan. Thiolated carrageenan (carrageenan-SH) was synthesized from kappa (κ)- and iota (ι)-carrageenan by bromine replacement of the hydroxyl moieties followed by substitution to thiol groups using thiourea. Thiolated κ- and ι-carrageenan exhibited 176.57 ± 20.11 and 109.51 ± 18.26 μmol thiol groups per gram polymer, respectively. The resazurin test in Caco-2 cells revealed no toxic effect of both thiolated carrageenans at a concentration below 0.1% (w/v). Regarding efflux pump inhibitory effect, cellular accumulation of multidrug-resistance protein 2 substrate, sulforhodamine 101, was 1.38- and 1.35-fold increased in cells treated with thiolated κ- and ι-carrageenan, respectively. Modification of κ- and ι-carrageenan led to 3.9- and 2.0-fold increase in dynamic viscosity of mucus-thiolated carrageenan mixture within 4 h. Furthermore, residence time of κ- and ι-carrageenan-SH on porcine intestinal mucosa was 6.4- and 1.8-fold prolonged, respectively, as demonstrated by rotating cylinder method, indicating improved mucoadhesive properties. Hence, thiolation of carrageenans led to novel pharmaceutical excipients for various applications.

Multifunctional adhesive polymers: Preactivated thiolated chitosan-EDTA conjugates

Aim The aim of this study was to synthesis preactivated thiolated chitosan‐EDTA (Ch‐EDTA‐cys‐2MNA) conjugates exhibiting in particular high mucoadhesive, cohesive and chelating properties. Methods Thiol groups were coupled with chitosan by carbodiimide reaction and further preactivated by attachment with 2‐mercaptonicotinic acid (2MNA) via disulfide bond formation. Determinations of primary amino and sulfhydryl groups were performed by TNBS and Ellman’s tests, respectively. Cytotoxicity was screened by resazurin assay in Caco‐2 cells. Mucoadhesive properties and bivalent cation binding capacity with Mg2+ and Ca2+ in comparison to chitosan‐EDTA (Ch‐EDTA) and thiolated Ch‐EDTA (Ch‐EDTA‐cys) were evaluated. Results Determination of 2MNA and total sulfhydryl groups indicated that 80% of thiol groups were preactivated. The results from cytotoxicity studies demonstrated that Ch‐EDTA‐cys and Ch‐EDTA‐cys‐2MNA were not toxic to the cells at the polymer test concentration of 0.25% (w/v) while cell viability decreased by increasing the concentration of Ch‐EDTA. Although EDTA molecule was modified by thiolation and preactivation, approximately 50% of chelating properties of the conjugates were maintained compared to Ch‐EDTA. Ch‐EDTA‐cys‐2MNA adhered on freshly excised porcine intestinal mucosa up to 6 h while Ch‐EDTA adhered for just 1 h. Conclusion According to the combination of mucoadhesive and chelating properties of the conjugates synthesized in this study, Ch‐EDTA‐cys‐2MNA might be useful for various mucosal drug delivery systems. Graphical abstract Figure. No Caption available.

Totally S-protected hyaluronic acid: Evaluation of stability and mucoadhesive properties as liquid dosage form

AIM It is the aim of this study to synthesize hyaluronic acid (HA) derivatives bearing mucoadhesive properties and showing prolonged stability at pH 7.4 and under oxidative condition as liquid dosage form. METHODS HA was modified by thiolation with l-cysteine (HA-SH) and by conjugation with 2-mercaptonicotinic acid-l-cysteine ligand to obtain an S-protected derivative (HA-MNA). The polymers were characterized by determination of thiol group content and mercaptonicotinic acid content. Cytotoxicity, stability and mucoadhesive properties (rheological evaluation and tensile test) of the polymers were evaluated. RESULTS HA-SH and HA-MNA could be successfully synthesized with a degree of modification of 5% and 9% of the total moles of carboxylic acid groups, respectively. MTT assay revealed no toxicity for the polymers. HA-SH resulted to be unstable both at pH 7.4 and under oxidative conditions, whereas HA-MNA was stable under both conditions. Rheological assessment showed a 52-fold and a 3-fold increase in viscosity for HA-MNA incubated with mucus compared to unmodified HA and HA-SH, respectively. Tensile evaluation carried out with intestinal and conjunctival mucosa confirmed the higher mucoadhesive properties of HA-MNA compared to HA-SH. CONCLUSIONS According to the presented results, HA-MNA appears to be a potent excipient for the formulation of stable liquid dosage forms showing comparatively high mucodhesive properties.

Novel in vitro transport method for screening the reversibility of P-glycoprotein inhibitors

The purpose of this study was to establish a novel in vitro method for screening reversibility of P-glycoprotein (P-gp) inhibitors. Caco-2 cells with 21days of cultivation were used as an in vitro model. Transport of rhodamine 123 in the presence of various inhibitors and after removing of inhibitors was determined. Transport of rhodamine 123 at 4°C and in the secretory direction assured that Caco-2 cells exhibited P-gp function at all time of experiment. The apparent permeability coefficient (Papp) of rhodamine 123 in the presence of verapamil, cyclosporin A, ritonavir, quinidine, N-ethylmaleimide, Cremophor® EL, Tween 80 and poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-cys-2MNA) was 2.3-, 3.8-, 2.3-, 3.1, 7.5-, 2.1-, 2.9- and 2.5-fold higher than Papp of rhodamine 123 alone. After removing of the inhibitors, Papp decreased to the same range of control except in the case of N-ethylmaleimide which was 2.4-fold higher than the control. These results revealed a reversible inhibition of verapamil, cyclosporin A, ritonavir, quinidine, Cremophor® EL, Tween 80 and PAA-cys-2MNA and an irreversible inhibition of N-ethylmaleimide for P-gp. Thus, this novel established that in vitro method might be an effective tool for screening the reversibility of inhibition of P-gp inhibitors.

Zeta-potential-changing nanoparticles conjugated with cell-penetrating peptides for enhanced transfection efficiency

AIM The aim of this study was to develop zeta-potential-changing nanoparticles (NPs) combining cell-penetrating peptides for gene delivery. METHODS & MATERIALS NPs were formed using phosphorylated carboxymethyl cellulose-glucosamine 6-phosphate (CMC-G6P) and polyethylene imine-polyarginine conjugates. Phosphate release was evaluated using intestinal alkaline phosphatase and cell lines. Transfection studies with plasmid DNA were then performed. RESULTS The zeta potential of CMC-G6P/branched PEI NPs was -3 mV and switched to +4 mV after intestinal alkaline phosphatase cleavage. The released phosphate in human colon adenocarcinoma cell line was more pronounced than human embryonic kidney cell line 293. Transfection studies demonstrated the greatest expression of plasmid DNA when being incorporated into CMC-G6P/polyethylene imine-polyarginine NPs. CONCLUSION Novel zeta potential changing NPs combining cell-penetrating peptides are a promising tool to deliver DNA drugs to target cells.

Design and evaluation of buccal-adhesive system made of modified xanthan

BACKGROUND Introducing the concept of mucoadhesion in the 1980s, application of mucoadhesive polymers for buccal drug delivery has been the subject of pharmaceutical interest. AIM The purpose of this study was to modify well-known xanthan in order to remarkably boost its suitability for buccal adhesiveness. METHOD Xanthan (X) was chemically modified by covalent binding of L-cysteine (SH) exhibiting sulfhydryl groups on the polymeric backbone via amide bond formation. Cytotoxicity, stability properties and mucoadhesiveness, respectively, of sulfhydryl-modified xanthan (H-SH) was investigated compared with unmodified xanthan (X). RESULTS According to cell viability studies X-SH was safe to use. X-SH revealed 1.61-fold higher stability in comparison to unmodified xanthan. Thereafter, mucoadhesion of X-SH augmented 8.35-fold in case of rotating cylinder assay. Tensile study showed 2.65-fold higher total work of adhesion when comparing X-SH with X. CONCLUSION In completion, novel sulfhydryl-modified xanthan proved itself being a useful and promising excipient for buccal-adhesive delivery systems.