Warayuth Sajomsang

Antibacterial activity of quaternary ammonium chitosan containing mono or disaccharide moieties: preparation and characterization.

The 9 quaternary ammonium chitosans containing monosaccharides or disaccharides moieties were successfully synthesized by reductive N-alkylation then quaternized by N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat-188). The chemical structures of quaternary ammonium chitosan derivatives were characterized by ATR-FTIR and (1)H NMR spectroscopy. The degree of N-substitution (DS) and the degree of quaternization (DQ) were determined by (1)H NMR spectroscopic method. It was found that the DS was in the range of 12-40% while the DQ was in the range of 90-97%. The results indicated that the O-alkylation was occured in this condition. Moreover, all quaternary ammonium chitosan derivatives were highly water-soluble at acidic, basic, and neutral pH. Minimum inhibitory concentration (MIC) antibacterial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria compared to quaternary ammonium N-octyl and N-benzyl chitosan derivatives. The quaternary ammonium mono and disaccharide chitosan derivatives showed very high MIC values which were in the range of 32 to >256 microg/mL against both bacteria. Also it was found that the antibacterial activity decreased with increasing the DS. This was due to the increased hydrophilicity of mono and disaccharide moieties. On the other hand, the low MIC values (8-32 microg/mL) were obviously observed when the DS of quaternary ammonium N-octyl and N-benzyl chitosan derivatives was lower than 18%. The results showed that the presence of hydrophobic moiety such as the N-benzyl group enhanced the antibacterial activity compared to the hydrophilic moiety against both bacteria.

Antifungal property of quaternized chitosan and its derivatives.

Five water-soluble chitosan derivatives were carried out by quaternizing either iodomethane or N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat188) as a quaternizing agent under basic condition. The degree of quaternization (DQ) ranged between 28±2% and 90±2%. The antifungal activity was evaluated by using disc diffusion method, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) methods against Trichophyton rubrum (T. rubrum), Trichophyton mentagrophyte (T. mentagrophyte), and Microsporum gypseum (M. gypseum) at pH 7.2. All quaternized chitosans and its derivatives showed more effective against T. rubrum than M. gypseum and T. mentagrophyte. The MIC and MFC values were found to range between 125-1000 μg/mL and 500-4000 μg/mL, respectively against all fungi. Our results indicated that the quaternized N-(4-N,N-dimethylaminocinnamyl) chitosan chloride showed highest antifungal activity against T. rubrum and M. gypseum compared to other quaternized chitosan derivatives. The antifungal activity tended to increase with an increase in molecular weight, degree of quaternization and hydrophobic moiety against T. rubrum. However, the antifungal activity was depended on type of fungal as well as chemical structure of the quaternized chitosan derivatives.

Water-soluble β-cyclodextrin grafted with chitosan and its inclusion complex as a mucoadhesive eugenol carrier

Inclusion complex between water-soluble βCD-grafted chitosan derivatives (QCD-g-CS) and eugenol (EG) was investigated as a new type of mucoadhesive drug carrier. The QCD-g-CSs were synthesized with various βCD moieties ranging from 5 to 23%. Spontaneous inclusion complex of these derivatives and EG were found and confirmed by FTIR and simulation study. Self-aggregated formations of QCD-g-CS were found, according to fluorescence and TEM studies, where the formations were preferable for QCD11g-CS and QCD5-g-CS. EG can be included in both βCD hydrophobic cavity and hydrophobic core of QCD-g-CS self-aggregates, resulting in varying entrapment efficiencies. Degree of QCD substitution on QCD-g-CS plays an important role on their physical properties, due to steric hindrance. The QCD11-g-CS showed excellent mucoadhesion, compared to the QCD5-g-CS and QCD23-g-CS. Moreover, the inclusion complex between QCD-g-CS and EG tend to express higher antimicrobial activities against Candida albicans, Streptococcus oralis and Streptococcus mutans, than the native QCD-g-CS.

Chitosan-triphosphate nanoparticles for encapsulation of super-paramagnetic iron oxide as an MRI contrast agent

Super-paramagnetic iron oxide nanoparticles (SPIONPs) were encapsulated at various concentrations within chitosan-triphosphate (SPIONPs-CS) nanoparticles using an ionotropic gelation method. The encapsulation of SPIONPs within CS nanoparticles enhanced their dispersion ability in aqueous solution, with all particles being lower than 130 nm in size and having highly positive surface charge. The SPIONPs-CS nanoparticles exhibited crystalline structure and super-paramagnetic behavior, as seen in non-encapsulated SPIONPs. The morphology of SPIONPs-CS nanoparticles showed that they almost spherical in shape. The effect of phantom environments (culture medium and 3% agar solution) on either T1 or T2 weighted MRI was investigated using a clinical 1.5T MRI scanner. The results revealed that 3% agar solution showed relaxation values higher than the culture medium, leading to a significant decrease in the MR image intensity. Our results demonstrated that the SPIONPs-CS nanoparticles can be applied as tissue-specific MRI contrast agents.

Methylated N-(4-N,N-dimethylaminobenzyl) chitosan coated liposomes for oral protein drug delivery.

In the present study, methylated N-(4-N,N-dimethylaminobenzyl) chitosan (TM(56)Bz(42)CS) was synthesised and investigated for oral protein drug delivery by combining it with liposomes entrapped with fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA), a model protein. Liposomes (LPs) composed of 10:2 molar ratios of egg yolk phosphatidylcholine (EPC) and sodium oleate (NaO) were prepared by a thin film hydration method and coated with TM(56)Bz(42)CS. BSA-loaded, TM(56)Bz(42)CS-coated liposomes (TM(56)Bz(42)CS-coated FITC-BSA-LP) were evaluated for their protein transport efficiencies and cytotoxicities in Caco-2 cells. Moreover, the in vitro stabilities of the TM(56)Bz(42)CS-coated LP-BSA were determined by examining the degradation of the protein in simulated intestinal fluid containing 1% w/v pancreatin porcine pancreas. The mean particle size and zeta-potential of the TM(56)Bz(42)CS-coated LP-BSA were 128 ± 15 nm and 5.38 ± 1.66 mV, respectively. Additionally, the initial FITC-BSA to lipid ratio (2.5% w/w) showed the highest entrapment efficiency percentage (50.13%) and FITC-BSA content (8.08 mg/g of lipid) overall. The results of the FITC-BSA transport showed that the TM(56)Bz(42)CS-coated FITC-BSA-LP enhanced protein permeability across the Caco-2 cell monolayers with low cytotoxicity. In addition, these liposomes protected against protein degradation in pancreatin. Our studies demonstrated that TM(56)Bz(42)CS-coated liposomes have the potential to be used in oral protein drug delivery methods.

Methylated N-(4-N,N-dimethylaminobenzyl) chitosan for novel effective gene carriers

The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM(47)-Bz(42)-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM(43)-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM(47)-Bz(42)-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM(47)-Bz(42)-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM(43)-CS/DNA and CS/DNA were formed at a ratio of above 1. TM(47)-Bz(42)-CS showed superior transfection efficiency to TM(43)-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM(47)-Bz(42)-CS and TM(43)-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM(47)-Bz(42)-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.

Self-aggregates formation and mucoadhesive property of water-soluble β-cyclodextrin grafted with chitosan.

Water-soluble β-cyclodextrin grafted with chitosan (CD-g-CS) was carried out by quaternizing the CD-g-CS with glycidyltrimethyl ammonium chloride (GTMAC) under mild acidic condition, corresponding to the quaternized CD-g-CS (QCD-g-CS). The degrees of substitution (DS) and quaternization (DQ), ranging from 5% to 23% and 66% to 80%, respectively, were determined by (1)H NMR spectroscopy. Self-aggregates formation of all QCD-g-CSs were investigated in water using dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques. The result revealed that all QCD-g-CSs are able to form self-aggregates in water. Large particle sizes ranged from 800 to 3000nm were obtained by DLS while zeta-potentials were ranging from 25 to 40mV. AFM and TEM depicted a spherical shape with particle sizes ranging from 100 to 900nm. Mucoadhesive and cytotoxic properties of all QCD-g-CSs were evaluated using a mucin particle method and MTT assay compared to quaternized chitosan (QCS). It was found that the mucoadhesive property increased with decreasing DS due to less quaternary ammonium moiety into the chitosan backbone. On the other hand, the cytotoxicity increased with increasing DS even though the DQ is decreased.

Layer-by-layer engineered nanocapsules of curcumin with improved cell activity.

Nanocarriers based on electrostatic Layer-by-layer (LbL) assembly of CaCO3 nanoparticles (CaCO3 NPs) was investigated. These inorganic nanoparticles was used as templates to construct nanocapsules made from films based on two oppositely charged polyelectrolytes, poly(diallyldimethylammonium chloride), and poly (sodium 4-styrene-sulfonate sodium salt), followed by core dissolution. The naked CaCO3 NPs, CaCO3 NPs coated with the polyelectrolytes and hollow nanocapsules were found with hexagonal shape with average sizes of 350-400 nm. A reversal of the surface charge between positive to negative zeta potential values was found, confirming the adsorption of polyelectrolytes. The loading efficiency and release of curcumin were controlled by the hydrophobic interactions between the drug and the polyelectrolyte matrix of the hollow nanocapsules. The quantity of curcumin released from hollow nanocapsules was found to increase under acidic environments, which is a desirable for anti-cancer drug delivery. The hollow nanocapsules were found to localize in the cytoplasm and nucleus compartment of Hela cancer cells after 24 h of incubation. Hollow nanocapsules were non-toxic to human fibroblast cells. Furthermore, curcumin loaded hollow nanocapsules exhibited higher in vitro cell inhibition against Hela cells than that of free curcumin, suggesting that polyelectrolyte based-hollow nanocapsules can be utilized as new carriers for drug delivery.

Methylated N-(4-pyridinylmethyl) chitosan as a novel effective safe gene carrier.

The objective of this study was to study the transfection efficiency of quaternized N-(4-pyridinylmethyl) chitosan; TM-Py-CS, using the pDNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh 7 cells). The factors affecting the transfection efficiency, e.g. degree of quaternization (DQ), the extent of N-pyridinylmethyl substitution (ES) and weight ratio, have been investigated. The results revealed that TM-Py-CS was able to condense with pDNA. Illustrated by agarose gel electrophoresis, complete complexes of TM(69)Py(62)CS/DNA were formed at weight ratio above 1.1, whereas those of TM(53)Py(40)CS/DNA and TM(52)Py(13)CS/DNA were above 1.8 and 8, respectively. TM(69)Py(62)CS showed superior transfection efficiency to TM(53)Py(40)CS, TM(52)Py(13)CS, TM(65)CS and TM(43)CS at all weight ratios tested. The highest transfection efficiency of TM(69)Py(62)CS/DNA complexes was found at weight ratio of 4. The results indicated that the improved gene transfection was possibly due to 4-pyridinylmethyl substitution on CS which promoted the interaction and condensation with DNA as well as N-quaternization which increased CS water solubility. In cytotoxicity studies, high concentration of TM-Py-CS and TM-CS could decrease the Huh 7 cell viability. In conclusion, this novel CS derivative, TM(69)Py(62)CS, showed promising potential as a gene carrier by efficient DNA condensation and mediated higher level of gene transfection.

pH-Responsive polymeric micelles based on amphiphilic chitosan derivatives: Effect of hydrophobic cores on oral meloxicam delivery

The amphiphilic chitosan derivatives, N-naphthyl-N,O-succinyl chitosan (NSCS), N-octyl-N-O-succinyl chitosan (OSCS) and N-benzyl-N,O-succinyl chitosan (BSCS), were synthesized. Meloxicam (MX) was loaded into polymeric micelles (PMs), and the effects of hydrophobic moieties of the inner core segment on the loading efficiency, stability of MX-loaded PMs, cytotoxicity, drug release, and porcine small intestine permeation were investigated. Among the hydrophobic cores, the N-octyl moiety revealed the highest MX loading efficiency and most stable MX-loaded PMs compared to the other hydrophobic cores. All PMs were spherically shaped (size 213-282 nm) and had low toxicity against Caco-2 cells. The release of MX from PMs was found to be dependent on both hydrophobic cores and hydrophilic shells. In acidic medium at 0-2h, low cumulative MX release was obtained in the MX-loaded OSCS PMs compared to MX-loaded NSCS PMs and MX-loaded BSCS PMs as well as MX free drug. However, when the pH was increased to 6.8, the MX release significantly increased in all MX-loaded PMs. Furthermore, the intestinal permeation rates of MX from all MX-loaded PMs were not significantly different. These results suggest that MX was successfully incorporated into the PMs at high efficiency and good stability by optimizing the hydrophobic moieties of the inner core segments.