Somsak Saesoo

Influence of curcumin-loaded cationic liposome on anticancer activity for cervical cancer therapy.

The delivery of curcumin has been explored in the form of liposomal nanoparticles to treat various cancer cells. Since curcumin is water insoluble and an effective delivery route is through encapsulation in liposomes, which were modified with three components of DDAB, cholesterol and non-ionic surfactant. The purpose of this study was to establish a critical role of DDAB in liposomes containing curcumin at cellular response against two types of cell lines (HeLa and SiHa). Here, we demonstrate that DDAB is a potent inducer of cell uptake and cell death in both cell lines. The enhanced cell uptake was found on DDAB-containing liposome, but not on DDAB-free liposome. However, the cytotoxicity of DDAB-containing liposomes was high and needs to be optimized. The cytotoxicity of liposomal curcumin was more pronounced than free curcumin in both cells, suggesting the benefits of using nanocarrier. In addition, the anticancer efficiency and apoptosis effect of the liposomal curcumin formulations with DDAB was higher than those of DDAB-free liposomes. Therefore curcumin loaded liposomes indicate significant potential as delivery vehicles for the treatment of cervical cancers.

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.

Hydroxyapatite-hybridized chitosan/chitin whisker bionanocomposite fibers for bone tissue engineering applications

Biomimetic nanofibrous scaffolds derived from natural biopolymers for bone tissue engineering applications require good mechanical and biological performances including biomineralization. The present work proposes the utility of chitin whisker (CTWK) to enhance mechanical properties of chitosan/poly(vinyl alcohol) (CS/PVA) nanofibers and to offer osteoblast cell growth with hydroxyapatite (HA) mineralization. By using diacid as a solvent, electrospun CS/PVA nanofibrous membranes containing CTWK can be easily obtained. The dimension stability of nanofibrous CS/PVA/CTWK bionanocomposite is further controlled by exposing to glutaraldehyde vapor. The nanofibrous membranes obtained allow mineralization of HA in concentrated simulated body fluid resulting in an improvement of Youngs modulus and tensile strength. The CTWK combined with HA in bionanocomposite is a key to promote osteoblast cell adhesion and proliferation. The present work, for the first time, demonstrates the use of CTWKs for bionanocomposite fibers of chitosan and its hydroxyapatite biomineralization with the function in osteoblast cell culture. These hydroxyapatite-hybridized CS/PVA/CTWK bionanocomposite fibers (CS/PVA/CTWK-HA) offer a great potential for bone tissue engineering applications.

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.

Synthesis and characterization of pH-responsive N-naphthyl-N,O-succinyl chitosan micelles for oral meloxicam delivery

The aim of this study was to synthesize pH responsive chitosan and to evaluate the influence of drug-loaded micelle methods on loading efficiency, particle size and micelle stability. N-naphthyl-N,O-succinyl chitosan (NSCS) was successfully synthesized and meloxicam (MX) was loaded into the inner core of the NSCS micelles by physical entrapment methods (dialysis, O/W emulsion, dropping and evaporation) with a regular spherical shape (particle size 84-382nm). MX-loaded micelles by evaporation method showed the highest entrapment efficiency. The stability of the drug-loaded micelles depended on not only the methods but also the initial of drug. NSCS micelles are less toxic on Caco-2 cells. In acidic medium at 0-2h, percentage cumulative release of MX from MX-loaded micelles was similar to free drug. When the pH was adjusted to pH 6.8, the MX release was increased significantly. Therefore, this NSCS micelle would be desirable to develop MX carrier for oral drug delivery.

Synthesis and anticervical cancer activity of novel pH responsive micelles for oral curcumin delivery.

Curcumin (CM) has demonstrated safety and efficacy as a drug, but its pharmaceutical role is restricted as a result of extremely low aqueous solubility, rapid systemic elimination, inadequate tissue absorption and degradation at alkaline pH; properties that severely curtail its bioavailability. To address this issue, CM was encapsulated within pH responsive amphiphilic chitosan, resulting in the formation of 100 nm spontaneously self-assembled polymeric micelles in water. The amphiphilic chitosan, namely N-benzyl-N,O-succinyl chitosan (BSCS), was prepared by reductive N-benzylation and N,O-succinylation. The stability of micelles after being re-dispersed in water was investigated using glycine as a cryoprotectant, and the average sizes were shown to be maintained at a level lower than 200 nm for up to 4 months, at temperatures of 4°C and 25°C. In vitro drug release results showed that CM was slowly released from the micelles without any burst effect in the intestine (pH 5.5-7.4), with limited release in the stomach (pH 1.2). Cytotoxicity assays indicated that CM loaded micelles showed half maximal inhibitory concentrations (IC50) 4.7-, 3.6-, and 12.2-fold lower than that of free CM in HeLa, SiHa and C33a cervical cell lines, respectively. Cellular uptake of micelles was confirmed by confocal laser scanning microscopy and flow cytometry, with a 6-fold significant increase in the amount of CM loaded micelles compared to free CM in all cervical cancer cells. Notably, CM loaded micelles promoted an increase (30-55%) in the percentage of early apoptosis of HeLa, SiHa and C33a cells, compared to free CM. These results suggest that BSCS micelles may be a promising carrier for effective oral delivery of CM.

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells.

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(®), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.

The PEI-introduced CS shell/PMMA core nanoparticle for silencing the expression of E6/E7 oncogenes in human cervical cells.

In this study, we examined the potential of cationic nanoparticle - polyethyleneimine-introduced chitosan shell/poly (methyl methacrylate) core nanoparticles (CS-PEI) for siRNA delivery. Initially, DNA delivery was performed to validate the capability of CS-PEI for gene delivery in the human cervical cancer cell line, SiHa. siRNA delivery were subsequently carried out to evaluate the silencing effect on targeted E6 and E7 oncogenes. Physicochemical properties including size, zeta potential and morphology of CS-PEI/DNA and CS-PEI/siRNA complexes, were analyzed. The surface charges and sizes of the complexes were observed at different N/P ratios. The hydrodynamic sizes of the CS-PEI/DNA and CS-PEI/siRNA were approximately 300-400 and 400-500nm, respectively. Complexes were positively charged depending on the amount of added CS-PEI. AFM images revealed the mono-dispersed and spherical shapes of the complexes. Gel retardation assay confirmed that CS-PEI nanoparticles completely formed complexes with DNA and siRNA at a N/P ratio of 1.6. For DNA transfection, CS-PEI provided the highest transfection result. Localization of siRNA delivered through CS-PEI was confirmed by differential interference contrast (DIC) confocal imaging. The silencing effect of siRNA specific to HPV 16 E6/E7 oncogene was examined at 18 and 24h post-transfection. The results demonstrated the capacity of CS-PEI to suppress the expression of HVP oncogenes.