Nattika Saengkrit

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.

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.

Surface modification of PLGA nanoparticles by carbopol to enhance mucoadhesion and cell internalization

Mucoadhesive poly (lactic-co-glycolic acid) (PLGA) nanoparticles having a modified shell-matrix derived from polyvinyl alcohol (PVA) and Carbopol (CP), a biodegradable polymer coating, to improve the adhesion and cell transfection properties were developed. The optimum formulations utilized a CP concentration in the range of 0.05-0.2%w/v, and were formed using modified emulsion-solvent evaporation technique. The resulting CP-PLGA nanoparticles were characterized in terms of their physical and chemical properties. The absorbed CP on the PLGA shell-matrix was found to affect the particle size and surface charge, with 0.05% CP giving rise to smooth spherical particles (0.05CP-PLGA) with the smallest size (285.90 nm), and strong negative surface charge (-25.70 mV). The introduction of CP results in an enhancement of the mucoadhesion between CP-PLGA nanoparticles and mucin particles. In vitro cell internalization studies highlighted the potential of 0.05CP-PLGA nanoparticles for transfection into SiHa cells, with uptake being time dependent. Additionally, cytotoxicity studies of CP-PLGA nanoparticles against SiHa cancer cells indicated that low concentrations of the nanoparticles were non-toxic to cells (cell viability >80%). From the various formulations studied, 0.05CP-PLGA nanoparticles proved to be the optimum model carrier having the required mucoadhesive profile and could be an alternative therapeutic efficacy carrier for targeted mucosal drug delivery systems with biodegradable polymer.

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.

Multicarboxylic acids as environment-friendly solvents and in situ crosslinkers for chitosan/PVA nanofibers with tunable physicochemical properties and biocompatibility

Monocarboxylic acids are common solvents for chitosan to fabricate nanofibers however the unpleasant odor and the additional step of fiber stabilization using crosslinkers, which might cause toxicity, are always the points to be aware of. The present work demonstrates the potential use of multicarboxylic acids as environment-friendly solvents and in situ crosslinking agents for chitosan electrospinning. The use of these solvents leads to the tunable physicochemical properties, cellular compatibility, and cost effective production. By changing di-, to tri-, and tetracarboxylic acids combining with the simple thermal treatment, the stability and mechanical properties of the nanofibrous mats, especially the elastic modulus and elongation at break, can be altered. The resulting nanofibers exhibit biocompatibility favorable for proliferation and adhesion of the osteoblast cells. The multicarboxylic acids allow us lab-scale reproducibility and possibility to semi-production of nanofibrous chitosan using Nanospider™.

Synthesis of Carbohydrate Capped Silicon Nanoparticles and their Reduced Cytotoxicity, In Vivo Toxicity, and Cellular Uptake

The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate-mediated molecular recognitions using nano-vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells.

Phospholipid-chitosan hybrid nanoliposomes promoting cell entry for drug delivery against cervical cancer

This study emphasizes the development of a novel surface modified liposome as an anticancer drug nanocarrier. Quaternized N,O-oleoyl chitosan (QCS) was synthesized and incorporated into liposome vesicles, generating QCS-liposomes (Lip-QCS). The Lip-QCS liposomes were spherical in shape (average size diameter 171.5±0.8nm), with a narrow size distribution (PDI 0.1±0.0) and zeta potential of 11.7±0.7mV. In vitro mucoadhesive tests indicated that Lip-QCS possesses a mucoadhesive property. Moreover, the presence of QCS was able to induce the cationic charge on the surface of liposome. Cellular internalization of Lip-QCS was monitored over time, with the results revealing that the cell entry level of Lip-QCS was elevated at 24h. Following this, Lip-QCS were then employed to load cisplatin, a common platinum-containing anti-cancer drug, with a loading efficiency of 27.45±0.78% being obtained. The therapeutic potency of the loaded Lip-QCS was investigated using a 3D spheroid cervical cancer model (SiHa) which highlighted their cytotoxicity and apoptosis effect, and suitability as a controllable system for sustained drug release. This approach has the potential to assist in development of an effective drug delivery system against cervical cancer.

Physical and biological characterization of sericin-loaded copolymer liposomes stabilized by polyvinyl alcohol.

Sericin protein (SP) is widely used as a nutrient biomaterial for biomedical and cosmeceutical applications although it shows low stability to heat and light. To overcome these problems and add value to wastewater from the silk industry, sericin protein was recovered as sericin-loaded copolymer-liposomes (SP-PVA-LP), prepared through thin film hydration. The size and morphology of the liposomes were investigated using dynamic light scattering (DLS), and electron microscopy (SEM and TEM). The particle size, liposome surface morphology and encapsulation efficiency of SP were dependent on PVA concentration. The hydrodynamic size of the nanoparticles was between 200 and 400nm, with the degree of negative charge contingent on sericin loading. SEM and TEM images confirmed the mono-dispersity, and spherical nature of the particles, with FTIR measurements confirming the presence of surface bound PVA. Exposure of liposomes to 500ppm sericin highlighted a dependence of encapsulation efficiency on PVA content; 2% surface PVA proved the optimal level for sericin loading. Cytotoxicity and viability assays revealed that SP-loaded surface modified liposomes promote cellular attachment and proliferation of human skin fibroblasts without adverse toxic effects. Surface modified copolymer liposomes show high performance in maintaining structural stability, and promoting enhancements in the solubility and bio-viability of sericin. Taken together, these biocompatible constructs allow for effective controlled release, augmenting sericin activity and resulting in effective drug delivery systems.

Acceleration of gene transfection efficiency in neuroblastoma cells through polyethyleneimine/ poly(methyl methacrylate) core-shell magnetic nanoparticles

Background The purpose of this study was to demonstrate the potential of magnetic poly(methyl methacrylate) (PMMA) core/polyethyleneimine (PEI) shell (mag-PEI) nanoparticles, which possess high saturation magnetization for gene delivery. By using mag-PEI nanoparticles as a gene carrier, this study focused on evaluation of transfection efficiency under magnetic induction. The potential role of this newly synthesized nanosphere for therapeutic delivery of the tryptophan hydroxylase-2 (TPH-2) gene was also investigated in cultured neuronal LAN-5 cells. Methods The mag-PEI nanoparticles were prepared by one-step emulsifier-free emulsion polymerization, generating highly loaded and monodispersed magnetic polymeric nanoparticles bearing an amine group. The physicochemical properties of the mag-PEI nanoparticles and DNA-bound mag-PEI nanoparticles were investigated using the gel retardation assay, atomic force microscopy, and zeta size measurements. The gene transfection efficiencies of mag-PEI nanoparticles were evaluated at different transfection times. Confocal laser scanning microscopy confirmed intracellular uptake of the magnetoplex. The optimal conditions for transfection of TPH-2 were selected for therapeutic gene transfection. We isolated the TPH-2 gene from the total RNA of the human medulla oblongata and cloned it into an expression vector. The plasmid containing TPH-2 was subsequently bound onto the surfaces of the mag-PEI nanoparticles via electrostatic interaction. Finally, the mag-PEI nanoparticle magnetoplex was delivered into LAN-5 cells. Reverse-transcriptase polymerase chain reaction was performed to evaluate TPH-2 expression in a quantitative manner. Results The study demonstrated the role of newly synthesized high-magnetization mag-PEI nanoparticles for gene transfection in vitro. The expression signals of a model gene, luciferase, and a therapeutic gene, TPH-2, were enhanced under magnetic-assisted transfection. An in vitro study in neuronal cells confirmed that using mag-PEI nanoparticles as a DNA carrier for gene delivery provided high transfection efficiency with low cytotoxicity. Conclusion The mag-PEI nanoparticle is a promising alternative gene transfection reagent due to its ease of use, effectiveness, and low cellular toxicity. The mag-PEI nanoparticle is not only practical for gene transfection in cultured neuronal cells but may also be suitable for transfection in other cells as well.