Shixiang Hou

Trans-resveratrol loaded chitosan nanoparticles modified with biotin and avidin to target hepatic carcinoma.

Conventional liver targeted system focuses on delivering drugs to liver, bringing toxicity on hepatic normal tissues. The purpose of this study is to construct a new system capable of specially targeting to hepatic carcinoma instead of the whole liver. Based on the fact that nanoparticles (NPs) bound with either biotin or avidin tend to accumulate in tumors and avidin-attached reagents were quickly eliminated from blood circulation and assembled in liver, trans-resveratrol loaded chitosan nanoparticles (CS-NPs), CS-NPs with the surface modified either by biotin (B-CS-NPs) or by both biotin and avidin (A-B-CS-NPs) were prepared and their physiochemical properties were investigated. The in vitro release profiles of the three NPs all conformed to bioexponential equation. Pharmacokinetic experiment indicated that A-B-CS-NPs rapidly assembled in liver after injection, with the highest liver targeting index of 2.70, while the modification of biotin attenuated the liver targeting ability of NPs. Inhibitory study on HepG2 cells declared that compared to trans-resveratrol solution and CS-NPs, both B-CS-NPs and A-B-CS-NPs significantly improved the anticancer activity. When incubated with HepG2 cells at high concentration for longer time, A-B-CS-NPs exhibited superior cytotoxicity than B-CS-NPs. This study exclaims that A-B-CS-NPs may be a potent drug delivery vector specially targeting to hepatic carcinoma.

Receptor mediated gene delivery by folate conjugated N-trimethyl chitosan in vitro

Folate conjugated N-trimethyl chitosan (folate-TMC) that was used for intracellular delivery of protein before was studied as a gene delivery carrier in this study using N-trimethyl chitosan (TMC) as a reference. MTT assay indicated that the two polymers were much less toxic than PEI. Agarose gel electrophoresis indicated that the two polymers effectively condensed pDNA. TMC/pDNA complex and folate-TMC/pDNA complex were nano-scale spherical particles confirmed by atomic force microscopy. Cellular uptake of the folate-TMC/pDNA complex containing YOYO-1 labeled pDNA in KB cells was enhanced compared with that of the TMC/pDNA complex and was inhibited by free folate (1 mM) in the medium. Transfection efficiency of the folate-TMC/pDNA complex in KB cells and SKOV3 cells (folate receptor over-expressing cell lines) increased with increasing N/P ratio and were enhanced up to 1.6-fold and 1.4-fold compared with those of the TMC/pDNA complexes, however, there was no significant difference between transfection efficiencies of the two complexes in A549 cells and NIH/3T3 cells (folate receptor deficient cell lines). It was concluded that the increase in transfection efficiencies of the folate-TMC/pDNA complexes were attributed to folate receptor mediated endocytosis. Subcellular distributions of both of the complexes at different time points in the process of cellular uptake were examined by confocal laser scanning microscope, which suggested that different intracellular trafficking pathways were employed by the two complexes.

Preparation and characterization of folate conjugated N-trimethyl chitosan nanoparticles as protein carrier targeting folate receptor: in vitro studies.

Folate conjugated N-trimethyl chitosan (folate-TMC) was synthesized and characterized using Fourier transform infrared (FTIR) and 1H spectroscopy. The fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) loaded TMC-nanoparticle (FB-TMC-NP) and FITC-BSA loaded folate-TMC-nanoparticle (FB-f-TMC-NP) were prepared by ionic cross-linking of TMC or folate-TMC with sodium alginate. Single factor analysis method was used to optimize the formulation of nanoparticles. The encapsulating efficiencies of FB-TMC-NP and FB-f-TMC-NP produced by optimal formulation were 98.3 ± 1.9% and 98.7 ± 2.7% (n=3), respectively. In addition, the mean diameters of FB-TMC-NP and FB-f-TMC-NP were 184.3 ± 8.3 nm and 176.1 ± 5.0 nm (n = 3), respectively. Transmission electron microscope (TEM) showed that the nanoparticles were of spherical shapes. The intracellular uptake of FB-f-TMC-NP by SKOV3 cells (folate receptor overexpressing cells) was 3.7-fold more than that of FB-TMC-NP and could be inhibited by the presence of 1 mM folate in the culture medium, although there was no significant difference between the intracellular uptake of FB-f-TMC-NP in A549 cells (folate receptor–deficient cells) and that of FB-TMC-NP in the same cells. In conclusion, the enhancement of cellular uptake of FB-f-TMC-NP by SKOV3 cells in a specific way was attributed to the folate receptor–mediated endocytosis. FB-TMC-NP was a promising carrier for protein.

Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis.

To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

Quantification of aesculin in rabbit plasma and ocular tissues by high performance liquid chromatography using fluorescent detection: application to a pharmacokinetic study.

A simple and sensitive high performance liquid chromatography method with fluorescence detection (HPLC-FD) was described for the determination of aesculin (AL) at low concentrations in rabbit plasma and ocular tissues. After deproteinization by methanol using pazufloxacin mesilate (PM) as an internal standard (I.S.), supernatants were evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in mobile phase and a volume of 20μL was injected into the HPLC for analysis. Analytes were separated on an Ultimate XB-C18 column (250mm × 4.6mm i.d., 5μm particle size) and protected by a ODS guard column (10mm × 4.0mm i.d., 5μm particle size), using acetonitrile-0.1% triethylamine in water (adjusted to pH 3.0 using phosphoric acid) (12:88, v/v) as mobile phase with a flow rate of 1.0mL/min. The wavelengths of fluorescence detector (FD) were set at 344nm for excitation and 466nm for emission. The lower limit of quantitation (LOQ) for AL was 0.80ng/mL for plasma and vitreous body, 1.59ng/mL for aqueous humor, and 6.55ng/g for iris and 1.66ng/g for retina. The method was used in the study of AL concentrations in plasma and ocular tissues after topical administration of AL eye drops.

Receptor-mediated gene delivery by folate-poly(ethylene glycol)-grafted-trimethyl chitosan in vitro.

Folate-poly(ethylene glycol)-grafted-trimethyl chitosan (F-PEG-g-TMC) and methoxypolyethylene glycol-grafted-trimethyl chitosan (mPEG-g-TMC)/pDNA complexes were prepared and characterized concerning physicochemical properties including cytotoxicity, condensation efficiency, particle size, and zeta potential. Furthermore, cellular uptake and transfection efficiency of the complexes were evaluated in vitro and compared with that of folate-trimethyl chitosan (folate-TMC) synthesized by our group to elucidate the effect of PEGylation. The cellular uptake of the F-PEG-g-TMC/pDNA with a copolymer nitrogen-to-DNA phosphate ratio (N/P ratio) of 20 in KB cells was specifically increased up to 1.68-fold compared with that of the mPEG-g-TMC/pDNA (N/P ratio 20) resulting in 1.5-fold and 1.4-fold increased transfection efficiency in KB cells and SKOV3 cells (folate receptor-overexpressing cell lines), respectively. The intracellular uptake and transfection efficiency of the F-PEG-g-TMC/pDNA were significantly enhanced relative to the folate-TMC/pDNA in folate receptor-overexpressing cells due to stabilizing effect of PEGylation. Subcellular localization of the complexes in the process of intracellular transportation was observed by confocal laser scanning microscopy suggesting quicker association of the F-PEG-g-TMC/pDNA. In conclusion, the F-PEG-g-TMC/pDNA complexes are potential vehicles for improving the transfection efficiency and specificity of gene.

The effect of deacetylated gellan gum on aesculin distribution in the posterior segment of the eye after topical administration

The aim of the present work was to evaluate the effect of deacetylated gellan gum on delivering hydrophilic drug to the posterior segment of the eye. An aesculin-containing in situ gel based on deacetylated gellan gum (AG) was prepared and characterized. In vitro corneal permeation across isolated rabbit cornea of aesculin between AG and aesculin solution (AS) was compared. The results showed that deacetylated gellan gum promotes corneal penetration of aesculin. Pharmacokinetics and ocular tissue distribution of aesculin after topical administration in rabbit eye showed that AG greatly improved aesculin accumulation in posterior segmentsrelative to AS, which was probably attributed to conjunctivital/sclera pathway. The area-under-the-curve (AUC) for AG in aqueous humor, choroid-retina, sclera and iris-ciliary body were significantly larger than those of AS. AG can be used as a potential carrier for broading the application of aesculin.