Galactosylated chitosan-graft-polyethylenimine as a gene carrier for hepatocyte targeting.

Chitosans have been proposed as alternative, biocompatible cationic polymers for nonviral gene delivery. However, the low transfection efficiency and low specificity of chitosan need to be addressed before clinical application. We prepared galactosylated chitosan-graft-polyethylenimine (GC-g-PEI) copolymer by an imine reaction between periodate-oxidized GC and low-molecular-weight PEI. The molecular weight and composition were characterized using gel permeation chromatography column with multi-angle laser scattering and (1)H nuclear magnetic resonance, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. GC-g-PEI showed good DNA-binding ability and superior protection of DNA from nuclease attack and had low cytotoxicity compared to PEI 25K. GC-g-PEI/DNA complexes showed higher transfection efficiency than PEI 25K in both HepG2 and HeLa cell lines. Transfection efficiency into HepG2, which has asialoglycoprotein receptors, was higher than that into HeLa, which does not. GC-g-PEI/DNA complexes also transfected liver cells in vivo after intraperitoneal (i.p.) administration more effectively than PEI 25K. These results suggest that GC-g-PEI can be used in gene therapy to improve transfection efficiency and hepatocyte specificity in vitro and in vivo.

Receptor-mediated gene delivery using chemically modified chitosan.

Chitosan has been investigated as a non-viral vector because it has several advantages such as biocompatibility, biodegradability and low toxicity with high cationic potential. However, the low specificity and low transfection efficiency of chitosan need to be solved prior to clinical application. In this paper, we focused on the galactose or mannose ligand modification of chitosan for enhancement of cell specificity and transfection efficiency via receptor-mediated endocytosis in vitro and in vivo.

Water-soluble and low molecular weight chitosan-based plasmid DNA delivery.

PURPOSE: Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery because of its high positive charges and low cytotoxicity. In this study, low molecular weight chitosan (LMWC, molecular weight of 22 kDa) was characterized and evaluated as a gene carrier. METHODS: Plasmid/LMWC complex was analyzed in 1% agarose gel electrophoresis. To confirm that the LMWC protected plasmids from nuclease. DNase I protection assays were performed. pSV-beta-galactosidase plasmid/LMWC complex was transfected into 293T cells and transfection efficiency was evaluated by beta-galactosidase assay. Cytotoxicity of LMWC was determined by MTT assay. RESULTS: Unlike high molecular weight chitosan (HMWC), LMWC is highly water soluble, and can form complex with plasmids in physiological buffer. The plasmid DNA was completely retarded at a weight ratio of 1:2 (plasmid:LMWC) in 1% agarose gel. DNase I protection assay showed that plasmids were protected from DNase-I over 60 min. The most efficient transfection was obtained at a weight ratio of 1:3 (plasmid:LMWC). The transfection efficiency of LMWC was significantly higher than naked DNA and higher than poly-L-lysine (PLL). MTT assay showed that LMWC was less cytotoxic than PLL. CONCLUSIONS: LMWC is non-toxic and has higher transfection efficiency than PLL. Therefore, LMWC will be useful in the development of safe gene carriers.

Effect of solvent on the preparation of surfactant-free poly(DL-lactide-co-glycolide) nanoparticles and norfloxacin release characteristics.

The surfactant-free nanoparticles of poly(DL-lactide-co-glycolide) (PLGA) were prepared by dialysis method without surfactant and physicochemical properties such as particle size and drug contents were investigated against used initial solvent. The size of PLGA nanoparticles and drug contents were significantly changed by used initial solvent. The size of PLGA nanoparticles prepared from dimethylacetamide (DMAc), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) as a initial used solvent was smaller than that of acetone. Selected initial solvent used to dissolve the copolymer significantly affects the size of nanoparticles and drug contents. It was shown that PLGA nanoparticles have spherical shapes from the results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. It was thought that surfactant-free nanoparticles of PLGA entrapping norfloxacin (NFX) has nice drug loading capacity without free-drug on the surface of nanoparticles through the analysis of X-ray powder diffraction. From these results, it was showed the potential that the PLGA nanoparticles could be formed successively by dialysis method without surfactant. Release kinetics of NFX used as a model drug was governed by not only drug contents but also particle size parameter. The higher the drug contents and the larger the particle size resulted in slower the drug release.

Clonazepam release from core-shell type nanoparticles of poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) triblock copolymers.

The triblock copolymer based on poly(epsilon-caprolactone) (PCL) as hydrophobic part and poly(ethylene glycol) (PEG) as hydrophilic one was synthesized and characterized. Core-shell type nanoparticles of poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) (CEC) block copolymer were prepared by a dialysis technique. According to the amphiphilic characters, CEC block copolymer can self-associate at certain concentration and their critical association concentration (CAC) was determined by fluorescence probe technique. CAC value of the CEC-2 block copolymer was evaluated as 0.0030 g/l. CAC values of CEC block copolymer decreased with the increase of PCL chain length, i.e. the shorter the PCL chain length, the higher the CAC values. From the observation of transmission electron microscopy (TEM), the morphologies of CEC-2 core-shell type nanoparticles were spherical shapes. Particle size of CEC-2 nanoparticles was 32.3+/-17.3 nm as a monomodal and narrow distribution. Particle size, drug loading, and drug release rate of CEC-2 nanoparticles were changed by the initial solvents and the molecular weight of CEC. The degradation behavior of CEC-2 nanoparticles was observed by 1H NMR spectroscopy. It was suggested that clonazepam (CNZ) release kinetics were dominantly governed by diffusion mechanism.

Adriamycin release from flower-type polymeric micelle based on star-block copolymer composed of poly(gamma-benzyl L-glutamate) as the hydrophobic part and poly(ethylene oxide) as the hydrophilic part.

Star-block copolymer based on PBLG as the hydrophobic part and PEO as the hydrophilic one (as abbreviated GEG) was synthesized and characterized. Polymeric micelle was prepared by the diafiltration method. From the measurement of photon correlation spectroscopy, the nanoparticle sizes of GEG-1, GEG-2 and GEG-3 were 106.5+/-59.2, 43.8+/-0.7 and 13.5+/-1.0 nm in number average, respectively, indicating of the formation of polymeric micelle. Also, the nanoparticle sizes were dependent on the PBLG chain length, i.e. the more PBLG content in the copolymer, the larger the particle size. From the observation of transmission electron microscope(TEM), GEG-2 block copolymer had almost spherical shapes with size range about 20-70 nm, that was similar to particle size measurement. Fluorescence spectroscopy measurement indicated that GEG block copolymers associated in water to form polymeric micelles and critical micelle concentration (CMC) values of the block copolymers decreased with increasing PBLG chain length in the block copolymer. Characteristic peaks of the protons of the benzyl group in the PBLG and the methylene protons adjacent to the benzyl group of the PBLG segment in the GEG-2 nanoparticles appeared in 7.2 approximately 7.4 and 5.0 approximately 5.2 ppm, respectively, and disappeared in D(2)O, indicating the restricted motions of these protons within the micellar core and the very rigid structure of the PBLG core in the GEG polymeric micelles. Release of ADR from the polymeric micelles in vitro was slower in longer PBLG chain length and higher loading contents of ADR.

Self-assembling nanospheres of hydrophobized pullulans in water.

In this report, we have prepared self-assembling nanospheres of hydrophobized pullulans. Pullulan acetate as a hydrophobized pullulan was synthesized by acetylation of pullulan and characterized by Fourier transform infrared (FTIR) measurement. From the results of photon correlation spectroscopy (PCS), hydrophobized pullulans could be self-assembled in water as nanospherical aggregates, and their number-average particle size was 74.3 +/- 38.2 nm with a unimodal distribution. Also, morphological studies observed by transmission electron microscopy (TEM) showed that self-assembly of hydrophobized pullulans results in nice spherical shapes with a size range of about 50-100 nm, which was in accordance with PCS measurements. Their size and morphology have acceptable properties for intravenous injectable drug-targeting carriers. The fluorescence probe technique was used for self-association of hydrophobized pullulans in water using pyrene as a hydrophobic probe. From the fluorescence measurement, the fluorescence intensity of pyrene increased with increasing concentration of hydrophobized pullulans, which indicates self-assembly formation of hydrophobized pullulans in water. Also, in the fluorescence excitation spectrum, a red shift was observed with increasing concentration of hydrophobized pullulans. These results also revealed that hydrophobized pullulans could be self-assembled in water, and from the plot of I337/I334 versus log c of hydrophobized pullulans, the critical association concentration was 0.0022 g/l, which was considerably lower than that of low molecular weight surfactants or poloxamer. A drug loading study was performed using clonazepam (CNZ) as a hydrophobic model drug. We observed that the higher the feeding amount of drug was, the more the drug loading contents were, the lower the drug loading efficiency was, and the larger the particle size was. CNZ was released from nanospheres via pseudo-zero-order kinetics, and the increased drug loading contents led to slower release of the drug.

Clonazepam release from core-shell type nanoparticles in vitro.

Block copolymers consisting of poly(gamma-benzyl L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene oxide) (PEO) as the hydrophilic block were synthesized and characterized. Core-shell type nanoparticles of the block copolymers (abbreviated as GE) were prepared by the diafiltration method. The particle size diameter obtained by dynamic light scattering of GE-1 (PBLG content: 60.5 mol%), GE-2 (PBLG content: 40.0 mol %), GE-3 (PBLG content: 124.4 mol %) copolymer was 309.9 +/- 160.9, 251.9 +/- 220.6 and 200.5 +/- 177.1nm, respectively. The shape of the nanoparticles by SEM or TEM was almost spherical. The critical micelle concentration of the block copolymers obtained by fluorescence spectroscopy was dependent on the chain length of hydrophobic PBLG. The micelle structure of the copolymers nanoparticle was very stable against sodium dodecyl sulfate. Clonazepam (CZ) was loaded onto the core part of the nanoparticle as the crystalline state. Release of CZ from the nanoparticles in vitro was dependent on the drug loading contents and PBLG chain length.

Clonazepam release from poly(DL-lactide-co-glycolide) nanoparticles prepared by dialysis method.

Aim of this work is to prepare poly(DL-lactide-co-glycolide) (PLGA) nanoparticles by dialysis method without surfactant and to investigate drug loading capacity and drug release. The size of PLGA nanoparticles was 269.9 +/- 118.7 nm in intensity average and the morphology of PLGA nanoparticles was spherical shape from the observation of SEM and TEM. In the effect of drug loading contents on the particle size distribution, PLGA nanoparticles were monomodal pattern with narrow size distribution in the empty and lower drug loading nanoparticles whereas bi- or trimodal pattern was showed in the higher drug loading ones. Release of clonazepam from PLGA nanoparticles with higher drug loading contents was slower than that with lower loading contents.