Studies On Gene Delivery System Mediated By A Novel Chitosan Oligosaccharide Grafted Stearic Acid Micelles

With the implementation of Human Gene Project (HGP) and the further identification of genetical materials cause disease, gene therapy represents a new and promising therapeutics modality. The principal problem of gene therapy is focused on transferring genetic material into targeted cells. However, these genetic materials (e.g. DNA, RNA) process several inherent characteristics of hydrophilic, polyanionic and large macromoleculars, which restrict the poor permeation of cell membranes. In addition, the rapid degradation by nuclease leads to the short biological half-life. Due to these challenges, one of the most urgent tasks that scientists are devoted to is developing the gene delivery system. Efficient gene transfer is a key factor in gene therapy. Two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. In general, viruses are very efficient gene-transfer vehicles;however, the signification problem of security and immunogenicity caused by viral vectors are limited to their use. Non-viral gene delivery systems have recently received increasing interest due to safety concerns with viral vectors. A number of non-viral gene delivery vectors including cationic liposome, nature and synthetic polymers have been introduced as alternatives against viral vectors due totheir potential advantages of low immunogenicity and ease to preparation.Polymer micelles are formed by self-assembly of amphiphilic copolymer in aqueous solution, which followed by the development of emulsion, liposomes, microparticles and nanoparticles. Over the past decades, polymer micelles have been considered as attractive drug carriers, because of nanoscaled large and desirable features in drug delivery system-such as high drug-loading, controlled release, biocompatible, permeability to biological membranes. Polymer micelles have many advantages in the drug targets delivery system. Due to the leakiness of the tumor blood vessels (relative to the healthy blood vessels in most other organs), colloidal particles preferentially accumulate in the tumor through permeability and retention effect. After chemically bound ("grafted") by cell surface-binding ligand or antiboby (folic acid et al.), polymer micelles can target to particular tissues, cells (especially tumor cells).Chitosan oligosaccharide (CSO) was prepared by enzymatic degradation and ultrafiltrated separation. Mediated by a l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), stearic acid (SA) was covalently attached to CSO, and the graft polymer (CSO-SA) was obtained. The critical aggregation concentration (CAC) of the CSO-SA was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The micellar size distribution and Zeta-potential, measured by light scattering and electrophoretic mobility, was investigated. Using pulmonary epithelium cell A549 as model cell, viability determinations of carreries were performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyl-tetrazolium bromide (MTT) assay. The average hydrodynamic diameter and zeta potential of CSO-SA/DNA complex nanoparticles were measured by dynamic light scattering using a Zetasizer. The morphological examinations of Plasmid DNA, CSO-SAmicelle and CSO-SA/DNA complex nanoparticles were performed by atomic force microscopy (AFM). The in vitro transfection efficiency of CSO-SA micelles was investigated by using plasmid DNA (pEGFP-Cl). Covalent conjugation with fluorescein isothiocyanate (FITC) yielded FITC-labeled CSO-SA graft copolymer and covalent conjugation fluorescein Rhodamine(Rh) yielded Rh-labeled DNA to determine whether the FITC-CSO-SA/DNA-Rh complexes internalized in cells using fluorescence microscopy.After enzymatic hydrolysis and ultra-filtration by various molecular weight cut off membrane, chitosan oligosaccharide (weight average molecular weight, Mw 18.4 Kda and the polydispersity, (.Mw /Wn) 1.94 were obtained in these work. The hydrophobic chain provided by SA was randomly bound to a portion of the hydrophilic amido of CSO by l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) mediated coupling reaction. The degree of amino substitute are 5.0 %, 15.4 %, 17.5% , 25.3%, 40.0 % measured by TNBS method.By a fluorometry in the presence of pyrene as a fluorescent probe, the aggregation behavior of CSO-SA micelles in aqueous solution was investigated. The critical aggregating concentration of CSO-SA is 0.035 mg ? ml/1. When the concentration of CSO-SA is lmg ? ml/1 in distilled water, the zeta potential of micelles have positive charges and with the value of 46.4±0.1mV. Quasi-spherical shape of CSO-SA micelle in aqueous environment was observed by AFM.The in vitro transfection efficiency of CSO-SA micelles was investigated by using plasmid DNA (pEGFP-Cl). The micelles could compact the plasmid DNA to form micelle/DNA complexes nanoparticles, which can efficiently protect the condensed DNA from enzymatic degradation by DNase I. The volume average hydrodynamic diameter of CSO-SA micelle/DNA complex increased from 203 nm to 318 nm anddecreased to 102 nm due to the variation of zeta potential when the N/P (The N/P ratio was calculated from the number of unreacted free primary amines of CSO-SA and the number of phosphate groups of DNA) ratio increased from 0.25 to 3.6 and from 3.6 to 58. The transfection efficiency with CSO-SA/DNA (N/P ratio is 29) was increased with the post-transfection time (in 76 h), while the optimal transfection of Lipofectamine?2000/DNA was obtained at 24 h. The transfection of CSO-SA was not interfered in the presence of 10 % fetal bovine serum, which showed remarkable enhancement effect. The optimal transfection efficiency of CSO-SA micelles in A549 cells was about 15 %, which was higher than that of CSO (about 2%) and approach to that of Lipofectamine?2000 (about 20%).In particular, CSO and SA were chosen as hydrophilic and hydrophobic segments due to their biodegradable nature and low toxicity. The IC50 value of the CSO-SA micelle against A549 cells was 543.16 μg?mL"1, while the IC50 of Lipofectamine?2000 was about 6 μg ? ml/1, which indicated that the micelles have superiorities of security and low toxicity and can be used as a drug carrier.To determine whether the micelles internalize in cells, green-fluorescent micelles were employed in our work, which was made from FITC-labeled CSO-SA graft copolymer. Typical CSO chain consisted of 106 units of monosaccharide covalently graft by 16 units of SA and 0.8 units of fluorescein isothiocyanate, which means 80 per 100 the free anime groups of CSO segment was not substituted. Thus, FITC-labeled micelles still have positive charges and with the value of 41.3±0.6mV.To further clear the mechanism of CSO-SA-mediated gene transfer. In this study, we examined the intracellular trafficking of CSO-SA/DNA complexes by using A549 cells, fluorescent probe-labeled materials, and microscopy. We found that the CSO-SA/DNA complexes were taken up into cells at 15~30 min. After 1 h, some ofthe DNA and CSO-SA complex was preferentially transferred into the nucleus in the form of the complex. After 2 h, almost all of the CSO-SA/DNA complexes transferred into the nucleus in the form of the complex, and after 4 h, DNA became detached from CSO-SA in the nucleus.