| In this dissertation, a novel glycolipid compound, stearic acid grafted chitosan oligosaccharide (CSO-SA) was synthesized by the chemical reaction between the amino group of chitosan oligosaccharide and the carboxyl group of stearic acid, based on the research of stearic acid solid lipid nanoparticles drug delivery system, and the nuclear transporting tendency of polysaccharide. The micelles in aqueous medium were prepared by the self-aggregation of the CSO-SA availing its amphiphility. The research of micelle/protein drug complex drug delivery system was conducted by utilizing the cationic property of CSO-SA micelle. Lipophilic anti-tumor drug was then loaded into CSO-SA micelle base on the hydrophobic characteristic of its core, and the drug release behavior was controlled by the modification of micellar surface. After the mechanism research of the cellular uptake and cellular nuclear import of CSO-SA micelle, the investigation of anti-tumor activity and reversing drug resistance of tumor cell were further performed.Stearic acid (SA) nanostuctured lipid carriers (NLC) with various oleic acid (OA) content were successfully prepared by solvent diffusion method in an aqueous system. The size and surface morphology of nanoparticles were significantly influenced by OA content. As OA content increased up to 30wt%, the obtained particles showed pronounced smaller size and more regular morphology in spherical shape with smooth surface. Compared with solid lipid nanoparticles (SLN), NLC exhibited improved drug loading capacity, and the drug loading capacity increased with increasing OA content. These results were explained by differential scanning calorimetry (DSC) investigations.The addition of OA to nanoparticles formulation resulted in massive crystal order disturbance and less ordered matrix of NLC, and hence, increased the drug loading capacity. The drug in vitro release behavior from NLC displayed biphasic drug release pattern with burst release at the initial stage and prolonged release afterwards, and the successful control of release rate at the initial stage can be achieved by controlling OA content.Stearic acid grafted chitosan oligosaccharide (CSO-SA), which synthesized by an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-mediated coupling reaction, was demonstrated to form micelle like structure by self-aggregation in aqueous solution. The amino substituted degree of CSO in CSO-SA could be controlled, after changing the charge ratio of SA to CSO and the reaction temperature. The critical micelle concentration (CMC) of CSO-SA with 15.4% amino substituted degree of CSO was about 0.035 mg ? mL-1. The micelles with 1.0 mg ? mL-1 concentration of CSO-SA had 70.6 nm volume average diameter with a narrow size distribution and 46.4±0.1 mV of zeta potential.The CSO-SA micelle/bovine serum albumin (CSO-SA/BSA) complex nanoparticles were prepared by the ionic interaction between CSO-SA micelle and BSA. The size, zeta potential and the morphology of formed CSO-SA/BSA complex nanoparticles were characterised by zetasizer and transmission electron microscopy (TEM). It was found that the sizes of complex nanoparticles were above 100 nm, and were bigger than the size of CSO-SA micelle. It means the complex nanoparticles were consisted of several CSO-SA micelles and proteins. The size of CSO-SA/BSA complex nanoparticles depended on the pH values of the dispersed aqueous vehicle, and the size diminished when the pH values of the dispersed medium decreased, meanwhile, the BSA encapsulation efficiency enhanced. The BSA release from complex nanoparticles decreased when the pH values of the delivery medium decreased in the range from 7.2 to 5.8, due to the stronger protonation of CSO-SA.In order to increase the stability of the micelle in vivo and controlled drug release, the surface of CSO-SA micelles were cross-linked by glutaraldehyde, and the effect of glutaraldehyde concentration on the size and zeta potential of formed nanoparticles was investigated. The drug-loaded CSO-SA micelle was prepared by dialysis method using camptothecin and adriamycin as anti-tumor drug model, respectively. The sizes and zeta potentials of drug loaded CSO-SA micelle with or without modification were determined by zetasizer. The drug encapsulation efficiency was performed using ultrafiltration separation and HPLC or fluorescence spectroscopy measurement. The in vitro drug release was characterized by the corresponding method. After the modification of the CSO-SA micelle surface, the size of formed nanoparticle was smaller then that of the micelle, the drug encapsulation efficiency of formed nanoparticle was improved, and the drug release rate from nanoparticle become slower. By increasing the amino substituted degree of CSO-SA, the drug encapsulation efficiency of formed nanoparticle could be further increased, and the drug release rate from nanoparticle were further slower.After the CSO-SA micelle was further labeled by fluorescein isothiocyanate, the properties, cellular uptake and the nuclear import of CSO-SA micelle were investigated, by using chitosan oligosaccharide nanoparticles and stearic acid solid lipid nanoparticles as control. It was found, the cellular uptake of chitosan oligosaccharide nanoparticles was the slowest, and the stearic acid solid lipid nanoparticles only could be reached at cytoplasm. After 2 h incubation of cells with FITC-CSO-SA micelles, the higher fluorescence intensity was dramatically concentrated in cellular nucleus, meanwhile, only lower fluorescence intensity was observed in cytoplasm, and the cellular uptake was depend on the energy and the concentration of CSO-SA. To further definitize the nuclear import pathway of FITC-CSO-SA micelles, the nuclear import studies of CSO-SA micelles were performed using digitonin-permeabilized cells, and the nuclear localization signal inhibitor (N-ethlmaleimide) and neoglycoproteins inhibitor (wheat germ agglutinin) were used, respectively. It was found the nuclear import could not be inhibited by N-ethlmaleimide, and could be inhibited by wheat germ agglutinin. The result means the nuclear import pathway was the glyco-dependent nuclear import.To improve the anti-tumor activity, the folated CSO-SA was further synthesized by the 1-ethyl-3-(3-dimethyIaminopropyl)carbodiimide-mediated coupling reaction. The drug-loaded micelle was prepared by employing motomycin C as a model drug. The properties of drug-loaded micelle was investigated. By using human lung cell line A549 as model cell, and motomycin C solution as control, the anti-tumor activity of CSO-SA micelles and folated CSO-SA micelles were determined by 3-(4,5-dimethyl -thiazol-2-yl)-2,5-diph-enyl-tetrazolium bromide method (MTT method). The effect of carrier concentration and the incubation time on the anti-tumor activity was investigated. It was found the CSO-SA micelle could import drug in to cellular nucleus, and markedly increased the anti-tumor activity of drug. The folated CSO-SA micelle had higher drug encapsulation efficiency. The folate modification of CSO-SA micelle could enhance the cellular uptake. These results led the further enhanced anti-tumor activity than CSO-SA micelle system.The adriamycin loaded CSO-SA micelles were prepared by using adriamycin as a model drug, and the properties of drug-loaded micelle was characterized. The drug encapsulation efficiency was above 50%, and the in vitro drug release was completed in 12 h. The inhibitor effects of adriamycin on the growth of adriamycin resistance MCF-7 cells and adriamycin sensitive MCF-7 cells were determined by MTT method. The drug resistance of drug resistance cells was 46.61 times higher than drug sensitive cells. The inhibitor effects of adriamycin loaded CSO-SA micelle on the growth of adriamycin resistance MCF-7 cells and adriamycin sensitive MCF-7 cells were also determined by MTT method. The drug resistance of drug resistance cells only was 2.7 times higher than drug sensitive cells. The increase of anti-tumor activity may produced by the increased drug concentration in nucleus importing by CSO-SA micelle. The adriamycin loaded CSO-SA micelle could reverse the drug resistance, and could increase the cytoxicity of drug resistance cells about 87.84 times.
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