Preparation Of Self-aggregated Nanoparticles Of Cholesterol-bearing Chitosan Derivatives And The Primary Study On Using Them As The Novel Carriers Of Drugs

Hydrophobically modified polysaccharides, also named hydrophobized polysaccharides, are amphiphilic in nature due to their hydrophilic backbones and hydrophobic pendants. This novel kind of surfactants can form hydrogel nanoparticles by the intra-and/or intermolecular hydrophobic interaction among the hydrophobic pendants in aqueous media. Because these hydrogel nanoparticles have potential uses in biotechnology and medicine due to their unique supra-molecular structures, various hydrophobized polysaccharides have been designed and synthesized, and their physicochemical characteristics have been studied extensively. The cationic polysaccharide chitosan has many good properties, such as biocompatibility, biodegradability, non-toxicity and bioadhesivity that make it suitable for use as a biomedical material. However, because of the complexity of chitosan in terms of its sources, quality standards and measurements, the study of hydrophobized chtiosan derivatives is in the primary stage, and there are many problems have to be resolved.This dissertation designed a series of hydrophobized chitosan derivatives that had the unique chemical structures to prepare self-aggregated nanoparticles. Moreover, this dissertation also studied the relationships between the chemical structures of hydrophobized chitosan derivatives and the morphologies of their self-aggregated nanoparticles to deduce the self-assembly mechanism. The self-aggregated nanoparticle systems that had good morphology and stable property were chosen to be used as carriers of model hydrophobic and protein drugs to assess their potentials as drug delivery systems. The main investigations of this dissertation are shown as follows.A series of cholesterol-bearing O-carboxylmethyl chitosan(CCMC) conjugates with different degrees of substitution(DS) of the cholesterol moieties and cholesterol-bearing chitosan(CHCS) were synthesized by the succinyl linkages and characterized by Fourier transform infrared(FTIR), Proton Nuclear Magnetic Resonance (~1H NMR) and elemental analysis. These hydrophobized chitosan derivatives were amphiphilic in nature and their self-aggregation behavior in aqueous media was evaluated by the fluorescence probe technique. CCMC and CHCS self-aggregated nanoparticles were prepared by probe sonication in water and analyzed by dynamic laser light-scattering(DLLS), zeta potential and transmission electron microscopy(TEM) technologies. CHCS self-aggregated nanoparticles had a roughly spherical shape, a mean diameter of 417.2 nm, and their zeta potential was+33.5 mV. CCMC self-aggregated nanoparticles were almost spherical in shape, and their size, ranging from 180 nm to less than 50 nm, could be controlled by DS of the cholesterol moieties. The zeta potentials of CCMC self-aggregated nanoparticles were negative, and the absolute values decreased with increased DS of the cholesterol moieties. The comparison of the morphologies between CHCS and CCMC self-aggregated nanoparticles showed that the introduction of negatively charged carboxymethyl groups were easier to form well-shaped and more stable self-aggregated nanoparticles.Coomassie brilliant blue G-250 loaded CCMC-2 self-aggregated nanoparticles were prepared by mixing-gel chromatographic separation method; the maximum amount of drug-loading content reached to 18.8％, suggesting a loading efficiency of 67.1％. The release of coomassie brilliant blue G-250 from CCMC-2 self-aggregated nanoparticles was very slow, and the total release amount was about 3.73％in 72 h. Epirubicin(EPB), as a model anticancer drug, was physically entrapped inside the CHCS self-aggregated nanoparticles by remote loading method. EPB-loaded CHCS self-aggregated nanoparticles were almost spherical in shape and their size increased from 338.2 nm to 472.9 nm with the EPB-loading content increasing from 7.97％to 14.0％. Compared with blank CHCS self-aggregated nanoparticles, EPB-loaded CHCS self-aggregated nanoparticles had a smaller size and a smoother surface. We believed this was perhaps because EPB had the amphiphilic property and operated the function of surfactant in the drug loading process. The release behavior of EPB from CHCS self-aggregated nanoparticles was studied in vitro by dialysis method and the results showed that EPB release rate decreased with the pH of the release media increasing. In acetate buffer(pH=3.5), the EPB release from CHCS self-aggregated nanoparticles was very fast, but in the distilled water(PBS, pH=5.8) and in phosphate buffered saline (PBS, pH=7.4), the EPB release was very slow and the total release amount was about 34.7％and 24.9％in 48 h. This release property is advantageous to controlled and sustained drug release.The stable complex of CCMC-2 and bovine serum albumin (BSA) was formed by the mixing method, and the complexation process contains three stages, the disaggregation of CCMC-2 self-aggregated nanoparticles, the formations of the loose structural complex and the tight structural complex. The molecular complexation constant between CCMC-2 and BSA was determined by fluorescence method and its value was 6.0×10~4. The secondary structures of BSA and CCMC-2-BSA complex were studied by circular dichroism(CD) measurement, and the results showed that theα-helix content decreased with the concentration of CCMC-2 increasing. Compared with free BSA, CCMC-2-BSA complex had stranger ability against a denaturant such as urea, what indicated CCMC-2 self-aggregated nanoparticle system had potentials as a carrier of protein drugs.