Preparation Of Modified Chitosan And Its Application In Gene Transfection

Nowadays, gene therapy has been widely accepted as an important medical technology. Different from general treatments, gene therapy is highly specific and effective especially for diseases cased by genetic defects, and is known as a minimally invasive method. From presented in 1967 on, gene therapy is developing at a rather high speed, and has attracted more and more attentions for its promising application. The main way for gene therapy is to delivery desired gene into specific cells to replace the unnormal gene, and achieve therapeutic effect. In the whole process, the most important and decisive step is to carry desired gene into cells. The most appropriate method for gene delivery is using gene carriers. Thus, an ideal gene carrier play a key role in therapeutic effect and the prospect of gene therapy.In this paper, the main research point is preparing suitable gene vectors. Tranditional vectors for gene therapy are formed by viral carriers, liposomes or polycations. They are effective but highly toxic, useless in clinical application. Natural polysaccharose chitosan is highly biocompatible, no side effects, and should be use in genetic engineering. However, the transfection efficiency of chitosan is rather low. In this thesis, at one hand, common modifications of chitosan are used to improve the transfection efficiency and make chitosan functional for more gene therapy applications. On the other hand, novel methods are tried to improve the transfection efficiency. This work consists of four parts of contents:the preparation of chitosan-based core-shell structure particles for gene sustained release; the preparation of core-shell structure particles of modified chitosan-based materials for in vivo sustained release; the scission of chitosan by 60Co γ-ray radiation and improving its transfection efficiency; the preparation of poly(tributyl-(4-vinylbenzyl) phosphonium) grafted chitosan with higer charge density. The main contents and results are as follows:1. The synthesis of thiolated N-alkylated chitosan and hydroxybutyl chitosan have been done by chemical modification of chitosan. The preparation of TACS/pEGFP core particles is achieved through electrostatic adsorption, and wrapped with HBC to form TACS@HBC core-shell particles. Transmission electron microscope (TEM) shows that the size of core-shell particles is 120 nm. Besides, the core-shell partilces are non-toxic with a cell viability of more than 95%. Meanwhile, sustained release and transfection experiments against HEK 293T cells and Hela cells show the good capacity of sustained release and high transfection efficiency (38.99%).2. HBC is further modified with polyethylene glycol (PEG) to prepare EG-HBC. The pEGFP was firstly combined with the thiolated and N-alkylated chitosan (TACS). Then, hydroxybutyl chitosan grafted with poly(ethylene glycol) (EG-HBC) was coated on the pEGFP-loaded TACS particles to fabricate TACS@EG-HBC particles. Nano Particle Analyzer shows the sizes of new core-shell particles are 200 nm. The in vitro and in vivo gene transfection experiments indicate that the pEGFP-loaded TACS@EG-HBC particles possess a better sustainable gene transfection capacity and a high transfection efficiency, which should be attributed to the biodegradation of the CS-based shell, the thiolation and N-alkylation modification on CS cores, and the grafted PEG chains with better biocompatibility. Moreover, the in vivo transfection of TACS@EG-HBC could persist up to even 60 d at a high level.3. The preparation of low molecular weight (MW) chitosan is performed by radiation scission. Different absorbed doses lead to various MW, when absorbed dose changed from 0 to 50 kGy, MW decreases from 350,000 to 50,000 g · mol-1. Dynamic light scattering (DLS) shows that the zeta potentials, sizes of particles formed by chitosan irradiated, and complex particles turn smaller with the decrease of MW. Hela cell transfection assay indicates the improvement of transfection ability both by fluorescence microscope and flow cytometry, from 4.76% to 11.1% in value.4. The fabrication of poly(tributyl-(4-vinylbenzyl) phosphonium) grafted chitosan (CS-P) is performed by radiation grafting technique. It was confirmed by 13C-NMR that the benzene ring and P atom appear on chitosan molecules, demonstrating a successful preparation of CS-P. The grafting yield is 2.51% calculated from the results of thermogravimetric analysis (TG). The zeta potential of particles formed by CS-P and plasmid (41.7±6.10 mV) is much higer than that of CS (27.7±5.29 mV). The higher transfection efficiency was confirmed as 32.8% compared to 4.59% of CS, with no influence on cell toxicity (cell viability is 93.1 ± 1.61%).