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Layer-by-Layer (LbL) gene delivery system is fabricated by polycation and plasmid DNA alternative deposition based on electrostatic force between each other. This dissertation explored the relationship between LbL film assembly and disassembly. Demonstrated that the film disassemble process is closely related to film interior structure. LbL interlayer diffusion, which is caused by the polyelectrolytes own properties, such as polymer chain length, molecular weight, and charge density, can influence film growth mode. The interlayer diffusion is the main reason to lead fast and bulk DNA releasing of bioreducible LbL film. Preventing interlayer diffusion by inserting small diffusion coefficient polymer, such as Polyethylenimine (PEI), can effectively achieve sustained and sequential DNA releasing. This dissertation also developed a series of poly (amido amine) s (PAAs) with high transfection efficiency and …