Construction Of Ultrathin DNA Multilayers For Controlled Local Release Of DNA

It is well known that gene therapy is a potential powerful tool to deal with many severe diseases such as cancer. Therefore it has been extensively researched worldwide. Effective gene delivery is the main objective in gene therapy. Toward the goal of constructing novel gene delivery system, two kinds of ultrathin DNA film via electrostatic layer-by-layer (LbL) self-assembly technique have been constructed.Firstly, (DNA/poly-L-(lysine) (PLL)) ultrathin film is constructed via LbL self-assembly technique. The PLL is chosen as model polycation because of its biocompatibility and biodegradability. Therefore, the PLL could endow the film with special ability of enzyme sensitivity. Both UV-vis spectrum and atomic force microscope (AFM) measurements show a typical LbL deposition of DNA and PLL. It is found that the salt concentration of the deposition solution has significant effect on the construction of the films, which might be attributed to the effect of salt ions on the conformation of polyelectrolytes and interaction between PLL and DNA molecules. Therefore, through the controlling salt concentration, the film's thickness and adsorbed amount of polyelectrolytes could be modulated in nano-scale. The AFM measurement and ethidium bromide (EtBr) adsorption show that the PLL/DNA complexes could be formed during the deposition of film. Such complexes might serve a mechanism to protect DNA from the attack of EtBr. Further experiment of enzyme degradation experiments are carried out to investigate the enzymatically sensitive ability of the film. The data of UV-vis spectrum and AFM show that the (PLL/DNA) film is degraded under enzyme solution and then the DNA is released from the film. After that, it is found that, through the method of glutaraldehyde (GA) cross-linking, the stability of the film could be controlled. More important, it significantly affects the profiles of DNA release.The polycation-based non-viral DNA nanoparticles, which is proved to be effective both in vitro and in vivo, is receiving increasing attention because of several advantages, such as ease of manipulation, low cost, and safety. Second part of our researches, toward the goal of construction of novel delivery system for polycation-based non-viral DNA nanoparticles, (DNA nanoparticles/polyanion) film is constructed via LbL self-assembly technique. Special DNA nanoparticles are prepared with the diameter of 10-30 nm and zeta potential value of 31.6 ± 6.09 mV.Poly(ethyleneimine) (PEI) is chosen as polycation to condense the DNA to nanoparticles. AFM and UV-vis measurements show that the DNA nanoparticles can be successively adsorbed onto the polyanion {poly-L-(glutamic acid) (PGA) or poly(styrene sulfonate) (PSS)} layer to form ultrathin films. The transmittance electron microscope (TEM) measurement clearly shows the cross-sectional films. The average one bilayer of (DNA nanoparticles/PGA) and (DNA nanoparticles/PSS) films is about 6 and 18 nm, respectively. The well-structured, easy processed multilayers with the non-viral DNA nanoparticles may provide a novel approach to precisely control delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc.Further, the cells culture experiments are carried out to investigate the transfection efficiency of both (PLL/DNA) and (DNA nanoparticles/polyanion) films. Firstly, experiment data confirm that the DNA that is released from the (PLL/DNA) film through enzymatic degradation can transfect the cells very well with the PEI vector, which means that the construction and deconstruction procedure will not affect the transfection ability of DNA. Secondly, through the enzymatic degradation of (DNA nanoparticles/PGA) film, the DNA nanoparticles can be released from the film and remain its complex configuration. Therefore, these released DNA nanoparticles can directly transfect the cells without help of other vector any more. An in situ cell culture experiment suggests that (DNA nanoparticles/polyanion) films have the ability of transfecting cells in situ. Because of the higher DNA nanoparticles density, film based on PSS leads to higher transfection efficiency in situ than that of film based on PGA. Through constructing the (DNA nanoparticles/PGA) film onto the PLA tissue engineering scaffold, the cells transfection on the scaffold can be achieved.In brief, the DNA or DNA nanoparticles contained films could be served as a novel gene delivery system. More important, these kinds of film could be deposited onto almost any kind and any shape of matrix surface, such as tissue engineering scaffold and interventional therapy devices, and therefore be served as a bridge between gene therapy and biomedical devices. The researches might not only figure out the problems induced by biomedical devices itself, but also advance the clinical application of biomedical devices in the field of gene therapy.