Studies On Preparation Of Magnetic Targeted Gene Transfection Composite And Application

Current methodological approaches for disease treatment are not perfect and have inherent limitations. As genetic information technology develops quickly, researchers have comprehensively understood many disease mechanisms and come up with a novel gene therapy. Recent years, gene therapy has been widely applied in the treatment of many genetic diseases. Non-viral vectors have some unique characteristics, such as simple preparation method, low immunogenicity and high genetic loading capacity, and has been widely used in gene therapy. Polyethyleneimine (PEI) is a typical cationic polymer non-viral vector with high transfection efficiency, and has been regarded as the gold standard for non-viral vector gene transfection. With the rapid development of nano-materials, magnetic nanoparticles have received extensive attention due to their good magnetism performance. Magnetic ferroferric oxide (Fe3O4) nanoparticles have excellent chemical stability and excellent magnetic responsiveness, and have been significantly applied in disease diagnosis and therapy. The initiation of this thesis is to use magnetic nanoparticles (MNPs) and PEI to fabricate a PEI-based magnetic targeted gene transfection composite, and subsequently the composite was investigated to check the possibility to find application in treating cardiovascular diseases. The main contents are as follows:1. Preparation of Fe3O4@PAA magnetic nanoparticlesHigh-temperature decomposition was used to decompose acetylacetone ironto prepare magnetic Fe3O4 nanoparticles, and then polyacrylic acid (PAA) was modified on the surface. Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FT-IR), size and zeta potential analysis were used to characterize products. The results indicated that PAA was successfully modified on the surface of the Fe3O4 nanoparticles. The prepared Fe3O4@PAA magnetic nanoparticles had uniform particle size about 8 nm, considerable negative surface charge, good dispersion and superparamagnetism.2. Establishment and transfection performance evaluation of PEI/DNA complexesBiocompatibility tests, DNA-binding ability analysis, size and zeta potential measurements were used to explore the feasibility of PEI as gene vectors. Transfection conditions were optimized. The results showed that PEI had low cytotoxicity at low concentrations, and it can be used for cell experiment. It had very strong DNA condensing ability, and can combine and condense DNA to form stable complexes at low N/P ratios. PEI/DNA complexes presented small size in the range of 145 to 160 nm with considerable positive surface charge. The highest transfection efficiency of PEI/DNA complexes achieved about 94.9%(N/P=10) in serum-free conditions.3. Establishment and transfection performance evaluation of MNPs/PEI/DNA complexesMNPs/PEI/DNA complexes were assembled by MNPs, PEI and DNA via electrostatic self-assembly. The feasibility of MNPs/PEI as gene vectors was also evaluated by many measurements including biocompatibility, DNA-binding ability, and size and zeta potential values, and transfection conditions were optimized. The results indicated that MNPs/PEI had low cytotoxicity at low concentrations. The cytotoxicity of MNPs/PEI was a little higher than that of PEI, but in the acceptable range, so that MNPs/PEI can be used for cell experiment. The DNA condensing ability of MNPs/PEI was a little weaker than PEI, but it still had considerable DNA binding ability, and can be used as gene vectors. MNPs/PEI/DNA complexes presented small size between 174 and 196 nm with significant positive surface charge. Magnetic field can enhance the transfection efficiency of MNPs/PEI/DNA complexes. The optimal transfection conditions were PEI/DNA (N/P) of 10, MNPs/DNA (v/w) of 0.25, serum-free conditions, and 20 min duration of magnetic field. Under these conditions, complexes achieved the highest transfection efficiency.4. Studies on Magnetic targeted knockout of PCSK9 geneProprotein Convertase Subtilisin/Kexin type 9 (PCSK9) gene related to cardiovascular diseases. It regulated the level of Low Density Lipoprotein Cholesterol (LDL-C) via mediating degradation of Low Density Lipoprotein Receptor (LDLR). The optimized PEI-based magnetic targeted gene transfection composite was combined with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems (CRISPR/Cas9) genome editing technology. Through designing target sequence-specific gRNA sequences to guide Cas9, the above composite was checked to investigate the possibility to knock the PCSK9 gene. However, after a series experiments, we failed at knocking the PCSK9 gene in our prelimary investigation. The reasons may be that CRISPR/Cas9 genome editing tool had relatively high off-target effects and double-strand breaks stimulated cellular repair systems. Further investigation is under progress in detail.