Synthesis Of Functionalized Fe₃O₄ Magnetic Nanoparticles And Its Applications In Hakai Plasmid DNA Separation And Purification And Loading

As a new type of function materials,magnetic nanoparticles?MNPs?are widely used in the field of biomedicine due to their numerous excellent properties.This paper mainly studies the application of functionalized Fe₃O₄ MNPs in nucleic acid separation and purification and gene delivery,including three parts:?1?Research on Hakai protein as a new target for lung cancer treatment was investigated.We first found that Hakai protein was highly expressed in non-small cell lung cancer?NSCLC?cell lines,and knocking down Hakai expression by siRNA could significantly inhibit the proliferation,migration and invasion of NSCLC cells,suggesting that Hakai protein plays an important role in the occurrence and development of NSCLC,and could be used as a potential new therapeutic target.The above results laid the foundation for our subsequent research of functionalized Fe₃O₄magnetic nanoparticles for the separation,purification and loading of Hakai plasmid DNA.?2?The synthesis of ethylenediamine-modified sodium hydrogen tartrate-coated Fe₃O₄ nanoparticles?EDA-SHT-MNPs?was carried out and its application in nucleic acid separation and purification was further studied.The microstructure,surface potential and surface modification of the material were characterized by transmission electron microscopy?TEM?,dynamic light scattering?DLS?and X-ray photoelectron spectroscopy?XPS?.Subsequently,on one hand,we systematically explored the application of EDA-SHT-MNPs for the extraction of bacterial Hakai plasmid DNA and analyzed the biological activity of the purified DNA products;On the other hand,in order to verify whether DNA extraction via EDA-SHT-MNPs had broad-spectrum properties,we further tested its application for mammalian cell genomic DNA extraction.The results showed that EDA-SHT-MNPs synthesized in this study can efficiently extract plasmid DNA and genomic DNA without affecting the biological activity of DNA;In addition,compared with the commercial kit,EDA-SHT-MNPs had advantages in both total DNA extraction and purity,and shows potential application value.?3?Preliminary studies on novel magnetic nanoparticle for gene transfection or gene therapy were performed.As mentioned above,in the first part of our work,we found that Hakai protein could be served as a potential therapeutic target in NSCLC,and in the second part,we have demonstrated that surface-functionalized magnetic nanoparticles presented better ability to bind DNA.In this part of the work,combining the polyamidoamine dendrimer?PAMAM?with good biocompatibility and the magnetic nanoparticles we obtained in the second part,we redesigned and synthesized a new type of magnetic nanoparticle,and the Hakai gene was used as a research object to explore the application of this material in gene transfection and gene therapy.We firstly synthesized third-generation polyamidoamine dendrimer?G3?by Michael addition reaction and iterative method with ethylenediamine as the core and methyl acrylate as raw materials,then directly encapsulated Fe₃O₄ nanoparticles or modified Fe₃O₄ nanoparticles coated with sodium hydrogen tartrate with G3 to obtain two kinds of surface cationic polymer modified magnetic nanoparticles,named as G3-MNPs and G3-SHT-MNPs,respectively.Through TEM,DLS and other methods,we characterized the microstructure,surface potential and size of the material.Furthermore,using the in vitro nucleic acid binding experiment and cell cytotoxicity analysis,we examined the Hakai plasmid DNA binding abilities and cell cytotoxic effects of the two materials.Preliminary results showed that,compared with G3-MNPs,G3-SHT-MNPs had better binding effect with plasmid DNA in vitro,followed with lower cytotoxicity,showing better biocompatibility.The above results indicate that G3-SHT-MNPs can be used as potential gene transfection and therapeutic vectors,and with further development in the future,the synthesized magnetic materials are expected to be used for gene therapy based on Hakai or other targets.In summary,we firstly proved that Hakai protein could be used as a potential new target for NSCLC treatment,and taking Hakai gene as the research object,EDA-SHT-MNPs and G3-SHT-MNPs were successfully synthesized,and the former could efficiently extract both Hakai plasmids DNA in bacterial and cellular genomic DNA,which could be used for subsequent applied research of nucleic acid extraction;the latter had the potential function of gene transfection and therapy,and exhibited important application prospects.