Synthesis And Biological Performation Of Novel Amphiphile/DNA Complexes

Gene therapy has been regarded as a potential treatment for a range of disorders with genetic anomaliea or deficiencies that involve the introduction of encoded therapeutic proteins or suppreeion of abnormally expressed proteins. Generally, genes are delivered either by viral or nonviral systems. Viral vectors still face various challenges including the cellular immune response, limited cargo capacity, targeting deliver into aimed cells and cost. Alternatively, numerous synthetic materials have been developed as nonviral vectors. Cationic surfactants and amphiphilic peptides which carry positive charges could condense and stabilize DNA through attractive electrostatic and hydrophobic interactions, and protect nucleic acids from enzymatic degradation, and release nucleic acids into the cyroplasm by the hydrophobic segment. Moreover, because cationic surfactants and amphiphilic peptides are chemically similar to lipid molecules and could be versatiles by designable synthesis, surfactant or peptides-based vectors have been considered as a promising potential nonviral gene vector strategy. Therefore, it is important to design and synthesize novel surfactants and amphiphilic peptides as nonviral vectors, and to investigate the interaction with DNA, for gene therapy application. The imidazolium Gemini surfactants [Cn-s-Cnim]Br2display better interfacial and self-assembly properties than those traditional monovalent surfactants, owning to the properties of traditional Gemini surfactant and single-chain imidazolium surfactant, which may condense DNA effectly.We synthesized a series of imidazoliu Gemini surfactants with a four-methylene spacer group ([Cn-4-Cnim]Br2, n=10,12, and14). Herring sperm DNA is chosen as a model gene system for short DNA, microRNA, and so on. We investigated the interactions between herring sperm DNA and surfactants by CD, ethidium bromide displacement assay, and discussed the DNA condensation mechanism. Upon addition of [Cn-4-Cnim]Br2, DNA molecules undergo the process from DNA compaction to multi-molecular DNA condensation accompanied by conformation change.[Cn-4-Cnim]Br2as novel Gemini surfactants can interact with DNA via electrostatic, hydrophobic and π-π interactions. Moreover, the stronger interaction between DNA and [Cn-4-Cnim]Br2with longer tail demonstrates the important contribution of the hydrophobic interaction.Furthermore, we investigated the morphologies, cytotoxicity, and gene transfection of plasmid DNA/[C12-4-C12im]Br2complexes at structural, phase properties, and cellular levels, respectively. Homogeneous DNA/[C12-4-C12im]Br2nanoparticles are formed with a diameter of approximately100nm and investigated by using dynamic light scattering (DLS) and atomic force microscopy (AFM). DNA condensates evolve from supercoiled DNA molecules, to individual toroids, to close-packed particles. Successfully EGFP gene transfection in vitro is demonstrated by using fluorescence microscopy in HEK293and HeLa cells. Quantitative analysis results of gene transfection by flow cytometry show that the efficiency of DNA/[C12-4-C12im]Br2is86.0%. The uniform condensate nanoparticles with nanometer size and spherical shape, high gene tranfection efficiency, moderate toxicity demonstrate that imidazolium Gemini surfactants are a promising nonviral vector in gene therapy.Gold nanoparticles have promising application in the fields of gene therapy, duing to their unique optical and photothermal properties, the availability of synthetic protocols that can tune the size and shape of the particles, the ability to modify the surface, and the relative biocompatibility. Novel imidazolium surfactants functionalized gold nanoparticles are expected to use as excellent agents for controlled DNA condensation, and significantly high gene expression, as well as reducing cytotoxicity. The seed growth method and [C12-4-C12im]Br2modified gold nanoparticles surface method were used, respectively, to get uniform size and shape, high yield gold nanoparticles. The gold nanoparticles which are modified [C12-4-C12im]Br2could concentrate [C12-4-C12im]Br2molecules, and exhibit the characteristies of multivalent ions, increasing the effect of DNA condensation as a result. The size distribution and uniform degree are determined by the spectroscopy and microscopy technology, and the gene transfection and the toxicity are evaluated at cellular level. The moderate cytotoxicity of [C12-4-C12im]Br2will limit its application in the field of gene therapy and biomedicine. Thus, we designed a series of amphiphilic peptides with excellent biocompatibility and degradability properties (choose the appropriate amino acids). We demonstrate the visualization of chiral hierarchical assembly from molecular level to assembly level and bulk solution level for amphiphilic peptide alone. Moreover, we also confirm that amphiphilic peptides interact and condense DNA through electrostatic and hydrophobic forces, owing to the properties of surfactants and peptides. The effect of amphiphilic peptides on cell viability is evaluated with an MTT assay, indicating the low cytotoxicity. The investigation of amphiphilic peptides could benefit their application in gene therapy, and provide basic data and theoretical guidance.