| With the development of interdisciplinary crossing, scientists can repair the genome in biological body at the molecular level and gene therapy is gradually expanding from in vitro experiments to in vivo clinical experiments. In gene therapy, specific genes are rearranged in vitro and loaded into the genetic carrier, which are then injected into the biology body and repaired the missing genome in cells through gene expression. As vehicles for gene transfer, gene vectors play vital roles in gene therapy. Gene vectors can generally be divided into viral vectors and non-viral ones. The advantage of the former is their high efficiency of gene expression. However, they suffer from difficult industrial production, uncertain immunization safety and risk of genetic variations, which greatly limits their applications. Hence, scientists began to exhaustively study the non-virus carriers, especially chemical gene carriers. Researches in recent years have proved that cationic liposomes and cationic surfactants can interact with negatively-charged DNA molecules through coulomb attraction, and effectively compact DNA molecules. Due to the biological toxicity of cationic surfactants as well as the uncontrollability of the binding behavior between cationic liposomes or cationic surfactants with DNA molecules, however, the extensive applications of these cationic species are still restricted. Alkyldimethylamine oxide has the characteristics of pH responsiveness and low toxicity, making it an ideal candidate to be used as a gene vector. Unfortunately, although investigations on controlling the interaction between the cationic alkyldimethylamine oxide with DNA by varying pH have appeared, research in this direction is still quite limited. In this thesis, we give a detailed study on DNA/alkyldimethylamine oxide binary mixture in aqueous solutions, with an emphasis of developping effective loading and release of DNA by changing the aggregation states of alkyldimethylamine oxide. The main contents of this doctoral dissertation can be divided into four parts, as can be seen below.Chapter I is a comprehensive introduction of the research background, inwhich the concept of gene therapy, types of gene vectors together with their advantages and disadvantages are introduced. Various characterization methods of the interaction between gene vectors and DNA molecules, developpment of new gene vectors are reviewed in detail. The objective and the scientific significance of this doctoral dissertation are also pointed out at the end of this part.In Chapter II, controlled compaction and decompaction of DNA by zwitterionic surfactants, alkyldimethylamine oxides (CnDMAO, n=10,12, and 14), were investigated in detail. It was found that DNA could be effectively compacted by cationic micelles of CnDMAOH+ which was produced from CnDMAO via protonation (CnDMAO+H+?CnDMAOH+) at acidic solution. During this process, water-insoluble CnDMAOH+/DNA complexes formed. At a pH of 4-5, DNA molecules were effectively compacted when the concentration of C10DMAOH+, C12DMAOH+ and C14DMAOH+ reached 8.0,1.6 and 0.9 mmol·L-1, respectively. Interestingly, it was found that at pH?4 the precipitates can be redissolved by increasing the concentration to 40,9.0 and 1.8 mmol·L-1 for C10DMAOH+, C12DMAOH+ and C14DMAOH+, respectively, which indicates that DNA molecules were released from CnDMAOH+/DNA complexes. This phenomenon was attributed to the formation of hydrogen bonds between CnDMAOH+ and its neutral form, which screens the electrostatic interaction between the positively-charged CnDMAOH+ micelles and negatively-charged backbones of DNA. Our results could demonstrate that the release of DNA from the CnDMAOH+/DNA precipitates depends on the concentration of cationic CnDMAOH+ and pH. Compared with conventional release of DNA by the addition of ?-CD and SDS, the present strategy allows for controlled release by a specific signal, which favors the penetration of DNA into cells and protects it from nucleases degradation.In Chapter III, the thermo-reversible capture and release of DNA were developed based on the protonation and deprotonation of alkyldimethylamine oxide (CnDMAO) in tris-HCl buffer solutions. DNA/C14DMAO in tris-HCl buffer solution (pH=7.2) is transparent at 25 C, indicating DNA exist mainly in individuals and the binding of C14DMAO is weak. With increasing temperature, the pH of the buffer solution continuously decreases, which leads to protonation of C14DMAO. This induces an obvious increase of the turbidity of the samples, indicating a stronger binding of the protonated C14DMAOH+ (C14DMAO+H+?C14DMAOH+) to DNA. Further investigations revealed the formation of DNA/C14DMAOH+ complexes, in which the stretched DNA molecules are effectively compacted as evidenced from UV-vis absorptions, circular dichroism (CD) measurements, atomic force microscopy (AFM) observations, dynamic light scattering (DLS) measurements and agarose gel electrophoresis (AGE). Interestingly, when the temperature is turned back to 25?, the compacted DNA molecules can fully recover to the stretched state, and this cycle can be repeated several times without obvious loss of efficiency. The effect of chain length of CnDMAO has also been investigated. When C14DMAO was replaced by C12DMAO, similar phenomena can be observed with a slightly higher solution temperature for DNA compression and a slightly lower pH of tris-HCl buffer solution (6.8) used for sample preparation. For systems containing C10DMAO, however, no DNA compact can be observed in a further low pH of tris-HCl buffer solution (6.6) and a much higher surfactant concentration (up to 40 mmol-L"1), because of much higher critical micelle concentration (cmc) of the shorter chain length C10DMAOH+, cationic C10DMAOH+ micelles can not form under the studied condition to compact DNA. These results indicate that besides the electrostatic attraction between the negatively-charged DNA and the positively-charged CnDMAOH+, the thermo-reversible capture and release of DNA is also driven by the hydrophobic-hydrophobic interaction of the surfactants. The strategy may provide an efficient and alternative approach for stimuli-responsive gene therapy and drug release.In Chapter IV, rich phase behavior was observed in a salt-free cationic/anionic (catanionic) surfactant mixtures of lauric acid (LA) with a nonionic surfactant, tetradecyl dimethylamine oxide (C14DMAO) in water. The phase behavior and microstructures in LA/C14DMAO/H2O were investigated by freeze fracture-transmission electron microscope (FF-TEM), polarized optical microscope (POM), differential scanning calorimetry (DSC), rheological measurements and 2H NMR. A variety of self-assembled microstructures were formed, including micelles (L1 phase), lamellae (L?1 phase), vesicles (Lav phase) and gels. Therein, utilizing the L1 phase and L?1 phase as the templates, gold nanoparticles could be well constructed and confirmed by transmission electron microscope (TEM), and energy dispersive spectrometer (EDS). Compared with the traditional method of preparation of gold nanomaterials in aqueous solutions, this method can avoid the addition of NaBH4 as reducing agent. The sample solution plays the roles as a template and a reductant. Moreover, the reduction process does not destroy the original self-assembled microstructures in the solution. Hence, by controlling the aggregate structures of the template solution, one can achieve the goal of regulating the morphology of gold nanomaterials, which provides a new path for preparation of noble metal nanostructured materials with different shapes and structures. The results of MTT assay of HK-2 cells shows that, as a gene carrier, spherical gold nanoparticles prepared in micellar phase possess the characteristics of higher loading efficiency and lower toxicity than those obtained in the traditional surfactant systems, demonstrating the potential applications in gene therapy. |
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