Preparation And Properties Of DNA/Cationic Surfactant Assemblies

Gene therapy, which can be realized by transporting functional DNA fragments to target cells for helping the repair of the disabled gene to function well, is of great importance in curing genetic diseases and cancer, and culturing specific functional cells, etc. Because of electrostatic or conformational reasons, DNA cannot cross cell membranes independently. Thus, one of the major challenges in gene therapy is to find proper tools or vectors for effectively transporting functional DNA fragments to target cells and achieve an efficient gene delivery in cells.Many synthetic materials can be used to assemble nanoscale aggregates with DNA and promote the delivery of it. Cationic surfactants have been used with some success for transporting DNA. The binding of cationic micelles or vesicles on DNA backbones can reduce the charge repulsion between adjacent phosphate groups, and make them approach each other, leading to the so-called "compaction" of DNA. The reduction both in the size of DNA molecules and the repulsion with phospholipid bilayers produce more opportunities for them to cross cell membranes. Under controlling conditions such as formation of cationic and anionic (catanionic) surfactant micelles, adding β-CD or salts, the binding of cationic micelles or vesicles can be disrupted, the captured DNA will be released, and the aim of gene delivery can be realized.The physicochemical properties of cationic surfactant/DNA complexes are important in probing their potential applications in pratical gene therapy. In this thesis, we focus on the self-assembly of DNA and cationic surfactant aggregates including cationic micelles, catanionic vesicles and cationic surfactant-coated gold nanoparticles in solution, as well as the physicochemical properties of DNA/surfactant complexes. The content of this doctoral dissertation are as follows:In Chapter 1, the properties of DNA as well as the background of gene therapy were firstly descried. Then the research advances and prospects on non-viral gene vectors including cationic surfactant aggregates (micelles, lipids and liposomes), nanoparticles, polymers in gene delivery were introduced. Next we discussed the interaction behavior of cationic surfactant aggregates and DNA, as well as the physicochemical properties of DNA/cationic surfactant complexes in detail. Finally, we demonstrated the purpose and significance of this thesis.In Chapter 2, a systematic work concerning the DNA compaction and decompaction controlled by cationic surfactants with different counterions was performed. We discovered that cationic surfactants with complex counterions, [FeCl3Br]-, cannot promote the decompaction of DNA like those with Br- and Cl- as counterions. This interesting finding could provide a better understanding of the interaction behavior of DNA and cationic surfactants. We conclude that the fundamental reason of the DNA decompaction lies upon the electrostatic competition between the counterions and DNA for associating withthe cationic aggregates. At a high concentration, the binding of counterions to cationic aggregates is promoted, which weakens and screens the electrostatic attraction between DNA and cationic aggregates. This could cause the decompaction of DNA. Our data revealed the fundamental reason of the compaction and decompaction behavior of DNA induced by cationic surfactants independently, a reasonable three-step model of the conformational changes of DNA controlled by different amounts of cationic surfactants was presented. The current work could provide guidance in gene delivery, gene therapy and biomedicine fields.In Chapter 3, a dual-responsive cationic surfactant, which contains both a light-responsive moiety azobenzene and a paramagnetic counterion, [FeCl3Br]-, was designed and synthesized. Not only does this cationic surfactant abundantly utilize inexhaustible and clean sources, i.e., light and magnetic field, but it also serves as a powerful dual-switch molecule for effectively controlling the capture and release of DNA. It was proved that the light switch can independently realize a reversible DNA compaction. The introduction of a magnetic switch can significantly enhance the compaction efficiency, help compact DNA with a lower dosage and achieve a magnetic field-based targeted transport of DNA. In addition, the light switch can make up the irreversibility of magnetic switch. This kind of self-complementation makes the cationic azoTAFe be useful as a potential tool that can be applied to the field of gene therapy and nanomedicine.In Chapter 4, we construct for the first time ordered surfactant-DNA hybrid nanospheres of double-strand (ds) DNA and cationic surfactants with magnetic counterion, [FeCl3Br]-. The specificity of the magnetic cationic surfactants that can compact DNA at high concentrations makes it possible for building ordered nanospheres through aggregation, fusion, and coagulation. Cationic surfactants with conventional Br- cannot produce spheres under the same condition because they lose the DNA compaction ability. When a light-responsive magnetic cationic surfactant is used to produce nanospheres, a dual-controllable drug delivery platform can be built simply by the applications of external magnetic force and alternative UV and visible light. These nanospheres obtain high drug absorption efficiency, slow release property, and good biocompatibility. There is potential for effective magnetic-field-based targeted drug delivery, followed by photocontrollable drug release. We deduce that our results might be of great interest for making new functional nucleic acid-based nanomachines and be envisioned to find applications in nanotechnology and biochemistry.In Chapter 5, the interaction of DNA with salt-free tetradecyltrimethylammonium hydroxide and lauric acid lamellar vesicles with positive charges was investigated to probe potential applications of vesicles in DNA transfection. The aggregation morphology of the vesicles changes greatly with the addition of DNA due to the dissociation of anionic surfactants, as indicated by 1H nuclear magnetic resonance, and the expelled surfactant molecules self-assemble into micelles at high concentrations of DNA. Salt-free catanionic vesicles have a much higher binding saturation point with DNA at R=2.3 (the ratio of DNA to the excess positive charge in vesicles) than formerly reported salt-containing systems, implying high transfection efficiency. DNA retains its native stretched state during the interaction process. This very interesting result shows that catanionic vesicles could help transport undisturbed and extended DNA molecules into the target cells, which is of importance in gene delivery, nanomedicine field, and controlling the formation of certain morphological aggregates.In Chapter 6, catanionic vesicles were constructed from the mixtures of sodium laurate (SL) and alkyltrimethylammonium bromide (CnTAB, n=12,14, and 16) and were used to control the loading capacity of DNA. The binding saturation point (BSP) of DNA to catanionic vesicles increases with the chain length of cationic surfactants, which is at 1.0,1.3 and 1.5 for CnTAB with n=12,14, and 16, respectively. Our measurements showed that the loading capacity and affinity of DNA can be controlled by catanionic vesicles. It increases with the chain length of cationic surfactants. Because of a large reduction in surface charge density, catanionic vesicles are prone to undergo re-aggregation or fusion with the addition of DNA. DNA molecules can still maintain original coil state during the interaction with catanionic CnTAL vesicles.1H NMR data reveals that the obvious dissociation of anionic ions, L", from catanionic C14TAL and C16TAL vesicles is due to the interaction with DNA; however, this phenomenon cannot be observed in C12TAB-SL vesicles. Agarose gel electrophoresis (AGE) results demonstrate that the electrostatic interaction between the two oppositely charged cationic and anionic surfactants is stronger than that between DNA and cationic surfactant, CnTAB (n=12,14, and 16). Not only is the dissociation of L- simply determined by the charge competition, but it also depends largely on the Variations in the surface charge density as well as the cationic and anionic surfactant competing ability in geometry configuration of catanionic vesicles. The complicated interaction between DNA and catanionic vesicles induces the deformation of cationic vesicles. Our results should provide guidance for choosing more proper vectors for DNA delivery and gene therapy in cell experiments.In Chapter 7, we produce a novel light-responsive and magnetic catanionic bilayer vesicles with specific tubular morphology. The formation of these microstructures is a result of the self-assembly of a synthetic dual-responsive cationic surfactant azoTAFe (C2H5O-azobenzene-OC2H4N+(CH3)3[FeCl3Br]-) and an anionic surfactant Texapon N70 (CH3(CH2)11(CH2CH2O)2.5SO3-Na+). These vesicles are demonstrated to be effective vectors to control the loading, migration and release of DNA. Their specific tubular morphology allows them to possess the highest DNA loading capacity among all the reported salt-containing catanionic vesicles. Their strong magnetism not only enables them to regulate an efficient magnetic field-based targeted transport of DNA, but also significantly enhances their DNA loading capacity in the presence of an external magnetic field. The light-responsive property can be used to control the formation of vesicles, which can help to realize a controllable capture and release of DNA. Cytotoxicity assay shows that these catanionic vesicles have good biocompatibility and are suitable for bio-related applications.In Chapter 8, a novel strategy for preparing magnetic gold nanoparticles that have high sensitivity and high efficiency by means of one-step modification with a paramagnetic cationic surfactant CTAFe (C16H33N+(CH3)3[FeCl3Br]-) was reported. The Au@CTAFe nanoparticles can effectively compact DNA and bind to proteins through the electrostatic interaction and make the resulting nanoparticle-biomacromolecule complexes be controlled by an external magnetic field. The native conformation of proteins and DNA can be adjusted suitably for transport at the cellular level without denaturation. This novel guidance for preparing magnetic gold nanoparticles and controlling the delivery of biomolecules could be applied in a diverse range of systems efficiently and non-invasively in nanotechnology and biotechnology.In Chapter 9, a low strength (0.25 T) magnetic field-based highly efficient DNA and proteins delivery platform was constructed based on one-step modified ultrafine (<2 nm) magnetic gold nanoparticles (AuNPs). These magnetic AuNPs were produced by using large amount of weak oxidizing paramagnetic surfactants, CTACe (C16H33N+(CH3)3[CeCl3Br]-) or CTAGd (C16H33N+(CH3)3[GdCl3Br]-), as surface modified compounds. The ultrafine magnetic AuNPs are very rare and can be served as vectors that highly concentrate the charge of cationic surfactants on their surfaces to significantly enhance the electrostatic interaction between cationic surfactants and negatively charged DNA and proteins, this gives rise to a very low critical concentration of this kind of agent in compacting DNA or precipitating proteins. Meanwhile, the large amounts of cationic surfactants endow AuNPs with strong magnetism, which makes these magnetic AuNPs magnetize and migrate these biomacromolecules with an outstanding efficiency.