Self-Assembled Architecture of DNA-Lipid Complexes

• Main Authors: Ching-Mao Wu, 吳清茂
• Other Authors: Hsin-Lung Chen
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/7yzr63

博士 === 國立清華大學 === 化學工程學系 === 92 === Polyanionic DNA can bind electrostatically with cationic liposomes (CLs) to form the complex exhibiting rich self-assembled structures at various length scales. This class of bioassembly has been considered as a nonviral gene delivery system for gene therapy or as a template for nanostructure construction. Understanding the self-assembly of DNA/CL complexes is crucial both for building the detailed nucleic acid delivery mechanism and also for application of this class of materials as nanostructural templates. This dissertation investigates the self-assemblies of the bioassemblies of DNA with (1) a cationic lipid, cholesteryl 3β-N-((dimethylamino)et- hyl)carbamate (DC-Chol), (2) a zwitterionic lipid, 1,2-di(cis-9-octadecenoyl)-sn-glyc- ero-3-phosphocholine (DOPC), and (3) with both DC-Chol and DOPC. The self-assembled structure of DNA-DC-Chol complexes in excess water was first reported. Neat DC-Chol self-assembled into cylindrical micelles in aqueous media. These micelles aggregated and fused into multilamellar condensates or vesicles upon complexation with DNA, and DNA chains confined between the lipid bilayers formed closely packed arrays irrespective of overall lipid-to-base pair molar ratio. The complexation was found to be a highly cooperative process, where the complexes with nearly 1:1 stoichiometry were formed even when DNA was in excess of DC-Chol in terms of the overall ionic charge. As DC-Chol became in excess, the unbound lipid did not fully macrophase separate from the stoichiometric complex but segregated to form domains coexisting with the bound lipid domains in the lamellae. The presence of these unbound lipid domains reduced the persistence length of the membrane and consequently induced topological transformation of the multilamellar phase from platelike lamellae to circular lamellae observed by TEM at higher DC-Chol composition. The self-assembly of DNA-DC-Chol complexes in the bulk state (i.e., solid state) has been investigated by SAXS. Two multilamellar phases, LI and LII, characterized by distinct states of lipid stacking and DNA conformation were observed. LII phase, formed when the lipid was in excess of DNA in terms of overall ionic charge, composed of B-DNA confined between the bilayers with the lipid tails aligning normal to the lamellar interface. When DNA are in excess, LI phase, in which the A-DNA bound with the titled lipid chains adopts A-form conformation, was favored as it offered more economical binding between these two components. The aggregation of a zwitterionic liposome composed of DOPC induced by DNA without the presence of multivalent salt has been revealed. Our results demonstrate that only a small fraction (< 10%) of DNA can bind electrostatically with a portion of the liposomes. Such a low degree of binding however induces significant aggregation of these oligolamellar liposomes to yield large multilamellar particles in which the number of hydrophilic/hydrophobic layers along the lamellar stacking direction becomes sufficiently large to yield multiple diffraction peaks in the small angle X-ray scattering (SAXS) profile. Addition of monovalent salt such as NaCl tends to disrupt the multilamellar structure. Finally, a multilamellar DNA-DC-Chol-DOPC complex was adopted to study the effect of divalent metal ions on the packing density of DNA condensed on the surfaces of the rigid DC-Chol/DOPC membrane. This study is distinguished from the previous one demonstrating that divalent metal ions can induce further condensation of DNA lying on the surfaces of fluid cationic membranes in that the membrane system adopted here is a rigid one. Our results demonstrate that the divalent cations, Ca2+ and Ni2+, only exerted an electrostatic screening effect between DNA but did not induce collapse transition of the DNA into a condensed state.