Nucleolipid Self Assemblies for the Confinement and Delivery of Nucleic Acids

In this study we report a full investigation on a DNA delivery system based on anionic liposomes of the nucleolipid POP-Ade (1-palmitoyl-2-oleoylphosphatidyl-adenosine). We studied first DNA complexation process Ca2+-mediated by the designed delivery system. Quartz Crystal Microbalance and Neutron Reflectometry data on POP-Ade Supported Lipid Bilayers, as well as Dynamic Light Scattering and Zeta Potential data on POP-Ade vesicles highlighted the presence of a specific molecular recognition interaction beetween POP-Ade adenosine polar headgroup and nucleic acids' nucleobases, contributing both to single stranded and double stranded nucleic acids' binding. Molecular recognition contribution was thus found to increase nucleic acids' encapsulation efficiency by the vector, and to guarantee specificity in the binding process, according to the Watson-Crick nucleobases pairing model. Two POP-Ade-based formulations were developed as possible DNA vectors, by mixing POP-Ade with two different zwitterionic "helper" lipids, DOPE and POPC, in order to tune liposomes' surface charge density. Liposomes-Ca2+-DNA complexes were characterized in terms of their liquid crystalline structure through Small Angle X-ray Scattering (SAXS), resulting respectively in an inverted hexagonal phase (DOPE nucleolipoplexes) and in a lamellar phase (POPC nucleolipoplexes). Interestingly enough, it was possible to obtain the same structural information on nucleolipoplexes (but in this case as spatially resolved information) by monitoring the diffusion of a fuorescent lipid within nucleolipoplexes' lipid structure, through fluorescence correaltion spectroscopy (FCS). Finally, the interaction between nucleolipoplexes (with DOPE or POPC as helper lipid) and Giant Unilamellar Vesicles (GUVs) taken as cell membrane model systems was investigated through confocal laser scanning microscopy (CLSM) and FCS. We investigated the ability of nucleolipoplexes' in docking on GUVs' surface and fusing with GUVs' bilayer, and we determined the liquid crystalline structure of nucleolipoplexes and the surface charge density of the target GUVs' membrane as critical factors in the occurrence of membrane fusion. FCS was employed to monitor the release of a fluorescently-labeled DNA inside GUVs' lumen upon interaction with nucleolipoplexes, verifying thus the active role of nucleolipoplexes in the transport of the genetic payload inside GUVs' lumen.