טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentWeisman Sarah
SubjectMicrostructure Characterization of Nucleic Acid-Lipid
Systems
DepartmentDepartment of Chemical Engineering
Supervisor Professor Emeritus Yeshayahu Talmon


Abstract

The aim of this research was to characterize the supramolecular structure of complexes between cationic lipids and nucleic acids (lipoplexes).  Better understanding and control of the structure of these systems can help improve their efficiency as drug delivery vectors for gene therapy.

We studied the nanostructure of DNA lipoplexes shortly after component mixing by direct imaging using cryogenic transmission electron microscopy.  Micrographs show small local areas of the expected lamellar phase, combined with several other nanostructures, suggesting that the systems were not at equilibrium.  The images were compared with a literature model for the early stages of lipoplex formation, and excellent agreement is found.

The structure of ODN (oligodeoxynucleotide) lipoplexes is expected to differ from the structure of DNA lipoplexes, as ODN is a short, flexible molecule, while DNA is a long, rigid helix.  We conducted a systematic study of the nanostructure of ODN lipoplexes shortly after component mixing.  We captured direct images of various stages in the process of lipoplex development.  Based on this evidence, we propose a mechanism for the layer-by-layer formation of a novel ODN-lipid condensed lamellar phase.

We studied the nanostructure of DNA lipoplexes after short-term incubation in human serum, simulating the environment after intravenous injection.  The nanostructure of one lipid formulation studied seems to be stable in serum.  The nanostructure of a second lipid formulation largely disintegrates during incubation in serum.  These results can explain the differences in efficiency of the lipid formulations for in vivo gene delivery.

We studied the nanostructure of ODN lipoplexes after short-term incubation in serum.  The main features of lipoplex nanostructure were conserved. Anionic serum proteins do not penetrate liposome interiors, but they coat the outer surfaces of the cationic membranes, causing mild lipoplex aggregation.