טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentVilensky Mickheev Rita
SubjectNanostructural Characterization of Lipoplexes and Polyplexes
DepartmentDepartment of Chemical Engineering
Supervisor Professor Emeritus Yeshayahu Talmon
Full Thesis textFull thesis text - English Version


Abstract

Gene therapy has the potential to cure a broad range of inherited and acquired diseases. The basic concept of gene therapy is simple: introduce into target cells genetic material that results in either a cure or a slowdown of the progression of the disease. Naked DNA undergoes rapid in-vivo degradation, thus a delivery system capable of introducing the genetic material into a variety of cells is required. Non-viral gene delivery systems, based on cationic polymers and lipids, were shown to be potential candidates as gene delivery agents. The main objective of this work was to characterize the structure of complexes between cationic lipids/polymers and nucleic acids. Better understanding of the structure of these systems can help improve their efficiency as delivery vectors for gene therapy. The main experimental tool used in this study was cryo-TEM. We used direct-imaging cryogenic transmission electron microscopy (cryo-TEM) to directly visualize the microstructures and freeze-fracture-replication (FFR) to examine the structures that are too big for direct-imaging cryo-TEM. The amphiphilic character of lipids or polymers that contain distinct hydrophobic and hydrophilic moieties leads to their self-assembly into a variety of structures in aqueous solution. Materials used in this study were the cationic penta-block-copolymer diethylaminoethylmethacrylate (PDEAEM), poly(ethyleneoxide) (PEO) and poly(propyleneoxide) (PPO), PDEAEM-PEO-PPO-PEO-PDEAEM, homopolymer polyethyleneimine (PEI), and the polycationic lipid ceramide carbamoyl-spermine (CCS). The self-assembly of polymers/lipids in the absence of DNA was also studied. We showed that these materials aggregate in water to form different types of structures. The morphology and the size of these structures were shown to be pH and composition dependent. Then we showed how mixing cationic lipids/polymers with anionic DNA in aqueous solution led to spontaneous complexation to form lipoplexes/polyplexes. The morphology of the polyplexes based on cationic penta-block-copolymer, homopolymer (PEI) and lipoplexes based on lipid (CCS) was examined at different cationic agent-to-DNA charge ratios. Lipoplexes based on polycationic lipid adopted lamellar phase; the size of the lipoplexes strongly depended on the lipid-to-DNA charge ratio. In several systems we studied the effect of pH and blood serum on the morphology of the complexes. We showed that acidic conditions had no visible structural effect on the morphology of pentablock-copolymer-based polyplexes. Serum addition to that system led to extensive aggregation of polyplexes, but this effect is minimized by the addition of a Pluronic tri-block-copolymer (PEO-PPO-PEO).