טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGolan Sharon
SubjectNanostructre of Lipid/Polymers-Nucleic Acid complexes
DepartmentDepartment of Chemical Engineering
Supervisor Professor Emeritus Yeshayahu Talmon
Full Thesis textFull thesis text - English Version


Abstract

The efficiency of non-viral gene delivery (transfection) is strongly influenced by the physicochemical properties of the carrier-nucleic acid complex. The factors that govern the properties of these complexes are the lipid or polymer composition (the carriers), type of nucleic acids, and medium conditions. However, to date, no clear picture has been established how the transfection efficiency is influenced by those properties.

This research sought the correlation between transfection efficiency and the nanoscopic morphological characteristics of the carrier-nucleic acid complexes. We used cryogenic transmission electron microscopy (cryo-TEM) and other complementary methods to characterize those complexes.

We characterized the nanostructures of complexes based on different lipid mixtures (lipoplexes) and oligodeoxynucleotides (ODN). The first system is based on the anionic lipid cholesteryl hemisuccinate (CHEMS), and the second on the zwitterionic lipid 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (egg-PC). Although these lipids are significantly different, when complexed with ODN, they formed similar onion-like multilamellar nanostructures, suggesting that the multilamellar nanostructure is a general phenomenon when lamellar-forming lipids are used.

The second part of our research focused on the unique cationic lipid bis(11-ferroce- nylundecyl)dimethylammonium bromide (BFDMA). This lipid contains ferrocenyl groups at the end of its hydrophobic tails. Therefore, the net charge of the lipid can be cycled between (reduced) and (oxidized), which causes morphological changes its lipoplexes, thus, changing its ability to transfect cells. Therefore, this lipid is a good model for the study of structure-transfection correlation.

We characterized the BFDMA lipoplexes in the oxidized and reduced states of the lipid, and found significant structural differences. The reduced BFDMA lipoplexes formed onion-like nanostructures, while the oxidized BFDMA lipoplexes formed amorphous aggregates coexisting with other metastable morphologies. We also showed that in a cell-culture medium reduced BFDMA lipoplexes preserved their structural integrity, while oxidized BFDMA lipoplexes underwent structural changes.

We studied the effect of serum-containing media, which simulate the environment of in-vivo transfection experiments, on reduced BFDMA lipoplexes. We found that as the serum concentration increased, we observed enhanced levels of DNA disassociation, which correlated with decreased levels of in-vitro cell transfection.

In the last part of our research we characterized complexes formed between cationic lipids and the flexible negatively-charged polymer, poly(acrylic acid) (PAA). These complexes exhibited multilamellar onion-like nanostructures, similar to complexes of those lipids with nucleic acids. Our findings suggest that the multilamellar packing is energetically favorable for a wide range of polyion-liposome systems, from oligonucleotides, and DNA to PAA.