Cationic Liposome-DNA (CL-DNA) complexes are widely used in non-viral gene delivery. Some lipid vectors have been commercialized, and most are very effective in cultured mammalian cells, allowing for applications outside of therapeutic DNA delivery.  For CL-DNA complexes to become widely useful for gene therapeutic purposes, however, their efficiency (particularly in vivo) still needs to be improved.  In addition, the mechanism of transfection is still incompletely understood. Furthering this understanding will provide rational routes towards improving transfection efficiencies. I work on the synthesis of new lipids designed to address these issues.

A large number of structurally quite diverse cationic lipids have been synthesized in efforts to improve the efficiency of CL-DNA complexes. The early results of this research have been reviewed in detail, and several more recent reviews with a smaller scope have also appeared. A large amount of work has also been performed in order to understand formation of cationic lipid-DNA (CL-DNA) complexes, their structure and their mechanism of action.  It was shown that CL-DNA complexes form inverted hexagonal or lamellar liquid crystal (LC) phases. The complex structure depends on the neutral lipid and the composition,  with the vast majority of the complexes studied so far forming the lamellar phase. Significant differences in the transfection properties of hexagonal and lamellar complexes have been reported and models for their different modes of action have been proposed. 

Attempts to unveil structure-property relationships for cationic lipids in the classical sense, i.e. between the chemical structure of the (cationic) lipid and transfection efficiency, have met with little success, however. This is due to the complexity of the task of optimizing the transfection protocol for the lipid and the biological system under investigation. Important parameters that affect transfection by CL-DNA complexes are the lipid/DNA charge ratio, lipid composition, complex structure, cell type, and the structure of neutral and cationic lipid. Indeed, certain lipids and lipid mixtures which had been considered as inefficient turn out to be very active when lipid composition and lipid/DNA charge ratio and complex preparation protocols are optimized carefully. 

On the other hand, certain properties of the entire self-assembled vector are directly related to transfection efficiency through their effect on well known cellular barriers to transfection. My synthetic work therefore aims to produce tailored lipids that allow us to vary these properties to probe their effect on the transfection efficiency. For lamellar complexes, the average charge per area of the lipid membrane (the membrane charge density) has been identified as a universal, i.e. lipid-independent, parameter governing the transfection efficiency until a critical value is reached.  The related cellular barrier is escape of the complexes from the endosome. 

Using a building block approach, we have prepared heterobifunctional PEGs carrying a lipophilic group on one end and a hydroxyl group on the other. Cholesterol or two alkyl chains can serve as the lipophilic moiety. The hydroxyl group can be used to attach for example ornithine (corresponding to two cationic charges) and other function-bearing groups while the PEG-chains should provide enhanced resistance to the immune system. For relatively simple PEG-lipids, x-ray diffraction reveals a condensation of the DNA with increasing PEG-lipid concentration due to depletion-attraction interactions for long PEG chains. Transfection using PEG-lipid CL-DNA demonstrates that the PEG-lipids are also presented on the outside of the complexes.

I have also systematically varied the functional group on the PEG lipids to provide lipids with multiple charges. Interest in this comes from the fact that some multivalent cationic lipids have been reported to have extremely high transfection efficiencies. These lipids, however, were not available in sufficient quantities for the experiments our group was planning to do to unravel their mechanism of action, so I developed an efficient synthesis of a series of new multivalent lipids. X-ray scattering, microscopy and transfection experiments with these lipids show that a much smaller amount of them is required for high transfection. We attribute this to their ability to create a high membrane charge density in the complex with DNA, which in turn facilitates its escape from the endosome after uptake into the cell. 

Most recently, I have synthesized PEG-lipids carrying terminal peptide residues.  I have devised a synthetic scheme that allows for facile variation of the length of the PEG spacer and the use of solid phase synthesis to construct the peptide and the lipid.  So far, I have prepared lipids bearing peptide sequences for cell targeting (RGD-type), nuclear localization and buffering as well as a protein transduction sequence.  The investigation of DNA complexes of these lipids is currently ongoing.  

I am also involved in other projects in Prof. Safinya's group, synthesizing deuterated dendrimers and fluorescently labeled PEGs.