Custom Chemical Synthesis

Design of Novel Lipids for Gene Delivery



A key facet of the interdisciplinary research in our group is the capability to synthesize tailored lipids and other molecules to probe important structural, chemical and physico-chemical parameters. For example, cationic liposome-DNA (CL-DNA) complexes are widely used in nonviral gene delivery. Some lipid vectors have been commercialized, and many are quite effective in transferring DNA to 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. This will require a combination of approaches, engineering more virus-like CL-DNA vectors with tailored lipids based on an understanding of the delivery pathway. To date, the mechanism of gene delivery by CL-DNA complexes is not understood completely. The new lipids we synthesize are designed to help investigate the mechanism of gene delivery and provide rational routes towards improving transfection efficiencies by addressing the current shortcomings of CL-DNA complexes.

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. 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 (more information in the section about CL-NA complexes on this website). 

Multivalent Cationic Lipids         

On the other hand, certain properties of the self-assembled vector are directly related to transfection efficiency through their effect on well known cellular barriers to transfection. Our synthetic work aims to design and synthesize lipids that allow varying these important vector properties to probe their effect on transfection efficiency. An important example is the synthesis of a series of multivalent cationic lipids. Some multivalent cationic lipids had been reported to have extraordinarily high transfection efficiencies. These lipids, however, were not available in sufficient quantities to unravel their mechanism of action. Thus, we developed an efficient (gram scale) synthesis of a series of new multivalent lipids [1,2]. The headgroup charge of these lipids (termed MVLs for multivalent lipids) was systematically varied from +1 to +5 while adding only a minimal number of atoms to the structures. X-ray scattering, microscopy and transfection experiments with these lipids show that they are highly efficient even when present in a fairly low concentration in the lipid. Having access to a whole series of lipids with systematically varied headgroup charged allowed us to identify the average charge per area of the lipid membrane (the membrane charge density) as a universal, i.e. lipid-independent, parameter governing the transfection efficiency for lamellar complexes [2].  The related cellular barriers are escape of the complexes from the endosome (up to a certain, optimal charge density) and excessive stability of CL-DNA complexes at very high charge densities. 

Cationic Lipids with Highly Charged Dendritic Headgroups

The discovery that membrane charge density is a key parameter for transfection motivated us to further explore the chemical parameter of headgroup charge. Using the amino acid ornithine as an AB2 building block, we have constructed lipids with dendritic headgroups. Each dendritic generation doubles the number of amino groups, which can serve as charge bearing groups or to connect previously prepared multivalent building blocks (see Fig. 1). In this way, we have prepared a series of lipids bearing 4, 8 and 16 positive charges in the headgroup [3,4]. Again, an efficient synthesis had to be developed to allow gram scale synthesis of the lipids. The most highly charged lipid, named MVLBG2 (16+, see Fig.1)) forms a novel structure of lipid DNA complexes, containing cylindrical lipid micelles in a hexagonal array with DNA arranged around them in a honeycomb structure. DNA complexes of this lipid transfect mammalian cells well, even showing a much enhanced performance (comparred to the commercially available standard lipid DOTAP) in a cell line that is empirically known to be hard to transfect [3].


Figure 1. Synthesis and structure of the new lipid MVLBG2 and the structure of its complexes with DNA (when MVLBG2 is mixed with 75 mol% of the neutral lipid DOPC). See also [3].

Poly(ethylene glycol)-Lipids (PEG-lipids)      

For applications of CL-NA complexes in vivo, it is essential to shield the complexes from rapid clearance from the blood stream using a shell of a flexible hydrophilic polymer on the outside. This idea was first developed in liposomes for drug delivery (so called STEALTH liposomes). We have synthesized a series of PEG-lipids containing PEG chains of varied lengths in their headgroup and investigated the effect of incorporating thess lipids into CL-DNA complexes.

Cholesterol or two alkyl chains can serve as the lipophilic moiety on one end of the PEG chain, while the other bears a hydroxyl group. This in turn can be used to attach other moieties, such as the amino acid ornithine (corresponding to two cationic charges) [5].

Our work has shown how the internal structure of the complexes is affected by the presence of short and long polymer chains, and that the PEG-lipids (with PEG headgroups that exceed a certain chain length) are indeed capable of shielding the complexes [6]. Complex aggregation in salt-containing cell culture media is prevented but at the same time, transfection efficiency is reduced by the PEG shield. This is due to the reduced electrostatic interaction between the positively charged complexes and the negatively charged cell membranes. For gene delivery applications, this apparent drawback of adding a PEG shield actually presents an opportunity. Namely, the unspecific attachment of CL-NA complexes to any cell can be substituted with specific (receptor-mediated) interactions. To this end, we have recently synthesized PEG-lipids carrying terminal peptide residues that bind to specific receptors on the cell surface. Current research has shown that a whole system of molecualr "zip codes" exists which allows not only the targeting of a specific tissue, but also the distinction between healthy and diseased tissues [7]. Our synthetic scheme allows facile variation of the length of the PEG spacer and the use of solid phase synthesis to construct the peptide and the lipid.  The investigation of DNA complexes of these lipids is currently ongoing.  

For more information:

[1] Ewert, K.; Ahmad, A.; Evans, H. M.; Schmidt, H. W.; Safinya, C. R.: Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery. J. Med. Chem. 2002, 45, 5023-5029.   MVL5 is now commercially available from Avanti Polar Lipids.
[2] Ahmad, A.; Evans, H. M.; Ewert, K.; George, C. X.; Samuel, C. E.; Safinya, C. R.: New multivalent cationic lipids reveal bell curve for transfection efficiency versus membrane charge density: lipid-DNA complexes for gene delivery. J. Gene Med. 2005, 7, 739-748.
[3] Ewert, K. K.; Evans, H. M.; Zidovska, A.; Bouxsein, N. F.; Ahmad, A.; Safinya, C. R.: A columnar phase of dendritic lipid-based cationic liposome-DNA complexes for gene delivery: Hexagonally ordered cylindrical micelles embedded in a DNA honeycomb lattice. J. Am. Chem. Soc. 2006, 128, 3998-4006.

[4] Ewert, K. K.; Evans, H. M.; Bouxsein, N. F.; Safinya, C. R.: Dendritic cationic lipids with highly charged headgroups for efficient gene delivery. Bioconjugate Chem. 2006, 17, 877-888.
[5] Martin-Herranz, A.; Ahmad, A.; Evans, H. M.; Ewert, K.; Schulze, U.; Safinya, C. R.: Surface functionalized cationic lipid-DNA complexes for gene delivery: PEGylated lamellar complexes exhibit distinct DNA-DNA interaction regimes. Biophys. J. 2004, 86, 1160-1168.
[6] Schulze, U.; Schmidt, H. W.; Safinya, C. R.: Synthesis of novel cationic poly(ethylene glycol) containing lipids. Bioconjugate Chem. 1999, 10, 548-552.
[7] Ruoslahti, E.: Vascular zip codes in angiogenesis and metastasis. Biochem. Soc. Transact. 2004, 32, 397-402.