Charge-reversal lipids, peptide-based lipids, and nucleoside-based lipids for gene delivery

Abstract

Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy's appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individual's genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: (1) The release of the nucleic acid from the carrier will increase gene transfection. (2) The use of biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery. (3) Mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will produce unique supramolecular assembles with high transfection efficiencies. The results presented in this Account demonstrate that engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics can improve the cellular uptake and transfection efficacy of nucleic acids. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate a shift of the lipid from positive to negative net charge improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, or aromatic) between the cationic headgroup and the hydrophobic chains, we can tailor lipids to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. The introduction of a peptide headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in vitro successes that we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have used in our research to provide readers with the tools to characterize and engineer new vectors.

Figure 1

Schematic of the charge-reversal lipids transforming from a cationic to an anionic state by hydrolysis of the terminal ester linkages on the hydrophobic chains.

Figure 2

Charge-reversal lipids and their analogues, including DOTAP, under investigation for gene delivery.

Figure 3

Transfection efficiency of the charge-reversal lipid, 1, and its analogoues in CHO cells. TransFast was used as the positive control. N=3, Avg + SD.

Figure 4

DNA transfection (blue) and cell uptake (red) in CHO cells in the presence of endocytosis inhibitors. Results are shown as relative to percent transfection (or uptake) without inhibitors. N=3, Avg + SD.

Figure 5

Structures of the betaine charge-reversal amphiphiles with different spacers.

Figure 6

Structure of the charge-reversal helper lipid, 11, and DOPC.

Figure 7

(top) Structures of the peptide-based amphiphiles synthesized. (bottom) DNA transfection results in CHO cells. Lipofectamine 2000 was used as the positive control. N=3, Avg + SD.

Figure 8

Chemical structures of uridine-based and 3-nitropyrrole nucleolipids used for transfection studies. (left to right) N-[5’-(2’,3’-dioleoyl)uridine]-N’,N’,N’-trimethylammonium, DOTAU; O-ethyl-dioleylphosphatidylcholinium-uridine, O-Et-DOUPC; 1‘-(2‘,3‘-dioleyl-5‘-trimethylammonium-d-ribofuranosyl)-3-nitropyrrole.

Figure 9

(top) pEGFP transfection in HeLa and MCF-7 cells using DOTAU, charge ratio in parenthesis. Positive control is Lipofectamine2000. (bottom) β- gal transfection in CHO cells using O-Et-DOUPC, charge ratio in parenthesis. Positive control is TransFast. N=3, Avg + SD.

Figure 10

Chemical structure of an anionic nucleotide-lipid, the thymidine 3’-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (diC16-3’-dT), and a non-nucleotide lipid, DPPG.

Scheme 1

Synthetic route to the charge-reversal lipids possessing a simple quaternary amine and their counterparts.

Scheme 2

Synthetic route to the peptide-based lipids.

Scheme 3

Synthetic route to the zwitterionic and betaine charge-reversal lipids.

Scheme 4

Synthetic route to the cationic nucleoside-based lipids.

Scheme 5

Synthetic route to the anionic nucleotide-based lipids.