Gene therapy offers the potential to cure a wide-range of diseases by delivering a missing gene or a functional substitute of a defective gene. Presently, the gene transfection activity is low with synthetic vectors, reflecting inefficiencies in the overall transfection pathway that include: DNA-synthetic vector complexation, endocytosis, endosomal escape, nuclear entry, and finally expression. Our research effort is focused on improving the release of the negatively-charged DNA from the DNA-amphiphile supramolecular complex. We have reported a new approach which entails the use of a charge-reversal amphiphile that transforms from a cationic (+1) to an anionic amphiphile (-1) intracellularly. This functional synthetic vector performs two roles: first, it binds and then releases DNA, and second, as a multi-anionic amphiphile, it destabilizes bilayers. This gene delivery system which undergoes an electrostatic transition intracellularly shows enhanced gene transfection efficiency.
Specificially, we have synthesized and characterized a family of charge-reversal amphiphiles. The prototype amphiphile, 1, has a cationic ammonium head group to bind DNA, lipophilic acyl chains to form a bilayer, and benzyl esters at the terminus of the acyl chains for enzymatic hydrolysis. To assess the role of each structural component, we have prepared compounds. Transfection experiments using the reporter gene, b-galactosidase (b-gal, pVax-LacZ1, Invitrogen) were performed with Chinese hamster ovarian (CHO) cells. Transfection efficiencies were assessed after 48 h using the b-galactosidase enzyme assay. Amphiphiles 1, 5, and 6-10 show some level of transfection, but the efficiency is greatest when using amphiphile 1. Amphiphile 1 can also transfect HEK293 and K562 cells (data figure not shown). Cytotoxicity experiments were also performed with CHO cells and none of the amphiphiles showed significant cytotoxicity, with results similar to the non-treated cells.
The charge reversal amphiphile 1 has limited aqueous solubility, and thus hampers formulation as well as the extent to which its structure can be altered to identify an optimized synthetic vector. Thus we have investigated other head groups. For example, in biology the recognition of nucleic acids by proteins involves more than electrostatic interactions. Examination of these protein nucleic acid recognition motifs typically reveals structures rich in basic and aromatic amino acids that provide important electrostatic and stacking contributions to binding. With this inspiration in mind, we explored peptide-based amphiphiles for gene delivery. Consequently, we have synthesized and characterized 14 new peptide-based amphiphiles with varied head groups and chain lengths. These amphiphiles are also active transfection agents. We are currently synthesizing new amphiphiles that possess the favorable attributes of both the charge-reversal and peptide amphiphiles.