New Degradable Cationic Lipid-DNA Complexes for Gene Delivery

Abstract

Cationic lipids (CLs) continue to attract attention as synthetic nucleic acid (NA) vectors, and are broadly used for gene transfection and silencing including applications in clinical trials. However, these complexes exhibit suboptimal gene expression due to inefficiencies in overcoming the cellular barriers to transfection (Ahmad et al., J. Gene Med. 2005, 7, 739). After entry of the CL-DNA complex into the cell, two major barriers are efficient dissociation of DNA from the complex and the cytotoxicity of CLs. To address both these issues, we have synthesized a novel series of multi¬valent lipids (CMVLs) with degradable disulfide bonds linking the positively charged headgroups of the CMVLs to their hydrophobic tails. The linker is designed to be cleaved in the reducing milieu of the cytoplasm thus facilitating CL-NA complex degradation, reducing cytotoxicity and improving NA release. X-ray scattering demonstrates that CMVLs form lamellar complexes with DNA, which degrade in reducing environments mimicking the cytoplasmic milieu. For lipids with highly charged headgroups such as CMVL5 (5+), X-ray diffraction under reducing conditions shows DNA condensed by the cleaved headgroup. No such condensation is observed for smaller headgroup charge, as in CMVL2 (2+). Most significantly, we observed an unexpectedly large reduction in cytotoxicity of degradable CMVL-vectors compared to vectors prepared from corresponding lipids without degradable bonds (MVLs). This is of particular importance for applications in gene silencing, where the delivery of short interfering RNAs (siRNAs) requires large CL/NA charge ratios. Correspondingly, the transfection efficiency of CMVL-DNA complexes remains high for high CL/DNA charge ratios. In summary, the much reduced cytotoxicity of these new degradable multivalent lipids and their propensity for DNA release in the cytoplasm open the way for the development of efficient non toxic CL-vectors of NAs. Funding provided by NIH GM-59288.