Reducible cationic lipids for gene transfer

Abstract

One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization.

Functional lipids and lipoplexes for improved gene delivery

“…Scherman et al prepared a series of lipopolyamines that contain reducible disulfide bonds at various structural positions within the lipid in order to evaluate its consequence on transfection [193,194]. In one example, the disulfide group is located in the linker domain and in the other between the linker and an additional C5 or C12 hydrocarbon chain (Fig.15) [193,194].…”

“…Scherman et al prepared a series of lipopolyamines that contain reducible disulfide bonds at various structural positions within the lipid in order to evaluate its consequence on transfection [193,194]. In one example, the disulfide group is located in the linker domain and in the other between the linker and an additional C5 or C12 hydrocarbon chain (Fig.15) [193,194]. In vitro transfection experiments showed that the vector with the disulfide located in the linker domain results in a loss of activity, whereas the lipids with a disulfide connecting the additional side chain displays a substantial improvement in transfection efficiency.…”

“…Also, cleavage of one of the two fatty acid chains, which converts the lipid into a detergent, also led to higher transfection compared to a non-cleavable analogue. In a subsequent study [194] they synthesized additional vectors (shown in Fig. 16) including: 1) a symmetric lipid with the disulfide bond between the hydrophilic region and hydrophobic region (DSL-1); 2) a gemini lipid with the disulfide connecting the two identical lipopolyamines (DSL-2); 3) an asymmetric lipid with one short alkyl chain and 1 long alkyl chain that contains the disulfide bond (DSL-3); 4) a similar lipid to DSL-3 but with a long alkyl chain and the disulfide located in the short alkyl chain (DSL-4); and the non-reducible counterpart to DSL-3 (NDSL).…”

Addition of ascorbic acid to the extracellular environment activates lipoplexes of a ferrocenyl lipid and promotes cell transfection

“…Over time, however, the structures and properties of lipids used for cell transfection have evolved to include chemical functionality designed to overcome or address a variety of intracellular barriers that limit cell transfection efficiency. For example, lipids have been designed to form lipoplexes that are stable in complex extracellular environments (including in the presence of serum proteins) [9–11] and/or release their cargo (DNA) in response to intracellular stimuli, including the presence of reducing agents [12–16] (e.g., glutathione), changes in pH [15, 17–19], or the presence of specific enzymes [20, 21]. The design of lipids that promote more efficient intracellular trafficking of internalized DNA has contributed significantly to the development of lipoplex-based approaches to DNA delivery.…”

Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery

“…This mechanism is used by biological toxins [37] as well as in drug delivery and diagnostic imaging [38-42]. A few studies have also applied the concept to gene delivery vectors [43-46], including CL–DNA complexes [47-50]. …”