Highly Efficient Gene Silencing Activity of siRNA Embedded in a Nanostructured Gyroid Cubic Lipid Matrix

Abstract

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing pathway with wide-ranging applications in functional genomics, therapeutics, and biotechnology. Cationic liposome-small interfering RNA (CL-siRNA) complexes have emerged as vectors of choice for delivery of siRNA, which mediates RNAi. However, siRNA delivery by CL-siRNA complexes is often inefficient and accompanied by lipid toxicity. We report the development of CL-siRNA complexes with a novel cubic phase nanostructure, which exhibit efficient silencing at low toxicity. The inverse bicontinuous gyroid cubic nanostructure was unequivocally derived by synchrotron X-ray scattering, while fluorescence microscopy revealed co-localization of lipid and siRNA in complexes. We attribute the efficient silencing to enhanced fusion of complex and endosomal membranes, facilitated by the cubic phase membrane's positive Gaussian modulus which may enable spontaneous formation of transient pores. The findings underscore the importance of understanding membrane-mediated interactions between CL-siRNA complex nanostructure and cell components in developing CL-based gene silencing vectors.

Cationic liposome–nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing

“…Remarkably, this structure was found to improve TE in hard-totransfect mouse embryonic fibroblast cells. More recently, a novel bicontinuous gyroid cubic phase (figure 2b, labelled Q G,siRNA II ) with siRNA incorporated within its two water channels was reported [36][37][38]. This phase was discovered in mixtures of siRNA with CLs containing a cubic phase forming lipid (GMO: 1-monooleoyl-glycerol) and DOTAP or the multivalent cationic lipid MVL5.…”

“…This phase was discovered in mixtures of siRNA with CLs containing a cubic phase forming lipid (GMO: 1-monooleoyl-glycerol) and DOTAP or the multivalent cationic lipid MVL5. The gyroid Q G,siRNA II phase shows high silencing efficiency due to the tendency of cubic phase-forming lipids to induce pores in the endosomal membranes that envelop CL-siRNA complexes and thus facilitate cytoplasmic delivery [36]. Significantly, DOTAP/GMO-siRNA complexes in the gyroid cubic Q G,siRNA II phase, at low cationic lipid content (i.e.…”

Research progress on siRNA delivery with nonviral carriers

“…The results demonstrate that the systems mediate a maximal specific gene silencing and knockdown with significantly low toxicity to mammalians. Leal et al 42 reported the development of cationic liposome (CL)-siRNA complexes with novel cubic phase nanostructures that exhibit efficient silencing at low toxicity. This finding underscored the importance of understanding the membrane-mediated interactions between the CL-siRNA complex nanostructure and cell components in developing CL-based genesilencing vectors.…”

Cationic Liposomes in Different Structural Levels for Gene Delivery

“…It has been concluded that DOPE facilitates endosomal escape by forming an unstable inverted hexagonal phase at the endosomal pH that destabilizes both the complex and the endosomal membrane. But in a recent study (Leal et al, 2010), they developed CL-siRNA complexes with a novel cubic phase nanostructure exhibiting efficient silencing at low toxicity by using glycerol monooleate other than DOPE as the helper lipid. The inverse bicontinuous gyroid cubic nanostructure was unequivocally established from synchrotron X-ray scattering data, while fluorescence microscopy revealed colocalization of lipid and siRNA in complexes.…”