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

TLDR

RNA interference is a conserved gene‑silencing pathway, and cationic liposome‑siRNA complexes are common delivery vectors, yet they often suffer from low efficiency and lipid toxicity. The authors engineered a cubic gyroid nanostructure in CL‑siRNA complexes, confirmed by X‑ray scattering and fluorescence microscopy, and attribute its superior silencing to membrane fusion facilitated by the phase’s positive Gaussian modulus that promotes transient pore formation. The resulting cubic CL‑siRNA complexes achieve potent gene silencing with reduced toxicity, highlighting the critical role of membrane‑mediated interactions between nanostructure and cellular components.

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 established from synchrotron X-ray scattering data, while fluorescence microscopy revealed colocalization 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.