Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads

Abstract

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presented ; This work has been funded by the Spanish Ministry of Science, Innovation and Universities (MICIU) (Grant RTI2018-095844-B-I00 and CTQ2017-88948-P), the University Complutense of Madrid (Spain) (project number UCMA05-33-010), and the Regional Government of Madrid (Grant P2018/NMT-4389). P.T. thanks Agencia Estatal de Investigación (AEI) through the Project MAT2016-80266-R and Xunta de Galicia (Grupo de Referencia Competitiva ED431C 2018/26; Agrupación Estratégica en Materiales-AEMAT ED431E 2018/08). ERDF funds are all greatly acknowledged. The proteomic ...