Efficient Cellular Knockdown Mediated by siRNA Nanovectors of Gemini Cationic Lipids Having Delocalizable Headgroups and Oligo-Oxyethylene Spacers

Abstract

The use of small interfering RNAs (siRNAs) to silence specific genes is one of the most promising approaches in gene therapy, but it requires efficient nanovectors for successful cellular delivery. Recently, we reported liposomal gene carriers derived from a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl dimethyl imidazolium) oligo-oxyethylene series ((C16Im)2(C2H4O)nC2H4 with n = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine as highly efficient cytofectins for pDNA. On the basis of the satisfactory outcomes of the previous study, the present work focuses on the utility of coliposomes of these gemini lipids with the biocompatible neutral lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA cellular transport in the presence of serum. The (C16Im)2(C2H4O)nC2H4/MOG-siRNA lipoplexes were characterized through (i) a physicochemical study (zeta potential, cryo-transmission electron microscopy, small-angle X-ray scattering, and fluorescence anisotropy) to establish the relationship between size, structure, fluidity, and the interaction between siRNA and the GCL/MOG gene vectors and (ii) a biological analysis (flow cytometry, fluorescence microscopy, and cell viability) to report the anti-GFP siRNA transfections in HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological analysis confirms the cellular uptake and indicates that a short spacer, a very low molar fraction of GCL in the mixed lipid, and a moderate effective charge ratio of the lipoplex yielded maximum silencing efficacy. At these experimental conditions, the siRNA used in this work is compacted by the GCL/MOG nanovectors by forming two cubic structures (Ia3d and Pm3n) that are correlated with excellent silencing activity. These liposomal nanocarriers possess high silencing activity with a negligible cytotoxicity, which strongly supports their practical use for in vivo knockdown studies.

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

“…In the present work, the GCL C 3 (C 16 His) 2 was used in combination with the helper lipid MOG as a nanoplatform to introduce gene material into HeLa and T731 cancer cells in an efficient and safe manner. A molar fraction (α) with respect to the GCL of 0.2, which implies a larger content of the neutral helper lipid (MOG) in the mixture, was chosen since it was revealed as optimal in previous works with similar siRNA nanocarriers [ 38 , 43 ]. The efficiency of the siRNA nanovector relies on a siRNA packing-unpacking mechanism conducted by the lipid mixture, which is responsible for the compaction of siRNA and its subsequent delivery inside the cells.…”

“…This effect has also been observed in lipoplexes formed by the lipid mixture C 3 (C 16 His) 2 /DOPE and pDNA [ 26 ] and in others lipoplexes that contain GCL lipids bearing the imidazolium ring in their structure [ 56 , 66 ]. By contrast, the effective charge of siRNA is considered to be the same as its nominal one ( ), as reported in the literature for either linear DNA (with thousands of base pairs, as calf thymus or salmon sperm DNA) [ 56 , 57 , 58 ] or short RNAs (with 19–25 bp) [ 38 , 67 ]. Considering these results, it can be concluded that the formation of lipoplexes is mainly driven by electrostatic forces between the oppositely charged cationic lipid and the anionic siRNA molecules.…”

“…The insertion of amino acid moieties, bare or functionalized, in their structure has been generally more used in pDNA transfection [ 18 , 20 , 22 , 24 , 25 , 26 , 27 , 28 ] than in siRNA vectorization [ 29 , 30 , 31 , 32 ], despite the fact that amphiphilic imidazolium salt has already been physico-chemically presented as a new generation of reagents for RNAi [ 33 ]. Additionally, gemini cationic lipids (GCL) have demonstrated to be efficient gene nanovectors [ 34 , 35 , 36 ], especially when an imidazolium group was included in their structure [ 37 , 38 ]. Both synthetic strategies are focused on overcoming the biological barriers that the nanocarrier encounters once it enters the bloodstream [ 39 ], as well as on improving the endocytosis pathway [ 40 ].…”

“…The helper lipid habitually used in the gene-knockdown field is the 1-(cis-9-octadecenoyl)-rac-glycerol (MOG). Its biocompatibility and ability to induce different lyotropic liquid crystal phases make it a safe option in gene therapy [ 38 , 41 , 42 , 43 ].…”

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid

“…Numerous artificial systems formed by lipids [7][8][9], polymers [10][11][12], dendrimers [13,14], carbohydrates [15,16], cyclodextrins [17,18], polypeptides [19,20], and nanoparticles [21,22] (among others) have been studied to avoid the adverse effects produced by viral vectors in biological media, such as immunogenicity and inflammatory responses [23,24]. In particular, cationic lipids have been investigated in detail as useful tools [25,26] because they present low toxicity and are easily synthesized, as well as establish strong electrostatic interactions with DNA/RNA double strains through their positive charges affording lipoplexes. Furthermore, the nanometer size and net positive charge of these complexes favors their internalization across the cell membrane, allowing the delivery of the cargo in the cellular cytoplasm and resulting in moderate-to-high transfection efficacy.…”