Biocompatible Nanovector of siRNA Consisting of Arginine-Based Cationic Lipid for Gene Knockdown in Cancer Cells

Abstract

Despite the use of small interfering RNAs (siRNAs) as therapeutic agents through the knockdown expression of pathogenic proteins, transportation and delivery of such siRNAs into cells continue to be under investigation. Within nonviral vectors, cationic lipids that include amino acid residues in their structures, and that have already demonstrated their suitability as plasmid DNA nanocarriers, may be also considered as potential siRNA vehicles. A double-chain cationic lipid based on the amino acid arginine mixed with a helper lipid has been the object of this biophysical study. First, ζ-potential measurements and agarose gel electrophoresis experiments confirmed the siRNA compaction, while small-angle X-ray scattering analysis (SAXS) revealed the structural pattern of the lipoplexes. Two bicontinuous cubic phases were found to coexist: the double-gyroid phase (Q II G ) and the double-diamond phase (Q II D ), with Pn3m and Ia3d as crystallographic space groups, respectively; the siRNA is known to be located inside their bicontinuous aqueous channels. Second, in vitro studies in HeLa-green fluorescent protein (GFP) and T731-GFP cell lines (modified for GFP overexpression) showed moderate to high gene knockdown levels (determined by flow cytometry and epifluorescence microscopy) with remarkable cell viabilities (CCK-8 assay). Finally, nano-liquid chromatography/mass spectrometry (nanoLC-MS/MS) was used to identify the nature of the proteins adhered to the surface of the lipoplexes after incubation with human serum, simulating their behavior in biological fluids. The abundant presence of lipoproteins and serum albumin in such protein corona, together with the coexistence of the bicontinuous cubic phases, may be behind the remarkable silencing activity of these lipoplexes. The results reported herein show that the use of amino-acidbased cationic lipids mixed with a suitable helper lipid, which have already provided good results as DNA plasmid nanocarriers in cellular transfection processes, may also be a biocompatible option, and so far little investigated, in gene silencing in vitro strategies.

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.…”

“…SAXS diffractograms (plots of intensities vs. the momentum transfer vector, q) were obtained for C 3 (C 16 His) 2 /MOG-siRNA lipoplexes at ρ eff = 4 and 10, and are collected in Figure 2 a. These ρ eff values were chosen to assure a good compaction level of the siRNA (ρ eff > 1, see Figure 1 b) and also because they were tested with successful results in other nanocarriers of pDNA or siRNA previously reported by us [ 26 , 43 ]. As can be observed in Figure 2 a, the Bragg peaks can be correlated with the Miller indexes of a lamellar L α lyotropic liquid crystal phase ((hkl) = (100), (200) and (300)) in both conditions (blue labels in Figure 2 a).…”

“…The proteomic profile of proteins surrounding the surface of C 3 (C 16 His) 2 /MOG-siRNA lipoplexes was analyzed through nanoLC-MS/MS. The procedure followed was fully detailed in a previous work [ 43 ]. Briefly, the lipoplexes formed were incubated for 1 h at 37 °C in the presence of HS.…”

“…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 ].…”

“…Following this strategy, and taking into account previous studies of cationic lipids (CLs) incorporating amino acid derivatives in their structures such as lysine [ 18 , 53 ] and arginine derivatives [ 43 ], we have worked in this study with a nanovector based on a gemini cationic lipid with functionalized histidine residues on the head groups, the bis(N(τ),N(π)-bis(methyl)-histidine hexadecyl amide) propane—abbreviated as C 3 (C 16 His) 2 (see Scheme 1 a). This GCL has already been used as an interesting nanoplatform for pDNA delivery [ 26 ].…”