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

Abstract

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.

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

“…As seen in this table, the interlamellar distance is not affected by the lipoplex composition (ρ eff ). Thus, an average value of (6.1 ± 0.3) nm has been obtained, in agreement with those found for other multilamellar lipoplexes previously reported [ 18 , 26 ]. On the other hand, d m was estimated around 4.5 nm from cryo-TEM micrographs where liposomes were found (see Figure 2 d, as an example) and also with Tanford’s model [ 69 , 70 , 71 ].…”

“…Dendrimers [ 12 , 13 ], polymers [ 14 , 15 ] and lipids [ 16 , 17 ] are other nanocarriers among non-viral vectors that may incorporate natural amino acids in their structures to reduce cytotoxicity and improve cellular uptake. Lysine, arginine and histidine are some of the most commonly used amino acids because of their positive charge at physiological pH, which enables electrostatic interactions with the anionic nucleic acids and the negatively charged cellular membranes [ 16 , 17 , 18 , 19 , 20 ]. In particular, the histidine group also offers a mechanism for endosomal scape, known as the “proton sponge effect” [ 21 , 22 , 23 ].…”

“…This strategy has been used as a source of inspiration for histidine-based nanocarriers in the last decade [ 21 ], specifically in the case of cationic lipids (CL), which have received much attention as non-viral gene vectors thanks to their structural similarities with the cell membrane. 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 ].…”

“…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 ].…”

Interaction of Lipoplex with Albumin Enhances Gene Expression in Hepatitis Mice

“…Plasmid DNA (pDNA) is one of the typical non-viral gene vectors, but naked pDNA itself does not show sufficient transfection efficiency after intravenous injection with normal volume. Therefore, cationic liposome-pDNA complex (lipoplex) is often used in transfection [7,8]. The surface of lipoplex is positively charged because of cationic liposomes.…”