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*.
Highlights
Therapeutic agents capable of replacing/silencing damaged DNA are one of the principal areas of research in the branch of medicine known as gene therapy [1,2,3]
The zeta potential data plotted against the mL/mDNA =/mDNA mass ratios display the typical sigmoidal profile, where charge inversion can be observed at specificφ values corresponding to the electroneutrality ratios
The incorporation of a lysine-derived residue to a C12 alkyl chain affords a biocompatible cationic lipid that, when mixed with the well-known fusogenic dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE) lipid, forms a non-viral gene nanocarrier able to stabilize, compact, and protect two different kinds of plasmids by forming supramolecular LYCl/DOPE-pDNA lipoplexes. These lipoplexes are well organized in a Lα lamellar lyotropic liquid crystal phase characterized by a sandwich-type compaction pattern, with alternating bilayers of LYCl/DOPE mixed lipid (~4.5 nm width) and an aqueous monolayer containing the pDNA and counterions (~2 nm width)
Summary
Therapeutic agents capable of replacing/silencing damaged DNA are one of the principal areas of research in the branch of medicine known as gene therapy [1,2,3]. Since the presence of amino acid residues improves the biocompatibility and cationic lipids are able to establish electrostatic interactions with DNA, some studies have been reported on their combination to design and synthesize alternative candidates for plasmid DNA/small interfering RNA nanocarriers [33,34,35,36]. It has been already reported that gene delivery systems formed by amino acid-based cationic lipids are internalized by habitual endocytic uptake pathways. A lysine-derived residue was introduced in the cationic head Mixtures of this single-chain cationic lipid, (S)-5-acetamido-6-(dodecylamino) -N,N,N-trimethyl-6-oxohexan-1-ammonium chloride (LYCl), and a well-known fusogenic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), were used to compact, protect, and transfect two different plasmid DNAs: those encoding the green fluorescent protein GFP (pEGFP-C3) and luciferase (pCMV-Luc). Each zeta potential and particle size data point represent the average of 50 and 30 independent measurements, respectively
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