SYNTHESIS, CHARACTERIZATION, AND IN-VITRO EVALUATION OF LIPOPEPTIDE AND OLIGOPEPTIDE- BASED LIPOSOME FOR GENE DELIVERY VEHICLE CANDIDATE
Efficient and safe gene delivery into a target cell in gene therapy is one of the active areas in pharmaceutical research currently. Non-viral expression vectors are highly desirable due to their safety, stability, and suitability for mass production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in the application of non-viral gene delivery. Lipopeptide-based gene carriers generally have low cytotoxicity, are capable of cell membrane penetration, and relatively simple to manufacture. In an attempt to obtain an efficient with low cytotoxicity of the non-viral gene delivery vector, we have designed a serial short linear lipopeptide transfection agents (MW of 700-1300 Da), consisting of an alkyl chain of lauryl (C-12) or palmitoyl (C-16), one cysteine, 1 to 5 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization via cysteine residue, the DNA binding at a neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues via proton sponge-like mechanism). Molecular interaction and dynamic simulation were performed to study the possibility of the short lipopeptide in facilitating active nuclear active by interacting with a shuttle protein of Importin-?. In-vitro evaluation of the transfection agent/DNA complexes was performed for their biophysical properties and transfection efficiencies. The in-silico interaction showed that Lau-CK2H and Pal-CK2H2 hypothetically expedited nuclear uptake. Both lipopeptides had lower binding energy (-6.3 kcal/mol and -6.2 kcal/mol, respectively), compared to the native ligand, viz, NLS (-5.4 kcal/mol). The number and identity of amino acids incorporated in the short linear lipopeptide construct affected their lipopeptide/ DNA complex-forming capacity, protection from DNase degradation, and transfection efficiency. In-vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the short linear lipopeptides, was much higher than Poly(L-lysine), which has no endosomal escape system. The transgene expression (ng Luciferase/mg protein) facilitated by two lipopeptides (Pal-CK2H2 and Pal- CK3H2) was slightly higher than that of branched polyethylene-imine (PEI-25K). The transgene expression of short linear lipopeptide was also enhanced by chloroquine up to 8-fold; however, it dramatically reduced in the presence of an inhibitor vacuolar H (+)-ATPase of bafilomycin A1 and restored to the initial value when the chloroquine was added into transfection systems. Co-polymer PEI-25K enhanced the transgene expression of the short linear lipopeptide-mediated gene delivery up to 50-fold, probably by interfering complex particle sizes and involving in the intracellular trafficking. More extended lipopeptide of Pal-CK2H2 carrying peptide sequences of trans-activator of transcription (TAT: YGRKKRRQRRR) and nuclear localization sequence (NLS: PKKKRKV) have been constructed to increase the capability of the lipopeptide in condensing DNA and penetrating the cell membranes. The extended lipopeptide (MW 3.3 kDa) and oligopeptide of TAT-NLS (MW 3.0 kDa) were synthesized using a peptide synthesizer according to the Fmocbased solid-phase peptide synthesis method. The extended lipopeptide could bind, condense, and protect DNA from DNase degradation efficiently. The lipopeptide composed of TAT-NLS/DNA nanoparticles were stable for up to 20 days at 6-8 o C. Intriguingly, TEM images of the lipopeptide/DNA complexes at charge ratio 4.0 were homogenous with a compact size of ~120 nm. The lipopeptides were also considered less cytotoxic to CHO-K1 and HepG2 cells then Poly-L-Lysine and Lipofectamine TM -2000. Transfection study using GFP encoding gene (pCSII-EF- AcGFP) revealed that the protein expression mediated by the lipopeptide was observed at charge ratio 4.0-8.0. The oligopeptide of TAT-NLS based-liposome with a neutral DOPE has increased the GFP expression on HepG2 cells comparable or slightly higher than the most common commercially available Lipofectamine TM - 2000. These results indicated that the lipopeptide-based transfection agent and the oligopeptide-based liposome could condense and protect DNA efficiently, forms stable and homogenous nanoparticles, and displays favourable characteristics as the potential gene carrier candidate with low cytotoxicity. The peptide-based transfection agents need to be optimized further, including the option of the type of alkyl chains (saturated or non-saturated alkyl chains), the sequence of the amino acid residues (linear or branched) included in the transfection agents. The formulation of a liposome-derived transfection agent needs to be optimized by selecting a different kind of lipids and surfactants for efficient in-vitro and in-vivo gene delivery purposes.