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.