N4,N9Dimyristoyl Spermine is a NonViral Cationic Lipid Vector for Plasmid DNA Formulation Osama A. A - PowerPoint PPT Presentation

Title: N4,N9Dimyristoyl Spermine is a NonViral Cationic Lipid Vector for Plasmid DNA Formulation Osama A. A


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N4,N9-Dimyristoyl Spermine is a Non-Viral
Cationic Lipid Vector for Plasmid DNA
FormulationOsama A. A. Ahmed, Charareh Pourzand
and Ian S. BlagbroughDepartment of Pharmacy and
Pharmacology, University of Bath, Bath BA2 7AY,
U.K.prsisb_at_bath.ac.uk
Introduction Non-viral delivery systems can be
defined to include the use of plasmid DNA alone
(so-called naked DNA) as well as DNA complexed
to synthetic carriers such as cationic lipids or
polymers (Niidome and Huang, 2002 Templeton,
2002). The use of an efficient carrier for
nucleic acid delivery is considered to be a
determinant factor for the successful application
of gene therapy. Cationic lipids are major gene
carriers among non-viral delivery systems. They
have the ability to condense DNA into particles
that can be readily endocytosed by cultured
cells, and facilitate endosomal escape leading to
efficient delivery to the nucleus (Blagbrough et
al, 2003 Tranchant et al 2004). They can be
classified as liposomal and non-liposomal
non-viral delivery vectors.
Lipoplex Particle Size and Zeta Potential
Measurements N4,N9-Dimyristoyl spermine-pEGFP
complex at N/P ratio (/-) 15, shows an average
particle size of 62 nm and has a z-potential of
43 mV (at the optimum charge ratio of
transfection), after mixing with a vortex mixer.
All measurements were carried out on lipoplexes
with 5 mg/ml plasmid DNA in HEPES buffer at pH
7.4 and 20 ?C.
43 mV
diameter 62 nm
Fig. 4. Particle size and z potential of
N4,N9-dimyristoyl spermine-pEGFP complex.
Transfection and Cytotoxicity Six cell lines were
used in the transfection experiments, FEK4, FCP4,
FCP5, FCP7 and FCP8 cells are human primary
fibroblasts derived from newborn foreskin
explants. HtTA cells are a human cervical
carcinoma, HeLa derived and transformed cell
line.
Fig. 1. The process of non-viral gene therapy by
endocytosis and the barriers that must be crossed
by toroidal DNA nanoparticles are illustrated for
lipopolyamines in the formulation of lipoplexes
(condensed DNA nanoparticles) leading to DNA
delivery and ultimately to the goal of
intracellular protein synthesis.
Fig. 5. Lipofection of primary skin and cancer
cell lines transfected with pEGFP alone (naked)
or complexed with N4,N9-dimyristoyl spermine or
the commercially available lipopolyamine
Transfectam.
Fig. 6. Viability of primary skin cells and
cancer cell lines transfected with pEGFP (2
mg/ml) free (naked DNA) or complexed with either
N4,N9-dimyristoyl spermine (30 mg/ml) or
Transfectam (15 mg/ml) in the tested cells.
Conclusions A lipopolyamine vector,
N4,N9-dimyristoyl spermine, has been developed
for DNA delivery. This new non-liposomal
formulation has the ability to transfect both
primary skin cells and cancer cell lines
efficiently. Acknowledgements We acknowledge
the financial support of the Egyptian Government
(studentship to O.A.A.A.). We are grateful to
Prof R. M. Tyrrell (University of Bath) for the
FEK4 and HtTA cell lines.
Fig. 2. Synthesis of N4,N9-dimyristoyl spermine.
References Blagbrough I. S. Geall A. J. and Neal
A. P. (2003), Polyamines and novel polyamine
conjugates interact with DNA in ways that can be
exploited in non-viral gene therapy, Biochem.
Soc. Trans. 31397-406. Geall A. J. and
Blagbrough I. S. (2000), Rapid and sensitive
ethidium bromide fluorescence quenching assay of
polyamine conjugate-DNA interactions for the
analysis of lipoplex formation in gene therapy,
J. Pharm. Biomed. Anal., 22849-859. Niidome T.
and Huang L. (2002), Gene therapy progress and
prospects Nonviral vectors. Gene Ther.
91647-1652. Templeton N. S. (2002), Liposomal
delivery of nucleic acids in vivo. DNA Cell Biol.
21857-867. Tranchant I. Thompson B. Nicolazzi
C. Mignet N. and Scherman D. (2004),
Physicochemical optimisation of plasmid delivery
by cationic lipids. J. Gene Med. 6S24-S35.
Fig. 3. Comparison of ethidium bromide
fluorescence quenching assay (lexcit 260 nm and
lemiss 600 nm with slit width 5 nm) (Geall and
Blagbrough 2000) and light scattering assay (
relative maximum apparent absorbance at ? 320
nm) of N4,N9-dimyristoyl spermine complexed with
pEGFP.