Nonviral Formulation of DNA Using N2, N3Dioleoyl Spermine Osama A' A' Ahmed and Ian S' Blagbrough De

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Non-viral Formulation of DNA Using N2,
N3-Dioleoyl SpermineOsama A. A. Ahmed and Ian S.
BlagbroughDepartment of Pharmacy and
Pharmacology, University of Bath, Claverton Down,
Bath BA2 7AY, U.K.prsisb_at_bath.ac.uk
Table 1. Calf thymus DNA binding data.
Introduction Lipospermines as lipopolyamine
non-viral DNA carriers, with their characteristic
cationic (spermine) and lipophilic moieties, are
promising vectors for DNA delivery through the
formation of lipoplexes (Blagbrough et al 2003).
The design of an efficient formula for the
delivery of DNA requires a detailed understanding
of the mechanism of gene delivery to the nucleus.
Non-viral gene delivery vectors continue to show
promise as alternatives to viral vectors. For the
development and formulation of efficient
non-viral gene carriers, the main requirements
are 1. Condensation of DNA (as a prodrug) to a
small particle size, masking the negative charges
on the phosphate backbone to facilitate gene
delivery across the cell membrane 2. Protecting
the active ingredient (DNA) from degradation by
nucleases 3. Endosomal escape 4. Cytosolic
transport 5. Facilitating nuclear entry 6.
Decomplexation from the excipients
Polyamine binding affinity The binding constant
of polyamines with DNA were estimated (Table 1)
and compared using EthBr assay (Basu et al 1990
Geall et al 1998). The drug concentration
producing 50 inhibition of fluorescence is
approximately inversely proportional to the
binding constant (Morgan et al 1979).
Light scattering assay To investigate the
formation of particles by polyamines, the
apparent UV absorbance at 320 nm was measured
showing light scattering.
Fig. 5. Light scattering assay ( relative
maximum apparent absorbance at ? 320 nm) of
calf thymus DNA with PLL9.6k, PLL27k, PEI2k,
PEI60k, spermine and N2,N3-dioleoyl spermine.
Transfection and cytotoxicity The transfection
efficiency of pEGFP into FEK4 (primary skin
fibroplast, derived from a foreskin explant) cell
line and the cancer HtTA cells (HeLa derivative)
was studied using N2,N3-dioleoyl spermine and the
commercially available reagents (Lipofectin and
Lipofectamine?). The results showed a higher
transfection efficiency of N2,N3-dioleoyl
spermine (at the ammonium / phosphate (/-)
charge ratio 2.5) than the commercially available
Lipofectin formulation in the FEK4 primary skin
cell line.
1.
2.
3.
4.
Fig. 1. Cellular barriers to efficient DNA
delivery to the nucleus.
Fig. 6. Lipofection of FEK4 and HtTA cells
transfected with pEGFP complexed with
Lipofectin, Lipofectamine? or N2,N3-dioleoyl
spermine .
Synthesis of N2,N3-dioleoyl spermine Spermine was
protected on the primary amino groups with ethyl
trifluoroacetate. Oleic acid and DCC-HOBt were
added to the diprotected spermine to form
N4,N9-dioleoyl-N1,N12-ditrifluoroacetyl-1,12-diami
no-4,9-diazadodecane. For the removal of the di
trifluoroacetyl groups, N4,N9-dioleoyl-N1,N12-ditr
ifluoracetyl-1,12-diamino-4,9-diazadodecane was
dissolved in methanol and the pH of the solution
was increased to pH 11 by saturating with
ammonia gas (Fig.2).
CF3COOC2H5 Metanol 25 oC for 18 h
Oleic acid DCC, HOBt DCM-MeOH 11 25 oC for 18 h
Fig. 7. Viability of primary skin cells at
different concentrations of N2,N3-dioleoyl
spermine either free or complexed with pEGFP
NH3 Methanol pH 11
Conclusions The EthBr fluorescence quenching
assay was validated for the analysis of DNA
interactions with polyamines and lipopolyamines,
and was used to monitor DNA condensation. The
formation of DNA particles was monitored by light
scattering. N2,N3-Dioleoyl spermine (LipoGen?)
shows promising transfection results in tissue
cultured cancer and skin fibroblast primary cell
lines. 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 and to Dr C. Pourzand
(University of Bath) for help in the cell biology
studies and for useful discussions.
Fig. 2. Synthesis of N2,N3-dioleoyl spermine.
Ethidium bromide fluorescence quenching
assay Ethidium bromide (EthBr) a cationic dye
that displays a marked increase in the
fluorescence upon binding with DNA and RNA. The
increase in the fluorescence of EthBr upon
binding with DNA(lexcit 260 nm and lemiss 600
nm with slit width 5 nm) making EthBr a useful
probe to measure drug-DNA interactions (Geall and
Blagbrough 2000 Gershon et al 1993).
Fig. 3. EthBr displacement assay of calf thymus
DNA with PLL9.6k, PLL27k, PEI2k, PEI60k,
spermine and N2,N3-dioleoyl spermine.
References Basu H. S., Schwietert H. C. A.,
Feuerstein B. G. and Marton L. J. (1990), Effects
of variation in the structure of spermine on the
association with DNA and the induction of DNA
conformational-changes, Biochem. J.,
269329-334. 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., Taylor R.
J., Earll M. E., Eaton M. A. W. and Blagbrough I.
S. (1998), Synthesis of cholesterol-polyamine
carbamates pKa studies and condensation of calf
thymus DNA, Chem. Commun., 1403-1404. 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. Gershon H.,
Ghirlando R., Guttman S. B. and Minsky A. (1993),
Mode of formation and structural features of
DNA-cationic liposome complexes used for
transfection, Biochemistry, 327143-7151. Morgan
A. R., Lee J. S., Pulleyblank D. E., Murray N. L.
and Evans D. H. (1979), Ethidium fluorescence
assays. Part 1. Physicochemical studies, Nucleic
Acids Res., 7547-569.
Fig. 4. EthBr displacement assay of p ?-gal with
PLL9.6k, spermine and N2,N3-dioleoyl spermine.