In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups

Article abstract of DOI:10.1016/S0009-3084(03)00033-1

A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.

Full text of DOI:10.1016/S0009-3084(03)00033-1

Chemistry and Physics of Lipids 124 (2003) 49–61
In vitro lipofection with novel series of symmetric
1,3-dialkoylamidopropane-based cationic surfactants containing
single primary and tertiary amine polar head groups
Mohammad Sheikha, Jennifer Feiga, Becky Geeb, Song Lic, Michalakis Savvaa,∗
a
Division of Pharmaceutics, Arnold & Marie Schwartz College of Pharmacy and Health Sciences,
Long Island University, 75 Dekalb Avenue, Brooklyn, NY 11201, USA
Department of Chemistry and Biochemistry, Long Island University, 75 Dekalb Avenue, Brooklyn, NY 11201, USA
b
c
School of Pharmacy, Department of Pharmaceutical Sciences, Center for Pharmacogenetics,
University of Pittsburgh, Pittsburgh, PA 15213, USA
Received 27 November 2002; received in revised form 28 February 2003; accepted 7 March 2003
Abstract
A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthe-
sized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phospho-
ethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity.
This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both
dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic deriva-
tives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity.
Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two
requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the
lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.
© 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cationic liposome; Gene delivery; Gene transfection; Non-viral gene therapy; 1,3-Diaminopropanol
1. Introduction
Although gene therapy as such, holds great promise
in treating a variety of single gene genetic disorders
and other multifactorial diseases such as cancer, the
discovery of a safe and efficient gene delivery vehicle
remains an important barrier to the successful gene
therapy. The current delivery systems are divided into
two main categories: the viral and the non-viral ones.
Each of these delivery systems can attain certain levels
Abbreviations: DOPE, dioleoylphosphoethanolamine; lmp, lipid
monovalent primary; lmt, lipid monovalent tertiary; MTT, methy-
lthiazoltetrazolium; SFM, serum free media; TEA, triethyl amine
∗
Corresponding author. Tel.: +1-718-488-1471;
fax: +1-718-780-4586.
E-mail address: msavva@liu.edu (M. Savva).
0009-3084/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0009-3084(03)00033-1

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M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
of gene transfer in vitro and in vivo and they both
share their advantages and disadvantages.
by different investigators using different cell lines and
different marker genes. In this article we report the
synthesis and transfection activity of a series of novel
primary and tertiary monovalent cationic lipids with
diamido linkages between the 1,3-diamino-2-propanol
backbone and the hydrocarbon chains.
In viral delivery systems, a replication deficient
virus is usually used to introduce transgenes into the
host cells. Viral systems are more efficient than the
non-viral ones, but exhibit severe drawbacks such
as immunogenicity, resulting in inactivation of the
expression by the immune system, particularly upon
repeating administration.
2. Materials and methods
The acknowledged necessity of DNA compaction
has inevitably restricted the non-viral delivery sys-
tems to cationic polymers and cationic lipids. These
cationic reagents cannot achieve expression levels as
high as their viral counterparts, but they can potentially
overcome some of the problems associated with the
viral vectors. First, lipoplexes, polyplexes or lipopoly-
plexes being non-proteinaceous in nature exhibit
low immunogenicity thus, repeated administration of
these complexes is feasible (Scheule et al., 1997).
Second, these reagents are able to condense plasmid
DNA and form particles of submicron size, a prereq-
uisite for in vivo gene delivery (Ogris et al., 1998).
Third, plasmid DNA compacted by cationic reagents
is resistant to nuclease degradation. Fourth, transfer
of unlimited size genes is possible (Jong et al., 2001)
and fifth, tissue specific accumulation is also feasible.
Considerable effort has therefore been devoted to the
synthesis of new more efficient cationic lipids.
The basic structure of bilayer forming cationic
lipids embodies three regions, namely the hydrophilic
region, which includes the cationic polar head group,
the hydrophobic region that usually includes two fatty
acid chains, and the interfacial region composed of
the synthon or the backbone that joins together the
two incompatible regions of the surfactant molecule.
The exact contribution of each region on the overall
transfection activity is not fully understood. More
information is gathered though with regard to the
effect of the hydrophobic and hydrophilic regions,
rather than the effect of the backbone. Although a
vast number of glycerol and non-glycerol synthons
with diester, diether and carbon–carbon linkages,
between the hydrocarbon chains and the backbone
having variable transfection activities, have been de-
scribed since the development of the first cationic
lipid DOTMA by Felgner et al. (1994), cationic lipid
backbone structure–activity relationships is very dif-
ficult since it would require comparing the work done
2.1. Materials
1,3-Diamino-propan-2-ol 95%, lauroyl chloride
98%, myristoyl chloride 98%, palmitoyl chloride
98%, stearoyl chloride technical grade (∼85%), oleoyl
chloride technical grade (∼85%), 4-nitro phenyl chlo-
roformate 97%, ethylene diamine 99.5% redistilled,
N,N-dimethyl-ethylenediamine, were purchased from
ALDRICH (Sigma-Aldrich, St. Louis, MO). DOPE
was from AVANTI POLAR LIPIDS, Inc. (Alabaster,
AL). Triethylamine (TEA), pyridine and all other
reagents used were from VWR (Bridgeport, NJ).
2.2. Synthesis
2.2.1. N,Nꢀ-ditetradecanoyl-1,3-diaminopropan-2-ol
The intermediate compound was synthesized by a
procedure similar to that described by Mergen et al.
(1991). Briefly, to a solution of 1,3-diaminopropan-2-ol
(0.0133 mol) in 250 ml anhydrous THF, was added
TEA (0.0798 mol) and the mixture was stirred at
room temperature for 5 min. Dropwise addition of
myristoyl chloride (0.0266 mol) was followed over
a period of 10 min. The reaction was stopped after
10–15 min. After removal of the organic solvent, to
the crude material was added 50 ml of distilled water
and 250 ml of cold THF to suppress foaming and
facilitate filtration. The mixture was stirred on ice for
5–10 min and the precipitate was removed by suction
filtration and washed several times with ethyl ether
before drying for 1 h in the vacuum oven at 60 ◦C
and air-dried for 1 day. Crystallization of the material
from CHCl3:Et2O (3:1 v/v) afforded 5.24 g (77%)
of N,Nꢀ-ditetradecanoyl-1,3-diaminopropan-2-ol as
a white crystalline powder; MS (FAB) m/z 511.3
[M + H]+; 1H NMR (400 MHz, CdCl3, 20 ◦C, TMS)
δ 6.22 (m, 2H, HNCO), 3.76–3.74 (m, 1H, CH),
3.42–3.24 (m, 4H, CH2N), 2.23–2.19 (t, 4H, CH2CO),
1.65–1.60 (m, 4H, CH2CH2CO), 1.30–1.26 (coherent

M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
51
2.2.3. N,Nꢀ-ditetradecanoyl-1,3-diaminopropane-2-
carbamoyl-(aminoethane) (1,3lmp2)
peak, 40H, 10(CH2)2), 0.90–0.87 (t, 6H, CH3); 13C
NMR (100 MHz, CdCl3, 41 ◦C, TMS) δ 176.03
(NHCO), 72.08 (CH), 43.87, 37.85, 33.11, 30.87,
30.84, 30.80, 30.68, 30.51, 26.94, 23.88, 15.29.
N,Nꢀ-ditetradecanoyl-1,3-diaminopropane-2-(p-nit-
rophenyl)-carbonate (0.0016 mol) was dissolved in
25 ml anhydrous CH2Cl2 to which was added ex-
cess ethylene diamine (0.0081 mol). The reaction was
stirred at room temperature for 3 h. After CH2Cl2
evaporation, the dry crude was taken up with 140 ml
CHCl3 and washed once with 140 ml dilute brine
and dilute brine-NaHCO3, respectively. The organic
layer was collected, dried (MgSO4), concentrated
and purified by column chromatography (silica gel
1.5 cm × 20 cm). The column was eluted successively
with 100 ml CHCl3, 100 ml 2, 5, 8, 10, 12, 20 and
30% MeOH/CHCl3. The 12 and 20% fractions were
combined and concentrated to give 0.77 g (79%) of
N,Nꢀ-ditetradecanoyl-1,3-diaminopropyl-2-carbamoyl-
(aminoethane) as a white powder precipitated from
ether. Anal. calcd for C34H68N4O4 (MW 596): C,
68.45; H, 11.41; N, 9.39. Found: C, 68.25; H, 11.72;
N, 9.34. MS (FAB) m/z 597.3 [M + H]+, 493.4
[M+-OC(O)NHC2H4NH2]; 1H NMR (400 MHz,
CdCl3, 20 ◦C, TMS) δ 6.59 (sb, 2H, HNCO),
5.58 (sb, 1H, HNC(O)O), 4.69–4.67 (m, 1H, CH),
3.43–3.23 (m, 6H, CH2NHC(O), CH2NHC(O)O),
2.87 (t, CH2N), 2.21–2.17 (t, 4H, CH2CO), 1.63–1.60
(m, 4H, CH2CH2CO), 1.27–1.24 (coherent peak,
2.2.2. N,Nꢀ-ditetradecanoyl-1,3-diaminopropane-2-
(p-nitrophenyl)carbonate
N,Nꢀ-ditetradecanoyl-1,3-diaminopropan-2-ol
(0.0098 mol) was suspended in 200 ml anhydrous
THF at 50–55 ◦C. To the turbid mixture, was added
p-nitrophenyl chlorocarbonate (0.0108 mol) followed
by a dropwise addition of pyridine (0.0012 mol). Af-
ter 2.5 h the turbid mixture turned to a clear solution.
Additional p-nitrophenyl chloroformate (0.0108 mol)
and pyridine (0.0124 mol) were added and the re-
action was stirred for 2 h more (total of 4.5 h). The
reaction was allowed to cool at room temperature
and a thin precipitate, presumably pyridine HCl,
was removed by suction filtration. The filtrate was
collected and THF was evaporated with the aid of
a rotary evaporator under diminished pressure. The
crude material was dissolved in a minimum quan-
tity of CHCl3. Insoluble unreacted starting mate-
rial N,Nꢀ-ditetradecanoyl-1,3-diaminopropan-2-ol in
CHCl3, was removed by suction filtration. To the
filtrate, was added more CHCl3 (150 ml total), trans-
ferred into a separation funnel and washed once with
150 ml 0.25N HCl (pyridine removal). The aque-
ous phase was discarded. The organic phase was
concentrated and purified by column chromatog-
raphy (silica gel column 2.8 cm × 30 cm), eluting
with 100 ml CHCl3, 100 ml 1–3%, 200 ml 5 and
7% and 100 ml 12% MeOH/CHCl3. Fractions of
100 ml were collected. Fractions 4 and 5 were pooled
and concentrated to give a total of 6.14 g (93%) of
N,Nꢀ-ditetradecanoyl-1,3-diaminopropane-2-(p-nitro-
phenyl) carbonate as a white powder. MS (FAB) m/z
40H, 10(CH2)2), 0.88–0.85 (t, 6H, CH3); 13C NMR
(100 MHz, CdCl3, 20 ◦C, TMS) δ 175.20 (NHCO),
157.12 (HNC(O)O), 72.99 (CH), 44.44, 42.48, 40.47,
37.97, 33.15, 30.90, 30.78, 30.63, 30.61, 27.05, 23.94,
15.40.
2.2.4. N,Nꢀ-ditetradecanoyl-1,3-diaminopropyl-2-
carbamoyl-(N,N-dimethylaminoethane) (1,3lmt2)
To a solution of N,Nꢀ-ditetradecanoyl-1,3-diamino-
propane-2-(p-nitrophenyl)-carbonate (0.0038 mol) in
25 ml anhydrous CH2Cl2, were added N,N-dimethyl-
ethylenediamine (0.0046 mol) and TEA (0.0011 mol).
The reaction was stirred at room temperature for 3.5 h.
The solvent was evaporated and the crude material was
worked up to give a total of 1.70 g (72%) of analyti-
cally pure N,Nꢀ-ditetradecanoyl-1,3-diaminopropyl-2-
carbamoyl-(N,N-dimethylaminoethane) as a waxy
material. Anal. calcd for C36H72N4O4 (MW 624):
C, 68.25; H, 11.72; N, 9.34. Found: C, 68.45; H,
676.0 [M + H]+; H NMR (400 MHz, CdCl3, 20 ◦C,
1
TMS) δ 8.29–8.26, 7.43–7.26 (two d, each J = 9.1 Hz,
4H,C6H4), 6.40–6.37 (m, 2H, HNCO), 4.78–4.76 (m,
1H, CH), 3.60–3.50 (m, 4H, CH2N), 2.26–2.22 (t, 4H,
CH2CO), 1.65–1.60 (m, 4H, CH2CH2CO), 1.28–1.24
(coherent peak, 40H, 10 (CH2)2), 0.88–0.85 (t, 6H,
CH3); 13C NMR (100 MHz, CdCl3, 20 ◦C, TMS)
δ 175.86 (NHCO), 156.32 (OC(O)O), 152.52 (0-C,
C6H4), 146.28 (C-NO2, C6H4), 125.34, 122.84, 76.11
(CH), 38.06, 36.69, 32.51, 31.90, 29.63, 29.47, 29.31,
29.25, 25.69, 22.67, 14.10.
11.41; N, 9.40. MS (FAB) m/z 625.5 [M + H]+; H
1
NMR (400 MHz, CdCl3, 20 ◦C, TMS) δ 6.47 (sb, 2H,

52
M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
Scheme 1. Synthetic scheme for the preparation of primary and tertiary 1,3-dialkoylamino derivatives.
HNCO), 5.65 (sb, 1H, HNC(O)O), 4.72–4.69 (m, 1H,
CH), 3.57–3.52 (m, 2H, CH2NHC(O)O), 3.27–3.20
(m, 4H, CH2NHC(O)), 2.43–2.40 (t, 2H, CH2N),
2.23 (s, 6H, N(CH3)2), 2.21–2.17 (t, 4H, CH2CO),
1.63–1.58 (m, 4H, CH2CH2CO), 1.28–1.24 (coher-
ent peak, 40H, 10(CH2)2), 0.88–0.85 (t, 6H, CH3);
13C NMR (100 MHz, CdCl3, 20 ◦C, TMS) δ 175.12
(NHCO), 157.07 (HNC(O)O), 72.82 (CH), 59.82,
46.22, 40.51, 39.51, 37.86, 33.10, 30.88, 30.87, 30.85,
30.74, 30.60, 30.56, 27.00, 23.89, 15.34.
(m, 4H, CH2CH2CO), 1.27–1.24 (coherent peak,
32H, 8(CH2)2), 0.88–0.85 (t, 6H, CH3); 13C NMR
(100 MHz, CdCl3, 20 ◦C, TMS) δ 175.32 (NHCO),
157.08 (HNC(O)O), 72.92 (CH), 42.23 (CH2N),
40.33, 37.99, 33.16, 30.90, 30.87, 30.79, 30.64, 30.60,
27.05, 23.96, 15.42.
2.2.6. N,Nꢀ-dilauroyl-1,3-diaminopropane-2-
carbamoyl-(N,N-dimethylaminoethane) (1,3lmt1)
Anal. calcd for C32H64N4O4 (MW 568): C, 67.61;
H, 11.27; N, 9.86. Found: C, 68.58; H, 11.60; N,
9.75. MS (EI) m/z 569.1 [M + H]+; 1H NMR
(400 MHz, CdCl3, 20 ◦C, TMS) δ 6.40 (sb, 2H,
HNCO), 5.49 (sb, 1H, HNC(O)O), 4.73–4.70 (m, 1H,
CH), 3.58–3.53 (m, 2H, CH2NHC(O)O), 3.28–3.21
(m, 4H, CH2NHC(O)), 2.45–2.42 (t, 2H, CH2N),
2.24 (s, 6H, N(CH3)2), 2.21–2.18 (t, 4H, CH2CO),
1.64–1.58 (m, 4H, CH2CH2CO), 1.29–1.25 (coher-
ent peak, 32H, 8(CH2)2), 0.89–0.85 (t, 6H, CH3);
13C NMR (100 MHz, CdCl3, 20 ◦C, TMS) δ 174.96
(NHCO), 156.71 (HNC(O)O), 72.92 (CH), 59.07
(N(CH3)2), 46.15 (CH2N), 40.56 (CH2NHC(O)),
39.39 (O(O)CNHCH2), 38.04, 33.15, 30.87, 30.86,
30.75, 30.76, 30.58, 30.56, 27.02, 23.95, 15.41.
All other compounds were synthesized using anal-
ogous procedures as described above for the dimyris-
toyl derivatives (Scheme 1).
2.2.5. N,Nꢀ-dilauroyl-1,3-diaminopropane-2-
carbamoyl-aminoethane (1,3lmp1)
Anal. calcd for C30H60N4O4 (MW 540): C, 66.67;
H, 11.11; N, 10.37. Found: C, 67.44; H, 11.34;
N, 10.26. MS (FAB) m/z 541.5 [M + H]+, 437.4
[M+-OC(O)NHC2H4NH2]; 1H NMR (400 MHz,
CdCl3, 20 ◦C, TMS) δ 6.75 (sb, 2H, HNCO), 5.79 (sb,
1H, HNC(O)O), 4.68–4.66 (m, 1H, CH), 3.48–3.29
(m, 6H, CH2NHC(O)O, CH2NHC(O)), 2.92–2.91 (bs,
2H, CH2N), 2.22–2.18 (t, 4H, CH2CO), 1.62–1.59

M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
53
2.2.7. N,Nꢀ-dipalmitoyl-1,3-diaminopropane-2-
carbamoyl-aminoethane (1,3lmp3)
2.2.10. N,Nꢀ-dioleoyl-1,3-diaminopropane-2-
carbamoyl-aminoethane (1,3lmp5)
Anal. calcd for C38H76N4O4 (MW 652): C, 68.25;
H, 11.72; N, 9.34. Found: C, 68.45; H, 11.41; N, 9.40.
MS (FAB) m/z 653.1 [M + H]+, 1H NMR (400 MHz,
CdCl3, 20 ◦C, TMS) δ 6.56 (sb, 2H, HNCO), 5.54 (sb,
1H, HNC(O)O), 4.68 (m, 1H, CH), 3.45–3.20 (m, 6H,
CH2NHC(O)O, CH2NHC(O)), 2.92 (bs, 2H, CH2N),
2.22 (b, 4H, CH2CO), 1.60 (m, 4H, CH2CH2CO),
1.22 (coherent peak, 48H, 8(CH2)2), 0.87–0.83 (t, 6H,
CH3).
Anal. calcd for C42H80N4O4 (MW 704): C, 71.59;
H, 11.36; N, 7.95. Found: C, 70.45; H, 11.58; N,
7.95. MS (FAB) m/z 705.5 [M + H]+, 601.4 [M+-
OC(O)NHC2H4NH2]; 1H NMR (400 MHz, CdCl3,
20 ◦C, TMS) δ 7.37–7.35 (sb, 2H, HNCO), 6.50 (sb,
==
1H, HNC(O)O), 5.38–5.33 (m, 4H, CH CH), 4.70
(m, 1H, CH), 3.42–3.20 (m, 6H, CH2NHC(O)O,
CH2NHC(O)), 2.81 (sb, 2H, CH2N), 2.62 (sb, 2H,
NH2) 2.22–2.18 (t, 4H, CH2CO), 2.01–1.96 (m,
==
8H, CH2CH CHCH2), 1.61 (sb, 4H, CH2CH2CO),
1.30–1.26 (coherent peak, 40H, 10(CH2)2), 0.89–0.85
(t, 6H, CH3); 13C NMR (100 MHz, CdCl3, 20 ◦C,
TMS) δ 175.14 (NHCO), 157.36 (HNC(O)O), 131.43–
2.2.8. N,Nꢀ-dipalmitoyl-1,3-diaminopropane-2-
carbamoyl-(N,N-dimethylaminoethane) (1,3lmt3)
Anal. calcd for C40H80N4O4 (MW 680): C, 70.59;
H, 11.76; N, 8.23. Found: C, 70.77; H, 12.11; N,
8.16. MS (EI) m/z 681.2 [M + H]+; 1H NMR
(400 MHz, CdCl3, 20 ◦C, TMS) δ 6.42–6.40 (sb,
2H, HNCO), 5.49 (sb, 1H, HNC(O)O), 4.73–4.70
(m, 1H, CH), 3.60–3.56 (m, 2H, CH2NHC(O)O),
3.27–3.21 (m, 4H, CH2NHC(O)), 2.44–2.42 (t, 2H,
CH2N), 2.24 (s, 6H, N(CH3)2), 2.21–2.18 (t, 4H,
CH2CO), 1.64–1.60 (m, 4H, CH2CH2CO), 1.24 (co-
herent peak, 48H, 12(CH2)2), 0.89–0.85 (t, 6H, CH3);
13C NMR (100 MHz, CdCl3, 20 ◦C, TMS) δ 175.00
(NHCO), 156.73 (HNC(O)O), 72.90 (CH), 59.21
(N(CH3)2), 46.31 (CH2N), 40.56 (CH2NHC(O)),
39.49 (O(O)CNHCH2), 38.04, 33.17, 30.95, 30.91,
30.77, 30.62, 30.57, 27.03, 23.96, 15.42.
==
130.57 (C C), 72.87 (CH), 65.34, 44.51, 42.49,
40.57, 37.77, 33.77, 33.08, 33.05, 30.94, 30.90, 30.84,
30.79, 30.71, 30.66, 30.62, 30.53, 30.50, 30.40, 30.38,
30.32, 30.27, 30.12, 28.40, 28.38, 27.02, 23.88, 15.33.
2.2.11. N,Nꢀ-dioleoyl-1,3-diaminopropane-2-
carbamoyl-(N,N-dimethylaminoethane) (1,3lmt5)
Anal. calcd for C44H84N4O4 (MW 732): C,
72.13; H, 11.47; N, 7.65. Found: C, 70.89; H,
11.55; N, 7.64. MS (FAB) m/z 733.7 [M + H]+,
705.8 [M+-(CH3)2]; 1H NMR (400 MHz, CdCl3,
20 ◦C, TMS) δ 6.53 (sb, 2H, HNCO), 5.74 (sb, 1H,
==
HNC(O)O), 5.38–5.34 (m, 4H, CH CH), 4.68–4.67
(m, 1H, CH), 3.56–3.36 (m, 2H, CH2NHC(O)O),
3.32–3.19 (m, 4H, CH2NHC(O)), 2.62–2.59 (t, 2H,
CH2N), 2.25 (s, 6H, N(CH3)2), 2.21–2.18 (t, 4H,
2.2.9. N,Nꢀ-distearoyl-1,3-diaminopropane-2-
==
CH2CO), 2.01–1.96 (m, 8H, CH2CH CHCH2),
1.60–1.57 (m, 4H, CH2CH2CO), 1.26 (coherent peak,
carbamoyl-(N,N-dimethylaminoethane) (1,3lmt4)
Anal. calcd for C44H88N4O4 (MW 736): C, 71.74;
H, 11.96; N, 7.61. Found: C, 70.89; H, 11.55; N, 7.64.
MS (FAB) m/z 737.5 [M + H]+, 709.4 [M+-(CH3)2];
1H NMR (400 MHz, CdCl3, 20 ◦C, TMS) δ 6.81–6.78
(sb, 2H, HNCO), 6.20 (sb, 1H, HNC(O)O), 4.68–4.67
(m, 1H, CH), 3.65–3.46 (m, 2H, CH2NHC(O)O),
3.22–3.19 (m, 4H, CH2NHC(O)), 2.38–2.35 (t, 2H,
CH2N), 2.18 (s, 6H, N(CH3)2), 2.16–2.14 (t, 4H,
CH2CO), 1.60–1.58 (m, 4H, CH2CH2CO), 1.21 (co-
herent peak, 56H, 14(CH2)2), 0.85–0.82 (t, 6H, CH3);
13C NMR (100 MHz, CdCl3, 20 ◦C, TMS) δ 175.11
(NHCO), 157.00 (HNC(O)O), 72.87 (CH), 59.34
(N(CH3)2), 46.26 (CH2N), 40.54 (CH2NHC(O)),
39.53 (O(O)CNHCH2), 37.93, 33.14, 30.93, 30.88,
30.77, 30.62, 30.58, 27.03, 23.93, 15.38.
40H, 10(CH2)2), 0.88–0.85 (t, 6H, CH3); 13C NMR
(100 MHz, CdCl3, 20 ◦C, TMS) δ 175.13 (NHCO),
==
157.46 (HNC(O)O), 131.50–130.62 (C C), 72.87
(CH), 65.34, 59.32, 46.26, 40.89, 39.57, 37.97, 33.77,
33.08, 33.05, 30.94, 30.90, 30.84, 30.79, 30.71, 30.66,
30.62, 30.53, 30.50, 30.40, 30.38, 30.32, 30.27, 30.12,
28.40, 28.38, 27.02, 23.88, 15.33.
It is important to note that the 1,3lmp5 and 1,3lmt5
are not pure dioleoyl derivatives, but isomers (as much
as 50%) as determined with 13C NMR.
2.3. Preparation of lipid dispersion
Cationic lipids alone or with DOPE and cholesterol
were dissolved in chloroform and transferred into

54
M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
borosilicate glass tubes. The organic solvent was re-
moved under a stream of nitrogen gas and completely
eliminated with overnight vacuum desiccation. The
dried lipids were hydrated with distilled water or
appropriate buffer. Lipid dispersions were prepared
with vortexing and sonication at a final cationic lipid
concentration of 0.6 mM.
0.024 mM. The following parameters were kept con-
stant:
(1) Number of cells per well was 50,000.
(2) +/− charge ratio was 2.
(3) Amount of plasmid DNA was 1 ␮g/well.
(4) Cationic lipid/DOPE was 6:4 mol/mol.
(5) Cationic lipid/DOPE/cholesterol was 6:4:2 mol/
mol.
2.4. Plasmids
(6) Volume added per well was 250 ␮l.
The plasmid DNA consisted of a beta-galactosidase
(β-gal) reporter gene driven by the human cy-
tomegalovirus (CMV) immediate-early promoter,
cloned into a pUC19 vector (Laboratory of Professor
Leaf Huang, Center for Pharmacogenetics, University
of Pittsburgh).
A pictorial representation of the transfection pro-
tocol along with relevant calculations is shown in
Scheme 2.
Approximately 50,000 cells were seeded into 1 mm
wells (48-well plate) 12 h before transfection. The
cells (60–70% confluency) were exposed to lipoplexes
in the absence or presence of serum for 3–4 h, after
which time the lipoplexes were removed and fresh
RPMI medium supplemented with 10% FBS was
added. The cells were incubated for a total of 48 h be-
fore being assayed for β-gal levels. Briefly, the cells
were washed twice with phosphate-buffered saline
(PBS) and lysed with 150 ␮l of lysis buffer (Tris
0.1 M; pH 8.0, EDTA 0.002 M, Triton × 0.05%). The
β-gal activity per well was estimated by adding 50 ␮l
of substrate solution (1.33 mg/ml ONPG) prepared in
0.2 M sodium phosphate, pH 7.2 and 2 mM magne-
sium phosphate, to the lysate in a 96-well plate. A cali-
bration curve constructed with pure commercial β-gal
enzyme (Sigma-Aldrich) was used to convert OD at
405 nm to units of activity (Srilakshmi et al., 2002).
Escherichia coli DH5␣ competent cells were trans-
formed with pUC19 CMV-β-gal using standard proto-
cols (Sambrook et al., 1989). Colonies were selected
and grown in terrific broth for 18–22 h. Alkaline and
detergent lysis of the bacteria was performed using a
procedure similar to that described by Maniatis and
coworkers (Sambrook et al., 1989) and the plasmid
was then purified using gel permeation chromatog-
raphy (Sepharose 4B) with 2.5 M ammonium acetate
as the mobile phase. The quality and purity of the
plasmid was verified by agarose gel electrophore-
sis. Three distinct bands for the supercoiled, nicked
and linear DNA were identified. Concentration of
plasmid DNA was determined spectrophotometri-
cally (1 OD260 = 50 ␮g/ml of plasmid DNA). The
OD260/OD280 ratio was 1.88 suggesting high qual-
ity plasmid, no protein contamination and absence
of salts.
2.6. β-Gal histochemistry
Cells were washed three times with PBS pH 7.2,
fixed for 5 min in 0.25% glutaraldehyde at 4 ◦C in
PBS and incubated in 250 ␮l of 2 mg/ml X-gal, 1 mM
MgCl2, 150 mM NaCl, 3.3 mM K4Fe(CN)6, 3.3 mM
K3Fe(CN)6, 60 mM Na2HPO4, 40 mM NaH2PO4, pH
7.2, for 4–6 h.
2.5. In vitro transfection studies
B16F0 mouse melanoma cells were purchased
from the American Type Culture Collection (ATCC,
CRL-6322) and cultured in RPMI media supple-
mented with 0.5 U/ml penicillin, 1 ␮g/ml strepto-
mycin, 4 mM l-glutamine, 1 mM sodium pyruvate
and 10% fetal bovine serum (FBS). The cells were
maintained and grown at 37 ◦C in a humidified atmo-
sphere containing 5% CO2.
2.7. Toxicity assay
Lipoplex cytotoxicity was determined on B16F0
cells using a modified MTT assay described elsewhere
(Savva et al., 1999). Briefly, cells were cultured as
described before for in vitro transfection studies. Af-
ter 42 h of incubation, 50 ␮l of MTT (5 mg/ml) were
In a typical transfection experiment, lipoplexes were
prepared by mixing cationic lipids and DNA in serum
free media to a final cationic lipid concentration of

M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
55
Scheme 2. Pictorial representation of the transfection protocol for 6 wells. (A) For serum free media (SFM) and (B) in the presence of
5% FBS. The volume of FBS added was calculated using: final volume × 0.05 = 1525 ␮l × 0.05 = 76.25 ␮l.
added in each well and the cells were incubated for an
additional 4 h. The supernatant was discarded, the cells
were washed once with cols PBS and 250 ml of DMSO
was added in each well. Measuring the absorbance
at 630 nm using a multiscan plate reader quantitated
the amount of purple formazan derivative due to the
cleavage of tetrazolium ring. The absorption obtained
with reduced formazan with cells in the absence of
lipoplexes was taken to be 100%.
(5–10 s, 14,000 rpm), incubated at room temperature
for approximately 30 min and subsequently they were
loaded and run on an 0.8% agarose gel, containing
0.5 ␮g/ml ethidium bromide, in a 16 mM TAE buffer
pH 7.4 or/and pH 8.0 for 0.5–1 h (5 V/cm).
3. Results and discussion
The transfection assay was specifically designed to
identify first, the cationic lipids that elicit high trans-
fection efficiencies in the absence of helper lipids,
second to identify the cationic lipids that require the
presence of DOPE for transfection activity and third,
to investigate the effect of inclusion of cholesterol in
the lipoplex formulation on the transfection activity,
especially in the presence of serum proteins. Careful
literature survey with regard to double-chained mono-
valent cationic lipid requirements for optimal in vitro
transfection has resulted into several conclusions.
First, with no exception cationic lipids elicit optimal
2.8. Agarose gel electrophoresis
Lipoplexes were prepared by mixing 0.2 ␮g of plas-
mid DNA with the appropriate amounts of cationic
lipids in a microfuge tube to a total volume of 8 ␮l TE
buffer 40 mM pH 7.2. Buffers were adjusted to pH val-
ues with the aid of a strong base or a strong acid. The
ionic strengths of buffers are uncorrected. The molar
concentration of DNA phosphate was calculated by
using the average molecular weight of the nucleotide
monomer as 330. Lipoplexes were briefly centrifuged

56
M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
gene delivery with minimum toxicity at 1 < +/− ratio
< 4 (Srilakshmi et al., 2002; Gaucheron et al., 2002;
Laxmi et al., 2001; Rosenzweig et al., 2000; Wheeler
et al., 1996; Akao et al., 1994; Balasubramaniam et al.,
1996; Ito et al., 1990). For this reason the +/− charge
ratio was fixed to 2. Second, the requirement for DOPE
in the lipoplex formulation can be ascertained at mo-
lar ratios 8:2 < cationic lipid/DOPE < 1:1 (Felgner
et al., 1994; Zuidam et al., 1999; Gaucheron et al.,
2002; Balasubramaniam et al., 1996). DOPE is known
to favor formation of reverse hexagonal phases (Hii),
at physiological pH, that are implicated in membrane
fusion processes (Siegel, 1999). Inclusion of DOPE
will potentially enhance the fusion properties and
endocytosis efficiency of the lipoplexes. On the other
hand, several reports argue that in-serum gene delivery
is most efficient with multilamellar structure-forming
lipids (Boukhnikachvili et al., 1997). Therefore, in
cases where the double-chained cationic lipids form
lamellar structures in isolation, it is wise to employ
cationic lipid/DOPE molar ratios higher or equal to 1.
At close to 1:1 ratio, lipoplexes exist as lamellar phases
while there is sufficient amount of Hii-forming lipid
to induce fusion upon “sticky” collision with cells.
Third, studies performed on egg lecithin/cholesterol
and pure DPPC/cholesterol bimolecular layers have
shown that cholesterol stabilizes bilayer vesicles. Al-
though the exact mechanism of stabilization is not
known, the studies have clearly shown that cholesterol
increases the thickness of the bilayer by stretching the
phospholipid acyl chains (Levine and Wilkins, 1971).
Cholesterol also increases the bilayer compressibil-
ity, thus favoring formation of small size dynamic
vesicles with high curvatures (Rand et al., 1980). It
furthermore stabilizes the liposomes by increasing
the bilayer separation, a phenomenon that is probably
due to improved hydration (Rand et al., 1980; Levine
and Wilkins, 1971). For cholesterol to function as
a stabilizer, a concentration higher than 11% and
lower than 50% relative to the phospholipid concen-
tration is required (Rand et al., 1980). To this end, we
have fixed the cationic lipid/DOPE/cholesterol mo-
lar ratios at 6:2:2. Lastly, the decision to use B16F0
melanoma cells to preliminary screen the transfec-
tion activity of lipoplex formulations was based on
their high replication rates that not only provides the
large number of cells needed for the assay but it also
increases the sensitivity of the assay, because the
uptake of exogenous DNA is facilitated by the nu-
clear envelope breakdown that occurs during mitosis
(Mortimer et al., 1999; Zauner et al., 1999; Bally et al.,
1999).
Fig. 1 illustrates the transfection efficiency of pri-
mary and tertiary 1,3-dialkoylamido cationic lipid
derivatives against B16F0 murine melanoma cells
in the absence of serum. All lipids, but the dioleoyl
derivatives 1,3lmp5 and 1,3lmt5 elicited no activ-
ity when used alone. The not statistically significant
slightly higher activity of the primary dioleoyl deriva-
tive, 1,3lmp5 relative to that of the corresponding
tertiary dioleoyl analog, as identified in the ONGP
assay, was also seen with X-gal staining (compare
Fig. 3D and Fig. 4E). The monovalent primary and
tertiary dilauroyl 1,3lmp1 and 1,3lmt1 showed no
activity in all different formulations (Fig. 1, Fig. 3A,
and Fig. 4A). The dipalmitoyl and distearoyl deriva-
tives behaved similarly, that is, they have elicited
insignificant to low levels of gene expression in the
absence of cholesterol and have shown moderate
levels of activity when cholesterol was included in
the formulation. Specifically, the β-gal activity lev-
els of 1,3lmp3/d/ch, 1,3lmt3/d/ch and 1,3lmt4/d/ch
was 1.00 0.07 units/ml/well, 0.32 0.18 units/ml/
well and 0.65
0.16 units/ml/well, respectively.
The dimyristoyl derivatives 1,3lmp2/d, 1,3lmp2/d/ch,
1,3lmt2/d and 1,3lmt2/d/ch attained average β-gal ac-
tivity levels at 1.38, 1.67, 2.99 and 3.66 units/ml/well,
respectively. Formulations composed of the di-
oleoyl primary derivative 1,3lmp5 have induced
enzyme activity levels, in the presence or absence
of DOPE and cholesterol, in the neighborhood of
1.80–2.41 units/ml/well (Fig. 1). Their transfection
activity seemed to be unaffected by the presence
of helper lipids, as also suggested by histochemical
staining (Fig. 3D). Contrary to that, the transfection
activity of the corresponding tertiary dioleoyl ana-
logue, 1,3lmt5, was higher in the presence of DOPE
and reached highest levels at 3.89 0.35 units/ml/well
in the presence of DOPE and cholesterol. Consid-
ering that the total volume of cell lysate per well
was 0.150 ml, the above levels would correspond to
583.5 munits/well.
In vitro lipoplex cytotoxicity, was evaluated
against B16F0 cells using an MTT assay (Fig. 5).
The 1,3lmp1, 1,3lmp3, 1,3lmp3/d and 1,3lmt1/d/ch
showed the highest toxicity with approximately 40%

M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
57
Fig. 1. Transfection activity of cationic lipids in B16F0 murine melanoma cells. Abbreviations: lmp/d, lmp/DOPE; and lmp/d/ch,
lmp/DOPE/cholesterol.
of the treated cells being affected. All other formu-
lations were well tolerated by the cells with cell
viability ranging between 70–100%.
In vitro transfection experiments of lipoplex formu-
lations in the presence of 5% FBS were not consis-
tent (not shown) and no conclusion could therefore be
made as to the role of cholesterol in stabilizing the
transfection activity of lipoplexes in the presence of
serum.
Agarose gel electrophoresis of lipoplexes was used
to appraise electrostatic and hydrophobic interaction
of cationic lipids with plasmid DNA under the influ-
ence of an electric field. Fig. 2 illustrates the effect of
monovalent cationic lipid formulations on the plasmid
DNA migration. In the absence of DOPE, primary
amine derivative 1,3lmp5 reduced most effectively
the plasmid DNA migration. Complete retardation of
plasmid DNA with 1,3lmp5 was observed above +/−
charge ratio 1. In agreement with others (Eastman
et al., 1997), a slight excess of positive charge is
needed to completely inhibit plasmid DNA migration.
Inclusion of DOPE and cholesterol essentially had
no effect on the DNA binding (Fig. 2D). This could
explain why the transfection activity of this lipid was
not affected by the presence of helper lipids (Fig. 1).
Contrary to that, the corresponding dioleoyl tertiary

58
M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
Fig. 2. Gel retardation assay. From left to right: (A) 1,3lmp1, 1,3lmp1/d, 1,3lmp1/d/ch; (B) 1,3lmp2, 1,3lmp2/d, 1,3lmp2/d/ch; (C) 1,3lmt2,
1,3lmt2/d, 1,3lmt2/d/ch; (D) 1,3lmp5, 1,3lmp5/d, 1,3lmp5/d/ch; and (E) 1,3lmt5, 1,3lmt5/d, 1,3lmt5/d/ch. Details of the assay are as
described in Section 2.
Fig. 3. Transfection activity of cationic lipids against B16F0 murine melanoma cells, in the absence of serum (n = 6, two independent
experiments). From left to right, lipid, lipid/DOPE (3:2 mol/mol), lipid/DOPE/cholesterol (3:2:1 mol/mol). The +/− charge ratio was fixed
to 2. (A) 1,3lmp1, R = C11H23; (B) 1,3lmp2, R = C13H27; (C) 1,3lmp3, R = C15H31; and (D) 1,3lmp5, R = C17H31. R1 = R2 = H.

M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
59
Fig. 4. Transfection activity of cationic lipids against B16F0 murine melanoma cells, in the absence of serum. From left to right, lipid,
lipid/DOPE (3:2 mol/mol), lipid/DOPE/cholesterol (3:2:1 mol/mol). The +/− charge ratio was fixed to 2. (A) 1,3lmt1, R = C11H23; (B)
1,3lmt2, R = C13H27; (C) 1,3lmt3, R = C15H31; (D) 1,3lmt4, R = C17H35; and (E) 1,3lmt5, R = C17H31. R1 = R2 = CH3.
amine derivative 1,3lmt5 failed to inhibit complete
DNA migration at all ratios but inhibited DNA mi-
gration quite effectively at +/− charge ratio ≥ 2, in
the presence of helper lipids. Interestingly, the trans-
fection activity of this lipid was significantly higher
in the presence of helper lipids (Fig. 1). Similarly to
the 1,3lmt5, the tertiary amine dimyristoyl analogue,
1,3lmt2 did not retard plasmid DNA migration at all
ratios when used alone, but bound to plasmid DNA
quite avidly in the presence of helper lipids (Fig. 2C).
The corresponding dimyristoyl primary amine deriva-
tive, 1,3lmp2, failed to associate with DNA at all
ratios in the absence of helper lipids but also failed
to promote complete retardation of plasmid DNA in
Fig. 5. Toxicity of lipoplexes prepared at 2:1 +/− ratio, on B16F0 cells. The data presented are the average of three independent experiments.

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M. Sheikh et al. / Chemistry and Physics of Lipids 124 (2003) 49–61
the presence of helper lipids at ratios relevant to the
transfection experiments, although helper lipids do
promote DNA binding of this lipid at higher +/− ra-
tios. In general, DNA retardation experiments suggest
that primary ammonium lipids do not interact with
DOPE as well as the corresponding tertiary amine
derivatives, especially in the case of dimyristoyl and
dioleoyl analogues. The results presented in Fig. 2
could fully explain the lack of activity of 1,3lmt2 in
the absence of helper lipids, but could not account for
the high transfection levels elicited by the 1,3lmp2 in
the presence of DOPE and cholesterol. The opposite
phenomenon (DNA binding accompanied with no bi-
ological activity) was observed with 1,3lmp1/d/ch. As
shown in Fig. 2A, 1,3lmp1/d/ch binds avidly to the
DNA but the corresponding lipoplex elicits no activity
(Fig. 1). An explanation for the lack of activity of di-
lauroyl derivatives could be found in the early work of
Minsky and coworkers (Ghirlando et al., 1992). They
have suggested that fatty acid chains of 12 carbons
are not long enough to create the hydrophobic envi-
ronment required for efficient DNA compaction. Dis-
tearoyl and dipalmitoyl derivatives behaved similarly
to the corresponding dimyristoyl analogs (not shown).
Molecular migration in gel electrophoresis is de-
pendent on both molecular charge and size. Inclusion
of DOPE reduced the amount of cationic lipid needed
to induce retardation of plasmid DNA, especially in
the case of monovalent 1,3lmt series. At present, it
is not known what effect DOPE has on the DNA
compaction. However, highly fluorescent DNA pre-
cipitated in the wells of the gel (intercalated ethid-
ium bromide) with formulations containing DOPE
suggests more efficient DNA encapsulation and
less efficient DNA compaction. This could explain
the complete DNA retardation observed with the
1,3lmt2/d and 1,3lmt5/d at +/− ratios less than 2.
In summary, a novel series of primary and ter-
tiary dialkoylamido monovalent cationic lipids were
synthesized and evaluated for their transfection ac-
tivities. In the absence of the helper lipid DOPE,
only the dioleoyl derivatives 1,3lmp5 and 1,3lmt5
elicited transfection activity. This is in sharp contrast
with previous work done with 1,2 glycerol-based
monovalent cationic lipids that found both dioleoyl
and dimyristoyl derivatives the most active transfec-
tion reagents (Felgner et al., 1994; Wheeler et al.,
1996).
Transfection is a multi-step process with the DNA
complexation with cationic lipid being only the first
step of it. The gel electrophoresis data alone cannot
provide explanations for all the different transfec-
tion activities exhibited by cationic lipids. It is clear
though, that tertiary amine cationic derivatives bear-
ing saturated hydrocarbon chains do not associate
with the plasmid DNA in the absence of DOPE and
not surprisingly do not elicit any transfection activity,
with the exception of 1,3lmt5. The ionic strength of
the running buffer was very low (16 mM) and it is not
expected to affect lipid binding to the DNA. Although
the pKas for primary and tertiary amines are similar
(the calculated pKas for 1,3lmp2, 1,3lmt2, 1,3lmp5
and 1,3lmt5 was 9.25, 9.04, 9.25 and 9.06, respectively
(ACD/pKa DB, Advanced Chemistry Development
Inc., CANADA)) their interaction with plasmid DNA
is amazingly different (Fig. 2). This could be primarily
for three reasons, that is, steric hindrance provided by
the methyl groups attached on the tertiary amine, re-
duced hydration and thus, reduced protonation of the
tertiary amine head group relative to the primary amine
polar head group and reduced overall hydration of the
cationic lipids that possess long saturated hydrocarbon
chains. All the above are related to the solid-to-liquid
crystalline phase transition temperature that is in turn
associated with fusion properties of lipid assemblies.
In the presence of DOPE and cholesterol, all five lipids
were active. It appears that at least in vitro, in addition
to plasmid DNA compaction, the physical properties
of the lipid layers surrounding the plasmid DNA play
a very important role for optimal transfection.
Acknowledgements
We thank Dr. Leaf Huang in the Center for Phar-
macogenetics, School of Pharmacy, University of
Pittsburgh, Pittsburgh, PA, for his gift of the pUC19
CMV-β-gal.
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