Synthesis, in vitro transfection activity and physicochemical characterization of novel N,N′-diacyl-1,2-diaminopropyl-3-carbamoyl- (dimethylaminoethane) amphiphilic derivatives

Article abstract of DOI:10.1016/j.chemphyslip.2004.09.017

A novel series of N,N′-diacyl-1,2-diaminopropyl-3-carbamoyl- (dimethylaminoethane) cationic derivatives was synthesized and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and cholesterol. Physicochemical properties of lipid-DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance technique at 23°C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a ±charge ratio of 2. In agreement with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A?² and 38.7 mN/m, respectively. This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved transfection activity.

Full text of DOI:10.1016/j.chemphyslip.2004.09.017

Chemistry and Physics of Lipids 133 (2005) 135–149
Synthesis, in vitro transfection activity and physicochemical
characterization of novel N,Nꢀ-diacyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane) amphiphilic
derivatives
Ahmad Aljaberi, Pensung Chen, Michalakis Savva∗
Division of pharmaceutical sciences, Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island
University, 75 DeKalb Avenue, Brooklyn, NY 11201, USA
Received 2 August 2004; received in revised form 21 September 2004; accepted 21 September 2004
Available online 11 November 2004
Abstract
A novel series of N,Nꢀ-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives was synthesized
and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and
cholesterol. Physicochemical properties of lipid–DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy
and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance
technique at 23 ◦C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in
vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a charge ratio of 2. In agreement
with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel
electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by
fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid
2
˚
that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A and 38.7 mN/m, respectively.
This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In
conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved
transfection activity.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cationic lipids; Gene delivery; Lipofection; 1,2-Diaminopropyl; Monolayer studies; Interfacial compressibility
Abbreviations: DOPE, dioleoylphosphatidylethanoleamine; lmt, lipid monovalent tertiary; 1,2lmt[1], dilauroyl derivative; 1,2lmt[2], dim-
yristoyl derivative; 1,2lmt[3], dipalmitoyl derivative; 1,2lmt[4], distearoyl derivative; 1,2lmt[5], dioleoyl derivative; MmA, mean molecular
are∗a
Corresponding author. Tel.: +1 718 488 1471; fax: +1 718 780 4586.
E-mail address: msavva@liu.edu (M. Savva).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.09.017

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A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
1. Introduction
in designing new lipids with enhanced transfection
activity.
In the current report, a novel series of cationic lipids
Cationic lipid-mediated gene delivery represents a
safer route for delivering plasmid DNA into somatic
cells compared to the immunogenic viral vectors. In
addition to patient safety, advantages include (1) ef-
ficient condensation and protection of plasmid DNA
from enzymatic degradation, (2) feasibility of repeat-
ing administration, (3) feasibility of targeted delivery
and tissue specific accumulation as well as (4) a cer-
tain level of simplicity in the handling techniques when
comparedwiththebiohazardsofviralvectors. Unfortu-
nately, their use as the gene delivery vector of choice is
limited by their low efficiency in cell transfection. This
serious drawback motivated researchers to investigate
the factors controlling the outcome of the lipofection
process in order to achieve transfection efficiencies that
can be clinically useful.
has been synthesized and tested for their ability to me-
diate transfection in vitro in the presence and absence
of helper lipids. The physicochemical properties of the
lipids in isolation and their lipoplexes were studied us-
ing various techniques. The interfacial properties of
these lipids were studied using the Langmuir film bal-
ance technique from which a correlation between these
properties and the transfection behavior of these new
lipids was determined.
2. Experimental
2.1. Materials
In an effort to generate a meaningful and practical
structure–activity relationship, various cationic lipids
were synthesized (Ilies et al., 2002; Miller, 1998).
Their physicochemical properties in isolation and af-
ter they were complexed with DNA were investigated
and correlated with their transfection activity. Some
of the properties identified to date as being impor-
tant for transfection activity are fluidity (Akao et al.,
1994; Singh et al., 2002) and hydration (Bennett et al.,
1997) of cationic lipids and particle size (Rejman et
al., 2004; Ross and Hui, 1999), charge density (Lin et
al., 2003; Zhang and Anchordoquy, 2004) and struc-
ture (Safinya, 2001; Congiu et al., 2004) of lipoplexes.
On the other hand, not much has been done with re-
spect to the interfacial properties of cationic lipids.
Wang et al. were the first to attempt to correlate such
properties of long chain alkyl acyl carnitine esters with
their transfection activities (Wang et al., 1998). More
recently, the interfacial interactions between DOTAP
and DPPC were reported (Bordi et al., 2003). Charac-
terization of the interfacial properties, together with
other physicochemical properties, of cationic lipids
in isolation is believed to provide important insights
into the various lipid–lipid interactions that take place
whether between cationic lipids and helper lipids in
the formulation or those between the aforementioned
lipids and the natural lipids in the cell and endo-
somal membranes. Therefore, understanding the in-
terfacial properties of cationic lipids and correlating
them to their transfection activity will inevitably help
2,3-Diaminopropionic acid monohydrochloride
(98%), lauroyl chloride (98%), myristoyl chloride
(97%), palmitoyl chloride (98%), stearoyl chloride
(99%), oleoyl chloride technical grade (85%), hy-
drogen chloride (>99%), lithium borohydride 2.0 M
solution in THF, triethylamine (99.5%), pyridine
(>99%), anh. THF (99.9%), 4-nitrophenyl chlo-
roformate (97%), N,N-dimethylethylenediamine
(95%), tris (99.8+%), cholesterol (>99%), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolam (MTT)
and o-nitrophenyl ␤-d-galactopyranoside were
purchased from Sigma–Aldrich (St. Louis, MO).
Anh. methanol and amylene-stabilized chloroform
were from VWR (Bridgeport, NJ). DOPE was from
AVANTI Polar Lipids Inc. (Alabaster, AL). Agarose,
ethidium bromide, RPMI medium, fetal bovine serum,
sodium pyruvate and trypsin–EDTA 1× (10 mg/ml)
were from Invitrogen Life Technologies (Carlsbad,
CA).
Column chromatography was performed with sil-
˚
ica gel, 70–230 mesh, 60 A (Sigma–Aldrich) and
KONTES chromatography columns (VWR). Thin
layer chromatography was developed on 0.25 mm sil-
ica gel plates (Fisher Scientific) using the following
solvent systems: (A) CHCl3/CH3OH (20:1, v/v) and
(B) CHCl3/CH3OH/aqueous NH4OH (66:33:1, v/v).
1H NMR spectra were recorded on a Varian Inova
400 MHz spectrometer. High-resolution mass spec-
troscopy was performed by the Department of Chem-
istry at Ohio State University. Elemental analysis (C, H,

A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
137
N) was performed by Robertson Microlit Laboratories
Inc. (Madison, NJ). 40 mM Tris buffer, pH 7.2 was used
in all the studies carried out in this paper unless other-
wise stated. Water for buffer preparation was obtained
from a Barnstead NANOpure ultrapure water system
(Barnstead, Dubuque, IA). The resistivity and surface
tension of the ultrapure water were 18 × 106 ꢀ cm and
72.5 mN/m, respectively.
4 and 5 were pooled and evaporated under diminished
pressure. Precipitation from methanol afforded 2.39 g
of compound 2 as a white powder (Yield = 60.2%).
Rf = 0.85 (A). 1H NMR (400 MHz, CDCl3, 20 ◦C,
TMS), δH (ppm): 6.71–6.69 [d, 1H, CO NH CH ],
6.02–5.95 [t, 1H, CO NH CH2 ], 4.60–4.58 [m,
1H, CH COO ], 3.73 [s, 3H, OCH3], 3.62–3.58
[m, 2H, NH CH2 CH ], 2.22–2.10 [m, 4H,
CO CH2 ], 1.62–1.50 [m, 4H, CO CH2 CH2 ],
1.30–1.09 [coherent, 40H, (CH2)10 ], 0.85–0.82 [m,
6H, CH3].
2.2. Synthesis
2.2.1. Methyl 2,3-diaminopropionate
dihydrochloride (1)
2.2.3. N,Nꢀ-Dimyristoyl-1,2-diaminopropan-3-ol
(3)
Fig. 1 represents the synthetic route used to
synthesize the various analogs of N,Nꢀ-diacyl-1,2-
diaminopropyl-3-carbamoyl-(dimethylaminoethane).
Intermediates 1, 2 and 3 were synthesized using a
modified procedure initially described by Sunamoto
et al. (1990). Briefly, 2,3-diaminopropionic acid
monohydrochloride (1 g, 7.11 mmol) was dissolved
in 50 ml of absolute methanol saturated with gaseous
hydrogen chloride and the reaction was refluxed at
95 ◦C for 6 h (yield = 90%, calculated from NMR).
1H NMR (400 MHz, D2O, 20 ◦C, TMS), δH (ppm):
4.32–4.28 [t, 1H, CH CO ], 3.63 [s, 3H, OCH3],
3.40–3.25 [m, 2H, CH2 ].
Compound 2 (2.3 g, 4.28 mmol) was dissolved in
50 ml anhydrous THF. LiBH4 (12.84 mmol) was added
and the reaction was stirred overnight at 60 ◦C. THF
was evaporated under diminished pressure and the dry
crude was taken up with 100 ml CHCl3 and washed
with 100 ml 1N HCl aqueous solution, 100 ml 1N
Na2CO3 aqueous solution and finally with 100 ml
brine. The organic layer was collected, concentrated
and subjected to column chromatography. The col-
umn was eluted and processed as described in the
previous step. Fractions 6 and 7 were pooled and
evaporated under diminished pressure to give 1.9 g
of compound 3 as a white powder (yield 88.3%).
Rf = 0.33 (A). 1H NMR (400 MHz, CDCl3, 20 ◦C,
TMS), δH (ppm): 6.41–6.35 [d, 1H, CO NH CH ],
6.28–6.15 [t, 1H, CO NH CH2 ], 3.82–3.6 [m, 2H,
NH CH2 CH ], 3.55–3.46 [m, 2H, CH2 OH],
3.24–3.18 [m. 1H, CH CH2 OH] 2.22–2.15 [m, 4H,
CO CH2 ], 1.61–1.53 [m, 4H, CO CH2 CH2 ],
1.32–1.18 [coherent, 40H, (CH2)10 ], 0.86–0.82 [m,
6H, CH3].
2.2.2. Methyl (N,Nꢀ-dimyristoyl)
2,3-diaminopropionate (2)
Compound 1 was dissolved in 150 ml dry DMF.
TEA (3.6 g, 35.5 mmol) was added followed by drop-
wise addition of myristoyl chloride (3.5 g, 14.22 mmol)
while stirring at 65 ◦C. After 3 h, additional TEA (3.6 g,
35.5 mmol) and myristoyl chloride (3.5 g, 14.22 mmol)
were added and the reaction was stirred for an addi-
tional 12 h at the same temperature. Precipitated tri-
ethylammonium chloride salt was removed by suc-
tion filtration. DMF was evaporated under dimin-
ished pressure and the crude product was taken up
with 100 ml CHCl3, transferred to a separation fun-
nel and washed with 100 ml 1N HCl aqueous solu-
tion and 100 ml brine. The organic layer was collected,
concentrated and subjected to column chromatogra-
phy. The column was eluted successively with 100 ml
CHCl3, 100 ml of 0.5%, 1%, 1.5%, 2%, 3%, 5%,
7%, 10%, 15%, 20% CH3OH:CHCl3. One hundred
milliliter fractions were collected and tested for the
presence of the product with TLC and NMR. Fractions
2.2.4. N,Nꢀ-Dimyristoyl-1,2-diaminopropane-3-
(4-nitrophenyl)carbonate (4)
Compound 3 (1.9 g, 3.8 mmol) was dissolved in
50 ml amylene-stabilized CHCl3. 4-Nitrophenyl-
chloroformate (0.766 g, 3.8 mmol) was dissolved
in 5 ml amylene-stabilized CHCl3 and added to the
reaction mixture dropwise followed by addition of
pyridine (0.31 ml, 3.8 mmol) while stirring at room
temperature (Sheikh et al., 2003). After 2 h, additional
4-nitrophenylchloroformate (0.383 g, 1.9 mmol) and
pyridine (0.155 ml, 1.9 mmol) were added and the
reaction was stirred overnight. The reaction mixture

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A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
Fig. 1. General synthetic scheme of N,Nꢀ-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives. Briefly, the car-
boxylic group of the synthon 2,3-diaminopropionic acid monohydrochloride was methyl-protected via fisher esterification to yield methyl 2,3-
diaminopropionate dihydrochloride (1). Acylation of (1) with varied length hydrophobic fatty acid derivatives resulted in methyl (N,Nꢀ-diacyl)
2,3-diaminopropionate(2). Reductionof(2)tothecorrespondingalcohol, N,Nꢀ-diacyl-1,2-diaminopropan-3-ol(3)waseffectedwithLiBH4 under
anhydrous conditions in organic solvent. The exposed hydroxyl group of (3) was further activated with 4-nitrophenyl chloroformate to the corre-
sponding N,Nꢀ-diacyl-1,2-diaminopropane-3-(4-nitrophenyl)carbonate (4). Finally, nucleophilic attack of (4) by N,N-dimethylethylenediamine
resulted into the final tertiary cationic surfactant molecules (5–9).
was diluted with additional CHCl3 to100 ml total and
washed once with 100 ml 1N HCl aqueous solution,
five times with 100 ml 1N Na2CO3 aqueous solution
and finally with 100 ml alkaline brine. The organic
layer was collected, concentrated and subjected to
column chromatography. The column was eluted and
processed as described for product 3. Fractions 4
and 5 were pooled and evaporated under diminished

A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
139
(ESI) m/z 591.5 [M + Na]+; 1H NMR (400 MHz,
CDCl3, 20 ◦C, TMS), δH (ppm): 6.81–6.79
pressure to give 2.2 g of compound 4 as a white powder
(yield = 75%). Rf = 0.56 (A). 1H NMR (400 MHz,
CDCl3, 20 ◦C, TMS), δH (ppm): 8.27–8.24, 7.35–7.28
[2d, 4H, C6H4], 6.63–6.61 [d, 1H, CO NH CH ],
[d, 1H,
CO NH CH2 ], 5.58–5.56 [t, 1H, OCONH ],
4.12–4.09 [m, 3H, CH CH2 OCON ],
3.4–3.30 [m, 2H, NH CH2 CH ], 3.24–3.19
CO NH CH ], 6.62–6.59 [t, 1H,
6.07–6.04 [t, 1H,
CO NH CH2 ], 4.36–4.20
[m, 3H, NH CH CH2 OCONH ], 3.60–3.37
[m, 2H, NH CH2 CH ], 2.20–2.13 [m, 4H,
CO CH2 ], 1.64–1.57 [m, 4H, CO CH2 CH2 ],
1.30–1.09 [coherent, 40H, (CH2)10 ], 0.88–0.82
[m, 6H, CH3].
[m, 2H, OCONH CH2 ], 2.41–2.37 [m, 2H,
CH2 N(CH3)2], 2.22–2.10 [m, 10H, CO CH2
and
N(CH3)2], 1.60–1.50 [m, 4H,
CO
CH2 CH2 ], 1.30–1.18 [coherent, 32H, (CH2)8 ],
0.85–0.82 [m, 6H, CH3].
2.2.5. N,Nꢀ-Dimyristoyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane), 1,2lmt[2] (5)
Compound 4 (1 g, 1.15 mmol) was dissolved in
50 ml CHCl3. N,N-Dimethylaminoethane (0.4 g, 4.54
mmol) was added to the reaction mixture and the
solution was stirred at room temperature. After 3 h, the
reaction mixture was diluted with 50 ml CHCl3 and
washed three times with 100 ml alkaline brine. The or-
ganic layer was collected, concentrated and subjected
to column chromatography. The column was eluted
with100 mlCHCl3, 100 mlof1, 2, 3, 5, 7, 10, 15, 20and
30% CH3OH:CHCl3. Fractions 7 and 8 were pooled
and evaporated under diminished pressure to give
0.55 g of 1,2lmt[2] as a white powder (yield = 58.3%).
Rf = 0.67 (B). Anal. calcd for C36H72N4O4 (MW
624): C, 69.23; H, 11.54; N, 8.97. Found: C, 69.38;
H, 11.72; N, 8.94. MS (ESI) m/z 647.6 [M + Na]+;
1H NMR (400 MHz, CDCl3, 20 ◦C, TMS), δH (ppm):
6.82–6.80 [d, 1H, CO NH CH ], 6.62–6.59 [t, 1H,
CO NH CH2 ], 5.55–5.52 [t, 1H, OCONH ],
2.2.7. N,Nꢀ-Dipalmitoyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane), 1,2lmt[3]
(7)
Yield = 62%. Rf = 0.66 (B). Anal. calcd for
C40H80N4O4 (MW 680): C, 70.59; H, 11.75; N,
8.24. Found: C, 69.99; H, 11.91; N, 8.10. MS
(ESI) m/z 703.6 [M + Na]+; 1H NMR (400 MHz,
CDCl3, 20 ◦C, TMS), δH (ppm): 6.70–6.68
[d, 1H,
CO NH CH2 ], 5.37–5.35 [t, 1H, OCONH ],
4.10–4.08 [m, 3H, CH CH2 OCON ],
3.43–3.32 [m, 2H, NH CH2 CH ], 3.22–3.19
[m, 2H, OCONH CH2 ], 2.41–2.38 [m,
2H, CH2 N(CH3)2], 2.22–2.10 [m, 10H,
CO NH CH ], 6.48–6.45 [t, 1H,
CO CH2 and N(CH3)2], 1.60–1.52 [m, 4H,
CO CH2 CH2 ], 1.30–1.18 [coherent, 48H,
(CH2)12 ], 0.85–0.82 [m, 6H, CH3].
2.2.8. N,Nꢀ-Distearoyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane), 1,2lmt[4]
(8)
Yield = 32.9%. Rf = 0.64 (B). Anal. calcd for
C44H88N4O4 (MW 736): C, 71.74; H, 11.96;
N, 7.61. Found: C, 71.6; H, 1214; N, 7.27. MS
(ESI) m/z 759.7 [M + Na]+; 1H NMR (400 MHz,
CDCl3, 20 ◦C, TMS), δH (ppm): 6.81–6.79
4.10–4.08
3.43–3.32 [m, 2H, NH CH2 CH ], 3.30–3.20
[m, 2H, OCONH CH2 ], 2.42–2.38 [m,
2H, CH2 N(CH3)2], 2.22–2.10 [m, 10H,
[m,
3H,
CH CH2 OCON ],
CO CH2 and N(CH3)2], 1.60–1.52 [m, 4H,
CO CH2 CH2 ], 1.30–1.18 [coherent, 40H,
(CH2)10 ], 0.85–0.82 [m, 6H, CH3].
All the subsequent compounds were synthesized us-
ing analogous procedures as described above for the
dimyristoyl derivatives.
[d, 1H,
CO NH CH2 ], 5.58–5.55 [t, 1H, OCONH ],
4.10–4.08 [m, 3H, CH CH2 OCON ],
3.43–3.32 [m, 2H, NH CH2 CH ], 3.22–3.20
[m, 2H, OCONH CH2 ], 2.42–2.38 [m,
2H, CH2 N(CH3)2], 2.22–2.10 [m, 10H,
CO NH CH ], 6.60–6.58 [t, 1H,
2.2.6. N,Nꢀ-Dilauroyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane), 1,2lmt[1]
(6)
CO CH2 and N(CH3)2], 1.60–1.52 [m, 4H,
CO CH2 CH2 ], 1.30–1.18 [coherent, 56H,
(CH2)14 ], 0.85–0.82 [m, 6H, CH3].
Yield = 72.9%. Rf = 0.69 (B). Anal. calcd for
C32H64N4O4 (MW 568): C, 67.60; H, 11.26; N,
9.86. Found: C, 67.89; H, 11.40; N, 9.65. MS

140
A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
2.2.9. N,Nꢀ-Dioleoyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane), 1,2lmt[5]
(9)
to be 1.88 suggesting a high quality plasmid free from
protein and salt contamination.
2.5. In vitro transfection assay
Yield = 49.4%. Rf = 0.65 (B). Anal. calcd for
C44H84N4O4 (MW 732): C, 72.13; H, 11.48; N, 7.65.
Found: C, 71.84; H, 11.86; N, 7.62. MS (ESI) m/z
755.6 [M + Na]+; 1H NMR (400 MHz, CDCl3, 20 ◦C,
TMS), δH (ppm): 6.78–6.70 [d, 1H, CO NH CH ],
6.55–6.50 [t, 1H, CO NH CH2 ], 5.48–5.42 [t, 1H,
Cationic lipids formulated in the presence and ab-
sence of the helper lipids were screened for activity
against B16-F0 mouse melanoma cells (ATCC, CRL-
6322) at the following cationic lipid/ DNA ( ) charge
ratios: 1/1, 2/1 and 4/1. All calculations were based
on the nucleotide average molecular weight taken to
be 330, and assuming complete protonation of the ter-
tiary amine group. Transfection experiments were car-
ried out as described previously. Briefly, approximately
5 × 104 cells were seeded in 1 mm wells (48-well plate)
12 h before transfection. Lipoplexes were prepared
30 min prior to transfection by mixing appropriate
amounts of the cationic lipid aqueous dispersions with
DNA in serum free media. The cells were transfected
with 250 ␮l of lipoplex dispersions and incubated at
37 ◦C for 4 h. The lipoplexes were then aspirated and
fresh RPMI medium containing 10% FBS was added
to each well. Cells were tested for ␤-galactosidase
activity 48 h after transfection using a 96-well mi-
croplate colorimetric assay that employed the sub-
strate o-nitrophenyl ␤-d-galactopyranoside (Sheikh et
al., 2003).
OCONH ], 5.33–5.25 [m, 4H,
CH CH ],
4.10–4.07 [m, 3H, CH CH2 OCON ],
3.43–3.32 [m, 2H, NH CH2 CH ], 3.25–3.18
[m, 2H, OCONH CH2 ], 2.40–2.35 [m, 2H,
CH2 N(CH3)2], 2.22–2.10 [m, 10H, CO CH2–
and N(CH3)2], 2.00–1.90 [m, 8H, CH2 CH CH
CH2 ], 1.62–1.50 [m, 4H,
CO CH2 CH2 ],
1.35–1.10 [coherent, 40H, (CH2)10 ], 0.85–0.70 [m,
6H, CH3].
2.3. Preparation of cationic aqueous dispersions
Particle size measurements, gel electrophoresis,
ethidium bromide displacement assay and cell trans-
fection studies were conducted with aqueous disper-
sions prepared at 0.6 mM cationic lipid concentra-
tion in Tris buffer. Cationic lipid/DOPE and cationic
lipid/DOPE/cholesterol molar ratios were fixed at 6:4
and 6:4:2, respectively. Briefly, aliquots of lipid so-
lutions in chloroform were transferred to borosilicate
glass test tubes. Chloroform was first evaporated under
a stream of nitrogen and completely removed by high
vacuum desiccation for 4 h. Dry lipid films were then
hydrated with 1 ml of Tris buffer at 55 ◦C for 30 min to
1 h with occasional vortexing, followed by sonication
for 5 min at room temperature.
2.6. MTT toxicity assay
Lipoplex cytotoxicity was assessed in B16-F0 cells
using a modified colorimetric MTT toxicity assay de-
scribed elsewhere (Savva et al., 1999). Briefly, cells
were cultured as described before for the in vitro trans-
fection assay. After 44 h of incubation, 50 ␮l of MTT
solution in PBS (5 mg/ml) was added to each well and
cells were incubated for an additional 4 h at 37 ◦C. The
supernatant was discarded and 250 ␮l of DMSO was
added to each well with gentle agitation in order to sol-
ubilize the formed purple formazan crystals. Absorp-
tion was measured at 630 nm using a multiscan plate
reader. The absorption obtained for cells treated with
DNA alone was taken to be 100%.
2.4. Plasmid preparation
A pUC19 plasmid DNA vector containing the beta-
galactosidase (␤-Gal) reporter gene driven by the hu-
man cytomegalovirus (CMV) immediate-early pro-
moter was isolated and purified as described else-
where (Sheikh et al., 2003). The concentration of plas-
mid DNA was determined spectrophotometrically at
260 nm using 1 OD = 50 ␮g/ml of plasmid DNA. The
purity and quality of the plasmid DNA was verified by
gel electrophoresis and the A260/A280 ratio was found
2.7. Agarose gel electrophoresis
Lipoplexes at charge ratios of 0.5:1, 1:1, 2:1,
4:1, 6:1 and 8:1 were prepared in microfuge tubes by

A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
141
mixing 0.2 ␮g of DNA with the appropriate amount
of the cationic lipid aqueous dispersions. The to-
tal volume of each microfuge tube was brought to
11 ␮l with 40 mM TAE buffer, pH 7.4. The lipoplexes
were briefly centrifuged at 14,000 rpm and incubated
at room temperature for approximately 30 min be-
fore they were loaded into a 0.8% agarose gel con-
taining 0.5 ␮g/ml of ethidium bromide. Naked DNA
was loaded on the outermost lanes as a negative con-
trol. The samples were subjected to an electrophoretic
field of 5 V/cm for 35 min in 16 mM TAE buffer,
pH 7.4. The migration of lipoplexes was visual-
ized using a Biorad mini-transilluminator and pictures
were taken with a Biorad photodocumentation system
(VWR).
that was run side by side to the original experiment
indicated that light scattering effects caused less than
2% change in the fluorescence signal (Borenstain and
Barenholz, 1993). Thus, the data presented are raw data
that has not been corrected for any light scattering ef-
fects. To provide the relative ethidium bromide dis-
placement from plasmid DNA, the fluorescence signal
of the ethidium bromide blank solution was subtracted
from all measurements and the fluorescence intensity
obtained from the plasmid DNA solution containing
ethidium bromide in the absence of lipid (F0) was as-
signed a value of 100. The resulting fluorescence in-
tensity after addition of the cationic lipids (F complex)
was normalized as follows:
Fcomplex
% displacement =
× 100.
F0
2.8. Dynamic light scattering
Particle size distribution of lipid aqueous disper-
sions and lipoplexes was determined at room tempera-
ture using a Malvern Zetasizer Nano ZS particle sizer
(Malvern Instruments Inc., MA) operating at 532 nm,
at a 90◦ fixed angle. The instrument was validated
with polystyrene microspheres (Bangs Laboratories
Inc., Fishers, IN). Care was taken during preparation
and dilution of the samples to prevent dust contami-
nation. Lipoplexes were freshly prepared by diluting
200 ␮l of 0.6 mM lipid dispersion to a final volume
of 1.5 ml with Tris buffer followed by addition of the
appropriate amount of DNA to the desired charge ra-
tio. Data presented are the mean raw intensity-weighed
particle size distributions obtained from the Gaussian
analysis.
2.10. Monolayer studies
A computer controlled KSV Minitrough film bal-
ance (KSV instruments Ltd., Finland) equipped with
two polyacetal made barriers and a platinum Wilhelmy
plate was used to construct the pressure–area isotherms
of monolayers of the different cationic lipids at the
air/water interface. The subphase buffer was a 40 mM
Tris buffer, pH 7.2 and was prepared as previously
described in the materials section. After each experi-
ment, the Teflon made trough (364 mm × 75 mm, to-
tal surface area 24,275 mm2) and the barriers were
cleaned by rinsing them thoroughly with purified wa-
ter. Lipid solutions (0.4–0.7 mg/ml) were prepared in
chloroform, stored at −20 ◦C and used within 3 days
of preparation. All experiments were carried out with
140 ml of the subphase buffer with the subphase tem-
perature maintained at 23 ◦C by an external water
bath circulator. Each experiment was repeated three
to six times in an open-air vibration-free environ-
ment with occasional variation in lipid amount spread
to ensure isotherms reproducibility (Welzel et al.,
1998).
2.9. Ethidium bromide displacement assay
Fluorescence measurements were collected with
a Cary Eclipse fluorescence spectrophotometer (Var-
ian Inc., CA.) at an excitation wavelength of 515 nm
(slit width 5 nm) and an emission wavelength of
595–605 nm (slit width 2.5 nm). Briefly, 22.5 ␮g
of plasmid DNA and 0.8 ␮g of ethidium bromide
(DNA:ethidium bromide complex ratio 34:1) were
added to a quartz cuvette and diluted to 3 ml with Tris
buffer. Cationic lipids were added in aliquots under
continuous stirring at room temperature and fluores-
cence was monitored continuously until the reading
became stable. A sample without ethidium bromide
Briefly, 20–50 ␮l aliquots of cationic lipid-
spreading solution were applied carefully drop by
drop to the surface of the aqueous subphase with
the aid of a Hamilton glass micro syringe. After an
initial delay period of 20 min, to ensure chloroform
evaporation, the barriers were closed at a constant
speed of 9.99 mm/min. Analysis of the constructed π/A

142
A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
isotherms was carried out with WinLB 1.61 software
provided by the manufacturer and surface pressure (π)
was plotted as a function of the mean molecular area
2
˚
(A ). Monolayer phase transitions and collapse were
identified using the first derivative of surface pressure
with respect to mean molecular area. All transitions
and collapse areas reported here are onset parameters.
Monolayer compressibility modulus at a given surface
pressure was calculated from the π/A isotherms using a
two dimensional form of the bulk compressibility mod-
ulus equation (Ali et al., 1998):
ꢀ
ꢁ
dπ
Kπ = (−Aπ)
dAπ
where K is the compressibility modulus in mN/m and
2
˚
Aπ the mean molecular area in A at a certain surface
pressure π.
Fig. 2. In vitro transfection activity of 1,2lmt[5] formulations at 1:1,
2:1 and 4:1 charge ratios in B16-F0 mouse melanoma cells as com-
pared to DC-Chol/DOPE (6/4) at 2:1 charge ratio. All formulations
were tested on the same day and the data presented are the average of
three wells. ␤-galactosidase levels are expressed as mU/well. Each
well is equivalent to 150 ␮l of cell lysate.
3. Results
3.1. In vitro transfection activity and toxicity
with a cell survival percentage exceeding 87%. Formu-
lations containing DOPE resulted in a minor decrease
in the cell survival percentage. It is worth noting that all
formulations showed a charge and dose dependent cy-
totoxicity. In other words, increasing the cationic lipid
content in the lipoplex resulted in an increased cyto-
toxicity.
The aqueous dispersions of the N,Nꢀ-diacyl-1,2-
diaminopropyl-3-carbamoyl-(dimethylaminoethane)
analogs were tested for their ability to deliver the
pUC19 plasmid DNA containing the ␤-galactosidase
reporter gene in B16-F0 cells. All analogs but the
dioleoyl derivative 1,2lmt[5] elicited no activity at
any of the three charge ratios studied in the presence
or absence of the helper lipids (Fig. 2). Lipoplexes
made with 1,2lmt[5] in the absence of helper lipids
resulted in a moderate increase of ␤-galactosidase
levels compared with the DNA control. Inclusion of
the helper lipid DOPE resulted in enhanced expression
levels of the reporter enzyme with the highest level
occurring around a charge ratio of 2. Similarly,
maximum activity in the presence of cholesterol was
observed at a charge ratio of 2. However, the activity
was much lower compared to the cationic lipid/DOPE
formulation.
The cytotoxicity of lipoplexes prepared from the dif-
ferent formulations of the five analogs was evaluated
in B16-F0 cells using the MTT cytotoxicity assay. No
significant cytotoxicity was detected for lipoplexes pre-
pared with the saturated fatty acyl chain analogs (data
not shown). As indicated in Fig. 3, lipoplexes prepared
with 1,2lmt[5] alone were well tolerated by the cells
Fig. 3. Cytotoxicity of 1,2lmt[5] formulations at 1:1, 2:1 and 4:1
charge ratios in B16-F0 mouse melanoma cells as compared to
DC-Chol/DOPE (6/4) at 2:1 charge ratio. Data presented are the
average of three wells. Results are expressed as percentage survival
of treated cell taking cells treated with DNA alone to be 100%.

A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
143
Fig. 4. Gel electrophoresis of lipoplexes prepared with different lipid formulations at the following charge ratios: 0.5, 1, 2, 4, 6 and 8,
respectively. The outermost wells were loaded with plasmid DNA as a negative control. (A) Lipoplexes prepared with cationic lipids alone, (B)
cationic lipid/DOPE (6:4 molar ratios) and (C) cationic lipid/DOPE/cholesterol (6:4:2 molar ratios).
3.2. Agarose gel electrophoresis
act with and completely retard plasmid DNA at a
charge ratio of 4. Interestingly, in the presence
of DOPE or DOPE and cholesterol, DNA migration
was completely inhibited at a charge ratio of 2.
Cationic lipids that failed to mediate transfection ac-
tivity also failed to complex with DNA at all charge
ratios.
The ability of the N,Nꢀ-diacyl-1,2-diaminopropyl-
3-carbamoyl-(dimethylaminoethane) cationic deriva-
tives to complex with plasmid DNA was investi-
gated by a gel electrophoresis retardation assay. As
seen in Fig. 4, only 1,2lmt[5] was able to inter-
Fig. 5. Cationic lipid induced DNA condensation monitored by an ethidium bromide displacement assay.

144
A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
Table 1
Particle size distribution of 1,2lmt[5] liposomal formulations and their lipoplexes
1,2lmt[5]
1,2lmt[5]/DOPE
1,2lmt[5]/d/chol
Mean diameter (nm)
P.I.a
Mean diameter (nm)
P.I.a
Mean diameter (nm)
P.I.a
Liposomes
156.7
194.8
2941
Ab
0.315
0.252
0.557
–
125.4
268.1
1216
275
0.224
0.33
1.0
136.9
248.2
2360
215
0.250
0.314
0.671
0.241
Lipoplexes (1:1)
Lipoplexes (2:1)
Lipoplexes (4:1)
0.382
Particle size measurements were carried out in 40 mM Tris buffer pH 7.2 as described in the experimental section. Lipid dispersions were
prepared at 0.6 mM, which is well above their CMC.
a
Values of polydispersity index (P.I.) above 0.18 denote polydisperse samples. Ab denotes aggregates of size above 6 ␮m. All other lipid
formulations showed aggregates >6 ␮m with polydispersity indices exceeding 1.
3.3. Ethidium bromide displacement assay
lipoplexes prepared at a 1:1 charge ratio. Increasing
the charge ratio resulted in a tremendous increase in
particle size for 1,2lmt[5] lipoplexes in the absence
of the helper lipids. Contrary to this, 1,2lmt[5]/DOPE
and 1,2lmt[5]/DOPE/chol lipoplexes aggregated at 2:1
charge ratio but exhibited a small particle size distribu-
tion when the charge ratio was increased to 4:1.
Agarose gel electrophoresis assay shows whether or
not the cationic lipid is capable of complexing plasmid
DNA, but it does not give any information about the ef-
ficiency with which the lipid condenses DNA. Further
characterization of the size of the complex and the ex-
tent of condensation of DNA requires the use of other
techniques. In order to investigate the extent of cationic
lipid induced condensation of plasmid DNA, reduction
of ethidium bromide fluorescence signal upon titrating
the plasmid DNA with aliquots of cationic lipid aque-
ous dispersions was monitored. All cationic deriva-
tives, including the active 1,2lmt[5] analog, with or
without the helper lipid DOPE displaced less than 10%
of ethidium bromide at a charge ratio of 2 (Fig. 5).
Similar results were obtained when an aliquot of either
1,2lmt[5] or 1,2lmt[5]/DOPE equivalent to a charge
ratio of 2 was added all at once to the plasmid DNA so-
lution suggesting that DNA condensation by 1,2lmt[5]
is not efficient.
3.5. Monolayer studies
The interfacial properties of the 1,2lmt cationic
lipids in isolation were investigated using the Lang-
muir film balance technique. Aliquots of cationic lipids
in CHCl3 were spread on the surface of the same
buffer system used to prepare the aqueous disper-
sions for transfection and for the other physicochem-
ical characterization studies, i.e. 40 mM Tris buffer,
pH 7.2. Representative compression isotherms of the
five analogues are shown in Fig. 6. At 23 ◦C, only
the unsaturated derivative 1,2lmt[5] formed monolay-
ers that existed in an all liquid-expanded state with
2
˚
molecular collapse area and pressure of 59.47 A and
3.4. Dynamic light scattering
38.67 mN/m, respectively (Fig. 7E and Table 2). On
the other hand, monolayers of the saturated deriva-
tives either exhibited an all liquid-condensed state or
a two-dimensional phase transition toward a liquid-
condensed state in their π/A isotherms. More specif-
ically, the compression isotherm of the shortest deriva-
tive, 1,2lmt[1], exhibited a typical liquid-expanded to
The particle size distribution of the dioleoyl deriva-
tive dispersions and lipoplexes is summarized in
Table 1. Liposomes prepared with the dioleoyl deriva-
tive exhibited a particle size distribution below 0.2 ␮m,
while the other transfection-inefficient lipids resulted
in aggregates of particle sizes well above the measur-
able range of the instrument. Neither prolonged incu-
bation nor incubation at higher temperatures (∼80 ◦C)
affected the particle size of the aggregates. Upon com-
plexing 1,2lmt[5] formulations with plasmid DNA, no
significant increase in particle size was observed with
liquid-condensed transition observed at a molecular
2
˚
area and pressure of 65.74 A and 21.63 mN/m, respec-
tively. Further reduction of the surface area resulted
in two overlapping maximums in the 1st derivative
graph (Fig. 7A). The onset of the first maximum is at
2
2
˚
˚
39.27 A and 42.13 mN/m, and the second at 32.9 A

A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
145
tions (Fig. 7B). Upon compressing the film beyond
that transition, surface pressure rose only marginally
2
˚
within an approximate area range 105−75 A , after
which, the surface pressure sharply increased indicat-
ing the presence of a chain ordered state. Monolayer
collapse was reached at a molecular area and pres-
2
˚
sure of 40.74 A and 40 mN/m, respectively (Table 2).
The 1,2lmt[3] and 1,2lmt[4] analogs formed all liquid-
condensed state monolayers at 23 ◦C with a collapse
2
˚
molecular area of 39.78 and 42.13 A and pressure of
39.46 and 39.79 mN/m, respectively (Fig. 7C and D,
Table 2).
Interfacial compressibility moduli (K) values of
monolayers prepared from the different 1,2lmt cationic
lipids, calculated at molecular collapse area are pre-
sented in Table 2. Comparison of the K values of the
different analogues revealed that 1,2lmt[5] monolayers
exhibited the greatest compressibility with a K value
of 80.36 10.4. For all other saturated derivatives, K
was found to increase with increasing chain length,
the maximal value being that of the distearoyl deriva-
tive 1,2lmt[4]. However, K values cannot be used to
judge compressibilities of lipids that have very differ-
ent molecular dimensions. The final K values could
sometimes be misleading in the sense that these val-
ues are the slope dΠ/dA multiplied by the area. In
Fig. 6. Representative π/A isotherms of 1,2lmt cationic derivatives
at the air/water interface at 23 ◦C. Tris buffer (40 mM, pH 7.2)
was used as the subphase. Isotherms were constructed using KSV
computer-controlled Minitrough film balance with a compression
rate of 9.99 mm/minute. (1) 1,2lmt[1]; (2) 1,2lmt[2]; (3) 1,2lmt[3];
(4) 1,2lmt[4]; and (5) 1,2lmt[5].
and 54 mN/m molecular area and surface pressure, re-
spectively. Monolayer collapse was most probably ini-
tiated at the first maximum and further increase in sur-
face pressure beyond that area is due to a multilayer,
the formation of which was completed at a molecu-
2
˚
lar area of 32.9 A . The presence of two maximums
in the dΠ/dA versus mean molecular area (MmA) plot
was very clear for 1,2lmt[1] and less evident for the
other long chain derivatives at the temperature studied
(Fig. 7).
The compression isotherm of 1,2lmt[2] exhibited
a similar two-dimensional phase transition but at a
much lower surface pressure (Fig. 6, isotherm 2). In
this case, the transition was broad and its onset was
difficult to determine by the first derivative calcula-
Table 2 as well as in Fig. 7, it can be seen that
2
˚
1,2lmt[5] monolayer collapsed at an area that is 20 A
larger than that for 1,2lmt[1]. In spite of the smaller
slope of 1,2lmt[5], its larger collapse area resulted
in a K value close to that of 1,2lmt[1]. For compar-
ison purposes between monolayers with varying col-
lapse areas, one should also pay attention to the rate of
change of surface pressure with respect to the change of
Table 2
Monolayer parameters of 1,2lmt amphiphilic seriesa
2
2
˚
˚
MmA (A )
Πc (mN/m)
K (mN/m)
dΠ/dAb
Phase state
L2
‘Lift off’ area (A )
1,2lmt[1]
39.27 1.6
42.13 1.47
97.83 15.54
60.26c
2.48 0.39
0.91c
127.99 7.19
65.47c
1.03
21.63c
1.24
3.88
0.09
1,2lmt[2]
1,2lmt[3]
1,2lmt[4]
1,2lmt[5]
a
40.74 0.42
39.78 0.78
42.13 0.62
59.47 2.17
40.00 1.28
39.46 1.15
39.79 2.06
38.67 0.74
134.67 18.15
157.75 8.62
160.2 20.8
80.36 10.4
3.2 0.35
3.85 0.27
3.75 0.56
1.33 0.18
L2
L2
L2
L1
113 3.76
68.71 4.45
78.04 6.22
151.89 10.08
Measured in 40 mM Tris buffer (pH 7.2) at 23 ◦C. All parameters are ‘onset’ transition values. MmA denotes Mean molecular Area expressed
2
˚
in A per molecule. L1 denotes liquid-expanded state (Adamson, 1982). L2 denotes liquid-condensed state.
b
Values calculated at monolayer collapse and at other two-dimensional phase transitions.
Phase transition denoted by a maximum in the dΠ/dA plot as a function of mean molecular area.
c

146
A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
Fig. 7. Surface pressure (Π) and first derivative of surface pressure (dΠ/dA) as a function of mean molecular area of different 1,2lmt cationic
derivatives. (A) 1,2lmt[1]; (B) 1,2lmt[2]; (C) 1,2lmt[3]; (D) 1,2lmt[4] and (E) 1,2lmt[5].
compressibility of the five analogues is in the order
of 1,2lmt[5] > 1,2lmt[1] > 1,2lmt[2] > 1,2lmt[3] ≥ 1,2-
lmt[4], with 1,2lmt[3] and 1,2lmt[4] films being the
least compressible.
MmA (shown in Table 2 as dΠ/dA) of the monolayer
as an indicator of monolayer state and compressibil-
ity. Thus, considering both the compressibility modu-
lus and the corresponding dΠ/dA values, the in-plane

A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
147
4. Discussion
tion was evident only at a charge ratio of 2. Since
this charge ratio was the optimum ratio for transfec-
tion activity, it appears that a particle size distribution
between 1 and 2 ␮m correlates with enhanced in vitro
activity.
The aim of this study was to delineate the
physicochemical properties of double-chained cationic
surfactants required for high plasmid DNA trans-
fection efficiencies. To this end, a new series
of cationic lipids, N,Nꢀ-diacyl-1,2-diaminopropyl-3-
carbamoyl-(dimethylaminoethane) derivatives were
synthesized by systemically varying the length and de-
gree of unsaturation of the hydrophobic anchor. The
fatty acyl chains were linked to the ionizable cationic
head group via amide bonds that are biodegradable but
significantly more stable than the easily hydrolyzed es-
ter bond found in natural phospholipids.
Besides physicochemical characterization of the
lipoplexes formed by these novel cationic lipids, the
Langmuir film balance technique was used in an at-
tempt to reveal the interfacial properties of cationic
lipids pertinent to efficient in vitro transfection activ-
ity. First derivative analysis of the π/A isotherms of
the saturated derivatives revealed that perturbation of
monomolecular films prepared from these lipids started
2
˚
at an area and pressure around 40 A and 40 mN/m,
In vitro transfection activity studies conducted
showed that only lipoplexes formulated with the di-
oleoyl derivative 1,2lmt[5] mediated significant in vitro
transfection activity. All the saturated derivatives were
transfection inefficient at all charge ratios in the pres-
ence and absence of the helper lipids. Optimum activity
of 1,2lmt[5] was obtained in the presence of DOPE at
respectively (Table 2). Interestingly, 1,2lmt[5] mono-
layer collapsed at a surface pressure similar to that ob-
served for the saturated analogues but its collapse area
2
◦
˚
was approximately 19–20 A larger at 23 C. Since all
five derivatives share the same head group composi-
tion, the origin of this difference in molecular collapse
areas between the dioleoyl derivative and the saturated
derivatives is the increased fluidity and hydration of
1,2lmt[5] due to the double bond it possesses in its acyl
chains. With regard to the saturated derivatives, strong
Van der Waals attractions between the acyl chains and
intermolecular hydrogen bonding between the polar
head group of adjacent lipid molecules in the highly
compressed film are expected to lead to removal of
the tightly bound interfacial water and tight packing of
the dehydrated molecules in the film. On the contrary,
the expanded unsaturated acyl chains of the dioleoyl
derivative will prevent proper ordering of film compo-
nents necessary for the exclusion of interfacial water
and the subsequent intermolecular hydrogen bonding
to take place (Ali et al., 1991). If a water molecule
at the interface occupies a surface area Aswater = 9.65
(Feng et al., 1994), then there is at least 2 extra water
molecules associated with each 1,2lmt[5] molecule at
the collapse relative to the other saturated derivatives.
The transfection potent lipid 1,2lmt[5] also formed the
most elastic monolayer at 23 ◦C, as indicated by its low
compressibility modulus. All other transfection ineffi-
cient saturated derivatives were characterized by large
compressibility moduli, which were found to be di-
rectly proportional to the chain length, being maximal
for 1,2lmt[4] (Table 2).
a
charge ratio of 2 (Fig. 2). In agreement with the
transfection results, 1,2lmt[5] was the only lipid found
to complex and retard DNA migration upon gel elec-
trophoresis (Fig. 4). Despite the efficient complexation,
no significant condensation of plasmid DNA took place
as indicated by fluorescence spectroscopy measure-
ments (Fig. 5). In vitro transfection, gel electrophoresis
and fluorescence spectroscopy suggest that inefficient
condensation of plasmid DNA by cationic lipids does
not abolish in vitro transfection activity, a phenomenon
that was previously observed by Banerjee et al. (2001).
Loose association of plasmid DNA with cationic lipids
is enough to promote significant in vitro transfection
activity. However, whether this is also valid for in vivo
transfection is doubtful since more stable lipoplexes are
required to overcome the inhibitory effect of serum.
To gain further insight on the transfection behavior
of the different analogues and their formulations, par-
ticle size distribution measurements were conducted.
Aqueous dispersions of the transfection inefficient
lipids exhibited particle size distributions > 6 ␮m. Such
large particle size distribution indicates that these lipids
failed to hydrate and form liposomes. On the other
hand, liposomes prepared from 1,2lmt[5] in the pres-
ence or absence of helper lipids exhibited particle size
distribution smaller than 200 nm. When these lipo-
somes were complexed with plasmid DNA, aggrega-
The correlation between molecular fluidity and im-
proved transfection activity was first introduced by

148
A. Aljaberi et al. / Chemistry and Physics of Lipids 133 (2005) 135–149
Akao et al. (1994). Their findings suggested that a
liquid-crystalline to gel phase transition temperature
below 37 ◦C is required for cationic lipid-mediated de-
livery of plasmid DNA in vitro. Besides molecular flu-
idity, Bennett et al. investigated the effect of molecular
hydration on in vivo transfection activity with a series
of dimyristoyl and dioleoyl derivatives possessing sev-
eral head group combinations (Bennett et al., 1997).
In the presence of DOPE, they found the highly hy-
drated dioleoyl derivatives to be superior for in vivo
transfection activity. They, along with others, have con-
cluded that 14-carbon or oleoyl chains satisfy the hy-
dration, fluidity and bilayer stability requirements for
efficient transfection efficiency (Felgner et al., 1994;
Ilies et al., 2004; Laxmi et al., 2001; Wang et al., 1998).
The results of the current study agree with the findings
stated above. The only lipid that satisfied the condi-
tions required for efficient transfection activity was the
dioleoyl derivative. On the other hand, the myristoyl
derivative 1,2lmt[2] was transfection inefficient even
in the presence of DOPE. This can be easily explained
by the rigidity and lack of minimum hydration of this
lipid aqueous dispersion. As mentioned earlier, par-
ticle size distribution measurements showed that this
lipid failed to form liposomes upon hydration. More-
over, Langmuir film balance studies performed with
this lipid showed that it formed films that exhibited
a liquid-condensed phase at a surface pressure value
comparable to that usually found in phospholipid bi-
layers, i.e. 30–35 mN/m (Ali et al., 1998 and references
therein). In part, this might be due to the introduction of
hydrogen bond functionalities in the polar part of the
1,2lmt[2] that significantly altered its phase behavior
making it a more rigid molecule.
Acknowledgment
The authors are grateful to Dr. Christopher M.
Hadad in the Department of Chemistry, Ohio State Uni-
versity, for performing the mass spectroscopy analysis.
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In summary, five different N,Nꢀ-diacyl-1,2-
diaminopropyl-cationic derivatives were synthesized
and tested for efficient in vitro transfection activity.
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an extensive investigation of their interfacial prop-
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