Báo cáo khoa học: NovelN,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery – synthesis,in vitro transfection activity, and physicochemical characterization docx

Novel N,N ¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent
cationic lipids for gene delivery – synthesis, in vitro
transfection activity, and physicochemical characterization
Michael Spelios and Michalakis Savva
Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island University, Brooklyn,
NY, USA
The development of highly potent and minimally toxic
cationic lipids for nucleic acid delivery depends on
generation of meaningful structure–activity relation-
ships. The rational design of efficacious transfection
amphiphiles is based on understanding the impact of
each of the lipid structural components on gene
Keywords
cationic lipid; elasticity; FRET; gene delivery;
lipoplex
Correspondence
M. Savva, Division of Pharmaceutical
Sciences, Arnold & Marie Schwartz College
of Pharmacy & Health Sciences, Long Island
University, 75 DeKalb Avenue, Brooklyn,
NY 11201, USA
Fax: +1 718 780 4586
Tel: +1 718 488 1471
E-mail:
(Received 23 September 2007, revised
5 November 2007, accepted 9 November
2007)
doi:10.1111/j.1742-4658.2007.06185.x
Novel N,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids
were synthesized and their physicochemical properties in lamellar assemblies
with and without plasmid DNA were evaluated to elucidate the structural

requirements of these double-chained pH-sensitive surfactants for potent
non-viral gene delivery and expression. The highest in vitro transfection
efficacies were induced at + ⁄ ) 4 : 1 by the dimyristoyl, dipalmitoyl and
dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion
of helper lipids. Transfection activities were reduced in the presence of either
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination
with cholesterol for all derivatives except 1,3lb5, which maintained reporter
gene expression levels at + ⁄ ) 4 : 1 and yielded increased lipofection activity
at a lower charge ratio of + ⁄ ) 2 : 1. Ethidium bromide displacement
indicated efficient plasmid DNA binding and compaction by the trans-
fection-competent analogs. Dynamic light-scattering and electrophoretic
mobility studies revealed lipoplexes of the active lipids with large particle
sizes (mean diameter ‡ 500 nm) and zeta potentials with positive values
(low ionic strength) or below neutrality (high ionic strength). Langmuir film
balance studies showed high in-plane elasticity of these derivatives in isola-
tion. In agreement with the monolayer experiments, fluorescence polariza-
tion studies verified the fluid nature of the highly transfection-efficient
amphiphiles, with gel-to-liquid crystalline phase transitions below physio-
logical temperature. The active compounds also interacted with endosome-
mimicking vesicles to a greater extent than the poorly active derivative
1,3lb4, as revealed by fluorescence resonance energy transfer experiments.
Taken together, the results suggest that well-hydrated and highly elastic
cationic lipids with increased acyl chain fluidity and minimal cytotoxicity
elicit high transfection activity.
Abbreviations
DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, 1,2-dioleoyl-3-
trimethylammonium-propane; DPH, 1,6-diphenyl-1,3,5-hexatriene; EGFP, enhanced green fluorescent protein; EtBr, ethidium bromide; FITC,
fluoroscein isothiocyanate; FRET, fluorescence resonance energy transfer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NBD-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); ONPG, 2-nitrophenyl b-
D-galacto pyranoside; PA,

1,2-dipalmitoyl-sn-glycero-3-phosphate; PC,
L-a-phosphatidylcholine (egg, chicken); pDNA, plasmid DNA; Rh-PE, 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); SFM, serum-free medium; TNS, 2-(p-toluidino)naphthalene-6-sulfonic acid.
148 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
transfer, namely the polar headgroup, the nonpolar tail
(either a cholesterol moiety or a pair of aliphatic
hydrocarbon chains), and the linker tethering both
regions together. Since the advent of lipofection
20 years ago [1], cationic lipids with new molecular
architectures have been developed and analyzed as
gene-delivery vectors, as seen in numerous recent
publications. Liu et al. [2] synthesized a series of 16
carbamate-linked cationic lipids, differing in their
hydrocarbon chain length, quaternary ammonium
head and counter-ion species, and examined their bio-
logical performance. Spacer modifications were studied
in cholesterol-based and aliphatic gemini cationic lipids
to determine their effects on the transfecting abilities
of these dimeric surfactants [3–5]. Rajesh et al. [6] were
the first to report the influence of linker orientation
reversal on the transfection efficiencies and physico-
chemical properties of two cationic amphiphiles with
identical hydrophilic and hydrophobic constituents.
Other groups have also recently described the design,
syntheses, physicochemical characterization and trans-
fection properties of novel cationic amphiphiles [7–11].
In an effort to further delineate the structural prop-
erties of these lipofection reagents that confer superior
transfection activity, a novel series of N,N¢-diacyl-1,3-
diaminopropyl-2-carbamoyl bivalent cationic lipids

was synthesized containing a symmetric bis-[2-dimeth-
ylamino-ethyl]-amine polar headgroup at the 2-posi-
tion and hydrophobic chains at the 1- and 3- positions
of the 1,3-diamino-2-propanol backbone (Fig. 1). The
series, designated 1,3lb, consists of four saturated lip-
ids, ranging in chain length from 12 to 18 carbons,
and a single monounsaturated derivative with a double
bond between the 9th and 10th carbons of each 18-car-
bon chain. Physicochemical characterization of the cat-
ionic lipids in lamellar assemblies with and without
plasmid DNA and in media of various ionic strengths
comprised a variety of studies and techniques, includ-
ing pK
a
determination, isothermal monolayer compres-
sion, fluorescence anisotropy, ethidium bromide
displacement, dynamic light scattering, determination
of zeta potential, and fluorescence resonance energy
transfer (FRET), and is indispensable for elucidating
the structural properties of these amphiphiles that
induce high transfection activity.
This work is a continuation of a recent study high-
lighting the superior gene delivery mediated by the di-
myristoyl derivative 1,3lb2 from the aforementioned
series as compared to two other cationic lipid vectors,
a conformational isomer and a monovalent analog
[12]. It was determined that a symmetrical bivalent
pH-expandable polar headgroup, in combination with
greater intramolecular space between the hydrophobic
chains, promotes highly efficacious in vitro lipofection

through efficient binding and compaction of pDNA,
increased acyl chain fluidity and high molecular elastic-
ity. The current study is a further examination of
the 1,3lb cytofectin involving systematic molecular
changes; specifically, determination of the effects of
hydrophobic chain length and degree of unsaturation
on target gene expression.
Results
Biological analysis
Lipoplexes of 1,3lb cationic lipids with and without
helper lipid(s) were examined at various + ⁄ ) charge
ratios for transfection activity. The shortest saturated
chain derivative 1,3lb1 was completely inefficient at pro-
moting lipofection at all charge ratios, both in the
absence and presence of neutral colipid(s). Formulations
lacking either 1,2-dioleoyl-sn-glycero-3-phosphoetha-
nolamine (DOPE) or phospholipid and cholesterol
induced the highest levels of reporter gene expression at
+ ⁄ ) 4 : 1, the exception being 1,3lb4 which exhibited
low activity throughout the range of charge ratios tested
(Fig. 2A). The dipalmitoyl derivative 1,3lb3 elicited
higher in vitro transfection activity than 1,3lb5, contrary
to findings that unsaturated derivatives are typically the
N
N
NCH
3
H
3
C

O
O
NH NH
R
O
O
R
H
3
C
H
3
C
The R group varies with the derivative:
C
11
H
23
for dilauroyl (1,3lb1)
C
13
H
27
for dimyristoyl (1,3lb2)
C
15
H
31
for dipalmitoyl (1,3lb3)
C

17
H
35
for distearoyl (1,3lb4)
C
17
H
31
for dioleoyl (1,3lb5)
Fig. 1. Structure of the 1,3lb derivatives.
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 149
most transfection-competent [13–15]. The activity of
b-galactosidase was approximately two- to threefold
greater than with 1,2-dioleoyl-3-trimethylammonium-
propane (DOTAP)-mediated gene delivery. Qualitative
analysis of transfection efficiency by detection of
enhance green fluorescent protein (EGFP), as seen in
Fig. 3, revealed the same activity trends as quantita-
tively determined by the 2-nitrophenyl b-d-galacto
pyranoside (ONPG) assay: 1,3lb3 > 1,3lb2 $ 1,3lb5 >
DOTAP > 1,3lb4.
Fluorescein covalently attached to plasmid allowed
visual tracking of cellular uptake. Internalization of
exogenous nucleic acid occurred to the greatest extent
with the aid of 1,3lb2, as indicated by the higher fluoro-
scein isothiocyanate (FITC)-plasmid DNA (pDNA)
intensity (green) when compared to the fluorescence
yields of labeled plasmid transported via other
derivatives (Fig. 4A). Lipoplexes formed with 1,2-di-

oleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl (Rh-PE)-labeled dispersions (red)
of 1,3lb2 were visualized as distinctly yellow spots, sug-
gesting well-associated complexes of nucleic acid and
lipid (Fig. 4B); overlaid FITC and rhodamine images
of cells transfected using the other transfection-active
analogs showed colocalization of plasmid and lipid to a
similar degree (not shown). In accordance with these
results, the dimyristoyl derivative was the most efficient
of the transfection-active compounds at condensing
pDNA as monitored by ethidium bromide (EtBr) dis-
placement. Images obtained after transfection with
1,3lb1 (results not shown) showed significantly fewer
cells, and fluorescent patches where no cells were pres-
ent, indicating large aggregates with a high affinity for
the plate surface that were not internalized and are
probably responsible for the elevated cytotoxicity. In
fact, except along the edges of the wells where cells
were densely packed and multilayered, accounting for
the 46% survival (data not shown), no viable cells were
detected after exposure to 1,3lb1. Dynamic light-
scattering studies revealed 1,3lb1-containing lipoplexes
(+ ⁄ ) 4 : 1) of the largest size with a mean diameter
around 1 lm.
Use of DOPE and cholesterol to enhance the gene-
delivery properties of cationic lipids has been exten-
sively documented [16–21]. For 1,3lb2 and 1,3lb3,
transfection activity was appreciably reduced at the
highest tested charge ratio by the incorporation of
DOPE, falling below levels reported for 1,3lb5; a simi-

lar result was observed when cholesterol was added
(Fig. 2B,C). The lipofection efficiency of 1,3lb5
increased significantly at + ⁄ ) 2 : 1, climbing above
that of commercially available DOTAP, and rose mod-
erately with the inclusion of cholesterol. The distearoyl
derivative continued to mediate low levels of transgene
expression at all charge ratios, even after the addition
of DOPE alone or in combination with cholesterol.
Increasing the + ⁄ ) charge ratio beyond 4 : 1 resulted
in decreased transfection activity for the most active
derivatives in all formulations (data not shown).
0
100
200
300
400
500
600
700
800
900
1000
1100
A
B
C
124
124
124
β-gal concentration (mU/well)

1,3lb2
1,3lb3
1,3lb4
1,3lb5
DOTAP
0
100
200
300
400
500
600
700
800
900
1000
1100
+/– charge ratio
+/– charge ratio
+/– charge ratio
β-gal concentration (mU/well)
1,3lb2/D
1,3lb3/D
1,3lb4/D
1,3lb5/D
DOTAP
0
100
200
300

400
500
600
700
800
900
1000
1100
β-gal concentration (mU/well)
1,3lb2/D/c
1,3lb3/D/c
1,3lb4/D/c
1,3lb5/D/c
DOTAP
Fig. 2. In vitro transfection activity of cationic lipids in the absence
of helper lipid(s) (A) and in the presence of DOPE (B) or DOPE and
cholesterol (C), as measured in a murine skin cell line (B16-F0 mela-
noma cells) by ONPG assay (n = 3).
Novel cytofectins for gene delivery M. Spelios and M. Savva
150 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny ltetra-
zolium bromide] reduction analysis revealed a low
cytotoxicity of lipoplexes at all charge ratios and
compositions, with cell viability greater than 60%,
except for formulations containing 1,3lb1, which were
poorly tolerated (data not shown).
A
B
C
D

E
F
Fig. 3. Fluorescence of EGFP in B16-F0 cells transfected with lipoplexes of (A) 1,3lb1, (B) 1,3lb2, (C) 1,3lb3, (D) 1,3lb4 and (E) 1,3lb5 at
± 4 : 1 in the absence of helper lipid(s), and with (F) DOTAP at ± 2 : 1. Images were acquired at 10 · magnification.
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 151
A
1,3lb2
1,3lb3
1,3lb4
1,3lb5
B
Novel cytofectins for gene delivery M. Spelios and M. Savva
152 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
Physicochemical characterization
pK
a
studies
Changes in the membrane surface charge of pH-stable
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ⁄ cho-
lesterol vesicles containing 5 mol% 1,3lb amphiphile
were monitored by measuring the fluorescence intensity
of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) in
the lipid bilayers as a function of pH. As expected, the
pH titration curves (not shown) of the derivatives
overlapped in accordance with their identical polar
headgroup. Curve fitting of the experimental data
revealed a pK
a
between 7.1 and 7.4, indicating that

only about 30–50% of the tertiary amine groups are
charged at physiological pH (Table 1). Higher values
were found for the isolated triamine (pK
a1
= 8.959,
pK
a2
= 9.592) [22], and may be attributed to reduced
hydration of the cationic lipids compared to the free
amine, as well as tight packing of the hydrophobic
chains, which promotes charge distribution over adja-
cent lipid molecules.
The pH titration curves were fitted to a modified
version of the Henderson–Hasselbach equation, which
contains an adjustable parameter C that affects the
slope of the transition region. In the original equation,
this parameter is equal to 1 for a univalent base (and
-1 for a monoprotic acid). The pK
a
values listed in
Table 1 for the bivalent lipids are the mean of two
acid dissociation constants, one for each of the ioniz-
able amino groups, resulting in pH titration curves
with slopes of lower steepness (C less than unity).
The TNS assay was used to ascertain the pK
a
values
of the cationic lipids in assemblies where they were
well separated from one another, precluding influences
of the hydrophobic anchors of the derivatives with

respect to the number of carbon atoms and double
bonds in the aliphatic chains. These pK
a
values deviate
from those obtained using vesicles where the deriva-
tives are in greater contact with each other, such as
the dispersions under investigation, and van der Waals
forces between adjacent cationic lipid molecules, as
dictated by their hydrophobic chain length and degree
of unsaturation, are a major contribution to the extent
of the bis-[2-dimethylamino-ethyl]-amine polar head-
group hydration, and, subsequently, protonation.
Thus, the acid dissociation constants in Table 1 are
molecular descriptors of the derivatives and do not
necessarily offer insight into the differences in transfec-
tion activities.
Table 1. Acid dissociation constants of cationic lipids as deter-
mined by nonlinear fitting of TNS fluorescence intensity–pH plots.
Lipid pK
a
C
a
Coefficient of
determination
b
% ionization
at pH 7.2
1,3lb1 7.24 0.40 0.991 41
1,3lb2 7.09 0.52 0.994 33
1,3lb3 7.37 0.40 0.984 48

1,3lb4 7.36 0.58 0.996 48
1,3lb5 7.42 0.53 0.995 51
a
C is an adjustable parameter affecting the slope of the transition
region of the fitted pH titration curves, as calculated from Eqn (1).
b
Goodness-of-fit statistics for pK
a
were assessed within a 95%
confidence interval.
40 mM Tris, pH7.2
0
20
40
60
80
100
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4
+/– charge ratio
EtBr fluorescence (% of max.)
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
SFM
0
20
40
60

80
100
0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 3.6 4
+/– charge ratio
EtBr fluorescence (% of max.)
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
Fig. 5. Percentage ethidium bromide displacement against the
charge ratio of lipoplexes in Tris buffer or SFM. Parabolic curve fits
of the experimental data (solid points) are shown as dashed lines.
Fig. 4. (A) Fluorescence images of B16-F0 cells treated with lipoplexes of FITC–pDNA. (B) FITC (top), rhodamine (center) and overlaid fluo-
rescence and brightfield (bottom) images of cells transfected using Rh-PE-labeled (1 mol%) dispersions of 1,3lb2. Lipoplexes were prepared
at a charge ratio of ± 4 : 1, and images were captured 4 h after transfection at 20 and 10 · magnification for (A) and (B), respectively.
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 153
Cationic lipid–pDNA binding studies
Binding curves of EtBr-intercalated pDNA titrated
with aliquots of cationic lipid dispersions are shown in
Fig. 5. With respect to the saturated derivatives, there
was a reduction in plasmid compaction efficiency with
increasing hydrophobic chain length. Interestingly, the
transfection-inactive lipid 1,3lb1 was the most efficient
at condensing pDNA, with complete charge neutraliza-
tion of the negative charges of pDNA at about
+ ⁄ ) 2.3 in low-ionic-strength medium. This finding is
contradictory to previous work suggesting inefficient
DNA condensation and dehydration by 12-carbon

fatty acid chains [23]. However, 1,3lb1 was also the
most toxic to the cells, accounting for its transfection
inactivity. The monounsaturated derivative 1,3lb5 dis-
played intermediate nucleic acid condensation efficacy,
with full EtBr exclusion at approximately + ⁄ ) 3.1.
Increasing the ionic strength resulted in complete
probe displacement at higher charge ratios for all lip-
ids, but there was no effect on the binding trends.
Langmuir monolayer studies
The surface properties of the cationic lipids were inves-
tigated using the Langmuir film balance technique.
Monolayers of the cationic lipids, with the exception
of the distearoyl derivative, exist in an all liquid-
expanded state at 23 °C. Monolayer collapse occurred
at lower mean molecular areas and higher surface pres-
sures as the acyl chain length increased from 1,3lb1 to
1,3lb14 (Table 2). Tighter lipid packing associated with
the additional van der Waals forces of longer hydro-
phobic chains more effectively excludes interstitial
water and reduces surface tension, allowing a greater
reduction in the available surface area of the mono-
layer prior to its collapse. The dimyristoyl and dioleoyl
derivatives 1,3lb2 and 1,3lb5, respectively, shared simi-
lar collapse parameters and comparable transfection
activities (Fig. 2A). The mixed-phase state of monolay-
ers composed of the poorly transfection-efficient deriv-
ative 1,3lb4 is evidenced by an L
1
-to-L
2

transition in
the compression isotherm. Specifically, liquid-expanded
behavior was observed up to a surface pressure and
mean molecular area of 24 mNÆm
)1
and 70 A
˚
2
, respec-
tively, at which point a transition occurred to a chain-
ordered phase. The pressure continued to rise upon
further surface area reduction until the monolayer
finally collapsed at 49 mNÆm
)1
and 39 A
˚
2
.At37°C,
the two-dimensional transition was absent and only a
liquid-expanded state was evident. For all derivatives,
molecular dimensions increased and compression
forces decreased at monolayer collapse in response to
elevated temperature. Differences between the mono-
layer collapse parameters of the cationic lipids were
not as apparent at 37 °C; the derivatives possessed
similar mean molecular areas at monolayer collapse,
and their collapse pressures were nearly identical.
Molecular elasticity is correlated with transfection
activity, and was assessed by first-derivative analysis of
the p–A isotherm; the smaller the slope at monolayer

collapse (dp ⁄ dA
c
), the higher the compressibility. The
value of dp ⁄ dA
c
was highest for 1,3lb4 at both 23 °C
and 37 °C, indicative of the lowest in-plane elasticity,
and lipoplexes containing the distearoyl analog con-
comitantly generated minimum reporter gene expres-
sion (Fig. 2A). The monounsaturated 1,3lb5 was found
to be the most elastic at 23 °C, with a dp ⁄ dA
c
value of
1mNÆm
)1
ÆA
˚
)2
.
Phase-transition temperature studies
DPH (1,6-diphenyl-1,3,5-hexatriene) was used to probe
for cationic lipid bilayer phase changes by monitoring
the depolarization of fluorescence of the extrinsic
fluorophore in response to temperature. The shortest
saturated chain derivative 1,3lb1 and the monounsatu-
rated analog 1,3lb5 displayed the most fluid behavior,
Table 2. Monolayer transition
a
and collapse parameters of the 1,3lb series. Measurements were performed using Tris buffer (40 mM,
pH 7.2) as the subphase. Values reported are the mean of n experiments ± standard deviation.

Mean molecular area (A
˚
2
) p (mNÆm
)1
)dp ⁄ dA
c
Phase state
b
23 °C37°C23°C37°C23°C37°C23°C37°C
1,3lb1 (n = 4,6) 59.27 ± 4.89 64.90 ± 3.22 37.75 ± 2.01 36.59 ± 1.34 1.27 ± 0.07 1.13 ± 0.08 L
1
L
1
1,3lb2 (n = 4,3) 55.98 ± 2.55 60.61 ± 2.29 41.62 ± 0.94 36.31 ± 1.37 1.55 ± 0.10 1.04 ± 0.04 L
1
L
1
1,3lb3 (n = 3,4) 46.91 ± 1.39 55.61 ± 3.41 40.29 ± 0.72 38.62 ± 0.75 1.32 ± 0.04 1.11 ± 0.03 L
1
L
1
1,3lb4 (n = 11,6) 38.70 ± 3.03
70.49 ± 3.43
a
56.06 ± 3.81 48.68 ± 6.71
23.73 ± 1.09
a
39.89 ± 0.80 2.29 ± 0.51 1.20 ± 0.11 L
2

L
1
L
1
1,3lb5 (n = 7,7) 54.08 ± 3.30 57.92 ± 6.41 39.04 ± 3.81 36.82 ± 2.54 1.00 ± 0.17 1.20 ± 0.14 L
1
L
1
a
Phase transition was determined by a discontinuity in the plot of dp ⁄ dA against mean molecular area (not shown).
b
L
1
and L
2
indicate the
liquid-expanded and liquid-condensed states, respectively.
Novel cytofectins for gene delivery M. Spelios and M. Savva
154 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
existing in a liquid crystalline state within the range
of temperatures scanned (Fig. 6A). A gel-to-liquid
crystalline phase transition was detected for all other
derivatives (temperature span 6–7 °C), and increased
in tandem with acyl chain length (Table 3). Only
1,3lb4 exhibited a three-dimensional phase transition
above physiological temperature, signifying an ordered
phase during transfection, with tight lipid packing, and
induced low reporter gene expression.
The behavior of the cationic lipids in two-dimen-
sional monolayers and three-dimensional bilayers

was compared. As shown in Table 3, a gel-to-liquid
crystalline transition temperature below 23 °C was
found for 1,3lb1, 1,3lb2 and 1,3lb5, coinciding with
the fluid state of these lipids as indicated by the p–A
isotherm at 23 °C and 37 °C. The three-dimensional
phase transition exhibited by 1,3lb4 at 45 °C complies
with monolayer compression data indicating the pres-
ence of a chain-ordered phase at 23 °C. Taking into
consideration the onset phase-transition temperature
determined from the first-derivative profile of the r–T
plot (Fig. 6B) instead of the transition midpoint, the
all liquid-expanded states at 23 °C and 37 °C of the
dipalmitoyl and distearoyl derivatives, respectively, in
monolayers complement the nature of these lipids in
bilayer assemblies at these temperatures.
Particle size and electrophoretic mobility studies
In vitro transfection activity was found to be a func-
tion of the size of the cationic lipid–pDNA complex.
At low ionic strength, lipoplexes of 1,3lb3 had the
largest particle size at + ⁄ ) 4 : 1, with a mean diame-
ter of approximately 740 nm (Fig. 7A). Cationic lipid-
mediated transfection with the dipalmitoyl derivative
yielded the highest levels of reporter gene expression at
this charge ratio without helper lipid(s), and could be
attributed, among other factors, to enhanced sedimen-
tation of larger particles onto cells [24,25]. Complexes
of plasmid and either 1,3lb2 or 1,3lb5 shared a similar
particle size ($ 0.5 lm), as well as transfection activ-
ity at the highest + ⁄ ) charge ratio tested. The
poorly transfection-competent lipids 1,3lb1 and 1,3lb4,

when complexed to pDNA at + ⁄ ) 4 : 1, generated a
particle size of the smallest mean diameter. In serum-
free medium, liposome and lipoplex particle sizes for
all lipids generally increased, and the overall particle
size trends between the derivatives were largely main-
tained, the most notable exception being lipoplexes of
1,3lb1 at + ⁄ ) 4 : 1 which exhibited a mean diameter
comparable with that of 1,3lb3-containing lipoplexes
(Fig. 7B).
The electrophoretic mobility of all samples was mea-
sured and converted to zeta potential. In low-ionic-
strength medium (Fig. 7C), the zeta potential remained
negative from + ⁄ ) 1 : 1 to 2 : 1, which hindered effi-
cient lipofection at these charge ratios (Fig. 2A).
Increasing the + ⁄ ) charge ratio to 4 : 1 resulted in
complete neutralization of the negative charges on
pDNA by cationic lipid dispersion of all derivatives
except 1,3lb4. Lipoplexes of the distearoyl analog con-
tinued to display a highly negative zeta potential, an
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
A
B

0 102030405060
0 1020304050
60
T (°C)
Anisotropy
1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5
T (°C)
dr /dT
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
Fig. 6. (A) Fluorescence anisotropy of DPH in cationic lipid bilayers
as a function of temperature, and (B) first-derivative data of r–T
plots. Dispersions were prepared with 40 m
M Tris, pH 7.2.
Table 3. Midpoint and onset phase-transition temperatures
obtained from curve fits and first-derivative analysis, respectively,
of the experimental data in Fig. 6.
Lipid
T
m
(°C)
Coefficient of
determination
a
T
onset
(°C)

T
offset
(°C) T
offset ) onset
1,3lb1 < 6 ND – – –
1,3lb2 16.8 0.998 13 20 7
1,3lb3 31 0.994 28 34 6
1,3lb4 45.1 0.988 42 48 6
1,3lb5 < 6 ND – – –
a
Best-fit parameters were assessed within a 95% confidence inter-
val.
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 155
indication that efficient complexation with and conden-
sation of pDNA did not occur, as verified by ethidium
bromide displacement (Fig. 5). Interestingly, 1,3lb1-
containing lipoplexes showed a positive zeta potential
of about 17 mV at + ⁄ ) 4 : 1, higher than and similar
to that for lipoplexes of transfection-active 1,3lb3 and
1,3lb5, respectively. At high ionic strength, only
negative values of zeta potential were observed for
lipoplexes at all charge ratios (Fig. 7D), suggesting a
minimal influence of electrostatic interactions on cell
internalization of 1,3lb–pDNA complexes in vitro.
Lipid-mixing studies
The membrane fusion activity of the cationic lipids via
electrostatic and hydrophobic interactions was mea-
sured by fluorescence resonance energy transfer using
the NBD-Rh FRET pair [26]. Lipid mixing occurs in

various cellular processes, including lipoplex internali-
zation and fusion of the internalized cationic lipid–
DNA complex with the endosome membrane. To sim-
ulate these processes in vitro, membrane fusion studies
were conducted at physiological temperature and ionic
strength, at pH 7.4 and 4.0, using endosome-mimick-
ing 1,2-dipalmitoyl-sn-glycero-3-phosphate : l-a-phos-
phatidylcholine (PC : PA) vesicles [27]. The fluorescent
lipid probes 1,2-dioleoyl-sn-glycero-3-phosphoethanol-
amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE)
and Rh-PE were incorporated into the lipid bilayer of
the anionic vesicle membranes, and the reduction in
fluorescence resonance energy transfer from nitro-
0
200
400
600
800
A
B
C
D
1 000
1
200
1
400
1
000
1

200
1
400
+/– 1:1 +/– 2:1 +/– 4:1 liposomes +/– 1:1 +/– 2:1 +/– 4:1 liposomes
+/– 1:1 +/– 2:1 +/– 4:1 li
p
osomes +/– 1:1 +/– 2:1 +/– 4:1 li
p
osomes
D (nm)D (nm)
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
0
200
400
600
800
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
–80
–60
–40
–20
–80

–60
–40
–20
0
20
40
60
80
Zeta potential (mV)Zeta potential (mV)
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
0
20
40
60
80
1,3lb1
1,3lb2
1,3lb3
1,3lb4
1,3lb5
Fig. 7. Particle size distribution (A,B) and zeta potential (C,D), as determined by dynamic light-scattering and electrophoretic mobility studies,
respectively, of cationic lipid dispersions and lipoplexes in 40 m
M Tris, pH 7.2 (A,C) and SFM (B,D).
0
10
20

30
40
50
60
1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5
% lipid mixing
pH 4.0
pH 7.4
Fig. 8. Lipid mixing, as assessed by FRET, of unlabeled 1,3lb dis-
persions with endosome-mimicking PC : PA (73 : 25) vesicles
labeled with the fluorescent lipid probes NBD-PE and Rh-PE
(1 mol% each), after 35 min. Studies were conducted at 37 °C and
154 m
M ionic strength at physiological and acidic pH.
Novel cytofectins for gene delivery M. Spelios and M. Savva
156 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
benzoxadiazole to rhodamine was monitored upon
probe dilution and increased fluorophore separation
achieved by fusion with unlabeled cationic liposomes.
Figure 8 shows the lipid mixing efficiency of 1,3lb dis-
persions. The percentage lipid mixing was nearly dou-
bled at pH 4.0 compared with pH 7.4 for all cationic
lipids except the least active 1,3lb4, which maintained
a constant and low membrane fusion efficacy irrespec-
tive of the pH. The trends were identical to those of
the binding studies (Fig. 5): 1,3lb1 > 1,3lb2 >
1,3lb5 > 1,3lb3 > 1,3lb4. The dimyristoyl derivative
exhibited the highest biomembrane fusogenicity of the
active analogs, and lipoplexes of this cationic lipid
were internalized to the greatest extent (Fig. 4). For

1,3lb2, the percentage lipid mixing was approximately
four times greater than for 1,3lb3, despite displaying
an approximately 1.4-fold lower transfection activity
than the most biologically active compound (Fig. 2A).
However, cell viability was compromised to a greater
degree with formulations of 1,3lb2 (63% survival com-
pared with 84% for 1,3lb3; data not shown). The same
holds true for 1,3lb5 in comparison with 1,3lb3.
Discussion
The current project is part of a greater endeavor to
understand the structural effects of double-chained
amphiphilic molecules and their aggregates, in the pres-
ence and absence of pDNA, on cationic lipid-mediated
gene delivery. In particular, the + ⁄ ) charge ratio, neu-
tral helper lipids, ionic strength, acyl chain length and
degree of unsaturation, the number of ionizable amines
in the polar headgroup, and the spatial arrangement of
the hydrophobic and hydrophilic regions within the lipid
molecule have been examined by our laboratory and
correlated with transfection efficiency in an exploration
of structure–function relationships that has spanned
several papers [12,28–31]. Generation of such relation-
ships is essential to the development of lipofection
reagents that are highly potent and minimally toxic.
Ewert et al. [32] identified the membrane charge den-
sity (r
M
) of cationic lipid vectors that form lamellar
complexes with DNA as a key universal parameter
governing their transfection efficiency. Our previous

work with monovalent cationic lipids also shows this
dependence on the average charge per unit area of the
membrane. Excluding helper lipids, only the dioleoyl
derivatives from a series of primary and tertiary
1,3-dialkoylamido monovalent cationic lipids [31], dif-
fering in molecular structure from the 1,3lb series by
only a single amine in the polar headgroup, elicited
transfection activity. Addition of a second amine
group and the subsequent increase in r
M
increased the
number of transfection-efficient derivatives to include
the dimyristoyl and dipalmitoyl analogs.
Increased fluidity, or a low gel-to-liquid crystalline
phase-transition temperature, of these lamellar assem-
blies under physiological conditions is another charac-
teristic of the lipid vesicles in isolation that has been
identified as critical for transfection activity [33–35].
An investigation was recently completed regarding the
transfection activity and physicochemical properties of
a 1,2-diamino-3-propanol series containing an attach-
ment of the same bivalent polar headgroup at the
3-position but with linkages of the acyl chains at the
1- and 2- positions of the 1,2-diamino-3-propanol
backbone [30]. The 1,3-dialkyl cationic amphiphiles
reported herein feature hydrophobic chains of greater
interchain distance than their 1,2-dialkyl counterparts,
and the impact this has biologically and physicochemi-
cally on these vectors is remarkable. Whereas only the
dioleoyl derivative of the 1,2lb series generated trans-

fection activity and efficiently bound and compacted
pDNA in the absence of helper lipid(s), increasing the
intramolecular space between the acyl chains activated
the dimyristoyl and dipalmitoyl derivatives. This struc-
tural modification also afforded these lipids higher
two-plane elasticity and increased fluidity relative to
their corresponding 1,2lb analogs, as indicated by the
lower compressibility moduli and reduced gel-to-liquid
crystalline phase-transition temperatures of the 1,3lb
derivatives.
Remarkably, many of the physicochemical proper-
ties of the dilauroyl derivative, which was found to be
a completely inefficient delivery system, are character-
istic of an ideal cytofectin. Dispersions of this lipid dis-
placed EtBr from pDNA and condensed plasmid to
the greatest extent, and were fusogenically superior in
lipid-mixing studies with endosome-mimicking vesicles.
1,3lb1 liposomes displayed the most fluid behavior out
of all the saturated derivatives, as indicated by the
absence of a gel-to-liquid crystalline phase transition.
In addition, lipoplexes containing 1,3lb1, due to better
hydration, possessed the highest zeta potential at the
+ ⁄ ) charge ratio with the highest reporter gene
expression. However, 1,3lb1 solubilizes cell membranes
and lyses cells in much the same way as strong micelle-
forming surfactants such as Triton X-100, and such
high cytotoxicity rendered the dilauroyl derivative
totally inactive.
Conclusion
Five cationic lipid derivatives, differing in the length

and degree of unsaturation of their hydrophobic chains,
were analyzed with reference to their gene-delivery
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 157
capabilities. Lipofection mediated by the dimyristoyl,
dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and
1,3lb5, respectively, resulted in the highest b-galactosi-
dase quantities at + ⁄ ) 4 : 1 in the absence of helper
lipids. Transfection activities were reduced in the pres-
ence of either DOPE alone or in combination with cho-
lesterol for all derivatives except 1,3lb5, which
maintained its reporter gene expression levels at
+ ⁄ ) 4 : 1 and yielded increased enzyme activity at a
lower charge ratio of + ⁄ ) 2 : 1. EtBr displacement
indicated efficient pDNA compaction by the transfec-
tion-competent analogs, irrespective of the ion concen-
tration in the dispersion medium (Tris buffer or
serum-free medium). Dynamic light-scattering and elec-
trophoretic mobility studies revealed lipoplexes of
active lipids with mean diameters of several hundred
nanometers and positive zeta potentials at low ionic
strength, or negative values in high-ionic-strength med-
ium. Langmuir film balance studies showed high hydra-
tion and in-plane elasticity of the active derivatives in
isolation. In agreement with the monolayer experi-
ments, fluorescence polarization studies verified the
fluid nature of the highly transfection-efficient lipids
with gel-to-liquid crystalline phase transitions below
physiological temperature. FRET experiments revealed
a greater degree, compared with the poorly active deri-

vative 1,3lb4, of lipid mixing of the active compounds
with anionic vesicles.
Lipids of the 1,3lb series were synthesized with the
intent of enhancing the transport of exogenous genetic
material into cells in vitro, and their design eliminates
the need for additional components in the gene-deliv-
ery system (i.e. DOPE and cholesterol), providing a
simpler yet more potent formulation. The evolution of
synthetic cationic lipid carriers in our laboratory and
the structure–activity data collected for the various ser-
ies contribute to the long-term goals of understanding
the mechanism of lipofection, and the rational design
of pharmaceutically sound and therapeutically superior
nonviral vectors for gene therapy in vivo.
Experimental procedures
Materials
Reagents and solvents were purchased from commercial
suppliers and were used without further purification.
Synthesis
N,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl-[bis-(2-dimethyl-
aminoethane)] derivatives were synthesized and identified to
purity > 99%, as described below.
Bis-(2-dimethylamino-ethyl)-amine
The synthesis was carried out as previously described [30].
1,3-Dimyristoylamidopropan-2-ol and 1,3-dimyristoyl-
amidopropane-2-(p-nitrophenyl) carbonate
The compounds were synthesized by a procedure similar to
those previously described [31].
1,3-Dimyristoylamidopropane-2-[bis-(2-dimethylamino-
ethane)] carbamate (1,3lb2)

To a solution of N,N¢-ditetradecanoyl-1,3-diaminopropane-
2-(p-nitrophenyl)-carbonate (0.0059 mol; 4.32 g) in 30 mL
anhydrous CH
2
Cl
2
were added bis-(2-dimethylaminoethyl)-
amine (0.0118 mol) and triethylamine (0.00118 mol). The
reaction was stirred at room temperature for 3.5 h, after
which time the solvent was evaporated under vacuum and
the reaction mixture transferred to a separation funnel with
100 mL of water and 100 mL of ethyl acetate. The aqueous
layer was discarded, and the organic phase was washed
three times with 100 mL saturated potassium bicarbonate
solution to remove the p-NO
2
-phenol. The oily residue that
resulted after concentrating the organic layers was dissolved
in a minimum amount of chloroform and loaded onto a sil-
ica gel column (26 · 2.8 cm). The column was eluted
sequentially with 100 mL chloroform, and 1, 3, 5, 7, 8, 9,
10, 12 (300 mL) and 15% methanol ⁄ chloroform. The 10%
and 12% fractions were combined and concentrated to give
2.79 g (68%) of N,N¢-ditetradecanoyl-1,3-diaminopropyl-2-
carbamoyl-[bis-(2-dimethylaminoethane)] as a waxy mate-
rial. The calculated composition for C
40
H
81
N

5
O
4
(relative
molecular mass 695) was C, 69.06; H, 11.65; N, 10.07. That
found was C, 68.54; H, 11.92; N, 9.96. MS (FAB) m ⁄ z
696.4 [M+H]
+
;
1
H NMR (400 MHz, CDCl
3,
20 °C, TMS)
d 6.86–6.83 (t, 2H, HNCO), 4.72–4.70 (m, 1H, CH), 3.45–
3.30 [m, 8H, (CH
2
)
2
NC(O)O, CH
2
NHC(O)], 2.41–2.35 [m,
4H, (CH
2
)
2
N], 2.21–2.20 [d, coherent peak, 12H, N(CH
3
)
2
],

2.19–2.11 (t, 4H, CH
2
CO), 1.58–1.53 (m, 4H, CH
2
CH
2
CO),
1.23–1.20 [coherent peak, 40H, 10(CH
2
)
2
], 0.84–0.81 (t, 6H,
CH
3
);
13
C NMR (100 MHz, CDCl
3,
20 °C, TMS) d 174.82
(NHCO), 156.37 [NC(O)O], 73.47 (CH), 59.28, 58.43,
46.98, 46.79, 40.66, 37.92, 33.10, 30.87, 30.84, 30.72, 30.59,
30.57, 30.55, 27.02, 23.89, 15.36.
1,3-Dilauroylamidopropane-2-[bis-(2-dimethylamino-
ethane)] carbamate (1,3lb1)
The calculated composition for C
36
H
73
N
5

O
4
(relative
molecular mass 639) was C, 67.61; H, 11.42; N, 10.95. That
found was C, 67.62; H, 11.57; N, 10.98. MS (EI) m ⁄ z
641.1. [M+H]
+
;
1
H NMR (400 MHz, CDCl
3,
20 °C,
TMS) d 6.79–6.70 (t, 2H, HNCO), 4.73–4.70 (m, 1H, CH),
3.58–3.29 [m, 8H, (CH
2
)
2
NC(O)O, CH
2
NHC(O)], 2.41–2.35
Novel cytofectins for gene delivery M. Spelios and M. Savva
158 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
[m, 4H, (CH
2
)
2
N], 2.22–2.21 [d, coherent peak, 12H,
N(CH
3
)

2
], 2.19–2.12 (t, 4H, CH
2
CO), 1.62–1.56 (m, 4H,
CH
2
CH
2
CO), 1.24–1.21 [coherent peak, 32H, 10(CH
2
)
2
],
0.84–0.81 (t, 6H, CH
3
).
1,3-Dihexanoylamidopropane-2-[bis-(2-dimethylamino-
ethane)] carbamate (1,3lb3)
The calculated composition for C
44
H
89
N
5
O
4
(relative
molecular mass 751) was C, 70.31; H, 11.85; N, 9.32. That
found was C, 68.97; H, 11.60; N, 9.74. MS (FAB) m ⁄ z
752.7 [M+H]

+
;
1
H NMR (400 MHz, CDCl
3
,20°C, TMS)
d 6.80 (bs, 2H, HNCO), 4.73 (m, 1H, CH), 3.45–3.31 [m,
8H, (CH
2
)
2
NC(O)O, CH
2
NHC(O)], 2.41–2.38 [m, 4H,
(CH
2
)
2
N], 2.23–2.21 [d, coherent peak, 12H, N(CH
3
)
2
],
2.17–2.13 (t, 4H, CH
2
CO), 1.60–1.57 (m, 4H, CH
2
CH
2
CO),

1.25–1.22 [coherent peak, 48H, 12(CH
2
)
2
], 0.86–0.83 (t, 6H,
CH
3
);
13
C NMR (100 MHz, CDCl
3,
20 °C, TMS) d 174.82
(NHCO), 156.41 [NC(O)O], 73.51 (CH), 59.41, 58.50,
47.11, 46.87, 40.70, 38.00, 33.14, 30.93, 30.89, 30.75, 30.62,
30.59, 27.05, 23.93, 15.39.
1,3-Distearoylamidopropane-2-[bis-(2-dimethylamino-
ethane)] carbamate (1,3lb4)
The calculated composition for C
48
H
97
N
5
O
4
(relative
molecular mass 807) was C, 71.37; H, 12.02; N, 8.67. That
found was C, 70.62; H, 12.18; N, 8.35. MS (FAB) m ⁄ z
808.6 [M+H]
+

,780.6 [M
+
-(CH
3
)
2
];
1
H NMR (400 MHz,
CDCl
3,
20 °C, TMS) d 6.90–6.88 (bs, 2H, HNCO), 4.72–
4.70 (m, 1H, CH), 3.47–3.29 [m, 8H, (CH
2
)
2
NC(O)O,
CH
2
NHC(O)], 2.40–2.36 [m, 4H, (CH
2
)
2
N], 2.21–2.20 [d,
coherent peak, 12H, N(CH
3
)
2
], 2.15–2.11 (t, 4H, CH
2

CO),
1.58–1.55 (m, 4H,
CH
2
CH
2
CO), 1.20 [coherent peak, 56H,
14(CH
2
)
2
], 0.84–0.81 (t, 6H, CH
3
);
13
C NMR (100 MHz,
CDCl
3,
20 °C, TMS) d 174.83 (NHCO), 156.40 [NC(O)O],
73.48 (CH), 59.38 [N(CH
3
)
2
], 58.49 [N(CH
3
)
2
], 47.07
[(CH
2

)
2
N], 46.87 [(CH
2
)
2
N], 40.67, 37.96, 33.13, 30.91,
30.87, 30.74, 30.62, 30.59, 27.03, 23.92, 15.33.
1,3-Dioleoylamidopropane-2-[bis-(2-dimethylamino-
ethane)] carbamate (1,3lb5)
The calculated composition for C
48
H
93
N
5
O
4
(relative molec-
ular mass 803) was C, 71.73; H, 11.58; N, 8.72. That found
was C, 70.45; H, 11.53; N, 8.38. MS (FAB) m ⁄ z 804.8
[M+H]
+
;
1
H NMR (400 MHz, CDCl
3,
20 °C, TMS) d 6.83
(bs, 2H, HNCO), 5.36–5.31 (m, 4H, CH=CH), 4.75–4.73
(m, 1H, CH), 3.56–3.52 [m, 4H, (CH

2
)
2
NC(O)], 3.37–3.29 [m,
4H,
CH
2
NHC(O)], 2.44–2.41 [m, 4H, (CH
2
)
2
N], 2.26–2.24
[d, coherent peak, 12H, N(CH
3
)
2
], 2.19–2.15 (t, 4H, CH
2
CO),
2.00–1.95 (m, 8H,
CH
2
CH=CHCH
2
), 1.61 (m, 4H,
CH
2
CH
2
CO), 1.29–1.25 [coherent peak, 40H, 10(CH

2
)
2
],
0.88–0.84 (t, 6H, CH
3
);
13
C NMR (100 MHz, CDCl
3
,20°C,
TMS) d 174.81 (NHCO), 156.38 [NC(O)O], 131.40–130.52
(C=C), 73.44 (CH), 65.34, 59.28, 58.46, 46.96, 46.77, 40.58,
37.86, 33.86, 33.77, 33.70, 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, 27.65, 23.87, 15.34.
Plasmids
pUC19-b-gal and pEGFP-N1 were propagated in DH5a-
competent cells and collected according to standard proto-
cols [36]. Plasmid DNA was purified by gel permeation
chromatography using a Sepharose 4B-packed column
equilibrated with 2.5 m ammonium acetate. Agarose gel
electrophoresis and the spectrophotometrically determined
A
260
⁄ A
280
ratio verified that the pDNA was of high quality
and purity.
Lipid dispersions and lipoplexes

Cationic lipids, DOPE and cholesterol were dissolved sepa-
rately in chloroform, and the appropriate volume of each
solution was added to 12 · 75 mm borosilicate glass dispos-
able culture tubes. The concentration of cationic lipid and
the molar ratio of cationic lipid : DOPE : cholesterol in the
final lipid formulations were maintained at 0.3 mm and
3 : 2 : 1, respectively. Complete evaporation of organic sol-
vent, first under a stream of nitrogen gas and then by high
vacuum desiccation, was followed by hydration of the dry
lipid films in Tris buffer (40 mm, pH 7.2) at elevated tem-
perature with periodic vortexing. Lipoplexes were prepared
at + ⁄ ) charge ratios of 1 : 1, 2 : 1 and 4 : 1 by pipetting a
constant volume of pDNA solution into an appropriate
amount of diluted liposome preparation.
In vitro transfection studies
Aliquots (250 lL) of lipoplexes in serum-free medium con-
taining 1 lg pUC19-b-gal plasmid DNA were added to
approximately 50 000 B16-F0 cells seeded into each well of
a 48-well tissue culture plate at least 12 h before transfec-
tion, and maintained in serum medium (Dulbecco’s modi-
fied Eagle’s medium supplemented with 10% fetal bovine
serum, 50 unitsÆmL
)1
penicillin and 50 lgÆmL
)1
streptomy-
cin). After incubation for 4 h at 37 °C in a 5% CO
2
in air
atmosphere, the lipoplexes were removed and replaced with

0.5 mL fresh serum medium. The cells were lysed after an
additional 44 h, and lipofection activity in terms of reporter
enzyme levels was quantified by a microplate colorimetric
assay utilizing the b-galactosidase substrate ONPG. Lipo-
plex cytotoxicity was assessed by MTT assay.
A similar procedure was followed for cells transfected
with pEGFP-N1 pDNA. Fluorescence images of intact
cells were acquired 48 h after lipofection using a Zeiss
Axiovert 200M inverted microscope (Carl Zeiss, Go
¨
ttingen,
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 159
Germany). For some experiments, a fluorescein label was
covalently attached to the pUC19 plasmid, with a labeling
efficiency of approximately one marker molecule every
20–60 nucleic acid base pairs (Mirus Label IT
TM
, Mirus
Bio Corporation, Madison, WI, USA), and lipoplexes were
formed with either unlabeled lipid dispersions or cationic
vesicles labeled with 1 mol% Rh-PE. Images were captured
after exchange of the lipoplexes for fresh serum medium.
pK
a
studies
Studies were performed at excitation and emission wave-
lengths of 321 and 445 nm, respectively. Excitation and emis-
sion slit widths were both 5 nm. Dry DOPC ⁄ cholesterol ⁄
cationic lipid films (0.95 ⁄ 0.95 ⁄ 0.1 molar ratio) were reconsti-

tuted with Tris buffer (40 mm, pH 7.2) to a final combined
lipid concentration of 2 mm. The aqueous dispersions were
dispensed as 100 lL aliquots into 4.5 mL plastic cuvettes
with four optical windows, and diluted to 2 mL with buffered
solutions (40 mm Tris, 40 mm Mes) of varying pH containing
TNS to give a lipid ⁄ probe molar ratio of 100 : 1. Samples
were stirred for 30 min before measurement of TNS fluores-
cence intensity. Nonlinear fitting of the pH titration curves
was performed using psi-plot (version 7.01, Poly Software
International, Pearl River, NY, USA) according to a modi-
fied version of the Henderson–Hasselbach equation
F ¼ A þ
B
1 þ 10
CðpH À DÞ
ð1Þ
where A is the minimum fluorescence, B is the difference
between the maximum and minimum emission intensities, C
is a parameter affecting the slope of the transition region,
and D is equal to the acid dissociation constant (pK
a
)of
the cationic lipid [12]. Goodness-of-fit statistics were
assessed within a 95% confidence interval.
Cationic lipid–pDNA binding studies
Plasmid DNA (22.5 lg) and EtBr (0.8 lg) were combined in
a quartz cuvette and diluted to 22.7 and 0.68 lm, respec-
tively, using Tris buffer or serum-free medium (SFM). Cat-
ionic lipids (1 mm) were added in 6.8 lL aliquots with
continuous stirring. The enhanced fluorescence intensity of

intercalated dye was measured at charge ratio increments of
+ ⁄ ) 0.2 : 1 at an excitation wavelength of 515 nm (slit
width 5 nm) and an emission wavelength between 595 and
605 nm (slit width 2.5 nm). The emission readings were trea-
ted as described previously [30] to generate plots of percent-
age ethidium bromide displaced from cationic lipid-bound
pDNA against the lipoplex charge ratio. The data was not
corrected for light-scattering effects, which caused a
change in the fluorescence signal of less than 2%, as
determined from samples lacking EtBr. The binding
profiles were modeled as parabolic functions of the form
y = ax
2
± bx.
Langmuir monolayer studies
Monomolecular cationic lipid films at the air–water inter-
face were studied using a computer-controlled KSV Minit-
rough (KSV Instruments Ltd., Helsinki, Finland) equipped
with a Wilhelmy plate electrobalance (KSV Instruments
Ltd.) to measure surface pressure and two symmetrically
moving hydrophilic Delrin barriers (Dupont, Wilmington,
DE, USA) to reduce the available surface area. Using a
gas-tight microliter syringe, 20 lL of cationic lipid solution
(0.75 mm in chloroform) were applied to the surface of
140 mL of the subphase (40 mm Tris, pH 7.2) within a
thermoregulated Teflon trough (364 · 75 mm effective film
area). After a time lag of 20 min to ensure complete evapo-
ration of organic solvent, the monolayer was compressed at
a constant rate of 10 mmÆmin
)1

. Plots of surface pressure
(p) against mean molecular area (A) were automatically
generated. Molecular compressibility was assessed from
first-derivative analysis of the p–A isotherm.
Phase-transition temperature studies
Cationic lipid dispersions (0.5 mm) were prepared with
1 mol% DPH, ensuring minimal membrane perturbation
by the fluorophore. Studies were conducted at an excitation
wavelength of 351 nm (slit width 5 nm) using a Cary
Eclipse spectrofluorometer (Varian Instruments, Walnut
Creek, CA, USA) equipped with motorized polarizers and
a temperature-controlled four-window cuvette holder.
Anisotropy (r) values of constantly stirred samples at vari-
ous temperatures were calculated using the Cary Eclipse
advanced reads application, software version 1.1 (Varian
Instruments), from fluorescence intensities of emitted light
at 430 nm (slit width 5 nm) polarized parallel and perpen-
dicular to the illuminating beam. Nonlinear fitting of the
r–T profiles was performed as described previously [30].
Particle size and electrophoretic mobility studies
Measurements were carried out at room temperature using
a Malvern Zetasizer Nano ZS system (Malvern Instru-
ments, Southboro, MA, USA) validated using polystyrene
microspheres (Duke Scientific Corporation, Palo Alto, CA,
USA) of 60 and 220 nm certified mean diameter. Lipoplex-
es were prepared with either Tris buffer (40 mm, pH 7.2),
filtered using a 0.2 lm filter, or with sterile SFM. Lipo-
somes were prepared with the same filtered buffer or SFM
by diluting cationic lipid dispersions (0.3 mm)to24nm.
Mean diameters of samples in disposable polymethylmeth-

acrylate cuvettes were obtained, using a 633 nm laser, from
Gaussian analysis of the intensity-weighted particle size
distributions using the instrument software (dispersion
technology software 3.00, Malvern Instruments Ltd.).
Electrophoretic mobility was measured in a folded capillary
Novel cytofectins for gene delivery M. Spelios and M. Savva
160 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS
cell (Malvern Instruments) using the laser Doppler veloci-
metry technique and converted by the software to zeta
potential.
Lipid-mixing studies
PC : PA vesicles (73 : 25) containing 1 mol% each of
NBD-PE and Rh-PE were prepared in phosphate buffer
(pH 7.4) and diluted to 3 mL (25 lm total lipid concentra-
tion) in a magnetically stirred quartz fluorescence cell, ther-
mostatically controlled at 37 °C, with either the same
buffer or acetate buffer pH 4.0. The ionic strength of buf-
fers was adjusted to 154 mm using NaCl. Negatively
charged liposomes were treated with a twofold molar excess
of cationic lipids, and emission scans (k
ex
= 475 nm) were
recorded at various times over a period of approximately
0.5 h. The extent of fusion between labeled and unlabeled
vesicles was calculated as
% lipid mixing ¼
F
t
À F
0

F
100
À F
0

 100 ð2Þ
where F
t
, F
0
and F
100
are the NBD ⁄ Rh ratios of maximum
probe fluorescence intensities at a particular time t, before
addition of cationic lipids, and in the presence of 0.1%
Triton X-100, respectively.
Acknowledgements
This work was supported in part by a pre-doctoral fel-
lowship in pharmaceutics from the PhRMA Founda-
tion (Washington, DC) and by National Institutes of
Health Grant EB004863.
References
1 Felgner PL, Gadek TR, Holm M, Roman R, Chan
HW, Wenz M, Northrop JP, Ringold GM & Danielsen
M (1987) Lipofection: a highly efficient, lipid-mediated
DNA-transfection procedure. Proc Natl Acad Sci USA
84, 7413–7417.
2 Liu D, Qiao W, Li Z, Cui X, Li K, Yu L, Yan K, Zhu
L & Cheng L (2007) Carbamate-linked cationic lipids
for gene delivery. Bioorg Med Chem, doi:10.1016/

j.bmc.2007.10.009
3 Bajaj A, Kondaiah P & Bhattacharya S (2007)
Synthesis and gene transfer activities of novel serum
compatible cholesterol-based gemini lipids possessing
oxyethylene-type spacers. Bioconjug Chem 18, 1537–
1546.
4 Bajaj A, Kondiah P & Bhattacharya S (2007) Design,
synthesis, and in vitro gene delivery efficacies of novel
cholesterol-based gemini cationic lipids and their serum
compatibility: a structure–activity investigation. J Med
Chem 50, 2432–2442.
5 Wettig SD, Badea I, Donkuru M, Verrall RE & Fold-
vari M (2007) Structural and transfection properties of
amine-substituted gemini surfactant-based nanoparti-
cles. J Gene Med 9, 649–658.
6 Rajesh M, Sen J, Srujan M, Mukherjee K, Sreedhar B
& Chaudhuri A (2007) Dramatic influence of the orien-
tation of linker between hydrophilic and hydrophobic
lipid moiety in liposomal gene delivery. J Am Chem Soc
129, 11408–11420.
7 Antipina MN, Schulze I, Dobner B, Langner A &
Brezesinski G (2007) Physicochemical investigation of a
lipid with a new core structure for gene transfection:
2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan-
1-ol. Langmuir 23, 3919–3926.
8 Chen H, Zhang H, McCallum CM, Szoka FC & Guo
X (2007) Unsaturated cationic ortho esters for endo-
some permeation in gene delivery. J Med Chem 50,
4269–4278.
9 Gardner RA, Belting M, Svensson K & Phanstiel O IV

(2007) Synthesis and transfection efficiencies of new
lipophilic polyamines. J Med Chem 50, 308–318.
10 Lamarche F, Mevel M, Montier T, Burel-Deschamps L,
Giamarchi P, Tripier R, Delepine P, Le Gall T, Cartier
D, Lehn P et al. (2007) Lipophosphoramidates as lipidic
part of lipospermines for gene delivery. Bioconjug Chem
18, 1575–1582.
11 Takahashi T, Kojima C, Harada A & Kono K (2007)
Alkyl chain moieties of polyamidoamine dendron-bear-
ing lipids influence their function as a nonviral gene
vector. Bioconjug Chem 18, 1349–1354.
12 Spelios M, Nedd S, Matsunaga N & Savva M (2007)
Effect of spacer attachment sites and pH-sensitive head-
group expansion on cationic lipid-mediated gene deliv-
ery of three novel myristoyl derivatives. Biophys Chem
129, 137–147.
13 Audouy S & Hoekstra D (2001) Cationic lipid-mediated
transfection in vitro and in vivo (review). Mol Membr
Biol 18, 129–143.
14 Tranchant I, Thompson B, Nicolazzi C, Mignet N &
Scherman D (2004) Physicochemical optimisation of
plasmid delivery by cationic lipids. J Gene Med 6, S24–
S35.
15 Wasungu L & Hoekstra D (2006) Cationic lipids, lipo-
plexes and intracellular delivery of genes. J Control
Release 116, 255–264.
16 Banerjee R, Mahidhar YV, Chaudhuri A, Gopal V &
Rao NM (2001) Design, synthesis, and transfection
biology of novel cationic glycolipids for use in liposo-
mal gene delivery. J Med Chem 44, 4176–4185.

17 Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai
YJ, Border R, Ramsey P, Martin M & Felgner PL
(1994) Enhanced gene delivery and mechanism studies
with a novel series of cationic lipid formulations. J Biol
Chem 269
, 2550–2561.
M. Spelios and M. Savva Novel cytofectins for gene delivery
FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 161
18 Ferrari ME, Rusalov D, Enas J & Wheeler CJ (2002)
Synergy between cationic lipid and co-lipid determines
the macroscopic structure and transfection activity of
lipoplexes. Nucleic Acids Res 30, 1808–1816.
19 Liu L, Zern MA, Lizarzaburu ME, Nantz MH & Wu J
(2003) Poly(cationic lipid)-mediated in vivo gene delivery
to mouse liver. Gene Ther 10, 180–187.
20 Narang AS, Thoma L, Miller DD & Mahato RI (2005)
Cationic lipids with increased DNA binding affinity for
nonviral gene transfer in dividing and nondividing cells.
Bioconjug Chem 16, 156–168.
21 Oudrhiri N, Vigneron JP, Peuchmaur M, Leclerc T, Lehn
JM & Lehn P (1997) Gene transfer by guanidinium-cho-
lesterol cationic lipids into airway epithelial cells in vitro
and in vivo. Proc Natl Acad Sci USA 94, 1651–1656.
22 Yperman J, Mullens J, Francois JP & Van Poucke LC
(1982) Calorimetric and potentiometric investigation of
diethylenetriamine and its N-methyl-substituted deriva-
tives in aqueous solution. J Phys Chem 86, 298–303.
23 Ghirlando R, Wachtel EJ, Arad T & Minsky A (1992)
DNA packaging induced by micellar aggregates: a novel
in vitro DNA condensation system. Biochemistry 31,

7110–7119.
24 Oberle V, Bakowsky U, Zuhorn IS & Hoekstra D (2000)
Lipoplex formation under equilibrium conditions reveals
a three-step mechanism. Biophys J 79, 1447–1454.
25 Ross PC & Hui SW (1999) Lipoplex size is a major
determinant of in vitro lipofection efficiency. Gene Ther
6, 651–659.
26 Struck DK, Hoekstra D & Pagano RE (1981) Use of
resonance energy transfer to monitor membrane fusion.
Biochemistry 20, 4093–4099.
27 Asokan A & Cho MJ (2005) Cytosolic delivery of mac-
romolecules 4. Head group-dependent membrane per-
meabilization by pH-sensitive detergents. J Control
Release 106, 146–153.
28 Aljaberi A, Chen P & Savva M (2005) Synthesis,
in vitro transfection activity and physicochemical
characterization of novel N,N¢-diacyl-1,2-diamino-
propyl-3-carbamoyl-(dimethylaminoethane) amphiphilic
derivatives. Chem Phys Lipids 133, 135–149.
29 Aljaberi A, Spelios M, Kearns M, Selvi B & Savva M
(2007) Physicochemical properties affecting lipofection
potency of a new series of 1,2-dialkoylamidopropane-
based cationic lipids. Colloids Surf B Biointerfaces 57,
108–117.
30 Savva M, Chen P, Aljaberi A, Selvi B & Spelios M
(2005) In vitro lipofection with novel asymmetric series
of 1,2-dialkoylamidopropane-based cytofectins contain-
ing single symmetric bis-(2-dimethylaminoethane) polar
headgroups. Bioconjug Chem 16, 1411–1422.
31 Sheikh M, Feig J, Gee B, Li S & Savva M (2003)

In vitro lipofection with novel series of symmetric
1,3-dialkoylamidopropane-based cationic surfactants
containing single primary and tertiary amine polar head
groups. Chem Phys Lipids 124, 49–61.
32 Ewert K, Slack NL, Ahmad A, Evans HM, Lin AJ,
Samuel CE & Safinya CR (2004) Cationic lipid–DNA
complexes for gene therapy: understanding the relation-
ship between complex structure and gene delivery path-
ways at the molecular level. Curr Med Chem 11, 133–
149.
33 Akao T, Nakayama T, Takeshia K & Ito A (1994)
Design of a new cationic amphiphile with efficient
DNA-transfection ability. Biochem Mol Biol Int 34,
915–920.
34 Boukhnikachvili T, Aguerre-Chariol O, Airiau M,
Lesieur S, Ollivon M & Vacus J (1997) Structure of
in-serum transfecting DNA–cationic lipid complexes.
FEBS Lett 409, 188–194.
35 Gaucheron J, Wong T, Wong KF, Maurer N & Cullis
PR (2002) Synthesis and properties of novel tetraalkyl
cationic lipids. Bioconjug Chem 13, 671–675.
36 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular
Cloning: A Laboratory Manual, 2nd edn. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
Novel cytofectins for gene delivery M. Spelios and M. Savva
162 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS