Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery

Article abstract of DOI:10.1021/ja0704683

A number of prior studies have demonstrated that the DNA-binding and gene transfection efficacies of cationic amphiphiles crucially depend on their various structural parameters including hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar headgroups connected by the same ester linker group. The only structural difference between the cationic amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2 cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene delivery efficacies of cationic liposomes.

Full text of DOI:10.1021/ja0704683

Published on Web 08/25/2007
Dramatic Influence of the Orientation of Linker between
Hydrophilic and Hydrophobic Lipid Moiety in Liposomal Gene
Delivery
Mukthavaram Rajesh,† Joyeeta Sen,†,§ Marepally Srujan,† Koushik Mukherjee,†,|
Bojja Sreedhar,‡ and Arabinda Chaudhuri*,†
Contribution from the DiVision of Lipid Science and Technology, Inorganic and Physical
Chemistry DiVision, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received January 22, 2007; E-mail: arabinda@iict.res.in
Abstract: A number of prior studies have demonstrated that the DNA-binding and gene transfection
efficacies of cationic amphiphiles crucially depend on their various structural parameters including
hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in
tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker
orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation
in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric
remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar
headgroups connected by the same ester linker group. The only structural difference between the cationic
amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene
transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings
in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant
size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence
resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably
higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher
biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence
anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes
than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the
higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of
lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2
cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor
a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-
binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene
delivery efficacies of cationic liposomes.
Introduction
a potentially replication competent virus through various
recombination events with the host genome; inducing inflam-
The clinical success of gene therapy, the modality to combat
a myriad of inherited diseases, continues to remain critically
dependent on the availability of safe and efficacious gene
delivery reagents, popularly known as transfection vectors.1
Broadly speaking, contemporary transfection vectors are clas-
sified into two major categories: viral and nonviral. Viral
vectors, although remarkably efficient in transfecting our body
cells, suffer from numerous biosafety related disadvantages. For
instance, viral vectors are capable of the following: generating
matory and adverse immunogenic responses; and producing
insertional mutagenesis through random integration into the host
genome; etc.2 More recently, it has been reported that retrovirus
vector insertion near the promoter of the proto-oncogene LMO2
in 2 human patients with X-linked severe combined immuno-
deficiency (SCID-XI) is capable of triggering deregulated
premalignant cell proliferation with unexpected frequency.3 In
addition, viral vectors have a low insert size limit for the
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Sci. U.S.A. 1994, 91, 4407-4411. (c) Knowles, M. R.; Hohneker, K. W.;
Zhou, Z.; Olsen, J. C.; Noah, T. L.; Hu, P. C.; Leigh, M. W.; Engelhardt,
J. F.; Edwards, L. J.; Jones, K. R.; Boucher, R. A. New Engl. J. Med.
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† Division of Lipid Science and Technology.
‡ Inorganic and Physical Chemistry Division.
§ Present address: School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599-7360, USA.
| Present address: Department of Chemistry and Division of Biological
Engineering, Massachusetts Institute of Technology, MA 02139, USA.
(1) (a) Verma, I. M.; Somina, M. Nature 1997, 389, 239-242. (b) Anderson,
W. F. Nature 1998, 392, 25-30. (c) Yla-Herttuala, S.; Martin, J. F. Lancet
2000, 355, 213-222.
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J. AM. CHEM. SOC. 2007, 129, 11408-11420
10.1021/ja0704683 CCC: $37.00 © 2007 American Chemical Society

Linker-Orientation Influence in Lipofection
A R T I C L E S
therapeutic genes they can pack inside. Consequently, an
increasing number of investigations are being reported on the
development of safe and efficacious nonviral alternatives
including cationic amphiphiles (also known as cationic trans-
fection lipids),4 cationic polymers,5 dendrimers,6 etc. Because
of their lesser immunogenic nature, robust manufacture ability
to deliver large pieces of DNA, and ease of handling and
preparation techniques, an upsurge of global interest has recently
been witnessed in developing efficacious cationic transfection
lipids for delivering genes into our body cells7 including our
own work.8
The molecular architectures of cationic amphiphiles consist
of a positively charged water-loving (hydrophilic) polar head-
group region and a nonpolar hydrophobic tail region (usually
consisting of either two long aliphatic hydrocarbon chains or a
cholesterol skeleton) often tethered together via a linker
functionality such as ether, ester, amide, amidine group, etc.
Understanding the structural parameters capable of influencing
the gene delivery efficiencies of cationic amphiphiles is essential
for rational design of efficient cationic transfection lipids. To
this end, the focus of many prior structure-activity investiga-
tions have been centered around probing the influence of each
of these three lipid structural components in modulating the gene
transfer efficacies of cationic amphiphiles. For instance, a
number of prior reports have demonstrated that the gene transfer
efficiencies of cationic amphiphiles critically depends on their
molecular architectures including hydrophobic alkyl chain
lengths,9 nature of headgroups8f,10 as well as on the nature of
linker and spacer functionalities used in covalent tethering of
the polar headgroups and the nonpolar tails of cationic
amphiphiles.8h,10,11 Recently, the in vitro gene transfer efficiency
of a cationic amphiphile with asymmetric hydrocarbon chains,
namely oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-
EPC), has been demonstrated to be about 50-fold superior to
that of its structurally very similar saturated asymmetric
counterpart stearoyldecanoyl-ethylphosphatidylcholine (C18:0/
C10-EPC).12 Toward addressing an hitherto unexplored issue
in liposomal gene delivery, namely, the influence of linker
orientation, in the present study, we have designed and
synthesized two structurally isomeric remarkably similar cationic
amphiphiles 1 and 2 (Scheme 1) bearing the same hydrophobic
tails and the same polar headgroups connected by the same ester
linker group. The sole structural difference between the cationic
lipids 1 and 2 is the orientation of the linker functionality (ester
group). Despite having such striking structural similarities, only
lipid 1 could efficiently deliver plasmid DNA encoding â-ga-
lactosidase enzyme into a number of cultured mammalian cells
including COS-1 (SV 40 transformed African green monkey
kidney cells), CHO (Chinese hamster ovarian cells), HepG2
(Human hepatocarcinoma cells), and A549 (human lung car-
cinoma cells). In sharp contrast, lipid 2 was found to be
essentially incompetent in delivering a â-galactosidase reporter
gene into any of these cells.
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The lipid/DNA complexes (lipoplexes) of both lipids 1 and
2 were found to be of similar size and morphology thereby ruling
out any major role of different lipoplex sizes and shapes behind
their remarkably contrasting gene delivery profiles. Findings
in the confocal microscopic experiments in representative
HepG2 cells using lipoplexes containing fluorescently labeled
plasmid DNA revealed a significantly higher cellular uptake of
lipid 1 associated DNA than that of lipid 2 associated DNA.
Fluorescence resonance energy transfer (FRET) studies revealed
higher membrane fusogenicity of the lipid 1/cholesterol lipo-
somes compared to the biomembrane fusogenicity of lipid
2/cholesterol liposomes. Studies on the thermotropic behaviors
of pure cationic liposomes of lipids 1 and 2 using techniques
of differential scanning calorimetry as well as fluorescence
anisotropy revealed a significantly lower gel to liquid crystalline
transition temperature for the lipid 1 liposomes than that for
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Lehn, P. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1651-1656. (m) Pitard,
B.; Oudrhiri, N.; Vigneron, J. P.; Hauchecorne, M.; Aguerre, O.; Toury,
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Lehn, P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2621-2626. (n) Pitard,
B.; Oudrhiri, N.; Lambert, O.; Vivien, E.; Masson, C.; Wetzer, B.;
Hauchecorne, M.; Scherman, D.; Rigaud, J. L.; Vigneron, J. P.; Lehn, J.-
M.; Lehn, P. J. Gene Med. 2001, 3, 478-487. (o) Vigneron, J. P.; Oudrhiri,
N.; Fauquet, M.; Fauguet, M.; Vergely, L.; Bradley, J.-C.; Basseleville,
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Chem. 2006, 17, 159-171. (b) Bharat, M. K.; Karmali, P. P.; Reddy, B.
S.; Chaudhuri, A. J. Med. Chem. 2005, 48, 3784-3795. (c) Sen, J.;
Chaudhuri, A. J. Med. Chem. 2005, 48, 812-820. (d) Sen, J.; Chaudhuri,
A. Bioconjugate Chem. 2005, 16, 903-912. (e) Mahidhar, Y. V.; Rajesh,
M.; Madhavendra, S. S.; Chaudhuri, A. J. Med. Chem. 2004, 47, 5721-
5728. (f) Majeti, B. K.; Singh, R. S.; Yadav, S. K.; Reddy, S. B.;
Ramkrishna, S.; Diwan,P. V.; Madhavendra, S. S.; Chaudhuri, A. Chem.
Biol. 2004, 11, 427-437. (g) Mahidhar, Y. V.; Rajesh, M.; Chaudhuri, A.
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Chaudhuri, A. J. Med. Chem. 2004, 47, 2123-2132. (i) Singh, R. S.;
Gonc¸alves, C.; Sandrin, P.; Pichon, C.; Midoux, P.; Chaudhuri, A. Chem.
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Chem. 2002, 45, 99-114. (b) Fichert, T.; Regelin, A.; Massing, U. Bioorg.
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A R T I C L E S
Scheme 1a
Rajesh et al.
a Reagents: (a) SOCl2 (1.5 equiv), pyridine (1.5 equiv), 0 °C-rt, DCM, 2 h; (b) N-methyl-N,N-diethanol amine (0.4 equiv), DMF, 0 °C-rt, 4 h, 1 N aq
NaOH/DCM (biphasic system); (c) 2-bromoethanol (1.5 equiv), 85 °C, 4 h; (d) Amberlyst A-26 chloride ion exchange resin; (e) TBDMS-Cl (1.5 equiv),
Imidazole (1.5 equiv), dry DCM 0 °C-rt, 12 h; (f) CH3(CH2)10CH2CH2OCOBr (2.5 equiv), K2CO3 (3 equiv), ethylacetate, reflux, 48 h; (g) TBAF (1.5
equiv), dry THF, 6 h; (h) MeI, DCM, 12 h; (i) Amberlyst A-26 chloride ion exchange; (j) SOCl2 (1.5 equiv), pyridine (1.5 equiv), CH3(CH2)11CH2OH (1.2
equiv), DCM, 0 °C-rt, 2 h.
lipid 2 liposomes. Findings in the dye entrapment experiment
were also consistent with the higher rigidity of lipid 2/cholesterol
(1:1 mole ratio) liposomes. Thus, the higher biomembrane
fusibility of lipid 1 liposomes than that of lipid 2 liposomes
presumably originates from the more rigid nature of lipid 2
cationic liposomes. Taken together, the present findings dem-
onstrate for the first time that even as minor a structural variation
as linker orientation reversal in cationic amphiphiles can
profoundly influence DNA-binding characteristics, membrane
rigidity, membrane fusibility, cellular uptake, and consequently
gene delivery efficacies of cationic liposomes.
2-bromoethanol and chloride ion exchange chromatography over
Amberlyst-A26 resin (Scheme 1A). Lipid 2 was prepared by
di-N-alkylation of O-tert-butyl-dimethylsilyl-2-aminoethanol
with n-tridecyl-3-bromopropanoate ester, followed by TBDMS-
deprotection with tetrabutylammonium fluoride, quaterization
with methyl iodide, and chloride ion-exchange chromatography
over Amberlyst A-26 (Scheme 1B). The n-tridecyl-3-bromopro-
panoate ester was synthesized by reacting 3-bromopropanoic
acid with thionyl chloride followed by esterification of the
resulting acid chloride with n-tridecyl alcohol (step j, Scheme
1C). The structures of all the intermediates (Scheme 1) were
1
Results
confirmed by H NMR spectral analysis, and the structures of
the final target lipids 1 and 2 were confirmed by both NMR
and mass spectral analysis. The purities of the final lipids were
confirmed by both elemental (C, H, N, Cl) and analytical HPLC
analysis in two different mobile phases. 1H NMR spectra of all
the intermediates I-V (Scheme 1), 1H NMR and mass spectra
of the final lipids 1 and 2, and the HPLC chromatograms for
Chemistry. Toward probing the influence of the structural
orientation of the linker functionality, we designed lipids 1 and
2 (Scheme 1) such that the two lipids architecturally differ only
in the orientation of their head-tail linker functionality (ester
group). Lipid 1 was synthesized by coupling myristic acid with
N-methyl-N,N-diethanolamine followed by quaternization with
9
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Linker-Orientation Influence in Lipofection
A R T I C L E S
Figure 1. In vitro gene delivery efficiencies of lipids 1 and 2 into COS-1 (A), CHO (B), HepG-2 (C), and A-549 (D) cells using cholesterol as co-lipid (at
a lipid/cholesterol mole ratio of 1:1). Units of â-galactosidase activity were plotted against the varying lipid-to-DNA (() charge ratios. Transfection experiments
were performed as described in the text. The transfection values shown are the average of triplicate experiments performed on the same day.
the final lipids 1 and 2 in two different mobile phases (100%
methanol and 95:5 methanol/water, v/v) are available in Figures
S1-S11 of the Supporting Information.
Physicochemical Characterizations of Liposomes and
Lipoplexes. Sizes and Zeta Potentials. The sizes and surface
potentials of the lipid 1 and lipid 2 lipoplexes (prepared in
DMEM) were measured using a dynamic laser light scattering
technique (Zetasizer 3000A, Malvern Instruments, U.K.). The
sizes of both lipids 1 and 2 lipoplexes were found to be similar
for lipoplexes having the same lipid/DNA charge ratios, and
interestingly, the sizes of both the lipoplexes steadily increased
from 200 to 600 nm as the lipid/DNA charge ratios increased
from 0.5:1 to 8:1(Table 1A).
Transfection Biology. A reporter gene expression assay was
used in evaluating the in vitro gene delivery efficacies of lipids
1 and 2 in four cultured mammalian cells including COS-1 (SV
40 transformed African green monkey kidney cells), CHO
(Chinese hamster ovary cells), HepG2 (human hepatocarci-
noma), and A549 (human lung carcinoma cells) using p-CMV-
SPORT-â-gal plasmid DNA as the reporter gene encoding the
enzyme â-galactosidase across the lipid/DNA charge ratio (()
range 8:1-0.5:1. Despite the opposite orientation of the linker
ester functionality being the only structural differences between
lipids 1 and 2, only lipid 1 was competent in delivering genes
into these four cells (Figure 1A-D). Lipid 1 showed its optimal
gene delivery efficacies at a lipid/DNA charge ratio of 1:1 in
COS-1, HepG2, and A549 cells (Figure 1A, C, and D,
respectively, filled bars) and at lipid/DNA charge ratios of 2:1
in CHO cells (Figure 1B, filled bars). In sharp contrast, lipid 2
turned out to be essentially incompetent in delivering genes into
any of these four cells across the entire lipid/DNA charge ratios
of 8:1-0.5:1 (Figure 1A-D, open bars). Equimolar amounts
of cholesterol (with respect to cationic lipids) were used as the
co-lipid (DOPE, the other commonly used co-lipid in liposomal
gene delivery, failed to impart gene transfer properties to both
lipids 1 and 2; data not shown). Thus, the relative transfection
profiles of lipids 1 and 2 summarized in Figure 1A-D
demonstrate that the orientation of the linker functionality used
in tethering the nonpolar tails and polar heads of cationic
amphiphiles is a crucial structural parameter in liposomal gene
delivery.
Findings in the transmission electron microscopic (TEM)
studies also revealed no significant size and morphological
differences between the liposomes and lipoplexes prepared with
lipids 1 and 2 (representative TEM pictures for lipoplexes
prepared using lipid/DNA charge ratios of 1:1 are shown in
Figure S12, Parts A-D, Supporting Information). Interestingly,
while the net surface potentials of lipid 1 lipoplexes remained
positive for the lipid/DNA charge ratios higher than 1:1, those
for lipid 2 lipoplexes remained negative across the entire lipid/
DNA charge ratio range 0.5:1-4:1 (Table 1B).
Lipid/DNA Binding Interactions and DNase I Sensitivities.
The electrostatic binding interactions between the plasmid DNA
and lipids 1 and 2 at varying lipid/DNA charge ratios were
measured by the conventional gel retardation assay. The
electrophoretic gel patterns revealed an interesting feature. While
both the lipids were capable of completely inhibiting the
electrophoretic mobility of plasmid DNA when lipoplexes were
prepared at a high lipid:DNA charge ratio of 8:1, significant
free DNA bands were found in both the lipoplexes at the lower
lipid/DNA charge ratios of 0.5:1 and 1:1 (Figure 2A). DNase I
sensitivity assays were carried out across the lipid/DNA charge
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A R T I C L E S
Rajesh et al.
Table 1. (A) Hydrodynamic Diameters of the Lipoplexes Across the Lipid/DNA Charge Ratios 0.5:1-8:1a and (B) Zeta Potentials (ê, mV) of
Lipoplexes
A. hydrodynamic diameters
lipid/DNA
lipid/DNA
lipid/DNA
lipid/DNA
lipid/DNA
(
±
)
(
±
)
(
±
)
(
±
)
(±)
lipid
(0.5:1)
(1:1)
(2:1)
(4:1)
(8:1)
lipid 1
lipid 2
size (nm)
size (nm)
(210 ( 8)
(214 ( 18)
(216 ( 2)
(227 ( 9)
(350 ( 13)
(346 ( 19)
(537 ( 17.)
(445 ( 36)
(615 ( 65)
(579 ( 21)
B. zeta potentials
lipid/DNA
lipid/DNA
lipid:DNA
lipid/DNA
lipid/DNA
(
±
)
(
±
)
(
±
)
(
±
)
(±)
(0.5:1)
(1:1)
2:1
(4:1)
(8:1)
lipid 1
lipid 2
-8 ( 1
-29.2 ( 4.2
-1.8 ( 0.8
-24.2 ( 2.2
13.2 ( 2.7
-20.9 ( 2.2
15.8 ( 1.8
-2.2 ( 2.5
21.6 ( 2.8
5.6 ( 1.7
a The sizes and the surface potentials of the lipoplexes prepared in the presence of plain DMEM were measured by the laser light scattering technique
using Zetasizer 3000A (Malvern Instruments, U.K.) as described in the text. Values shown are the averages obtained from three (sizes) and ten (zeta
potentials) measurements.
Cell Viabilities. MTT-based cell viability assays were
performed in representative CHO cells using lipoplexes of both
lipids 1 and 2 across the entire range of lipid/DNA charge ratios
used in the actual transfection experiments (8:1-0.5:1). Percent
cell viabilities were found to be significantly high across the
entire lipid/DNA charge ratios when cells were treated with
lipoplexes prepared with either lipid 1 or 2 (more than 80%,
data shown in Figure S13, Supporting Information).
Cellular Uptake Studies. The relative cellular uptake of
plasmid DNA were measured in representative HepG2 cells by
incubating lipoplexes (containing lipid/DNA charged ratio of
1:1 where lipid 1 showed its optimal transfection efficiency in
HepG2 cells) prepared with lipids 1 and 2 and a fluorescein-
labeled p-CMV-SPORT-â-Gal for 4 h at 37 °C. After washing
the cells with phosphate buffer saline, live HepG2 cells were
viewed with a confocal microscope. Although a number of
fluorescein-labeled cells were visible when the cells were treated
with lipid 1 lipoplex (Figure 3A-C), hardly any fluorescein-
labeled cells could be detected when cells were treated with
the lipoplex of lipid 2 (Figure 3D-F). Similarly, relative cellular
uptake of the Rho-PE labeled lipoplexes of lipids 1 and 2 were
studied in HepG2 cells. A number of HepG2 cells were found
to be labeled red when cells were incubated with Rho-PE labeled
lipid 1 lipoplexes for 4 h while hardly any HepG2 cells were
found to be labeled red upon the same treatment with Rho-PE
labeled lipid 2 lipoplexes (epifluorescence microscopic images
for cellular uptake experiments using Rho-PE labeled lipoplexes
are shown in Figure S14, Supporting Information).
Membrane Fusogenicities of Liposomes. Relative biomem-
brane fusogenicities of the liposomes prepared from lipids 1
and 2 (using equimolar amounts of cholesterol as co-lipid) were
Figure 2. Electrophoretic gel patterns for lipoplex-associated DNA in gel
assessed by using a fluorescence resonance energy transfer
retardation assay (A) and in DNase I sensitivity assay (B). The lipid/DNA
(FRET) assay. Because of spatial proximity, fluorescence energy
transfer ensues from N-(7-nitro-2-oxa-1,3-diazol-4-yl)-1,2-di-
hexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) (en-
ergy donor) to RhodamineRed-x-1,2-dihexadecanoyl-sn-glycero
3-phosphoethanolamine (Rho-PE) (energy acceptor) when the
biomembrane mimicking liposomal formulations (dioleyol-
phosphatidylcholine/dioleyol-phosphatidylethanolamine/dioleyol-
phosphatidylserine/cholesterol (DOPC/DOPE/DOPS/Chol) at
45:20:20:15, w/w) containing these two fluorophores are excited
at the excitation wavelength of NBD-PE (485 nm). Emission
at 595 nm (emission wavelength of Rho-PE) was measured.
charge ratios are indicated at the top of each lane. The details of the treatment
are as described in the text.
ratios 0.5:1-8:1. After the free DNA digestion by DNase I,
the total DNA (both digested and inaccessible DNA) was
separated from the lipids and DNase I (by extracting with
organic solvent) and loaded onto a 1% agarose gel. The band
intensities of inaccessible and therefore undigested DNA
associated with transfection incompetent lipid 2 were found to
be significantly less compared to those associated with trans-
fection efficient lipid 1 across the lipid/DNA charge ratios 4:1-
0.5:1 (Figure 2B).
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Linker-Orientation Influence in Lipofection
A R T I C L E S
Figure 4. Biomembrane fusogenicities of lipid 1/Chol and lipid 2/Chol
liposomes. Fusion was induced by adding the cationic lipid 1/Chol and
lipid 2/Chol liposomes to the double fluorophore labeled biomembrane
mimicking DOPC/DOPE/DOPS/Chol liposomal formulations. The values
shown are representative of three independent measurements. The details
of FRET experiments are as discussed in text.
(34 °C). The gel to liquid crystalline temperatures of the lipid
1 and lipid 2 liposomes were also studied by the technique of
fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene
(DPH) as the fluorescence probe. Consistent with the findings
in the DSC study (Figure 5A), the midpoint of transition (the
transition temperature) for the lipid 1 liposomes was found to
be close to 34 °C in the fluorescence anisotropy study (Figure
5B). However, no sharp midpoint transition temperature was
detected in the fluorescence anisotropy study for lipid 2
liposomes (Figure 5B).
Dye Entrapment Experiments. Finally, toward probing the
inherent membrane rigidity differences, if any, between the
cationic liposomes of lipids 1 and 2 containing equimolar
amounts of cholesterol (liposomes used in the actual transfection
experiments), a simple dye entrapment experiment was carried
out using cholesterol containing liposomes with entrapped 5/6-
carboxyfluorescein (CF), a fluorescent dye soluble in 10 mM
Tris‚HCl buffer at pH 8. The relative amount of CF released
(i.e., % permeation of CF) in 60 min from the lipid 2/cholesterol
liposome was found to be significantly less than that released
from the lipid 1/cholesterol liposomes (data shown in Figure
S16, Supporting Information). Such a remarkably decreased
release of entrapped CF from lipid 2/cholesterol liposomes
strongly supports the notion that the membrane rigidity of lipid
2/cholesterol (1:1 mole ratio) liposomes is significantly higher
than that of lipid 1/cholesterol (1:1 mole ratio) liposomes.
Figure 3. Confocal microscopic images of HepG2 cells transfected with
lipoplexes of lipid 1 (A-C) and lipoplexes of lipid 2 (D-F) containing
fluorescein-labeled plasmid DNA. Lipid/DNA charge ratios in both the
lipoplexes were maintained at 1:1. (A and D) Fluoroscent images. (B and
E) Overlay images. (C and F) Phase contrast bright field images. The details
of confocal microscopic experiments are as described in the text.
Fluorescence intensities of the double fluorophore labeled
biomembrane mimicking liposomes were also measured in the
presence of 1% Triton X-100 after ensuring complete mixing
of the lipids, and these intensities were used for normalization
(to 100% fusions) of measurements. The results summarized
in Figure 4 clearly demonstrate that the cationic liposomes of
lipid 1 possess about a 3.5-fold higher biomembrane fusogenicity
than that of the liposomes of lipid 2. These relative biomembrane
fusogenicity profiles for the cationic liposomes of lipids 1 and
2 did not change significantly when an FRET experiment was
conducted monitoring the increase in the emission intensity
NBD-PE (% normalized fluorescence recovery vs time plot
obtained by measurements of increasing emission intensities of
NBD-PE are shown in Figure S15, Supporting Information).
Thermotropic Phase Behaviors of Lipid 1 and Lipid 2
Liposomes by Differential Scanning Calorimetry and Fluo-
rescence Anisotropy. The thermograms obtained for the pure
cationic liposomes of lipids 1 and 2 (Figure 5A) in a differential
scanning calorimetric (DSC) study revealed a significantly
higher gel to liquid crystalline transition temperature (Tm) for
lipid 2 liposomes (49 °C) than that for lipid 1 liposomes
Discussion
Systematic structure-activity investigations aimed at under-
standing how variations in the lipid structural components
including polar headgroups, nonpolar hydrocarbon tails, and the
linker functionalities influence the gene transfer efficacies of
cationic amphiphiles continue to remain an actively pursued area
of research in nonviral gene delivery.8f,g,9a-f,10,11,13 The nature
(13) Niculescu-Duvaz, D.; Heyes, J.; Springer, C. J. Curr. Med. Chem. 2003,
10, 1233-1261.
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J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11413

A R T I C L E S
Rajesh et al.
Figure 5. DSC thermograms of cationic liposomes of lipids 1 and 2 (A); fluorescence anisotropy vs temperature plots for lipid 1 and lipid 2 liposomes (B).
Details of experiments are as described in text.
of the linker group in particular plays a dominant role in
modulating gene delivery efficiencies of cationic lipids. The
relative orientation of the cationic headgroup and hydrocarbon
anchor is governed by the nature of the linker bond bridging
them. The linker group controls the conformational flexibility,
degree of stability, biodegradibility, and, hence, the gene transfer
efficacy of a cationic amphiphile. Most commonly used linker
groups include ethers, esters, carbamates, amides, carbonates,
phosphonates, disulfides, etc. A number of prior reports
demonstrated advantages of one class of linker functionality over
the others. For instance, cholesterol-based cationic amphiphiles
with an ether linker functionality have been demonstrated to
exhibit superior in vitro gene transfer efficacies to their ester
counterparts.11a,c Ester and carbonate linkers are biodegradable
and less toxic but not stable chemically.14 The phosphate diester
bond (as in DOPE) is biodegradable and has a chemical stability
higher than that of esters but lower than that of amides. On the
other hand, phosphonates are hydrolytically more stable than
phosphates and presumably more biodegradable.15 The length
of the linker determines the level of hydration of a lipid.
Incorporating oxyethylene units between the cholesterol moiety
and the headgroup has been reported to increase the level of
hydration and decrease the transfection activity.11c However,
to date addressing the issue of linker orientation remains
unexplored in liposomal gene delivery. To this end, we have
designed in the present investigation two structurally very similar
cationic amphiphiles 1 and 2 (Scheme 1) differing only in the
orientation of their ester linker functionality. As demonstrated
by the transfection results described above, despite having such
close structural resemblances, the gene transfer efficiencies of
lipid 1 were found to be strikingly superior to those of the
essentially transfection incompetent lipid 2 in multiple cultured
cells.
In order to gain insights into the possible origin of such
contrasting transfection profiles of the two structurally seemingly
close cationic lipids 1 and 2, first we studied the size and
morphological characteristics of the lipoplexes of both lipids 1
and 2 using techniques of transmission electron microscopy
(TEM) and dynamic light scattering (DLS). Measurements of
the hydrodynamic diameters of both the lipoplexes (prepared
across the varying lipid/DNA charge ratios of 0.5:1-8:1) by
the dynamic light scattering technique revealed no significant
differences in lipoplex sizes. The sizes of both the lipoplexes
prepared with lipids 1 and 2 were found to similarly increase
with increasing lipid/DNA charge ratios from around 200 nm
(14) Aberle, A. M.; Tablin, F.; Zhu, J.; Walker, N. J.; Gruenert, D. C.; Nantz,
M. H. Biochemistry 1998, 37, 6533-6540.
(15) Duvaz-N. D.; Heyes, J.; Springer, C. J. Curr. Med. Chem. 2003, 10, 1233-
1261.
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Linker-Orientation Influence in Lipofection
A R T I C L E S
at the lipid/DNA charge ratio 0.5:1 to around 600 nm at the
lipid/DNA charge ratio 8:1 (Table 1A). Consistent with these
findings, transmission electron microscopic studies of repre-
sentative liposomes and lipoplexes (prepared with lipid/DNA
charge ratios of 1:1) of both lipids 1 and 2 revealed no
significant difference in their size and morphological charac-
teristics (transmission electron micrographs of these representa-
tive liposomes and lipoplexes are shown in Figure S12, Parts
A-D, Supporting Information). These findings ruled out the
possibility of lipoplex sizes playing any dominant role behind
the contrasting gene transfer properties of lipids 1 and 2.
lipid 2 lipoplexes (Figure 3A-F) could possibly be related to
any inherent biomembrane fusogenicity differences between
lipids 1 and 2 liposomes, we next evaluated the relative
membrane fusogenicities of both the lipid 1 and lipid 2
liposomes with a popular biomembrane mimicking liposomal
formulation (dioleyol-phosphatidylcholine/dioleyol-phosphati
dylethanolamine/dioleyol-phosphatidylserine/cholesterol at 45:
20:20:15, w/w) by using fluorescence resonance energy transfer
(FRET) technique pioneered by Struck et al.16 This technique
depends upon the interactions that occur between two fluoro-
phores when the emission band of one (the energy donor)
overlaps with the exicitation band of the second (the energy
acceptor) and when such fluorophores are in close physical
proximity. These conditions are satisfied when the biomembrane
mimicking liposomal formulations are prepared with both the
donor and the acceptor fluorophores such as N-(7-nitro-2-oxa-
1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoetha-
nolamine (NBD-PE) and Rhodamine Red-x-1,2-dihexadecanoyl-
sn-glycero 3-phosphoethanolamine (Rho-PE), respectively. In
such liposomes, the energy from a photon absorbed by the
energy donor, NBD-PE, is transferred to the energy acceptor,
Rho-PE, causing the latter to fluoresce as if it is excited directly.
Since the efficiency of the FRET between two such fluorophores
critically depends upon their spatial separation, any fusion event
of such double-fluorophore containing biomembrane mimicking
liposomes with cationic liposomes of lipids 1 and 2 (devoid of
any fluorophore) decreases the efficiency of resonance energy
transfer. In other words, the decrease in percent of normalized
fluorescence recovery (i.e., maximum fusion corresponding to
Rho-PE fluorescence emission intensity at 595 nm observed
when the double-fluorophore labeled biomembrane liposomal
formulation is completely disrupted with 1% Triton X-100)
provides evidence for reduced membrane fusion.
Lipid 1 lipoplexes were transfection efficient mostly at a lipid/
DNA charge ratio of 1:1 (Figure 1). Both gel retardation (Figure
2A) and DNase I sensitivity (Figure 2B) assays showed the
presence of a significant amount of free DNA in the lipid 1
lipoplex containing this lipid:DNA charge ratio of 1:1. Thus,
full association of lipid 1 to DNA and full protection from
degredation by DNase I appear to be irrelevant to the transfec-
tion activity of lipid 1. The surface potential of lipid 1 lipoplex
at the optimal lipid/DNA charge ratio 1:1 was found to be
slightly negative (-1.8 ( 0.8 mV, Table 1B). The electro-
phoretic gel patterns in the gel retardation assay and DNase I
sensitivity assay for the lipid 2 lipoplexes as well as the surface
potential of the lipid 2 lipoplex at a lipid/DNA charge ratio of
4:1 (-2.2 ( 2.5 mV, Table 1B) were comparable to those for
lipid 1 lipoplexes at its optimal lipid/DNA charge ratio of 1:1
(Figure 2A-B and Table 1B). Putting it differently, if lipid/
DNA binding interactions and lipoplex surface potentials are
key transfection modulating physicochemical parameters for the
present class of cationic amphiphiles, lipid 2 should have shown
comparable levels of transfection activity to those of lipid 1 at
the lipid/DNA charge ratio 4:1. However, as summarized in
the transfection results depicted in Figure 1, lipid 2 maintained
its transfection incompetent nature at the 4:1 lipid/DNA charge
ratio. Thus, the transfection results (Figure 1), lipoplex surface
potential characteristics (Table 1B), lipid/DNA binding interac-
tions (Figure 2A), and DNase I sensitivities of lipid associated
DNA (Figure 2B), taken together, are consistent with the notion
that some other physicochemical property (i.e., other than
lipoplex surface potentials, lipid/DNA binding interactions, and
the DNase I sensitivity of the lipid associated DNA) is likely
to play a dominant role behind the contrastingly different
transfection properties of lipids 1 and 2. Since more than 80%
CHO cells remained viable upon treatment with lipids 1 and 2
lipoplexes across the entire lipid/DNA charge ratios 8:1-0.5:1
(Figure S13, Supporting Information), varying cellular toxicities
were also ruled out as a possible factor behind the contrasting
transfection profiles of lipids 1 and 2.
The percent of normalized fluorescence recovery was found
to be about 3.5-fold higher when the double-fluorophore labeled
biomembrane mimicking liposomal formulations was incubated
with lipid 1/cholesterol liposomes for 30 min compared to the
normalized fluorescence recovery (%) upon treatment of the
biomembrane mimicking liposomes with lipid 2/cholesterol
liposomes for the same time period (Figure 4). Such FRET
results are consistent with significantly higher biomembrane
fusogenicities for lipid 1/cholesterol liposomes than those for
lipid 2/cholesterol liposomes. The remarkably higher biomem-
brane fusogenicity for lipid 1/cholesterol liposomes than those
for lipid 2/cholesterol liposomes (Figure 4) was fully consistent
with the findings in the cellular uptake study using with Rho-
PE labeled lipoplexes of lipids 1 and 2 in representative HepG2
cells. A number of Rho-PE labeled HepG2 cells were detected
in the inverted epifluorescence microscopic study when cells
were incubated with Rho-PE labeled lipid 1 lipoplexes while
hardly any Rho-PE cell could be seen upon similar treatment
of the cells with Rho-PE labeled lipoplexes of lipid 2 (the
epifluorescence fluorescence micrographs for cellular uptake
studies using Rho-PE labeled lipoplexes in HepG2 cells are
shown in Figure S14 of the Supporting Information). Thus, the
poor biomembrane fusibility of lipid 2 liposomes is likely to
play a major role in the severely compromised transfection
efficacies of lipid 2. Currently believed intracellular pathways
Toward probing the influence of relative DNA uptake profiles
in modulating the transfection properties of the lipid 1 and lipid
2 lipoplexes, representative HepG2 cells were treated with both
lipid 1 and lipid 2 lipoplexes prepared with a fluorescein-labeled
pCMV-SPORT-â-gal plasmid DNA and cellular uptake of
fluorescently labeled plasmid DNA was monitored by confocal
microscopy. The findings in the confocal microscopic studies
(Figure 3A-F) clearly demonstrated that the essentially trans-
fection incompetent nature of lipid 2 is likely to originate from
the extremely poor cellular uptake of plasmid DNA associated
with lipid 2. In order to gain insights into whether such
contrastingly enhanced DNA uptake profiles of the lipid 1 and
(16) Struck, D. K.; Hoekstra, D.; Pagano, R. E. Biochemistry 1981, 20, 4093-
4099.
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A R T I C L E S
Rajesh et al.
involved in cationic lipid mediated gene transfer (lipofection)
include the following: (a) endocytotic cellular uptake of the
lipid/DNA complex (lipoplex); (b) release of DNA from the
resulting endosomes into the cell cytoplasm; and (c) the nuclear
transport of the endosomally released DNA followed by
transcription and gene expression.17-20 Improved biomembrane
fusogenicity of cationic liposomes is expected to mediate not
only the enhanced cellular uptake of the cationic liposome
associated DNA (step a) but also the efficacy of step b. This is
because efficient release of DNA from endosomes into cell
cytoplasm (step b) is likely to depend on how efficiently the
cationic lipids can fuse with the anionic lipid components of
the endosomal membranes. Thus, enhanced biomembrane
fusogenicity of cationic liposomes are likely to correlate with
the transfection efficiency of cationic lipids as found in the
present study. Wang and MacDonald have also found a similar
correlation between membrane fusogenicity and transfection
efficacies of cationic liposomes.21
are consistent with the remarkable rigid nature of these
equimolar cholesterol containing cationic liposomes.
As discussed above, the findings in the DSC and fluorescence
anisotropy studies provided enough evidence for the existence
of higher membrane rigidity in pure cationic liposomes of lipid
2 compared to that for pure cationic liposomes of lipid 1.
However, the findings in these thermotropic studies were not
insightful enough to probe any inherent membrane rigidity
differences between the transfection efficient lipid 1/cholesterol
(1:1 mole ratio) liposomes and transfection incompetent lipid
2/cholesterol (1:1 mole ratio) liposomes. To this end, toward
qualitative probing of the differences in the membrane rigidity
of the equimolar cholesterol containing liposomes of lipids 1
and 2 (used in the actual transfection experiments), finally we
carried out a simple fluorescent dye entrapment experiment. A
significantly higher amount of fluorescent dye (CF) released
from lipid 1/cholesterol (1:1 mole ratio) in 60 min compared
to the amount of CF released from the lipid 2/cholesterol (1:1)
liposomes during the same time interval (Figure S16, Supporting
Information) provided strong evidence for the existence of
higher membrane rigidity in lipid 2/cholesterol. Use of cationic
liposomes with less rigid or more fluid membranes are known
to be an advantage in transfection.9e,22-24 Cationic liposomes
with fluid membranes are likely to fuse more efficaciously with
both cellular plasma membranes and endosomal membranes.
Thus, the significantly greater membrane fluidity of the lipid
1/cholesterol liposomes than that of lipid 2/cholesterol liposomes
is likely to play a dominant role in the strikingly superior
transfection efficacies of lipid 1/cholesterol liposomes. The
higher membrane rigidity of the lipid 2/cholesterol liposomes
presumably originate from enhanced headgroup hydration.
Hydration of ester carbonyl groups (through hydrogen bonding
with water) present near the polar headgroup region of am-
phiphilic molecules contributes to the headgroup hydration of
liposomal aggregates.25 With the covalent bond distance of the
ester carbonyl group from the positively charged nitrogen in
lipid 2 being shorter than that in lipid 1, the degree of interfacial
hydration is likely to be higher in lipid 2 liposomes. The
enhanced interfacical hydration in lipid 2 liposomes, in turn, is
expected to significantly shield the large Coulombic repulsion
among the positively charged headgroups thereby imparting
higher rigidity to the liposomal membrane (through more
compact packing of the amphiphiles in the liposomal ag-
gregates). Consistent with this supposition, the degree of
interfacial hydration of the transfection efficient cationic N-(1-
(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP)/cholesterol liposomes has indeed been shown to be
lower than that of its transfection incompetent DOTAP/DOPE
counterpart.26 Significantly more positive surface potentials of
lipid 1 lipoplexes compared to those for the lipid 2 lipoplexes
across the entire lipid/DNA charge ratio 0.5:1-8:1 (Table 1B)
is also likely to originate from possibly less hydrated and
therefore less shielded positive surface charges of lipid 1/cho-
lesterol liposomes. An important issue deserves mention at this
In order to understand whether the origin of the contrasting
biomembrane fusibility of lipid 1/cholesterol and lipid 2/cho-
lesterol liposomes (Figure 4) could possibly be related to the
differences in the inherent membrane fluidity differences of the
cationic liposomes of lipids 1 and 2, next we studied the
thermotropic behaviors of the pure cationic liposomes of lipids
1 and 2 by both differential scanning calorimetry (DSC) and
fluorescence anisotropy. DSC thermograms (Figure 5A) revealed
the existence of a significantly higher solid gel to fluid liquid
crystalline-like phase transition temperature (Tm, a property
typical of membranous liposomal aggregates) for lipid 2
liposomes (49 °C) compared to that for lipid 1 liposomes
(34 °C). This finding (Figure 5A) is consistent with a higher
inherent membrane rigidity of the lipid 2 liposomes (compared
to that of lipid 1 liposomes). The results in the fluorescene
anisotropy studies further confirmed the higher membrane
rigidity of lipid 2 liposomes. The fluorescence anisotropy values
(r) increase with increasing membrane rigidity of the liposomal
aggregates, and Tm values of liposomal aggregates are deter-
mined from the midpoints of the sigmoidal r vs temperature
plots. Consistent with the findings in the DSC study (Figure
5A), lipid 1 liposomes showed a clear midpoint transition
temperature (Tm) close to 34 °C in the fluorescence anisotropy
study (Figure 5B). However, the fluorescence anisotropy values
remained high (0.32-0.25) throughout the entire temperature
range (15-60 °C) thereby demonstrating a significantly higher
rigidity of the lipid 2 liposomes compared to lipid 1 liposomes
(Figure 5B). Interestingly, neither the DSC nor the fluorescence
anisotropy study revealed the existence of any clear gel to liquid
crystalline temperature or midpoint transition temperatures when
lipid 1 and lipid 2 liposomes containing equimolar amounts of
cholesterol (with respect to cationic lipids) were used instead
of using pure cationic liposomes (data not shown). Such
absences of any pronounced reduction in fluorescence anisotropy
values for the equimolar cholesterol containing liposomes of
lipids 1 and 2 across the entire temperature range of 15-60 °C
(22) Regelin, A. E.; Fankhaenel, S.; Gurtesch, L.; Prinz, C.; v. Kiedrowski, G.;
Massing, U. Biochim. Biophys. Acta 2000, 1464, 151-164.
(23) Savva, M.; Chen, P.; Aljaberi, A.; Selvi, B.; Spelios, M. Bioconjugate Chem.
2005, 16, 1411-1422.
(17) Bally, M. B.; Harvie, P.; Wong, F. M.; Kong, S.; Wasan, E. K.; Reimer,
D. L. AdV. Drug DeliVery ReV. 1999, 38, 291-315.
(18) Zabner, J.; Fasbender, A. J.; Moninger, T.; Poellinger, K. A.; Welsh, M. J.
J. Biol. Chem. 1995, 270, 18997-19007.
(24) Akao, T.; Osaki, T.; Ito, A.; Kunitake, T. Bull. Chem. Soc. Jpn. 1991, 64,
3677-3681.
(19) Friend, D. S.; Papahadjopoulos, D.; Debs, R. J. Biochim. Biophys. Acta
1996, 1278, 41-50.
(20) Xu, Y.; Szoka, F. C., Jr. Biochemistry 1996, 35, 5616-5623.
(21) Wang, L.; MacDonald, R. C. Gene Ther. 2004, 11, 1358-1362.
(25) Hu¨bner, W.; Blume, A. Chem. Phys. Lipids. 1998, 96, 99-123.
(26) Hirsch-Lerner, D.; Barenholz, Y. Biochim. Biophys. Acta 1999, 1461, 47-
57.
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Linker-Orientation Influence in Lipofection
A R T I C L E S
point of discussion. The distribution of cholesterol among
different cellular membranes is not uniform. At high concentra-
tions, free cholesterol can self-associate and form domains (rafts)
of condensed complexes of cholesterol and membrane phos-
pholipids.27 The existence of similar cholesterol domains is being
proposed within the membranes of cationic liposomes containing
high amounts of cholesterol.28 Since the cationic liposomes of
lipids 1 and 2 contain high amounts of cholesterol, the existence
of such cholesterol domains in these liposomal membranes
cannot be ruled out. In that case the remarkably higher
biomembrane fusogenicity of the less rigid lipid 1/cholesterol
may also be related to the smoother cholesterol partitioning into
the negatively charged biomembrane mimicking liposomal
formulations. Koynova et al. has recently demonstrated that
pronounced nonlamellar phase forming liposomal compositions
of a cationic amphiphile with two asymmetric hydrocarbon
chains, namely oleoyldecanoyl-ethylphosphatidylcholine (C18:
1/C10-EPC), is likely to act as a dominant factor behind their
high membrane fusogenic and high transfection properties
compared to its structurally very similar saturated asymmetric
counterpart stearoyldecanoyl-ethylphosphatidylcholine (C18:0/
C10-EPC).12 Clearly, similar biophysical investigations need to
be undertaken in the future to understand whether the remark-
ably high fusogenic as well as high gene delivery efficacies of
lipid 1/Chol liposomes compared to lipid 2/Chol liposomes also
correlates with its enhanced nonlamellar phase forming proper-
ties when mixed with cellular membranes.
liposomes than that of the lipid 2 liposomes. Findings in the
dye entrapment experiment are consistent with the significantly
less membrane rigidity for the transfection efficient lipid
1/cholesterol (1:1 mole ratio) liposomes. Enhanced biomembrane
fusogenicity and the contrastingly high transfection property of
lipid 1 liposomes presumably originate from the less rigid or
more fluid character of the lipid 1 liposomal membranes
compared to that of the lipid 2 liposomal membranes. Taken
together, the present findings demonstrate for the first time that
even as a minor structural variation linker orientation reversal
in cationic amphiphiles can profoundly influence DNA-binding
characteristics, membrane rigidity, membrane fusibility, cellular
uptake, and consequently gene delivery efficacies of cationic
liposomes.
Experimental Section
Materials and Methods. Mass spectral data were acquired by using
a commercial LCQ ion trap mass spectrometer (ThermoFinnigan,
SanJose, CA) equipped with an ESI source or micromass Quatro LC
triple quadrapole mass spectrometer for ESI analysis. 1H NMR spectra
were recorded on a Varian FT 200 MHz or AV 300 MHz NMR
spectrometer. 1-Tridecanol, n-tetradecanoic acid, tert-butyldimethylsilyl
chloride, tetra-n-butylammonium fluoride (1 M solution in THF), and
Amberlyst A-26 chloride ion exchange resin were purchased from
Lancaster (Morecambe, U.K.). Column chromatography was performed
with silica gel (Acme Synthetic Chemicals, India, 60-120 mesh).
p-CMV-SPORT-â-gal plasmid was a generos gift from Dr. Nalam
Madhusudhana Rao of the Centre for Cellular and Molecular Biology,
Hyderabad, India. Cell culture media, fetal bovine serum, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly-
ethylene glycol 8000, o-nitrophenyl-â-D-galactopyranoside, N-methyl-
n,n-diethanolamine, and cholesterol were purchased from Sigma-
Aldrich, St. Louis, MO, U.S.A. NP-40, antibiotics, and agarose were
procured from Hi-media, India. Unless otherwise stated all reagents
were purchased from local commercial suppliers and were used without
further purification. COS-1, CHO, HepG2, and A549 cells were
procured from the National Centre for Cell Sciences (NCCS), Pune,
India. Cells were grown at 37 °C in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified
atomosphere containing 5% CO2/95% air. Elemental analyses (C, H,
N, Cl) for lipids 1 and 2 were carried out in VIMTA LABS, Hyderabad,
Inida.
Conclusions
In the present investigation, toward addressing an hitherto
unexplored issue in liposomal gene delivery, namely, the
influence of linker orientation, we have designed and synthesized
two structurally isomeric very similar cationic lipids 1 and 2
(Scheme 1) bearing the same hydrophobic tails and the same
polar headgroups connected by the same ester (linker) group.
The only structural difference between the cationic amphiphiles
1 and 2 is the opposite orientation of the linker ester functional-
ity. While lipid 1 showed high efficacies in delivering reporter
genes into multiple cultured mammalian cells including CHO,
COS-1, HepG2, and A549, lipid 2 was found to be essentially
incompetent in transferring genes into any of these cells.
Findings in both transmission electron microscopic and dynamic
laser light scattering studies revealed no significant size differ-
ence between the liposomes and the lipoplexes of lipids 1 and
2. Confocal microscopic studies using lipoplexes containing
fluorescently labeled plasmid DNA demonstrated a significantly
higher cellular uptake of DNA complexed with lipid 1 lipo-
somes. Results of fluorescence resonance energy transfer
(FRET) studies using cationic liposomes of lipids 1 and 2 and
biomembrane mimicking liposomal systems (DOPC/DOPE/
DOPS/Chol) containing fluorescently labeled donor (NBD-PE)
and acceptor (Rho-PE) lipids are consistent with the significantly
higher biomembrane fusogenicity of lipid 1 liposomes. Studies
on the thermotropic phase behaviors of the pure cationic
liposomes of lipids 1 and 2 using the techniques of differential
scanning calorimetry (DSC) and fluorescence anisotropy re-
vealed significantly less membrane rigidity of the lipid 1
Syntheses of N,N-Di-[O-tetradecanoyl-2-hydroxyethyl]-N-hy-
droxyethyl-N-methylammonium Chloride (Lipid 1, Scheme 1A).
Steps a and b. Synthesis of N,N-Di-[O-tetradecanoyl-2-hydroxy-
ethyl]-N-Methyl Amine (I, Scheme 1A): In a 50 mL single neck round
bottomed flask pyridine (2.7 mL, 33 mmol) was added at 0 °C to a
solution of n-tetradecanonic acid (5 g, 22 mmol) in dry DCM. Thionyl
chloride (2.4 mL, 33 mmol) was added to the reaction mixture at 0 °C,
and the reaction mixture was stirred at room temperature for 2 h. The
unreacted thionyl chloride and pyridine were removed on a rotavapor
by repeated chasing with dry DCM and N-methyl-N,N-diethanolamine
(1 g, 8.4 mmol) dissolved in dry DMF was added to the residue at 0
°C. The temperature was gradually raised to room temperature, and
the reaction mixture was stirred at room temparature for an additional
6 h. The resulting hydrochloride salt was filtered and washed with 20
mL of dry ether. Finally recrystallization from 5:15 (v/v) methanol/
ethylacetate afforded a pure hydrochloride salt intermediate. The
recrystalized hydrochloride salt was stirred for 5 min in a DCM (25
mL)/1 M aqueous NaOH (10 mL) biphasic system. The organic layer
was separated, dried over anhydrous sodium sulfate, filtered, and the
filtrate was concentrated on a rotary evaporator. The residue upon
chromatographic purification on a 60-120 mesh silica gel column using
2% methanol/CHCl3 as eluent afforded the intermediate tertiary amine
(27) McConnell, H. M.; Radhakrishnan, A. Biochim. Biophys. Acta 2003, 1610,
159-173.
(28) Hungerford, G.; Baptista, A. L. F.; Coutinho, P. J. G.; Castanheira, E. M.
S.; Oliveira, E. C. D. R. J. Photochem. Photobiol. A 2006, 181, 99-105.
9
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A R T I C L E S
Rajesh et al.
N,N-di-[O-tetradecanoyl-2-hydroxyethyl]-N-methyl amine (I, Scheme
1A) as a white solid (1.55 g, 13% yield, Rf ) 0.6, 5% methanol/CHCl3).
1H NMR (200 MHz, CDCl3): δ ppm ) 0.9 (t, 6H, -(CH2)-CH3),
1.2-1.4 (m, 40H, -O-CO-CH2-CH2-(CH2)10-), 1.6-1.7 (m, 4H,
-O-CO-CH2-CH2-(CH2)10-), 2.2 (t, 4H, -O-CO-CH2-CH2-
(CH2)10-), 2.35 (s, 3H, H3C-N-CH2-CH2-), 2.65 (t, 4H, -N-CH2-
CH2-), 4.1 (t, 4H, N-CH2-CH2-O-CO-). ESIMS m/z: 540 [M +
1+] (calcd for C33H65NO4, 100%).
Steps c and d. In a 25 mL round bottomed flask intermediate I
prepared in step b above (0.5 g, 0.9 mmol) and 2-bromoethanol (0.2 g,
1.56 mmol) were stirred at 85 °C for 4 h. Crystallization from 20 mL
of 1:4 (v/v) benzene/n-pentane followed by column chromatographic
purification (using 60-120 mesh silica gel and 4% methanol/CHCl3,
v/v, as eluent) and chloride ion exchange on Amberlyst A-26 resin
(with methanol as eluent) afforded pure lipid 1 (0.06 g, 11% yield, Rf
) 0.4, 10% methanol/chloroform).
1H NMR (300 MHz, CDCl3): δ ppm ) 0.9 (t, 6H, -(CH2)10-CH3),
1.2-1.4 (m, 40H, -O-CO-CH2-CH2-(CH2)10-), 1.5-1.7 (m, 4H,
-O-CO-CH2-CH2-(CH2)10-), 2.35 (t, 4H, CH3(HOCH2-CH2)N+-
(CH2-CH2-O-CO-CH2-(CH2)10-CH3)2, 3.25 (s, 3H, CH3(HOCH2-
CH2)N+(CH2-CH2-O-CO-CH2-(CH2)10-CH3)2), 3.5-4.0 (m, 8H,
CH3(HOCH2-CH2)N+(CH2-CH2-O-CO-CH2-(CH2)10-CH3)2), 4.45
(m, 4H, CH3(HOCH2-CH2)N+(CH2-CH2-O-CO-CH2-(CH2)10-
CH3)2). ESIMS m/z: 585 [M + 1+] (calcd for C35H70NO5, 100%).
Synthesis of N,N-Di-(2-(n-tridecyloxycarbonyl)ethyl)-N-methyl,N-
(2-hydroxyethyl)ammonium Chloride (Lipid 2, Scheme 1B): Step
e. Synthesis of O-tert-Butyl-dimethylsilyl-2-ethanolamine (II, Scheme
1B): A solution of 2-aminoethanol (0.5 g, 8.2 mmol) dissolved in 2
mL of dry DCM was transferred to a 50 mL two-neck round-bottom
flask, and the solution was cooled to 0 °C. Imidazole (0.84 g, 12.3
mmol) dissolved in 3 mL of dry DCM was added to this cold solution
at 0 °C. tert-Butyl-dimethylsilylchloride (1.85 g, 12.3 mmol) in 5 mL
of DCM was added to the cold reaction mixture and stirred for 15 min
at 0 °C. The temperature was gradually raised to room temperature,
and stirring was continued for an additional 12 h. The reaction mixture
was then diluted with CHCl3 (100 mL) and washed with water (2 ×
50 mL). The organic layer was separated, dried over anhydrous Na2-
SO4, and concentrated on a rotary evaporator. The residue, upon
chromatographic purification on a 60-120 mesh size silica gel column
using 2% methanol/chloroform (v/v) as the eluent, afforded the title
compound as a light brown liquid (0.9 g, 64% yield, Rf ) 0.6, 5%
methanol/chloroform).
Step g. Synthesis of N-2-Hydroxyethyl-N,N-di-(2-tridecyloxycar-
bonyl)ethylamine (IV, Scheme 1B). Compoud III (0.15 g) prepared
in step f above was dissolved in 2 mL of THF at 0 °C in a 25 mL
round bottomed flask, and 2 mL of tetra-n-butyl ammonium (TBAF, 1
M in THF) were added to the cold solution. The temperature of the
reaction mixture was gradually raised to room temperature, and stirring
at room temperature was continued for 6 h. The reaction mixture was
concentrated on a rotavapor, and the residue was diluted with 50 mL
of CHCl3 and washed with water (2 × 25 mL). The organic layer was
separated, dried over anhydrous sodium sulfate, filtered, and concen-
trated. The residue upon column chromatographic purification using a
60-12 mesh size silica gel column and 15-20% ethylacetate/hexane,
v/v, as eluent afforded the pure title compound IV as a colorless liquid
(50 mg, 55% yield, Rf ) 0.5, 30% ethylacetate/hexane).
1H NMR (200 MHz, CDCl3): δ ppm ) 0.9 (t, 6H, -(CH2)-CH3),
1.2-1.4 (m, 40H, CO-O-CH2-CH2-(CH2)10-), 1.6-1.7 (m, 4H,
CO-O-CH2-CH2-(CH2)10-), 2.4-2.5 (t, 4H, N(-CH2-CH2-CO-
O-), 2.6 (t, 2H, -N-CH2-CH2-O-), 2.8 (t, 4H, -N-CH2-CH2-
CO-O-) 3.6 (t, 2H, -N-CH2-CH2-O-), 4.1 (t, 4H, N(-CH2-
CH2-CO-O-CH2-). ESIMS m/z: 570 [M + 1+] (calcd for C34H67NO5,
100%).
Steps h and i. Synthsis of N,N-di-(2-(n-tridecyloxycarbonyl)ethyl)-
N-methyl,N-(2-hydroxyethyl)ammonium Chloride (Lipid 2, Scheme
1B): Intermediate IV (50 mg) prepared in step g above was dissolved
in 1 mL of DCM in a 25 mL round-bottom flask, and 1 mL of methyl
iodide was added to the solution. The reaction mixture was stirred at
room temperature for 4 h and concentrated. Crystallization from 20
mL of 1:5 (v/v) ethylacetate/n-pentane followed by chloride ion
exchange on Amberlyst A-26 resin (using chloroform as eluent)
afforded pure lipid 2 as a white solid (0.04 g, 78% yield, Rf ) 0.3, 5%
methanol/chloroform).
1H NMR (200 MHz, CDCl3) : δ ppm ) 0.9 (t, 6H, -(CH2)10-
CH3), 1.2-1.4 (m, 40H, -CO-O-CH2-CH2-(CH2)10-), 1.6-1.7 (m,
4H, CO-O-CH2-CH2-(CH2)10-), 2.9 (t, 4H, N(-CH2-CH2-CO-
O-)), 3.4 (s, 3H, H3C-N+-CH2-CH2-), 3.8-3.9 (t, 2H, -N+-CH2-
CH2-OH), 4.1 (t, 4H, N(-CH2-CH2-CO-O-CH2-). ESIMS m/z:
585 [M + 1+] (calcd for C35H70NO5, 100%).
Step j. Synthesis of n-Tridecyl-3-bromopropionate (Scheme 1C):
A solution of 3-bromopropanoic acid (1 g, 6.5 mmol) in 5 mL of dry
DCM was taken in a 50 mL single-neck round-bottom flask, and the
solution was cooled to 0 °C. Sequentially pyridine (0.8 mL, 9.8 mmol)
and thionyl chloride (0.8 mL, 9.8 mmol) were added to the cold solution
at 0 °C. The temperature was gradually raised to room temperature,
and the reaction mixture was stirred at room temperature for 2 h. The
unreacted thionyl chloride and pyridine were removed on a rotavapor
by repeated chasing with dry DCM, and n-tridecyl alcohol (1.6 g, 8.0
mmol) dissolved in 5 mL of dry DCM was added to the residue. The
reaction mixture was stirred at room temperature for 6 h, diluted with
100 mL of CHCl3, and washed with water (2 × 50 mL). The organic
layer was separated, dried over anhydrous sodium sulfate, and
concentrated by rotary evaporator. The residue, upon column chro-
matographic purification over 60-120 mesh size silica gel column using
2% ethylacetate/hexane (v/v) as eluent, afforded the pure title compound
as a colorless liquid (1.2 g, 51% yield, Rf ) 0.7, 10% ethylacetate/
hexane).
1H NMR (200 MHz, CDCl3): δ (ppm) ) 0.1 (s, 6H, -Si-(CH3)2),
0.9 (s, 9H, Si-C(CH3)3), 2.7 (t, 2H, H2N-CH2-CH2-), 3.6 (t, 2H,
CH2-CH2-O-Si-).
Step f. Synthesis of N-2-tert-Butyl-dimethylsilyloxyethyl-N,N-di-
(2-n-tridecyloxycarbonyl)ethyl Amine (III, Scheme 1B): A mixture
of intermediate II prepared above in step e (0.15 g, 0.8 mmol), the
bromo-ester intermediate V (0.71 g, 2.12 mmol, prepared as described
below in step j), and K2CO3 (0.35 g, 2.55 mmol) in 5 mL of ethylacetate
was taken in a 25 mL round-bottom flask, and the reaction mixture
was stirred at 60 °C for 48 h. The reaction mixture was diluted with
50 mL of ethylacetate and washed with water (2 × 30 mL). The organic
layer was separated, dried over anhydrous sodium sulfate, filtered, and
concentrated on a rotavapor. The residue upon chromatographic
purification using a 60-120 mesh size silica gel column and 5%
ethylacetate/hexane, v/v, as eluent afforded the pure title compound as
a colorless liquid (0.15 g, 25% yield, Rf ) 0.4, 10:90 ethylacetate/
hexane).
1H NMR (200 MHz, CDCl3): δ (ppm) ) 0.9 (t, 3H, H3C-CH2-
(CH2)10-), 1.3-1.4 (m, 20H, H3C(-CH2)10-CH2-), 1.7 t, 2H, -CH2-
CH2-O-CO-), 2.9 (t, 2H, (-O-CO-CH2-CH2-), 3.7 (t, 2H, Br-
CH2-CH2-), 4.2 (t, CH2-CH2-O-CO-).
1H NMR (200 MHz, CDCl3): δ ppm ) 0.1 (s, 6H, -Si-(CH3)2-),
0.9 (s and t, 15H, Si-C(CH3)3, -CH2-(CH2)10-CH3), 1.2-1.4 (m,
40H, -CO-O-CH2-CH2-(CH2)10-CH3),1.6-1.7 (m, 4H, -CO-
O-CH2-CH2-(CH2)10-), 2.45 (t, 4H, N-(CH2-CH2-CO-O), 2.6
(t, 2H, N-(CH2-CH2-OH), 2.85 (t, 4H, N-CH2-CH2-CO-O), 3.6
(t, 2H, -N-CH2-CH2-OTBDMS), 4.1 (t, 4H, N-CH2-CH2-CO-
O-CH2-).
Preparation of Liposomes. The cationic lipid and cholesterol in
the appropriate mole ratios were dissolved in a mixture of chloroform
and methanol (3:1, v/v, 500 µL) in a glass vial. The solvent was
removed with a thin flow of moisture-free nitrogen gas, and the dried
lipid film was kept under high vacuum for 8 h. A 1 mL aliquot of
sterile deionized water was added to the vacuum-dried lipid film, and
the mixture was allowed to swell overnight. The vial was then vortexed
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11418 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007

Linker-Orientation Influence in Lipofection
A R T I C L E S
for 2-3 min at room temperature to produce multilamellar vesicles
(MLVs). MLVs were then sonicated in an ice bath until clarity using
a Branson 450 sonifier at 100% duty cycle and 25 W output power to
produce small unilamellar vesicles (SUVs).
Plasmid DNA. p-CMV-SPORT-â-gal plasmid was amplified in
DH5R-strain of Escherichia coli, isolated by alkaline lysis procedure
and finally purified by PEG-8000 precipitation as described previously.8a
The purity of plasmid was checked by A260/A280 ratio (around 1.9) and
1% agarose gel electrophoresis.
solution (24 µL) was loaded on each well. The samples were
electrophoresed at 80 V for 45 min, and the DNA bands were visualized
in the gel documentation unit.
DNase I Sensitivity Assay. Briefly, in a typical assay, pCMV-
SPORT-â-gal plasmid (1 µg) was complexed with a varying amount
of cationic lipids in a total volume of 30 µL in Hepes buffer, pH 7.40,
and incubated at room temperature for 30 min on a rotary shaker.
Subsequently, the complexes were treated with 10 µL of DNase I (at
a final concentration of 1 µg/mL) in the presence of 20 mM MgCl2
and incubated for 20 min at 37 °C. The reactions were then halted by
adding EDTA (to a final concentration of 50 mM) and incubated at
60 °C for 10 min in a water bath. The aqueous layer was washed with
50 µL of phenol/chloroform/isoamylalcohol (25:24:1mixture, v/v) and
centrifuged at 10 000 g for 5 min. The aqueous supernatants were
separated, loaded (20 µL) on a 1% agarose gel (prestained with
ethydium bromide), and electrophoresed at 100 V for 1 h.
Transfection of Cells. Cells were seeded at a density of 10 000
cells (for CHO and COS-1) and 15 000 cells (for A549 and HEPG2)
per well in a 96-well plate 18-24 h before the transfection. A 0.3 µg
amount of plasmid DNA was complexed with varying amounts of lipids
(0.45-7.2 nmol) in a plain DMEM medium (total volume made up to
100 µL) for 30 min. The lipid/DNA (() charge ratios were from 0.5:1
to 8:1 over these ranges of the lipids. The complexes were then added
to the cells. After 3 h of incubation, DMEM was removed, and a 10%
complete medium was added to the cells. The medium was changed
with a 10% complete medium after 24 h, and the reporter gene activity
was estimated after 48 h. The cells were washed with PBS (2 × 100
µL) and lysed with 50 µL of lysis buffer [0.25 M Tris-HCl pH 8.0,
0.5% NP40]. Care was taken to ensure complete lysis. The â-galac-
tosidase activity per well was estimated by adding 50 µL of 2X-substrate
solution [1.33 mg/mL of o-nitrophenyl-â-D-galactopyranoside (ONPG),
0.2 M sodium phosphate (pH 7.3), and 2 mM magnesium chloride] to
the lysate in a 96-well plate. Absorption at 405 nm was converted to
â-galactosidase units using a calibration curve constructed with pure
commercial â-galactosidase enzyme. The values of â-galactosidase units
in triplicate experiments assayed on the same day varied by less than
20%. The transfection experiment was carried out in duplicate, and
the transfection efficiency values shown in Figure 1 are the average of
triplicate experiments performed on the same day. The day-to-day
variation in the average transfection efficiency was found to be within
2-fold. The transfection profiles obtained on different days were
identical.
Transmission Electron Microscopy. Transmission electron mi-
croscopy was performed on a FEI Tecnai 12 TEM apparatus operated
at 100 kV. Lipoplex samples were transferred onto an ultrathin-carbon
coated copper grid by placing the grid on top of a 10 µL drop of the
sample for 1 min. After removing the excess fluid from one side, the
grid was placed on a 20 µL water drop for a 30 s wash. The excess
fluid was removed, and the grid was placed for 1 min on a 20 µL drop
of freshly filtered uranyl acetate (1.33%). Once again, the excess fluid
was wicked away, and the grid was air-dried.
DNA Uptake Study by Confocal Microscopy: For confocal
microscopy experiments, 100 000 cells were seeded in each well of a
24-well plate and cells were incubated with lipoplexes (with 1:1 lipid/
DNA charge ratio) of lipids 1 and 2 in 200 µL of DMEM containing
25 µg of fluorescein-labeled p-CMV-SPORT-â-gal plasmid DNA
(prepared by “random primed” fluoroscein-12-dUTP labeling of
p-CMV-SPORT-â-Gal following the manufacturer’s instructions avail-
able with Roche’s Fluoroscein High Prime Kit, Catalog No. 1 585 622)
for 4 h at 37 °C. DMEM was removed, and the cells were washed
with PBS (2 × 200 µL) and trypsinized with 0.1% Trypsin/EDTA
solution. A 500 µL aliquot of complete medium was added to the
trypsinized cell suspension, the suspension ws centrifuged for 3 min
at 2000 rpm, and the complete medium was removed. PBS (500 µL)
was added to the cells, and the cell suspension was once more
centrifuged for 3 min at 2000 rpm. The supernatant was removed, and
the cell pellets were resuspended in 500 µL of PBS. A 20 µL aliquot
of this cell suspension was applied to a glass slide, a cover slip was
put on the cells on the glass slide, and the slide was then mounted
onto the confocal microscope (Leica TCS SP2 organic laser scanner)
at a 200 magnification. The cells containing fluorescein-labeld DNA
were then viewed using the 488 nm excitation line of krypton/argon
laser, and the green fluorescences were detected at 514-550 nm.
FRET Assay. The membrane fusion activity of both Chol/lipid 1
(1:1 mole ratio) and Chol/lipid 2 (1:1 mole ratio) liposomes were
measured with the FRET assay essentially as described previously.12
NBD-PE and Rho-PE (Avanti-Polar Lipids, USA) were used as the
donor and acceptor fluorescent lipids, respectively. The liposome
DOPC/DOPE/DOPS/Chol (45:20:20:15, w/w ratio, the total lipid
concentration used was 0.5 mM) was used as the biomembrane
mimicking lipid formulation, and this liposomal formulation was labeled
with the donor and acceptor lipids. The concentrations of both donor
and acceptor lipids were 0.005 mM (i.e., 1% with respect to the total
biomembrane mimicking lipid content). The total lipid concentrations
used in both the Chol/lipid 1 (1:1 mole ratio) and Chol/lipid 2 (1:1
mole ratio) liposomes were the same as those of the biomembrane
micking lipid formulation (0.5 mM). Labeled model biomembrane
liposomal formulations were placed in an FLX 800 Microplate Fluo-
roscence Reader (BioTek Instruments Inc., U.K.) at room temperature,
and equimolar amounts of Chol/lipid 1 (1:1 mole ratio) and Chol/lipid
2 (1:1 mole ratio) liposomes were added. Fluorescence intensities were
recorded as a function of time with excitation at 485 nm and emission
Toxicity assay. Cytotoxicities of the lipids 1 and 2 were assessed
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reduction assay as described earlier.10a The cytotoxicity assay
was performed in 96-well plates by maintaining the same ratio of
number of cells to amount of cationic lipid, as used in the transfection
experiments. MTT was added 3 h after addition of cationic lipid to the
cells. Results were expressed as percent viability ) [A540(treated cells)
- background/A540(untreated cells) - background] × 100.
Zeta Potential (ê) and Size Measurments. The sizes and the surface
charges (zeta potentials) of liposomes and lipoplexes were measured
by photon correlation spectroscopy and electrophoretic mobility on a
Zeta sizer 3000HSA (Malvern UK). The sizes were measured in
deionized water with a sample refractive index of 1.59 and a viscosity
of 0.89. The system was validated by using the 200 nm + 5 nm
polystyrene polymer (Duke Scientific Corps. Palo Alto, CA). The
diameters of liposomes and lipoplexes were calculated by using the
automatic mode. The zeta potential was measured using the following
parameters: viscosity, 0.89 cP; dielectric constant, 79; temperature,
25 °C; F(Ka), 1.50 (Smoluchowski); maximum voltage of the current,
V. The system was validated by using DTS0050 standard from Malvern,
U.K. Measurements were done 10 times with the zero-field correction.
The potentials were calculated by using the Smoluchowski approxima-
tion.
DNA Binding Assay. The DNA binding ability of the cationic lipids
1 and 2 were assessed by their gel retardation assay on a 1% agarose
gel (prestained with ethydium bromide) across the varying lipid/DNA
charge ratios of 0.5:1 to 8:1. pCMV-â-gal (0.30 µg) was complexed
with the varying amount of cationic lipids in a total volume of 20 µL
in Hepes buffer, pH 7.40, and incubated at room temperature for 20-
25 min. A 4 µL aliquot of 6X loading buffer (0.25% Bromophenol
blue in 40% (w/v) sucrose in H2O) was added to it, and the resulting
9
J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007 11419

A R T I C L E S
Rajesh et al.
at 595 nm. Fusion (100%) was determined from the Rho-PE fluores-
cence intensity of the labeled biomembrane liposomal formulation in
the presence of 1% Triton X100.
at 25 °C by following the changes in fluorescence intensity of the
emitted light (due to leakage of CF from liposomes) in 60 min in an
FLX 800 Microplate Fluoroscence Reader (BioTek Instruments Inc.,
U.K.). The excitation and emission wavelengths used for this experiment
were 485 and 528 nm, respectively. Percentage CF releases were
calculated using the equation: Permeation of CF (%) ) [(Ft - Fi)/ (Ff
- Fi)] × 100 where Fi, Ft, and Ff are the fluorescence emission values
at time t ) 0, at time t, and the final fluorescence on complete disruption
of liposomal structures with the final concentration of 1% (v/v) Triton
X-100.
Differential Scanning Calorimetry. Lipids 1 and 2 (2.2 mg) were
dissolved in 150 µL of choloroform, and the solvent was removed by
chasing with a gentle flow of dry N2 gas. The residual solvent was
removed under high vacuum for 6-8 h. The dry lipid films were
hydrated with 250 µL of pure deionized water at room temperature
overnight, vortexed, and sonicated to clarity in a bath sonicator
(ULTRAsonik 28X). Differential scanning calorimetry (DSC) experi-
ments with the resulting liposomal samples (50 µL of liposomes
containing 0.43 mg of lipids) were performed on an 821e Mettler-Toledo
(Schwerzenbach, Switzerland) calorimeter. Sealed aluminum crucibles
of 50 µL capacity were used as sample holders. The scan rates for
heating were 5 °C/min. Thermograms were obtained between 0 and
70 °C and were referenced against deionized water.
Fluorescence Anisotropy Measurements. Lipids 1 and 2 (0.58 mg)
were dissolved in 100 µL of choloroform along with DPH at a lipid/
DPH mole ratio of 300:1 and dried under a gentle flow of dry N2 gas.
The residual solvent was removed under high vacuum for 6-8 h. The
dried lipid films were hydrated in 1 mL of buffer (Tris-HCl, pH 7.4,
100 mM) overnight, vortexed, and sonicated to clarity in a bath sonicator
(ULTRAsonik 28X). The resulting liposomal solutions were used for
anisotropy measurements. Anisotropy was measured by recording the
DPH fluorescence values (excitation at 354 nm and emission at 427
nm) in parallel and perpendicular polarizer positions in a Fluorolog
3-22 fluorescence spectrophotometer. The fluorescence intensity of the
emitted light polarized parallel (I|) and perpendicular (I ) to the excited
light across the temperature range 15-60 °C (scan rate 5 °C/min, the
samples were allowed to equilibrate for 5 min between the successive
scans). The fluorescence anisotropy values (r) were calculated by the
instrument software using the Perrin equation r ) (I|) - (GI )/(I|) +
(GI ) where G is the instrumental grating factor.
Acknowledgment. Financial support received from the
Department of Biotechnology, Government of India (to A.C.)
is gratefully acknowledged. M.R. thanks the Council of
Scientific and Industrial Research (CSIR), Government of India,
for his doctoral research fellowship; J.S. and K.M. thank the
University Grant Commission, Government of India, for their
doctoral research fellowships. We sincerely thank Dr. Anand
K. Kondapi and Mr. A. D. Saikrishna, University of Hyderabad,
for their help in conducting the confocal microscopic experi-
ments in their laboratory. We gratefully acknowledge the help
from Dr. N. M. Rao and Mr. Shoeb Ahmed, Centre for Cellular
and Molecular Biology, in taking fluorescence anisotropy
measurements in their laboratory.
Note Added after ASAP Publication. After this paper was
published ASAP on August 25, 2007, an error was corrected
in the heading for Scheme 1C. The corrected version was
published ASAP on August 31, 2007.
Supporting Information Available: 1H NMR (200 MHz)
spectra of all the synthetic intermediates shown in Scheme 1
as well as those of final target lipids 1 and 2, ESI mass spectra
for the final lipids 1 and 2, reversed phase HPLC chromatograms
for lipids 1 and 2 in two mobile phases and the details of the
HPLC conditions, TEM pictures of liposomes and lipoplexes,
percent cell viability data, epifluorescence micrographs for
cellular uptake experiments using labeled liposomes, FRET
results based on increasing NBD fluorescence, percent CF
leakage data in dye entrapment experiments, and elemental
analysis data for lipids 1 and 2. This material is available free
of charge via the Internet at http://pubs.acs.org.
Dye Entrapment Experiment. The protocols followed for liposomal
entrapment of 5/6-CF and for monitoring its subsequent release from
the liposomes were essentially as described previously.29 Briefly,
equimolar cholesterol containing liposomal suspensions of lipids 1 and
2 (1 mM total lipid concentration; 0.5 mM in both cationic lipid and
cholesterol) were prepared in Tris-HCl buffer (25 mM, pH 8.0)
containing 25 mM CF. The CF-loaded liposomes were separated from
the unentrapped free CF dye by passing through a sephadex G-50
column with an elution buffer of 50 mM Tris-HCl buffer (pH 8.0).
Transmembrane permeation of the liposomally loaded CF was measured
(29) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta 2000, 1467, 39-53.
JA0704683
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11420 J. AM. CHEM. SOC. VOL. 129, NO. 37, 2007