Design, syntheses, and transfection biology of novel non-cholesterol-based guanidinylated cationic lipids

Article abstract of DOI:10.1021/jm049417w

The design of efficacious cationic transfection lipids with guanidinium headgroups is an actively pursued area of research in nonviral gene delivery. Herein, we report on the design, syntheses, and gene transfection properties of six novel non-cholesterol-based cationic amphiphiles (1-6) with a single guanidinium headgroup in transfecting CHO, COS-1, MCF-7, A549, and HepG2 cells. The in vitro gene transfer efficiencies of lipids 1-6 were evaluated using both the reporter gene and the whole cell histochemical X-gal staining assays. The efficiencies of lipids 1-3, in particular, were found to be about 2- to 4-fold higher than that of commercially available LipofectAmine in transfecting COS-1, CHO, A-549, and MCF-7 cells. However, the relative transfection efficiencies of lipids 1-3 and LipofectAmine were found to be comparable in HepG2 cells. Cholesterol was found to be a more efficacious co-lipid than dioleoyllphosphatidyl ethanolamine (DOPE). In general, lipids 1-3 containing the additional quaternized centers were observed to be more transfection efficient than lipids 4-6 with less positive headgroups. MTT-assay-based cell viability measurements in representative CHO cells revealed high (>75%) cell viabilities of lipids 1-6 across the lipid/DNA charge ratios 0.1:1 to 3:1. Electrophoretic gel patterns observed in DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 might play some role in diminishing their in vitro gene transfer efficacies. Size and global surface charge measurement by a dynamic laser light scattering instrument equipped with ζ-sizing capacity revealed the nanosizes and surface potentials of both the transfection efficient and the incompetent lipoplexes to be within the range of 200-600 nm and +3.4 to -34 mV, respectively. To summarize, given the feasibility of a wide range of structural manipulations in the headgroup regions of non-cholesterol-based cationic amphiphiles, our present findings are expected to broaden the potential of cationic amphiphiles with guanidinium headgroups for use in nonviral gene therapy.

Full text of DOI:10.1021/jm049417w

8
12
J. Med. Chem. 2005, 48, 812-820
Design, Syntheses, and Transfection Biology of Novel Non-Cholesterol-Based
Guanidinylated Cationic Lipids
Joyeeta Sen and Arabinda Chaudhuri*
Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received July 22, 2004
The design of efficacious cationic transfection lipids with guanidinium headgroups is an actively
pursued area of research in nonviral gene delivery. Herein, we report on the design, syntheses,
and gene transfection properties of six novel non-cholesterol-based cationic amphiphiles (1-6)
with a single guanidinium headgroup in transfecting CHO, COS-1, MCF-7, A549, and HepG2
cells. The in vitro gene transfer efficiencies of lipids 1-6 were evaluated using both the reporter
gene and the whole cell histochemical X-gal staining assays. The efficiencies of lipids 1-3, in
particular, were found to be about 2- to 4-fold higher than that of commercially available
LipofectAmine in transfecting COS-1, CHO, A-549, and MCF-7 cells. However, the relative
transfection efficiencies of lipids 1-3 and LipofectAmine were found to be comparable in HepG2
cells. Cholesterol was found to be a more efficacious co-lipid than dioleoyllphosphatidyl
ethanolamine (DOPE). In general, lipids 1-3 containing the additional quaternized centers
were observed to be more transfection efficient than lipids 4-6 with less positive headgroups.
MTT-assay-based cell viability measurements in representative CHO cells revealed high (>75%)
cell viabilities of lipids 1-6 across the lipid/DNA charge ratios 0.1:1 to 3:1. Electrophoretic gel
patterns observed in DNase I protection experiments support the notion that enhanced
degradation of DNA associated with lipoplexes of lipids 4-6 might play some role in diminishing
their in vitro gene transfer efficacies. Size and global surface charge measurement by a dynamic
laser light scattering instrument equipped with ú-sizing capacity revealed the nanosizes and
surface potentials of both the transfection efficient and the incompetent lipoplexes to be within
the range of 200-600 nm and +3.4 to -34 mV, respectively. To summarize, given the feasibility
of a wide range of structural manipulations in the headgroup regions of non-cholesterol-based
cationic amphiphiles, our present findings are expected to broaden the potential of cationic
amphiphiles with guanidinium headgroups for use in nonviral gene therapy.
Introduction
DNA to the nucleus followed by its transgene ex-
pression.2
0-23
Thus, rational design of efficient cationic
Design of safe and efficacious gene transfer reagents
continues to remain as one of the key challenges in gene
therapy, the therapeutic modality to combat myriads
of inherited diseases, dreadful viral infections, and
cancer. Viral vectors, although remarkably efficient in
transfection lipids are often based on covalent grafting
of various structural elements (into the molecular
architecture of the cationic lipids) capable of favorably
modulating one or more of these mechanistic steps
involved in lipofection pathways.1
9,24-26
In 1996, a
transfecting body cells, suffer from numerous biosafety-
related disadvantages.1-3 Conversely, cationic am-
particularly elegant design of efficient cationic trans-
fection lipid was pioneered by Vigneron et al.27 The
distinguishing features of the guanidinium functionality
exploited in their design include: (a) the guanidinium
group remains protonated over a much wider range of
pH than other basic groups due to its remarkably high
pKa values (13.5); (b) it forms characteristic parallel
phiphiles, because of their least immunogenic nature,
robust manufacture, ability to deliver large pieces of
DNA, and ease of handling and preparation techniques,
are increasingly becoming the alternative nonviral
4
-14
transfection vectors of choice,
our own work in-
1
5-19
cluded.
+
-
zwitterionic hydrogen bonds N-H ‚‚‚O with phosphate
ions; (c) the guanidinium groups are also capable of
forming hydrogen bonds with nucleic acid bases; (d) the
guanidinium group of the arginyl residues plays a major
role in DNA-binding proteins such as histones and
protamines.27 The rationales for using cholesterol as the
hydrophobic tail were its membrane-compatible char-
acteristics and its ability to facilitate the cellular uptake
of various oligonucleotides and polar drugs.27 It is
pertinent to mention here that the design of an aliphatic-
hydrocarbon-tail-based cationic lipid with an amidine
headgroup, a headgroup structurally close to guanidine,
was reported earlier by Ruysschaert et al. in 1994.28
Design of an efficient cationic transfection lipid needs
strategies to deal with a multitude of cellular barriers.
Currently believed lipoplex (lipid/DNA complex)-medi-
ated intracellular transfection pathways involve: (a)
formation of lipid/DNA complexes and their initial
binding to the cell surface; (b) endocytotic internaliza-
tion of the lipid/DNA complexes; (c) trafficking in the
endosome/lysosome compartment and escape of DNA
from the endosome/lysosome compartment to the cell
cytoplasm; (d) transport of the endosomally released
*
Author to whom correspondence should be addressed. Phone: 91-
4
0-27193201. Fax: 91-40-27160757. E-mail: arabinda@iict.res.in.
1
0.1021/jm049417w CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/08/2005

Guanidinylated Cationic Lipids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 813
Chart 1. Structures of Novel Non-Cholesterol-Based
Guanidinylated Cationic Lipids 1-6
Scheme 1a
Ever since the development of the above-mentioned
cholesterol and aliphatic-hydrocarbon-tail-based cationic
transfection lipids with guanidinium and amidine head-
groups were reported, investigations aimed at broaden-
ing the scope of both cholesterol- and non-cholesterol-
based cationic transfection lipids with guanidine or
amidine headgroups have remained unabated.29-45
Herein, we report on the design, syntheses, and gene
transfection properties of six novel non-cholesterol-based
nontoxic cationic amphiphiles (1-6, Chart 1) with a
single guanidinium headgroup in transfecting CHO,
COS-1, MCF-7, A549, and HepG2 cells. As delineated
below, the efficiencies of lipids 1-3, in particular, were
found to be about 2- to 4-fold higher than that of
commercially available LipofectAmine in transfecting
COS-1, CHO, A-549, and MCF-7 cells. Given the feasi-
bility of a wide range of structural manipulations in the
headgroup regions of non-cholesterol-based cationic
amphiphiles, present findings are expected to further
broaden the potential of cationic amphiphiles with
guanidinium headgroups for use in nonviral gene
therapy.
a
Reagents and conditions: (i) HgCl2 (1 equiv), TEA (1 equiv),
DMF, N2 atmospheric pressure, 0 °C; (ii) MeI; (iii) TFA/DCM (1:
); (iv) Cl ion-exchange resin; (v) DIEA (1 equiv), DMF/DCM.
1
lating the gene transfer properties of non-cholesterol-
based guanidinylated cationic lipids. Structures of all
of the synthetic intermediates and the target lipids
shown in Scheme 1 were confirmed by H NMR and
LSIMS.
1
Transfection Biology. Figure 1 summarizes the
relative in vitro gene delivery efficacies of lipids 1-6 in
transfecting CHO, COS-1, HepG2, MCF7, and A549
cells. In these in vitro transfection biology experiments,
the cationic liposomes of lipids 1-6 were prepared in
combination with an equimolar amount of cholesterol
as the co-lipid, and pCMV-SPORT-â-gal plasmid DNA
was used as the reporter gene across the lipid/DNA
charge ratios of 9:1 to 0.1:1. The in vitro gene transfer
efficiencies of lipids 1-6 were evaluated using both the
reporter gene and the whole cell histochemical X-gal
staining assays. In the reporter gene assay, the in vitro
gene transfer efficiencies of lipids 1-3 with no free
tertiary amine base centers were found to be remark-
ably efficient, about 2- to 4-fold higher than that of
commercially available LipofectAmine in transfecting
COS-1, CHO, A-549, and MCF-7 cells (Figure 1, parts
A-D, respectively). However, the relative transfection
efficiencies of lipids 1 and 2 and LipofectAmine were
found to be comparable in HepG2 cells (Figure 1, part
E). Interestingly, for the present lipids, cholesterol was
found to be a more efficacious co-lipid than 1,2-dioleoyl-
sn-glycero-3-phosphoethanolamine (DOPE). Represen-
tative transfection profiles using DOPE as a co-lipid are
shown in Figure S7 of the Supporting Information. In
general, the dicationic guanidinylated lipids 1-3 con-
taining the additional quaternized centers were ob-
served to be more transfection efficient than lipids 4-6
with less positive headgroups, and the optimal trans-
fection properties were observed with the lipid/DNA
charge ratio of 1:1 and 3:1 (Figure 1). Interestingly, only
lipid 6 was found to be essentially incompetent in
transfecting all five of the cells across all the lipid/DNA
charge ratios (Figure 1). Similar transfection profiles
Results and Discussion
Chemistry. The hydrophobic mixed primary-ter-
tiary amine (I, Scheme 1, a readily available precursor
1
9
in our laboratory prepared as described previously )
was reacted with 1 equiv each of N,N-di-tert-butyloxy-
carbonyl thiourea (II, Scheme 1, prepared by reacting
thiourea with 2 equiv of Boc-anhydride (Boc ) tert-
butyloxycarbonyl) in the presence of 2 equiv of sodium
hydride in anhydrous tetrahydrofuran as described
4
6
previously ), mercuric chloride, and triethylamine in
anhydrous N,N-dimethylformamide/dichloromethane un-
der nitrogen atmosphere at 0 °C followed by the usual
workup. The resulting tertiary amine intermediate III
(
Scheme 1) upon quaternization with excess methyl
iodide followed by acid deprotection and chloride ion-
exchange chromatography afforded lipids 1-3 (Scheme
1, part A). The mono-guanidinylated cationic lipids 4-6
containing a free tertiary amine center were prepared
by reacting 1 equiv of the common mixed primary-
tertiary amine intermediate I (Scheme 1, part B) with
1
equiv of 1H-pyrazole-1-carboxamidine hydrochloride
(IV, Scheme 1, part B, prepared by reacting pyrazole
with 1 equiv of cyanamide in anhydrous p-dioxan
containing few drops of concentrated HCl) in the pres-
ence of 1 equiv of diisopropylethylamine in anhydrous
N,N-dimethylformamide/dichloromethane. Lipids 4-6
were designed with a view to probe the role of an
additional basic tertiary amine center, if any, in modu-

8
14 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Sen and Chaudhuri
Figure 1. In vitro transfection efficiencies of lipids 1-6 in COS-1 (A), CHO (B), A-549 (C), MCF-7 (D), and HepG2 (E) 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. The transfection efficiencies of the lipids were compared to that of commercially available
LipofectAmine 2000. Transfection experiments were performed as described in the text. The transfection values shown are the
average of duplicate experiments performed on the same day.
for lipids 1-6 were also observed in the whole cell
histochemical X-gal staining assay. A representative
X-gal staining pattern is shown in Figure 2 for trans-
fection efficient lipid 1 and transfection incompetent
lipid 6 in CHO cells. An important point seems ap-
propriate to bring at this point of discussion. Vigneron
et al. compared the transfection properties of their
cholesterol-based bis-guanidinylated lipids with that of
Lipofectin, one of the very first generations of liposomal
transfection kits then commercially availlable. Given
the usually higher in vitro transfection properties of
LipofectAmine-2000 (compared to Lipofectin) and the
superior transfection efficacies of lipids 1-3 to that of
LipofectAmine-2000 (Figure 1), the non-cholesterol-
based mono-guanidinylated cationic lipids described
herein will further broaden the versatility of using
guanidinylated cationic lipids in nonviral gene delivery.
In their original design of bis-guanidinium-tren-
cholesterol (BGTC), Vigneron et al.,27 the additional
tertiary amine center between the polar bis-guani-
dinium head and the hydrophobic cholesterol tail in the
lipid structure was grafted to ensure enhanced trans-
fection efficacies through buffering of the acidic endo-
some. The presence of an additional tertiary amine
center with pKa values much lower than that of guani-
dine was expected to buffer (to some extent) the acidic
environment of endosomes and thereby was expected
to protect the lipoplex associated DNA against assault
by lysosomal DNase. However, in our present study,
lipids 4-6 with an additional tertiary amine base were,
2
7

Guanidinylated Cationic Lipids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 815
Figure 2. Histochemical whole cell staining of transfected CHO cells with X-gal with lipid 1 and lipid 6 at lipid/DNA charge
ratios of 3:1 and 1:1. Cells expressing â-galactosidase were stained with X-gal as described in the text.
in general, found to be significantly less transfection
2) are unlikely to originate from varying cell cytotox-
icities of the lipids.
efficient than their quaternized counterparts lipids 1-3
(Figure 1). Such relative transfection profiles of lipids
Nanosizes and Global Surface Charges of the
Lipoplexes. Towards physicochemically characterizing
the present lipoplexes, the nanosizes and the global
surface charges of the representative lipoplexes pre-
pared with lipid 1 (highly transfection competent) and
lipid 6 (transfection incompetent) were measured using
a dynamic laser light scattering instrument equipped
with ú-sizing capacity across the lipid/DNA charge ratios
of 0.1:1 to 9:1 in the presence of Dulbecco’s modified
Eagle’s medium (DMEM). The nanosizes of lipoplexes
prepared from both the transfection efficient and the
incompetent lipids (1 and 6, respectively) similarly
increased with increasing lipid/DNA charge ratios within
the range 230-590 nm (Table 1). Interestingly, in the
presence of DMEM, although the global surface charges
of lipoplex 1 became less negative as the lipid/DNA
charge ratios increased from 0.1:1 to 3:1, that of the
lipoplexes prepared with lipid 6 remained remarkably
negative even at a lipid/DNA charge ratio of 3:1 (Table
1). Given that the biological membranes are also nega-
tively charged, such significantly high negative surface
potentials of lipoplex 6 may lead to some electrostatic
barrier to its cellular uptake process. Flow cytometric
studies will be needed in the future to gain better
mechanistic insights into whether the completely trans-
fection incompetent nature of lipid 6 originates from an
impeded cellular uptake process.
1
-3 and 4-6 indirectly imply that the presence of an
additional free tertiary amine center (with lower pKa
values) in the molecular structures of guanidinylated
cationic lipids is perhaps not needed for non-cholesterol-
based guanidinylated cationic lipids. Alternatively, en-
dosomal buffering for guanidinylated cationic transfec-
tion lipids may not be as important a mechanistic event
for non-cholesterol-based dicationic mono-guanidin-
ylated cationic transfection lipids as was originally
envisioned. for the cholesterol-based bis-guanidinylated
cationic lipids.27,33 Taken together, the results sum-
marized in Figures 1 and 2 convincingly demonstrate
the future potential of non-cholesterol-based mono-
guanidinylated cationic transfection lipids for use in
nonviral gene delivery.
MTT-based cell viabilities of both lipids 1-6 and
LipofectAmine were evaluated in representative CHO
cells across the entire range of lipid/DNA charge ratios
used in the actual transfection experiments. Cell vi-
abilities of both the lipids 1-3 and the lipids 4-6 were
found to be remarkably high (more than 75%) particu-
larly up to the lipid/DNA charge ratios of 3:1 (Figure
3
). However, except for lipid 6, high cell viabilities were
somewhat compromised at high lipid/DNA charge ratios
of 9:1 (Figure 3). Thus, the contrasting in vitro gene
transfer efficacies of lipids 1-3 and 4-6 (Figures 1 and

8
16 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Sen and Chaudhuri
Figure 3. MTT-assay-based cellular cytotoxicities of lipids 1-6 and LipofectAmine against representative CHO cells. The cell
viability values shown are the averages of duplicate experiments performed on the same day.
Table 1. Nanosizes (nm) and ú Potential (mV) of Lipoplexes in the Presence of Plain DMEMa
lipid/DNA charge ratio
sample code
9:1
3:1
1:1
0.3:1
0.1:1
sizes (nm)
lipid 1
lipid 6
lipid 1
lipid 6
585.3 ( 3.1
514.4 ( 7.0
1.7 ( 2.5
3.4 ( 3
408.7 ( 19.4
408.2 ( 11.3
-1.5 ( 2.5
229.1 ( 1.8
274.7 ( 3.6
-3.2 ( 2.7
-31.8 ( 2.2
216.2 ( 2.1
248.5 ( 3.8
-29.8 ( 1.6
-33.7 ( 0.8
233.8 ( 4.8
290.4 ( 3.6
-34.1 ( 1.3
-33.3 ( 0.8
ú potential(mV)
-23.7 ( 3.6
a
Sizes and ê potentials were measured by laser light scattering technique using ú sizer 3000A (Malvern Instruments, U.K.). Values
shown are the averages obtained from 3 (size) and 10 (ú potential) measurements.
Lipid/DNA Binding Interactions and Lipoplex
Sensitivities to DNase I. Finally, with a view to
characterize the electrostatic binding interactions be-
tween DNA and the present cationic liposomes as a
function of the lipid/DNA charge ratios, we performed
both the conventional electrophoretic gel retardation
assay and the DNase I sensitivity assays. Representa-
tive electrophoretic gel patterns observed in the simple
gel retardation assay for lipoplexes prepared from lipids
1, 3, 4, and 6 are shown in Figure 4. The lipids were
capable of completely inhibiting the electrophoretic
mobility of plasmid DNA from lipoplexes prepared at
high lipid/DNA charge ratios of 9:1 (Figure 4). However,
at lower lipid/DNA charge ratios of 3:1, more free DNA
was found to be present in lipoplexes of lipids 6
compared to the amount of free DNA present in the
lipoplexes prepared from lipids 1, 3, and 4 (Figure 4).
Such gel patterns are consistent with the notion that
relatively weaker lipid/DNA binding interactions might
play some role in abolishing the in vitro gene transfer
efficacies of lipid 6. However, at a lower range of lipid/
DNA charge ratios (1:1 to 0.1:1), all of the lipoplexes
showed the presence of a significant amount of free DNA
Figure 4. Electrophoretic gel patterns for lipoplex-associated
DNA in gel retardation (part A) for lipids 1 and 3 and (part
B) for lipids 4 and 6. The lipid/DNA charge ratios are indicated
at the top of each lane. The details of treatment are as
described in the text.
(
Figure 4).
The presence of relatively higher amounts of free
DNA in the lipoplexes prepared with relatively trans-
fection inefficient lipids 4 and 6 (compared to those in
lipoplexes 1 and 3) was more convincingly demonstrated
by monitoring the sensitivities of the lipoplexes upon

Guanidinylated Cationic Lipids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 817
taining the additional quaternized centers (absent in
lipids 4-6) were observed to be remarkably transfection
efficient. MTT-assay-based cell viability measurements
in representative CHO cells revealed high (>75%) cell
viabilities of lipids 1-6 across the lipid:DNA charge
ratios 0.1:1 to 3:1. Electrophoretic gel patterns observed
in DNase I protection experiments support the notion
that enhanced degradation of DNA associated with
lipoplexes of lipids 4-6 by cellular DNase I might
possibly play some role in diminishing their in vitro gene
transfer efficacies. Size and global surface charge mea-
surement by a dynamic laser light scattering instrument
equipped with ú-sizing capacity revealed the nanosizes
and surface potentials of both the transfection efficient
and the incompetent lipoplexes to be within the range
of 200-600 nm and +3.4 to -34 mV, respectively.
Clearly, further cell biology experiments including cel-
lular uptake studies, intracellular trafficking experi-
ments aimed at understanding subcellular localizations
of the lipid/DNA complexes, etc. need to be carried out
in the future to gain better mechanistic insights into
the origin of the contrasting transfection profiles of
lipids 1-3 and 4-6. To summarize, given the feasibility
of a wide range of structural manipulations in the
headgroup regions of non-cholesterol-based cationic
amphiphiles, the present findings further broaden the
potential of cationic amphiphiles with guanidinium
headgroups for use in nonviral gene therapy.
Figure 5. Electrophoretic gel patterns for lipoplex-associated
DNA with DNase I sensitivity assay (part A) for lipids 1 and
3
and (part B) for lipids 4 and 6. The lipid/DNA charge ratios
are indicated at the top of each lane. The details of the
treatment are as described in the text.
Experimental Section
General Procedures and Materials. Fast atom bombard-
ment mass spectrometry (FABMS) data were acquired by the
liquid secondary ion mass spectrometry (LSIMS) technique
using meta-nitrobenzyl alcohol as the matrix. LSIMS analysis
was performed in the scan range 100-1000 amu at the rate
treatment with DNase I. After the free DNA digestion
by DNase I, the total DNA (both digested and inacces-
sible DNA) was separated from the lipid and DNase I
1
of 3 scans/s. H NMR spectra were recorded on a Varian FT
(
1
by extracting with organic solvent) and loaded onto a
% agarose gel. Resulting electrophoretic gel pattens in
200 MHz, AV 300 MHz, or Varian Unity 400 MHz spectrom-
eter. 1-Bromotetradecane, 1-bromohexadecane, 1-bromoocta-
decane, n-tetradecylamine, n-hexadecylamine, n-octadecyl-
amine, pyrazole, and cyanamide were procured from Lancaster
(Morecambe, England). N,N-Diisopropyl ethylamine was pro-
cured from Aldrich (USA). Unless otherwise stated, all of the
reagents were purchased from local commercial suppliers and
were used without further purification. The progress of the
reactions was monitored by thin-layer chromatography on 0.25
mm silica gel plates. Column chromatography was performed
with silica gel (Acme Synthetic Chemicals, India, 60-120
mesh). p-CMV-SPORT-â-gal plasmid was a generous gift from
Dr. Nalam Madhusudhana Rao. LipofectAmine-2000 was
purchased from Invitrogen Life Technologies. (USA). Cell
culture media, fetal bovine serum, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), poly(ethylene gly-
col) 8000, o-nitrophenyl-â-D-galactopyranoside, and cholesterol
were purchased from Sigma (St. Louis, MO). NP-40, antibiot-
ics, and agarose were purchased from Hi-media (India). DOPE
and DOPC were purchased from Fluka (Switzerland). Unless
otherwise stated, all of the other reagents purchased from local
commercial suppliers were of analytical grades and were used
without further purification. COS-1 (SV 40 transformed african
green monkey kidney cells), CHO (Chinese hamster ovary),
HepG2 (human hepatocarcinoma), MCF-7 (human breast
adenocarcinoma cell), and A549 (human pulmonary carcinoma)
cell lines were procured from the National Centre for Cell
Sciences (NCCS), Pune, India. Cells were grown at 37 °C in
DMEM with 10% Fetal bovine serum (FBS) in a humidified
such DNase I protection experiments for representative
lipoplexes prepared with lipids 1, 3, 4, and 6 are shown
in Figure 5. Band intensities of inaccessible and there-
fore undigested DNA associated with transfection in-
competent lipoplexes 4 and 6 (lipoplex 6 in particular)
were significantly less than those associated with trans-
fection efficient lipoplexes 1 and 3 across the range of
lipid/DNA charge ratios of 3:1 to 0:1:1 (Figure 5). Such
gel patterns in DNase I sensitivity assays indicate that
the plasmid DNA associated with lipids 4 and 6 (par-
ticularly lipid 6) is likely to be more susceptible to
degradation by cellular DNase I than the DNA com-
plexed to lipids 1 and 3. Thus, findings in our DNase I
sensitivity assay are consistent with enhanced degrada-
tion of lipoplex-associated DNA by cellular DNase I
playing some important role behind the severely com-
promised transfection efficacies of lipid 6 in particular.
In conclusion, we have demonstrated that the pres-
ently described non-cholesterol-based cationic am-
phiphiles particularly lipids 1-3 (Chart 1) containing
a single guanidinium headgroup are remarkably effica-
cious in transfecting CHO, COS-1, MCF-7, and A549
cells with transfection efficiencies about 2- to 4-fold
higher than that of widely used commercially available
LipofectAmine-2000. For these new mono-guanidiny-
lated cationic transfection lipids, cholesterol was found
to be a more efficacious co-lipid than dioleoyllphosphati-
dyl ethanolamine (DOPE). In general, lipids 1-3 con-
2
atmosphere containing 5% CO /95% air. Purity of all of the
final lipids (1-6) was determined by analytical HPLC (Shi-
madzu model LC10A) using a PARTISIL 5 ODS-3 WCS
analytical column (4.6 mm × 250 mm, Whatman Inc., Clifton,
NJ) in two different mobile phases. One solvent system (A)

8
18 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Sen and Chaudhuri
was methanol/acetonitrile/water/trifluoroacetic acid in the ratio
5:10:25:0.05 (v/v) for 15 min with a flow rate of 0.8 mL/min.
NH
(Calcd for C36
2
+); 8.7 (bs, 1H, CH
2
NH). LSIMS (lipid 2) m/z: 565 [M+]
6
77 4
H N , 22%).
The other mobile phase (B) was methanol/water/trifluoroacetic
acid in the ratio 75:25:0.05 for 15 min with a flow rate of 0.8
mL/min. Peaks were detected by UV absorption at 219 nm.
All of the target lipids (1-6) showed more than 95% purity.
Typical retention times in mobile phase A were 3.56 min (lipid
N,N-Di-n-tetradecyl-N-[2-guanidinyl]ethyl-N-methyl-
1
ammonium Chloride (Lipid 1). H NMR (200 MHz, CDCl
δ (ppm): 0.9 (t, 6H, CH (CH 14); 1.2-1.3 (m, 44H, CH (CH
CH ); 3.1 (m, 4H,
); 3.5 (m, 2H, N CH
3
)
3
2
)
3
2
)11);
+
+
1
.5-1.7 [m, 4H, N (CH
2
2 2 3
) ); 3.0 (s, 3H, N CH
+
+
+
N (CH
CH CH
lipid 1) m/z: 510 [M + 1 ] (Calcd for C32
N,N-Di-n-octadecyl-N-[2-guanidinyl]ethyl-N-methyl-
ammonium Chloride (Lipid 3). H NMR (200 MHz, CDCl )
2
CH
2
2
)
2
2
CH
); 8.7 (bs, 1H, CH
, 82%).
2
NH); 3.7 (m, 2H, N -
1
), 3.59 min (lipid 2), 3.61 min (lipid 3), 3.57 min (lipid 4),
+
2
NH); 7.4 (bs, 4H, NH
2
2
NH). LSIMS
3.62 min (lipid 5), and 3.64 min (lipid 6).
+
(
69 4
H N
Synthesis of Lipids 1-3. Lipids 1-3 were synthesized
following the procedures depicted schematically in Scheme 1
1
3
(
part A). As representative details, synthetic procedures for
3 2 15 3 2 15
δ (ppm): 0.9 (t, 6H, CH (CH ) ); 1.2-1.3 (m, 60H, CH (CH ) );
lipid 2 are provided below.
1.5-1.7 (m, 4H, N
+
(CH CH ) ); 3.0 (s, 3H, N
+
CH ); 3.1 (m, 4H,
2
2 2
3
+
+
+
Synthesis of Lipid 2. Step i. Synthesis of N,N-Di-n-
hexadecyl-N-[2-(N′,N′-di-tertbutoxycarbonyl-guanidinyl]-
ethylamine (III, Scheme 1, Part A). Mercury chloride (0.28
g, 1.0 mmol) was added to a mixture of N-2-aminoethyl-N,N-
di-n-hexadecylamine (I, 0.49 g. 0.95 mmol), bis-N-Boc-thiourea
N (CH
CH CH
(lipid 3) m/z: 621 [M ] (Calcd for C40
2
CH
)
2 2
); 3.5 (m, 2H, N CH
2
CH
); 8.7 (bs, 1H, CH
, 40%).
2
NH); 3.7 (m, 2H, N -
+
2
2
NH); 7.4 (bs, 4H, NH
2
2
NH). LSIMS
+
85 4
H N
Synthesis of Lipids 4-6. Lipids 4-6 were synthesized
following the procedures depicted schematically in Scheme 1
(part B). As representative details, synthetic procedures for
lipid 5 are provided below.
(II, 0.26 g, 0.95 mmol, prepared by reacting 1 equiv of thiourea
with 2 equiv of Boc-anhydride in the presence of 2 equiv of
sodium hydride in anhydrous tetrahydrofuran as described
Synthesis of Lipid 5. Step v. Synthesis of N,N-Di-n-
hexadecyl-N-[2-guanidinyl]ethylamine (Lipid 5). Dry
N,N-dimethylformamide was added to N-2-aminoethyl-N,N-
di-n-hexadecylamine (I, 0.20 g, 0.39 mmol), 1H-pyrazole-1-
carboxamidine hydrochloride (0.06 g, 0.39 mmol, prepared by
reacting 1 equiv of pyrazole with 1 equiv of cyanamide in
anhydrous p-dioxan containing a few drops of concentrated
HCl), and N,N-di-isopropyl-N-ethyl amine (DIEA, 0.051 g, 68
µL, 0.39 mmol) to produce a final concentration of ap-
proximately 2 M reagents. The mixture was stirred at room
temperature under nitrogen for 14 h. The reaction mixture
was then taken in 100 mL of ethyl acetate, washed sequen-
tially with water (2 × 100 mL) and brine solution (2 × 100
mL), dried over anhydrous magnesium sulfate, and filtered.
Ethyl acetate was removed from the filtrate on a rotary
evaporator. Silica gel column chromatographic purification of
the resulting residue using 60-120 mesh silica gel size and
7-9% methanol in chloroform (v/v) as the eluent afforded 0.13
4
6
previously ), and triethylamine (0.21 g, 2.1 mmol) dissolved
in dry N,N-dimethylformamide (DMF, 5 mL) and dry dichlo-
romethane (DCM, 2 mL) at 0 °C with continuous stirring. The
resulting mixture was stirred at 0 °C under nitrogen for 40
min, diluted with ethyl acetate (20 mL), and filtered through
a pad of Celite. The filtrate was sequentially washed with
water (2 × 20 mL) and brine solution (2 × 20 mL), dried over
anhydrous sodium sulfate, and filtered, and the solvent from
the filtrate was removed by rotary evaporation. The residue
upon column chromatographic purification with 60-120 mesh
silica gel using 2-2.5% methanol/dichloromethane (v/v) as
eluent afforded 0.51 g of the pure title compound III (71%, R
f
)
0.8, 10% methanol/dichloromethane, v/v).
1
H NMR (200 MHz, CDCl
3
) δ (ppm): 0.9 (t, 6H, CH
3
(CH
2
)
13);
1
2
.2-1.4 (bs, 56H, (CH )14); 1.4-1.6 (2s, 18H, -CO-O-
C(CH
.6 (m, 2H, NCH
3 3
)
); 2.4-2.7 (bm, 6H, N(CH
2
CH
2 2
) ; NCH
2 2
CH NH); 3.4-
3
2
CH NH); 8.6 (t, 1H, CH
2
2
NH); 11.4 (s, 1H,
NHBoc).
f
g of pure lipid 5 (55% yield, R ) 0.7-0.8 using 20% methanol
chloroform as the TLC developer).
Step ii. Synthesis of N,N-Di-n-hexadecyl-N-[2-(N′,N′-
di-tert-butoxycarbonyl-guanidinyl]ethyl-N-methylam-
monium Iodide (Scheme 1, Part A). The intermediate III
obtained above in step i was dissolved in 3 mL of dichlo-
romethane/methanol (2:1, v/v), and 3 mL of methyl iodide was
added. The solution was stirred at room temperature over-
night. Solvent was removed on a rotary evaporator. The
residue upon column chromatographic purification with 60-
1
H NMR (200 MHz, CDCI
13); 1.5 (m, 4H, N(CH
); 2.6 (t, 2H, N(CH CH NH)); 3.25 (t, 2H, N(CH
); 8.1 (bs, 1H, CH NH). LSIMS
(lipid 5) m/z: 551 [M ] (Calcd for C35 , 100%).
N,N-Di-n-tetradecyl-N-[2-guanidinyl]ethylamine (Lip-
id 4). H NMR (200 MHz, CDCI ) δ (ppm): 0.9 (t, 6H, CH -
(CH ) ); 1.2-1.4 (m, 44H, (CH ) ); 1.5 (m, 4H, N(CH CH ) );
3
) δ (ppm): 0.9 (t, 6H, CH
3 2
(CH )13);
1.2-1.4 (m, 52H, (CH
N(CH CH
CH NH)); 7.4 (bm, 4H, NH
2
)
2
CH ); 2.4 (t, 4H,
2 2
)
2
2
)
2
2
2
2
-
+
2
2
2
+
75 4
H N
1
3
3
1
20 mesh size silica gel and 3% methanol in dichloromethane
v/v) as eluent afforded 0.47 g of the title compound (78% yield,
) 0.6, 10% methanol in dichloromethane, v/v).
2
11
2
11
2
2 2
(
2.4 (t, 4H, N(CH CH ) ); 2.6 (t, 2H, N(CH CH NH)); 3.25 [t,
2
2
2
2
+
2
R
f
2H, N(CH CH NH)]; 7.4 [bm, 4H, NH
2
]; 8.1 [bs, 1H,
2
2
1
+
H NMR (200 MHz, CDCl
.2-1.3 (m, 52H, CH (CH
C(CH ); 1.65 (m, 4H, N (CH
m,4H, N (CH
3
) δ (ppm): 0.9 (t, 6H, CH
3
(CH
2
)
14);
-CH
2
NH]. LSIMS (lipid 4) m/z: 496 [M + 1 ] (Calcd for
1
3
2
)
13); 1.4-1.6 (2s, 18H, -CO-O-
C
31
H
67
N
4
, 100%).
+
+
)
3 3
2
CH
2
)
2
); 3.3 (s, 3H, N CH
3
); 3.4
NH); 3.8 (m, 2H,
NH); 11.3 (s, 1H, NHBoc).
N, N-Di-n-octadecyl-N-[2-guanidinyl]ethylamine (Lip-
+
+
1
(
CH )
2 2 2
); 3.6 (m, 2H, N CH
2
CH
2
3 3
id 6). H NMR (200 MHz, CDCI ) δ (ppm): 0.9 (t, 6H, CH -
+
N CH
2
CH
2
NH); 8.4 (t, 1H, CH
2
2 15 2 15 2 2 2
(CH ) ); 1.2-1.4 (m, 60H, (CH ) ); 1.5 (m, 4H, N(CH CH ) );
Steps iii and iv. Synthesis of N,N-Di-n-hexadecyl-N-
2-guanidinyl]ethyl-N-methylammonium Chloride (Lip-
2.4 (t, 4H, N(CH
2H, N(CH CH NH)); 7.4 (bm, 4H, NH
LSIMS (lipid 6) m/z: 607 [M ] (Calcd for C39
2
CH
2
)
2
); 2.6 (t, 2H, N(CH
2
CH
); 8.1 (bs, 1H, CH
, 100%).
2
NH)); 3.25 (t,
+
[
2
2
2
2
NH).
+
id 2, Scheme 1, Part A). The intermediate obtained in step
83 4
H N
ii was dissolved in dry DCM (2 mL), and trifluoroacetic acid
TFA, 2 mL) was added to the solution at 0 °C. The resulting
Preparation of Liposomes. The cationic lipid and cho-
lesterol in a 1:1 mole ratio was dissolved in a mixture of
chloroform and methanol (3:1, v/v) in a glass vial. The solvent
was removed with a thin flow of moisture-free nitrogen gas,
and the dried lipid film was then kept under high vacuum for
8 h. Sterile deionized water (5 mL) was added to the vacuum-
dried lipid film, and the mixture was allowed to swell
overnight. The vial was then vortexed for 2-3 min at room
temperature to produce multilamellar vesicles (MLVs). MLVs
were then sonicated in an ice bath to clarity using a Branson
450 sonifier at 100% duty cycle and 25 W output power to
produce small unilamellar vesicles (SUVs).
(
solution was left stirred at room temperature overnight to
ensure complete deprotection. Excess TFA was removed by
flushing nitrogen to give the title compound as a trifluoro-
acetate salt. Column chromatographic purification using 60-
1
20 mesh size silica gel and 12-14% (v/v) methanol/chloroform
as eluent followed by chloride ion exchange chromatography
over amberlyst A-26 chloride ion exchange resin afforded 0.31
f
g of the pure lipid 2 (94% yield, R ) 0.3, 10% methanol in
chloroform, v/v).
1H NMR (200 MHz, CDCl
3
) δ (ppm): 0.9 (t, 6H, CH
3
(CH
13); 1.5-1.7 (m, 4H, N (CH CH
); 3.5 (m, 2H,
NH); 7.4 (bs, 4H,
2
)14);
+
1
.2-1.3 (m, 52H, CH
3
(CH
2
)
2
2
)
2
);
Preparation of Plasmid DNA. pCMV-SPORT-â-gal plas-
mid DNA was prepared by alkaline lysis procedure and
purified by PEG-8000 precipitation according to the protocol
+
+
3
.0 (s, 3H, N CH
3
); 3.1 (m, 4H, N (CH CH )
2 2 2
+
+
N CH
2
CH
2
NH); 3.7 (m, 2H, N CH
2
CH
2

Guanidinylated Cationic Lipids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 819
of DNase I (at a final concentration of 1µg/mL) in the presence
described earlier.15 The plasmid preparations showing a value
of OD260/OD280 more than 1.8 were used.
2
of 20 mM MgCl 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/isoamyl alcohol (25:24:1mixture, v/v) and
centrifuged at 10 000g for 5 min. The aqueous supernatants
were separated, loaded (25 µL) on a 1% agarose gel (prestained
with ethydium bromide), and electrophoresed at 100 V for 1
h.
Transfection Biology. Cells were seeded at a density of
2
0 000 cells (for CHO, MCF-7, A549, HepG2) and 15 000 cells
(
for COS-1) per well in a 96-well plate 18-24 h before the
transfection. Plasmid DNA (0.3 µg) was complexed with
varying amounts of lipids (0.09-8.1 nmol) in plain DMEM
medium (total volume made up to 100 µl) for 30 min. The
charge ratios were varied from 0.1:1 to 9:1 (() over these
ranges of the lipids. The complexes were then added to the
cells. After 3 h of incubation, 100 µL of DMEM with 20% FBS
was added to the cells. The medium was changed to 10%
complete medium after 24 h, and the reporter gene activity
was estimated after 48 h. The cells were washed twice with
phosphate-buffered saline (PBS, 100 µL each) and lysed in 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-galactosidase
activity per well was estimated by adding 50 µL of 2×
substrate solution (1.33 mg/mL of o-nitrophenyl-â-D-galacto-
pyranoside (ONPG), 0.2 M sodium phosphate (pH 7.3), and 2
mM magnesium chloride) to the lysate in a 96-well plate.
Adsorption 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
Figures 1 are the average of duplicate experiments performed
on the same day. Each transfection experiment was repeated
three times, and 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.
X-Gal Staining. Cells expressing â-galactosidase were
histochemically stained with the substrate 5-bromo-4-chloro-
3
-indolyl-â-D-galactopyranoside (X-gal) as described previ-
1
6
ously. Briefly, 48 h after transfection with lipoplexes in 96-
well plates, the cells were washed two times (2 × 100 µL) with
phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl,
1
2 4 2 4
0 mM Na HPO , 2 mM KH PO , pH 7.4) and fixed with 0.5%
glutaraldehyde in PBS (225 µL). After 15 min of incubation
at room temperature, the cells were washed again with PBS
three times (3 × 250 µL) and subsequently were stained with
1
3 6
.0 mg/mL X-gal in PBS containing 5.0 mM K [Fe(CN) ], 5.0
4 6 4
mM K [Fe(CN) ], and 1 mM MgSO for 2-4 h at 37 °C. Blue
colored cells were identified by light microscope (Leica, Ger-
many).
Acknowledgment. Financial support received from
the Department of Biotechnology, Government of India
(
to A.C.) is gratefully acknowledged. J.S. sincerely
thanks the University Grant Commission (UGC), Gov-
ernment of India, for her doctoral research fellowship.
We thank Shiva Prasad for helping us to take the gel
pictures.
Toxicity Assay. Cytotoxicities of the lipids 1-6 were
Supporting Information Available: Reverse phase HPLC
chromatograms for the lipids 1-6 in two mobile phases with
details of the HPLC parameters and representative reduced
transfection profiles of lipids 1-6 in CHO, HepG2, and A-549
cells. This information is available free of charge via the
Internet at http://pubs.acs.org.
assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
15
razolium bromide (MTT) reduction assay as described earlier.
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.
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(
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