Anchor dependency for non-glycerol based cationic lipofectins: Mixed bag of regular and anomalous transfection profiles

Article abstract of DOI:10.1002/1521-3765(20020215)8:4<900::AID-CHEM900>3.0.CO;2-X

Although detailed structure-activity, physicochemical and biophysical investigations in probing the anchor influence in liposomal gene delivery have been reported for glycerol-based transfection lipids, the corresponding investigation for non-glycerol based simple monocationic transfection lipids have not yet been undertaken. Towards this end, herein, we delineate our structure-activity and physicochemical approach in deciphering the anchor dependency in liposomal gene delivery using fifteen new structural analogues (lipids 1-15) of recently reported non-glycerol based monocationic transfection lipids. The C₁₄ analogues in both series 1 (lipids 1-6) and series 2 (lipids 7-15) showed maximum efficiency in transfecting COS-1 and CHO cells. However, the C₁₂ analogue of the ether series (lipid 3) exhibited a seemingly anomalous behavior compared with its transfection efficient C₁₀ and C₁₄ analogues (lipids 2 and 4) in being completely inefficient to transfect both COS-1 and CHO cells. The present structure-activity investigation also convincingly demonstrates that enhancement of transfection efficiencies through incorporation of membrane re-organizing unsaturation elements in the hydrophobic anchor of cationic lipids is not universal but cell dependent. The strength of the interaction of lipids 1-15 with DNA was assessed by their ability to exclude ethidium bromide bound to the DNA. Cationic lipids with long hydrophobic tails were found, in general, to be efficient in excluding EtBr from DNA. Gel to liquid crystalline transition temperatures of the lipids was measured by fluorescence anisotropy measurement technique. In general (lipid 2 being an exception), transfection efficient lipids were found to have their mid transition temperatures at or below physiological temperatures (37°C).

Full text of DOI:10.1002/1521-3765(20020215)8:4<900::AID-CHEM900>3.0.CO;2-X

FULL PAPER
Anchor Dependency for Non-Glycerol Based Cationic Lipofectins:
Mixed Bag of Regular and Anomalous Transfection Profiles**
Rajkumar Sunil Singh,[a] Koushik Mukherjee,[a] Rajkumar Banerjee,[a]
Arabinda Chaudhuri,*[a] Samik Kumar Hait,[b] Satya Priya Moulik,[b]
Yerramsetti Ramadas,[c] Amash Vijayalakshmi,[c] and Nalam Madhusudhana Rao*[c]
Abstract: Although detailed structure
activity, physicochemical and biophysi-
cal investigations in probing the anchor
influence in liposomal gene delivery
have been reported for glycerol-based
transfection lipids, the corresponding
investigation for non-glycerol based sim-
ple monocationic transfection lipids
have not yet been undertaken. Towards
this end, herein, we delineate our struc-
ture activity and physicochemical ap-
proach in deciphering the anchor de-
pendency in liposomal gene delivery
using fifteen new structural analogues
(lipids 1 15) of recently reported non-
glycerol based monocationic transfec-
tion lipids. The C14 analogues in both
series 1(lipids 1 6) and series 2 (lipids
7
15) showed maximum efficiency in
organizing unsaturation elements in the
hydrophobic anchor of cationic lipids is
not universal but cell dependent. The
strength of the interaction of lipids 1 15
with DNA was assessed by their ability
to exclude ethidium bromide bound to
the DNA. Cationic lipids with long
hydrophobic tails were found, in gener-
al, to be efficient in excluding EtBr from
DNA. Gel to liquid crystalline transition
temperatures of the lipids was measured
by fluorescence anisotropy measure-
ment technique. In general (lipid 2 being
an exception), transfection efficient lip-
ids were found to have their mid tran-
sition temperatures at or below physio-
logical temperatures (378C).
transfecting COS-1and CHO cells.
However, the C12 analogue of the ether
series (lipid 3) exhibited a seemingly
anomalous behavior compared with its
transfection efficient C10 and C14 ana-
logues (lipids 2 and 4) in being com-
pletely inefficient to transfect both
COS-1and CHO cells. The present
structure activity investigation also
convincingly demonstrates that en-
hancement of transfection efficiencies
through incorporation of membrane re-
Keywords: amphiphiles
liposomes structure activity
relationships ¥ transfection
¥ lipids ¥
¥
vectors are, in general, superior to non-viral vectors. How-
ever, concern on the safety issues in using viral vectors are
increasingly making the non-viral vectors as the alternative
Introduction
Use of efficient transfection vector is a prerequisite for
success in gene therapy. The gene delivery efficiencies of viral
vector of choice. Among the several existing non-viral
26]
vectors,[1, 2] amphiphilic cationic transfection lipids[3
un-
questionably hold promise in non-viral gene therapy. Robust
manufacture, simplicity of handling techniques, least immu-
nogenic response and ability to form stable injectable com-
plexes even with large DNA are some of the distinct
advantages associated with cationic transfection lipids.
Cationic transfection lipids are composed of three seg-
ments: a hydrophobic anchor, a linker and a head group.
Efforts to delineate the contribution of each of these
three segments on overall transfection efficiency is confound-
ed by the complexity of the transfection pathway includ-
ing formation of lipoplex along with the co-lipid, size and
surface properties of the complexes, uptake and subsequent
transport of the DNA into the nucleus. Different cell lines
and plasmid constructs add to the complexity and make
comparison of data obtained from different laboratories
arduous.
[a] Dr. A. Chaudhuri, R. S. Singh, K. Mukherjee, R. Banerjee[‡]
Division of Lipid Science and Technology
Indian Institute of Chemical Technology
Hyderabad 500 007 (India)
Fax : (‡91)40-7160757
E-mail: arabinda@iict.ap.nic.in
[b] S. K. Hait, S. P. Moulik
Centre for Surface Science, Department of Chemistry
Jadavpur University, Kolkata 700 032 (India)
[c] Dr. N. M. Rao, Y. Ramadas, A. Vijayalakshmi
Centre for Cellular and Molecular Biology
Hyderabad 500 007 (India)
Fax : (‡91)40-7171195
E-mail: madhu@ccmb.ap.nic.in
‡
[ ] Present Address: School of Pharmacy
University of Pittsburgh, Pennsylvania 19104 (USA)
[**] IICT Communication No. 4716.
900
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Chem. Eur. J. 2002, 8, No. 4

900 909
Ever since Felgner et al.[26] pioneered the use of glycerol-
backbone based cationic lipid (DOTMA) mediated gene
delivery in 1987, intense global efforts have been witnessed for
HO OH
HO OH
HO OH
Cl
R'
Br
N
Cl
N
N
(CH2)15
(CH2)15
developing newer and more efficient cationic transfection
RO
OR
Series 1
R
CH3
CH3
30]
lipids.[3
Interestingly, majority of the subsequently devel-
Series 2
DHDEAB
oped efficient cationic amphiphiles such as, DOTAP,[24]
DMDHP,[10] DMRIE,[22] retained the glycerol backbone of
DOTMA[26] (see Experimental Section for abbreviations) as
the linker between the cationic head group and the hydro-
phobic tail region. Recently, we reported four remarkably
efficient non-glycerol based nontoxic simple monocationic
transfection lipids namely, DHDEAB, MOOHAC, DOM-
HAC and DOHEMAB,[5] in which the hydrophobic n-alkyl or
n-alkenyl tails are covalently linked to the cationic head
groups either directly or through an ester group. The most
efficient one in this series is DHDEAB with two 2-hydroxy-
ethyl head groups and two n-hexadecyl hydrophobic tails
directly attached to the quaternary nitrogen atom. The
transfection efficiency of DHDEAB is better than that of
Lipofectamine, one of the most widely used commercial
transfection agent.
7: R' (= R-CH2CH2-) = n-C8H17
8: R' (= R-CH2CH2-) = n-C10H21
9: R' (= R-CH2CH2-) = n-C12H25
10: R' (= R-CH2CH2-) = n-C14H29
11: R' (= R-CH2CH2-) = n-C16H33
12: R' (= R-CH2CH2-) = n-C18H37
13: R' (= oleyl, R-CH2CH2-) = n-C18H35/n-C18H37
14: R' (= R-CH2CH2-) = oleyl
15: R' (= R-CH2CH2-) = n-C20H41
1: R = n-C8H17
2: R = n-C10H21
3: R = n-C12H25
4: R = n-C14H29
5: R = n-C16H33
6: R = n-C18H37
interaction of lipids 1 15 with DNA was assessed by their
ability to exclude ethidium bromide bound to the DNA.
Cationic lipids with long hydrophobic tails were found, in
general, to be efficient in excluding EtBr from DNA. Gel to
liquid crystalline transition temperatures of the lipids were
measured by fluorescence anisotropy measurement techni-
que. As outlined below, in general (lipid 2 being an
exception), transfection efficient lipids were found to have
their mid transition temperatures at or below physiological
temperatures (378C).
Detailed structure activity investigations in lipid mediated
gene delivery using glycerol,[10, 22, 29, 30] polyamine[8, 9] and
cholesterol[27, 28] based cationic transfection lipids with varying
chain lengths hydrophobic tails, ether linker regions, unsatu-
rated hydrophobic tails with membrane reorganizing capa-
bilities have been reported. However, corresponding study
using non-glycerol based cationic transfection lipids have not
yet been undertaken. Towards this end, herein, with a view to
probe the anchor-dependency profile in non-glycerol based
cationic lipid mediated gene delivery, we have synthesized
fifteen new (lipids 1 15) structural analogues of our pre-
viously reported non-glycerol based highly efficient cationic
transfection lipid DHDEAB.[5] The overall yields of the
present DHDEAB structural analogues have been signifi-
cantly improved in the new synthetic methods adopted in the
present work. In the first series, to investigate the role of an
additional ether linkage between the cationic head group and
the hydrophobic anchor in modulating the transfection
efficiencies of non-glycerol monocationic lipids, cationic
amphiphiles 1 6 containing hydrophobic anchors with vary-
ing chain lengths linked to the quaternary nitrogen atom by an
ether linkage were synthesized. In the second series, since
membrane reorganizing ability of the lipoplex formulation is
considered to be advantageous for transfection, chain lengths
and unsaturation properties of the hydrophobic anchor in
DHDEAB analogues were altered (amphiphiles 7 15).
As delineated below, the C14 analogues in both series 1
(lipids 1 6) and series 2 (lipids 7 15) showed maximum
efficiency in transfecting COS-1and CHO cells. However, the
Results and Discussion
Chemistry: The detailed synthetic procedures for the new
cationic amphiphiles 1 6 with ether linkers and the new
DHDEAB analogues 7 15 are outlined below in the
Experimental Section. Amphiphiles 1 6 were synthesized
by quaternizing the secondary amines containing the appro-
priate ether linkage (intermediate V, Scheme 1) with alkaline
2-chloroethanol. As outlined in Scheme 1, the necessary
secondary amine intermediates V were synthesized by cou-
pling the appropriate 2-aminoethyl n-alkyl ether (interme-
diate IV, Scheme 1) with the corresponding 2-bromoethyl-n-
alkyl ether (intermediate II, Scheme 1) in presence of base.
2-Aminoethyl n-alkyl ether intermediates IV (Scheme 1)
were conventionally synthesized from the 2-hydroxyethyl n-
alkyl ether (I, Scheme 1) in three steps by converting the
starting primary alcohol (I, Scheme 1) to the corresponding
primary bromide (intermediate II), reaction of the primary
bromide with sodium azide and finally reduction the resulting
primary azides (intermediate III, Scheme 1).
i)
ii)
RO
RO
RO
OH
Br
N3
(II)
(I)
(III)
H
N
iv)
iii)
RO
+ (II)
NH2
(IV)
RO
OR
(V)
C
12 analogue of the ether series (lipid 3) exhibited a seemingly
HO OH
anomalous behavior compared with its transfection efficient
C10 and C14 analogues (lipids 2 and 4) in being almost
completely inefficient to transfect both COS-1and CHO cells.
The present structure activity investigation also convincingly
demonstrates that enhancement of transfection efficiencies
through incorporation of membrane reorganizing unsatura-
tion elements in the hydrophobic anchor of cationic lipids is
not universal but cell dependent. The strength of the
1: R = n-C8H17
2: R = n-C10H21
3: R = n-C12H25
4: R = n-C14H29
5: R = n-C16H33
6: R = n-C18H37
v)
N
Cl
RO
OR
(VI)
Scheme 1. Syntheses of new ether based cationic amphiphiles
1 6.
i) CBr4, PPh3, imidazole, dichloromethane; ii) NaN3, dimethylformamide;
iii) PPh3, H2O, tetrahydrofuran; iv) K2CO3/dimethylsulfoxide; v) excess
chloroethanol, aqueous NaOH.
Chem. Eur. J. 2002, 8, No. 4
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901

A. Chaudhuri, N. M. Rao et al.
FULL PAPER
Much to our satisfaction, in the present work, we have
succeeded in altering our previous low yield method[5] for
synthesizing the DHDEAB analogues (lipids 7 15). As
outlined in Scheme 2, all the DHDEAB analogues were
synthesized by quaternizing the hydrophobic secondary
amine intermediates (III, Scheme 2) with 2-chloroethanol in
presence of base. The necessary secondary amine intermedi-
ates (III, Scheme 2) were conventionally synthesized in two
steps by coupling the primary amines (I) with the appropriate
aldehydes (II, Scheme 2) followed by sodium borohydride
reduction of the resulting imine.
H
Figure 1. Transfection efficiencies of lipids 1 15 on COS-1cells with
cholesterol as co-lipid. a) Reporter gene activities of lipids 1 6 were
N
R'
i)
RCH2 CH
O
H2N R'
+
~
~
&
&
*
*
R
plotted against charge ratios. 1 ( ), 2 ( ), 3 ( ), 4 ( ), 5 ( ) and 6 ( ).
(I)
(II)
(III)
b) Reporter gene activities of lipids 7 15 were plotted against charge
~
~
!
!
&
&
*
*
^
ratios. 7 ( ), 8 ( ), 9 ( ), 10 ( ), 11 ( ), 12 ( ), 13 ( ), 14 ( ) and 15 ( ).
The b-galactosidase activities in each well (mmol of ortho-nitrophenol
produced per hour) was converted to an absolute b-galactosidase milliunits
using standard curve obtained with pure (commercial) b-galactosidase. The
data shown are average values from four independent experiments (n ˆ 4).
HO OH
7: R' (= R-CH2CH2-) = n-C8H17
8: R' (= R-CH2CH2-) = n-C10H21
9: R' (= R-CH2CH2-) = n-C12H25
10: R' (= R-CH2CH2-) = n-C14H29
11: R' (= R-CH2CH2-) = n-C16H33
12: R' (= R-CH2CH2-) = n-C18H37
13: R' (= oleyl, R-CH2CH2-) = n-C18H35/n-C18H37
14: R' (= R-CH2CH2-) = oleyl
ii)
Cl
N
R'
R
(IV)
15: R' (= R-CH2CH2-) = n-C20H41
Scheme 2. Syntheses of new DHDEAB analogues. i) MgSO4 (1equiv),
dichloromethane; ii) NaBH4 (2 equiv), dichloromethane/MeOH; ii) excess
chloroethanol/aqueous NaOH.
Transfection studies: To assess the transfection efficiencies of
the amphiphiles 1 15, we have used a plasmid containing b-
galactosidase reporter gene under the control of a CMV
promoter on two cell lines namely COS-1(Figure 1) and CHO
(Figure 2). Lipoplexes (containing 1:1 mol ratios of lipids and
cholesterol) were prepared at charge ratios (‡/ À ) ranging
from 0.1to 9.0 and tested for their transfection efficiency.
Consistent with our previous observation,[5] the auxiliary lipid
DOPE conventionally used in cationic liposome mediated
gene delivery was found to be inefficient for the present
DHDEAB structural analogues too (data not shown). Inter-
estingly, without cholesterol as co-lipid, the present non-
glycerol based lipids were found to be very poor in trans-
fecting cells (as an representative example, the transfection
data for lipids 4 and 10 both in presence and absence of
cholesterol are shown in Figure 3). However, the exact role
played by cholesterol in modulating the transfection efficien-
cies of the present transfection lipids is still an open question
at this stage of investigation. In both COS-1and CHO cells,
Figure 2. Transfection efficiencies of lipids 1 15 on CHO cells with
cholesterol as co-lipid. a) Reporter gene activities of lipids 1 6 were
~
~
&
&
*
*
plotted against charge ratio 1 ( ), 2 ( ), 3 ( ), 4 ( ), 5 ( ) and 6 ( ).
(B) Reporter gene activities of lipids 7 15 were plotted against charge
~
~
!
!
&
&
*
*
ratios. 7 ( ), 8 ( ), 9 ( ), 10 ( ), 11 ( ), 12 ( ), 13 ( ), 14 ( )and 15
^
(
).The b-galactosidase activities in each well (mmol of ortho-nitrophenol
produced per hour) was converted to an absolute b-galactosidase milliunits
using standard curve obtained with pure (commercial) b-galactosidase. The
data shown are average values from four independent experiments (n ˆ 4).
both COS-1and CHO cells, the C 14 analogue of DHDEAB[5]
(lipid 10, series 2) showed maximum transfection efficiency at
lipid: DNA charge ratios 0.3:1and 1:1, respectively (Figures
1B and 2B). However, the chloride counterion analogue of
DHDEAB (lipid 11, series 2) was about threefold more
transfection efficient in COS-1cells than in CHO cells at a
‡/ À charge ratio of 1:1 (Figures 1B and 2B). Interestingly,
lipids 13 and 14 (series 2) having one and two unsaturated
double bonds, respectively, in the hydrophobic anchor were
efficient in CHO cells (Figure 2B) while the transfection
efficiencies of both were remarkably reduced in COS-1cells
(Figure 1B). This result convincingly indicates that the
presence of membrane reorganizing unsaturation elements
in the hydrophobic anchor of cationic lipids need not
necessarily improve the transfection efficiency of cationic
C
10 analogue of the ether based lipids (lipid 2, series 1) showed
its optimal transfection efficiency at lipid: DNA charge ratio
of 0.3:1whereas the equally efficient C analogue (lipid 4,
14
series 1) was most active at charge ratio (‡/ À ) of 1 :1
(Figures 1A and 2A). Surprisingly, the C12 analogue of the
ether based lipid (lipid 3, series 1) did not show any trans-
fection activity in both COS-1and CHO cells (Figures 1A and
2A).
Although lipids 1 6 (series 1) showed similar transfection
activity profiles in both COS-1and CHO cells (Figures 1A
and 2A), lipids 7 15 (series 2) showed differential activity
profiles in COS-1and CHO cells (Figures 1B and 2B). In
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Cationic Lipofectins
900 909
Table 1. Particle sizes (the hydrodynamic diameter, dh) of the representa-
tive liposomes and lipid/DNA complexes.
amphiphiles, the effect is rather cell dependent in in vitro in
liposomal gene delivery.
Lipid/DNA mol ratio
dh[a] [nm]
Transfection efficiencies of the present non-glycerol based
monocationic lipids (lipids 4 and 10) were observed to be
higher than those of commercially available glycerol based
monocationic transfection lipids such as, Lipofectin and
DMRIE. Figure 3 shows a representative comparative data
in COS-1cells. Both lipids 10 and 4 were more than twofold
and 3 4-fold transfection efficient than DMRIE and Lip-
ofectin, respectively, in presence of cholesterol (not DOPE)
as co-lipid (Figure 3). Pure lipids 10 and 4 (i.e., in absence of
lipid 4
1:0
3:173.4
1:1
0.3:136.3
lipid 10
1:0
3:1
1:1
0.3:121
43.4 Æ 6.5
Æ 11.0
89.5 Æ 13.4
Æ 5.45
229.3 Æ 33.4
110.3 Æ 16.5
258.8 Æ 38.8
Æ 32.8
8.7
cholesterol) were found to be less efficient. The transfection
[a] The DNA/lipid complexes were prepared by adding DNA to the
liposomes at the charge ratios indicated. The particle sizes of pure
liposomes and the lipid/DNA complexes were measured by a dynamic light
scattering spectrophotometer. The concentrations of cationic lipids 4 and
10 and DNA used in size measurement experiments were same as those
used in transfection experiments.
efficiencies of lipids 4 and 10 were also observed to be higher
than that of Lipofectamine, one of the most extensively used
commercially available lipopolyamine based transfection
lipids (Figure 3). However, the polycationic head group
to any selective biodistributions and/or organ specific trans-
gene expression in in vivo experiments needs to be inves-
tigated.
Toxicity studies: Cytotoxic effects of the lipoplexes made by
the series 1and 2 lipids were tested on COS-1cell lines
(Figure 4) using MTT assay.[32] The treatment protocols were
identical for both cytotoxicity and transfection assays. Series 1
lipids are safer on the cells since, except for C8 lipids, rest of
the lipids have <15% loss in viability up to 3:1 charge ratio.
In series 2, lipids with chain lengths less than C12 have
maximal cytotoxic effect, whereas rest of the lipids have
<20% loss of viability up to 3:1charge ratio. Lipids in either
series with shorter chain lengths may not form suitable
complexes and may behave more as cell lysing detergents,
hence their higher toxicity.
Figure 3. Comparison of the transfection efficiencies of lipid 4 and 10 with
and without co-lipids (DOPE and cholesterol) on COS-1cells. These
efficiences were compared with DMRIE, Lipofectin and Lipofectamine.
&
&
*
*
DMRIE ( ), Lipofectamine ( ), Lipofectin ( ), pure lipid 4 ( ), 4 with
~
~
!
!
DOPE ( ), 4 with cholesterol ( ), pure lipid 10 ( ), 10 with DOPE (
)
^
and 10 with cholesterol ( ).
nature of Lipofectamine (in contrast to monocationic head
group of the present series) makes this latter comparison less
relevant. Needless to say that the observed low cytotoxicities
of the present lipids (shown below) and the higher in vitro
transfection efficiencies of lipids 4 and 10 compared with
Lipofectin and DMRIE justifies further in-depth structure
activity investigations using various new structural analogues
of the present non-glycerol based transfection lipids. The
outcome of such study is likely to add promising new non-
glycerol based cationic amphiphiles to the arsenal of trans-
fection lipids for future use in the area of non-viral gene
therapy.
With a view to characterize the representative particle sizes,
we have determined the particle sizes of the most transfection
active lipoplexes made from lipids 4 and 10 using a dynamic
light scattering spectrophotometer. Interestingly, the sizes of
lipid 4/DNA complexes were observed to be remarkably
smaller than those of the lipid 10/DNA lipoplexes (Table 1).
Given similar transfection efficiences of lipids 4 and 10, the
lipoplex size data suggest that size does not have any
correlation with the transfection efficiency. At this point of
investigation, it is difficult to track the origin of such size
difference. Whether or not, this remarkable size difference
between the lipoplexes formed from lipids 4 and 10 will lead
Figure 4. Cytotoxicity of lipids 1 15 on COS-1cells. Toxicity, measured as
~
~
)
&
&
*
*
percent viability, was plotted in a) 1 ( ), 2 ( ), 3 ( ), 4 ( ), 5 ( ) and 6 (
~
~
!
!
&
&
*
*
^
and in b) 7 ( ), 8 ( ), 9 ( ), 10 ( ), 11 ( ), 12 ( ), 13 ( ), 14 ( ) and 15 ( ).
The absorption obtained using reduced formazon with cells in the absence
of lipids was taken to be 100. Results were expressed as per cent viability ˆ
[A540(treated cells) À background]/[A540(untreated cells) À background] Â
100.
DNA lipid interactions probed by ethidium bromide bind-
ing: The strength of the interaction of lipids 1 15 with DNA
was assessed by their ability to exclude ethidium bromide
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A. Chaudhuri, N. M. Rao et al.
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(EtBr) bound to the DNA. Detail titration curves of all fifteen
lipids with plasmid DNA indicated that ability of a lipid to
interact with the DNA and to exclude EtBr was strongly
dependent on the anchor chain length (Figure 5). Cationic
lipids with long hydrophobic tails were found, in general, to be
efficient in excluding EtBr from DNA. Results shown in
Figure 5 also demonstrate that the EtBr displacement effi-
ciencies of lipids 3 and 4 in series 1are remarkably compa-
rable to those of lipids 9, 10 and 11 of series 2. Similarly, lipids
5 and 6 in series 1and lipids 14 and 15 of series 2 exhibited
comparable EtBr displacement (Figure 5). Given that addi-
Anisotropy measurements: Membrane fluidity of the cationic
liposomes is known to be an advantage in transfection. Fluid
membranes may assist fusion of lipoplexes with the endo-
somes and release the DNA into the cytoplasm efficiently.
With a view to probe the role of membrane fluidity in
modulating the transfection efficiencies, we studied the gel to
liquid crystalline transition temperature of lipids 2 6 and 9
15 by fluorescence anisotropy measurement technique using
DPH as fluorescent probe. Lipids 1, 7 and 8 did not entrap any
DPH to take anisotropy measurements. The shorter chain
lengths of these lipids probably did not allow them to make
vesicles. This is also corroborated by their poor interactions
with DNA (Figure 5). The mid-points of transition for lipids 4,
5 and 6 in series 1were observed to be 23 8C, 388C and 508C,
respectively (Figure 6A). Such increase in transition temper-
atures was consistent with the increasing anchor chain lengths
for lipids 4 6 in series 1. Lipids 2 and 3 did not show any
transition in the observed temperature range but their high
anisotropy values indicate that the membrane of these
vesicles is rigid. Lipids 11, 12 and 15 of series 2 showed
transition temperature with mid-point of transition being
228C, 52 8C and 538C, respectively (Figure 6B). The mid-
point of transition of series 2 lipids were also observed to
increase with their chain length. Lipids 9 10 and 13 14 did
not show any transition and all of them have low anisotropy
values (Figure 6B) indicating the fluid nature of the liposome
membranes made from these lipids across the temperature
À À
tional -C C O- linkage present in the molecular architec-
tures of lipids 1 6 in series 1increases their effective anchor
lengths, these results are consistent with anchor chain length
dependent EtBr displacement. However, among all the lipids
1
15 studied in the present work, lipids 12 and 13 of series 2
turned out to be the most efficient in displacing EtBr from
DNA (Figure 5). Thus, in general, presence of saturated
hydrocarbon chain anchors containing 18 or less carbon atoms
significantly enhances the lipid/DNA interactions for non-
glycerol based monocationic transfection lipids containing
dihydroxyethyl head groups.
À
À
range tested. Given that -CH2 CH2 O- spacer for series 1
lipids are part of their hydrophobic tails, the anchor lengths of
lipids 2 and 9 are likely to be close. However, the fluorescence
anisotropy in liposomes made with lipid 2 was found to be
significantly higher than that of lipid 9 (Figure 6A and B).
Similarly, inspite of having similar anchor lengths, the
observed fluorescence anisotropy of liposomes made with
lipid 3 was remarkably higher than that of liposome made
from lipid 10 (Figure 6). Such unexpectedly higher membrane
anisotropy associated with lipids 2 and 3 indicate that the
ether link in lipids 2 and 3 may be involved in additional
interactions to rigidify their liposomal membrane. Unsatu-
rated hydrophobic chains present in lipids 13 and 14 increased
the membrane fluidity as expected.
When compared with the transfection efficiency, lipids that
have transition temperatures at or below physiological
temperatures (378C) are efficient. Lipid 2 was an exception
with mid-point of transition well above 378C though based on
chain length should have transition temperature below 378C.
Probably head-group interactions come to the fore in this
lipid. Our results are comparable to similar study with
analogues of DOTAP[29] in which myristoyl derivatives were
shown to have higher transfection efficiency than lauroyl,
palmitoyl, stearoyl or oleoyl derivatives of DOTAP using
cholesterol as co-lipid. Only myristoyl derivative of DOTAP
alone had mid-point of transition near 378C. Our studies
demonstrate that unsaturated chains do not impart any
transfection advantage compared with the saturated chains
(Figure 2). Liposomes in solid gel-like phase would be less
flexible in their interaction with the DNA or with the cell
membranes. Interestingly, liposomes made from lipid 3 did
not exhibit any well defined mid-point of temperature of
Figure 5. DNA and lipid interaction assessed by exclusion of ethidium
bromide from DNA: Exclusion of ethidium bromide, monitored as
decrease in fluorescence was plotted against the various charge raios of
lipids 1 6 (a) and 7 15 (b). The order of addition of ethidium bromide or
cationic lipids did not alter the final equilibrium fluorescence value.
904
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Chem. Eur. J. 2002, 8, No. 4

Cationic Lipofectins
900 909
(lipids 7 15) showed maximum efficiency in transfecting
COS-1and CHO cells. However, the C 12 analogue of the ether
series (lipid 3) exhibited a seemingly anomalous behavior
compared to its transfection efficient C10 and C14 analogues
(lipids 2 and 4) in being completely inefficient to transfect
both COS-1and CHO cells. Myristyl anchor containing lipids
were found to be optimal both in interacting with DNA and in
their transfection efficiency. The present structure activity
investigation convincingly demonstrates that enhancement of
transfection efficiencies through incorporation of membrane
reorganizing unsaturation elements in the hydrophobic an-
chor of cationic lipids is not universal but cell dependent.
Cationic lipids with long hydrophobic tails were found, in
general, to be efficient in excluding EtBr from DNA. In
general (lipid 2 being an exception), transfection efficient
lipids were found to have their mid transition temperatures at
or below physiological temperatures (378C). Lipids 10 and 4,
being more than twofold and 3 4-fold transfection efficient
than DMRIE and Lipofectin, respectively, are two new
promising cationic transfection lipids for future use in the
area of non-viral gene delivery.
Experimental Section
Abbreviations: DHDEAB: N,N-di-n-hexadecyl-N,N-dihydroxyethylam-
monium bromide; DMDHP: (Æ)-N,N-[Bis(2-hydroxyethyl)]-n-[2,3-bis-
(tetradecanoyloxy)propyl]ammonium chloride; DMRIE: 1,2-dimyristylox-
ypropyl-3-dimethyl-hydroethyl ammonium bromide; DOTAP: 1,2-dio-
leoyloxy-3-(trimethylamino)propane; DOTMA: 1,2-dioleyl-3-N,N,N-tri-
methylaminopropane chloride; DOPE: 1,2-dioleoyl-propyl-3-phosphatidy-
lethanolamine.
Figure 6. Temperature dependent anisotropy measurements of lipids 2
6
and 9 15: Parallel and perpendicular fluorescence of pure cationic lipid
vesicles preloaded with DPH was recorded at various temperatures.
Anisotropy was calculated as described in the Experimental Section for
lipids 2 6 (a) and lipids 9 15 (a). Lipids 1, 7 and 8 did not entrap any DPH
to take anisotropy measurements. Temperature in the cuvette was
measured with a precalibrated thermocouple.
General procedures and materials: The high-resolution mass spectrometric
(HRMS) analysis were performed on a Micromass AUTOSPEC-M mass
spectrometer (Manchester, UK) with OPUS V3.1X data system. Data were
acquired by liquid secondary ion mass spectrometry (LSIMS) technique
using meta-nitrobenzyl alcohol as the matrix. LSIMS analysis were
performed in the scan range 100 1000 amu at the rate of 3 scanssÀ1
.
transition within the experimental temperature range 108
658C (Figure 6) indicating the possible existence of a solid
gel-like membrane for these liposomes. It can not be
determined from the present data whether or not the
complete lack of transfection observed for lipid 3 in both
COS-1and CHO cell lines (Figures 1and 2) has its origin to
high membrane rigidity associated with liposomes prepared
using lipid 3.
1H NMR spectra were recorded on a Varian FT 200 MHz or Varian Unity
400 MHz. 1-Bromooctane, 1-bromodecane, 1-bromododecane, 1-bromo-
tetradecane, 1-bromohexadecane, 1-bromooctadecane, sodium hydride,
sodium borohydride and carbon tetrabromide were purchased from
Lancaster (Morecambe, England). Unless otherwise stated, all reagents
were purchased from local commercial suppliers and were used without
further purification. Column chromatography was performed with silica gel
(Acme Synthetic Chemicals, India, finer than 200 and 60 120 mesh).
pCMV.SPORT-b-gal, Lipofectamine, cell culture media and fetal calf
serum were purchased from GibcoBRL, Rockville, USA. 3-(4,5-Dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyethylene gly-
col 8000, NP-40, antibiotics, agarose, o-nitrophenyl-b-d-galactopyranoside
were purchased from Sigma, St. Louis, USA. DNA molecular weight
markers were purchased from Bangalore Genei, Bangalore, India.
Cholesterol and dioleoyl phosphatidyl ethanolamine (DOPE) were
purchased from Avanti Polar, Alabama, USA. COS-1cell line (SV 40
transformed African green monkey kidney, ATCCCRL 1650) and CHO
(chinese hamster ovary) was obtained from ATCC, Maryland, USA. Unless
otherwise stated, all reagents were purchased from local commercial
suppliers and were used without further purification.
Conclusion
In summary, the present investigation outlines structure ac-
tivity and physicochemical approach in deciphering the
anchor-dependency profile in in vitro liposomal gene delivery
using fifteen new structural analogues (lipids 1 15) of our
recently reported efficient non-glycerol based cationic trans-
fection lipid DHDEAB.[5] Present investigation demonstrate
that quaternization of hydrophobic secondary amines con-
taining the desired alkyl/alkenyl chains with alkaline 2-chlor-
oethanol is a very simple route for synthesizing non-glycerol
based transfection efficient DHDEAB-structural analogues.
The C14 analogues in both series 1(lipids 1 6) and series 2
Syntheses
All the 2-haloethyl n-alkyl ether starting materials used to synthesize the
cationic amphiphiles 1 6 were prepared following the same protocol
outlined in Scheme 1. As a representative example, synthesis of 2-bro-
moethyl n-dodecyl ether is described below in detail.
2-Bromoethyl n-dodecyl ether: 2-Hydroxyethyl n-dodecyl ether (1g,
4.34 mmol, prepared conventionally by reacting 1-bromododecane with
Chem. Eur. J. 2002, 8, No. 4
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905

A. Chaudhuri, N. M. Rao et al.
FULL PAPER
ethylene glycol and NaH at 808C in dry dimethylformamide under N2 for
CH2-CH2-CH2-], 3.35 [t, 2H, N3-CH2-CH2-O-CH2-], 3.45 [t, 2H, N3-CH2-
CH2-O-], 3.60 [t, 2H, N3-CH2-CH2-O-].
2
3 d), triphenylphosphine (2.28 g, 8.69 mmol) and imidazole (591.4 mg,
8.69 mmol) were dissolved in a 50 mL two-necked round-bottomed flask in
dry dichloromethane (3 mL) under nitrogen atmosphere. The solution was
cooled to 08C, carbon tetrabromide (2.88 g, 8.69 mmol, dissolved in 2 mL
dry dichloromethane) was added dropwise to the cold solution and the
temperature of the reaction mixture was gradually (1h) raised to room
temperature. The reaction mixture was then stirred for 20 h at room
temperature, concentrated and column chromatographic purification
(using 60 120 mesh size silica gel and 5 7% ethyl acetate in hexane as
the eluent) of the residue afforded the pure title compound as a colorless
liquid (800 mg, 78.5%). Rf ˆ 0.50 (hexane/ethyl acetate 95:5). 1H NMR
(200 MHz, CDCl3): d ˆ 0.90 [t, 3H, CH3-(CH2)9-], 1.20 1.42 [m, 18H,
-(CH2)9-], 1.50 1.65 [m, 2H, Br-CH2-CH2-O-CH2-CH2-(CH2)8-], 3.35
3.50 [m, 4H, Br-CH2-CH2-O-CH2-], 3.70 [t, 2H, Br-CH2-CH2-O-].
2-Bromoethyl n-octyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90[t, 3H,
CH3-(CH2)5-], 1.20 1.40 [m, 10H, -(CH2)5-], 1.50 1.65 [m, 2H, Br-CH2-
CH2-O-CH2-CH2-], 3.35 3.50 [m, 4H, Br-CH2-CH2-O-CH2-], 3.70 [t, 2H,
Br-CH2-CH2-O-].
2-Bromoethyl n-decyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.88 [t, 3H,
CH3-(CH2)7-], 1.15 1.40 [m, 14H, -(CH2)7-], 1.50 1.65 [m, Br-CH2-CH2-
O-CH2-CH2-], 3.35 3.50 [m, 4H, Br-CH2-CH2-O-CH2-], 3.70 [t, 2H, Br-
CH2-CH2-O-].
2-Azidoethyl octadecyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 3H,
CH3-(CH2)15-], 1.15 1.40 [m, 30H, -(CH2)15-], 1.50 1.65 [m, 2H, -O-CH2-
CH2-CH2-], 3.35 [t, 2H, N3-CH2-CH2-O-CH2-], 3.45 [t, 2H, N3-CH2-CH2-
O-], 3.60 [t, 2H, N3-CH2-CH2-O-].
All the 2-aminoethyl n-alkyl ether intermediates used to synthesize the
cationic amphiphiles 1 6 were prepared following the same protocol
outlined in Scheme 1. As a representative example, synthesis of 2-amino-
ethyl-n-dodecyl ether is described below in detail.
2-Aminoethyl n-dodecyl ether: In a 25 mL round-bottomed flask, a
mixture of 2-azidoethyl n-dodecyl ether (330 mg, 1.29 mmol) and triphenyl
phosphine (508.7 mg, 1.94 mmol) dissolved in tetrahydrofuran (2 mL) was
stirred at room temperature. Effervescence was observed. After 10 min,
water (three drops) was added and the reaction mixture was kept under
stirring for 38 h. The reaction mixture was then concentrated on a rotary
evaporator and column chromatographic purification of the residue (using
60 120 mesh size silica and 5 10% methanol in chloroform as the eluent)
afforded the pure title compound (280.5 mg, 94.6%) as a pale yellow
colored liquid. Rf ˆ 0.30 (chloroform/methanol 90:10); 1H NMR (200 MHz,
CDCl3): d ˆ 0.90 [t, 3H, CH3-(CH2)9-], 1.15 1.40 [m, 18H, -(CH2)9-], 1.50
1.65 [m, 2H, -O-CH2-CH2-CH2-], 2.90 [t, 2H, NH2-CH2-CH2-], 3.35 3.55
[m, 4H, -CH2-O-CH2-], 3.60 [brs, 2H, NH2-].
1
2-Bromoethyl n-tetradecyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)11-], 1.20 1.40 [22H, -(CH2)11-], 1.57 [q, 2H, -O-CH2-CH2-
(CH2)11-], 3.37 3.50 [m, 4H, Br-CH2-CH2-O-CH2-], 3.70 [t, 2H, Br-CH2-
CH2-O-].
2-Aminoethyl n-octyl ether: H NMR (200 MHz, CDCl3): d ˆ 0.88 [t, 3H,
CH3-(CH2)5-], 1.15 1.40 [m, 10H, -(CH2)5-], 1.45 1.62 [m, 2H, -O-CH2-
CH2-CH2-], 3.00 [m, 2H, NH2-CH2-CH2-]; 3.45 [t, 2H, NH2-CH2-CH2-O-
CH2-], 3.55 [t, 3H, NH2-CH2-CH2-O-], 4.68 [brs, 2H, NH2-].
2-Aminoethyl n-decyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.88 [t, 3H,
CH3-(CH2)7-], 1.15 1.40 [m, 14H, -(CH2)7-], 1.45 1.60 [m, 2H, -O-CH2-
CH2-CH2-], 2.87 [t, 2H, NH2-CH2-CH2-], 3.00 [brs, 2H, NH2-], 3.33 3.45
[m, 4H, -CH2-O-CH2-].
2-Bromoethyl n-hexadecyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)11-], 1.18 1.40 [m, 26H, -(CH2)13-], 1.50 1.62 [m, 2H,
-O-CH2-CH2-(CH2)11-], 3.38 3.50 [m, 4H, Br-CH2-CH2-O-CH2-], 3.70 [t,
2H, Br-CH2-CH2-O-].
2-Aminoethyl n-tetradecyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)11-], 1.15 1.40 [m, 22H, -(CH2)11-], 1.45 1.60 [m, -O-CH2-
CH2-CH2-], 2.90 [brs, 2H, NH2-CH2-CH2-], 3.35 3.50 [m, 4H, -CH2-O-
CH2-], 3.55 [brs, 2H, NH2-].
2-Aminoethyl n-hexadecyl ether: 1H NMR (400 MHz, CDCl3): d ˆ 0.88 [t,
3H, CH3-(CH2)13-], 1.20 1.40 [m, 26H, -(CH2)13-], 1.50 1.62 [m, 2H, -O-
CH2-CH2-CH2-], 2.83 [t, 2H, NH2-CH2-CH2-O-], 3.38 3.45 [m, 4H, -CH2-
O-CH2-].
1
2-Chloroethyl n-octadecyl ether: H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)15-], 1.15 1.40 [m, 30H, -(CH2)15-], 3.47 [t, 2H, Cl-CH2-
CH2-O-CH2-], 3.55 3.70 [m, 4H, Cl-CH2-CH2-O-].
All the 2-azidoethyl-n-alkyl ether intermediates used to synthesize cationic
amphiphiles 1 6 were prepared following the same protocol outlined in
a representative example, synthesis of 2-azidoethyl-n-
dodecyl ether is described below in detail.
Scheme 1. As
Synthesis of 2-azidoethyl n-dodecyl ether: In a 15 mL round-bottomed
flask, NaN3 (443.3 mg, 6.82 mmol) was added to 2-bromoethyl n-dodecyl
ether (400 mg, 1.36 mmol) dissolved in dry dimethylformamide (2 mL)
under nitrogen and the temperature of the reaction was raised to 608C. The
reaction mixture was kept under stirring at 608C for 16 h. The temperature
of the reaction mixture was gradually lowered to room temperature, water
(15 mL) added and the reaction mixture extracted with ethyl acetate (3 Â
20 mL). The organic phases were then washed with saturated brine solution
(3 Â 15 mL), dried with anhydrous Na2SO4, filtered and the filtrate was
concentrated on a rotary evaporator. Column chromatographic (using 60
120 mesh size silica and 5 7% (v/v) ethyl acetate in hexane as eluent) of
the residue afforded the pure title compound as a colorless liquid (345 mg,
1
2-Aminoethyl n-octadecyl ether: H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)15-], 1.15 1.40 [m, 30H, -(CH2)15-], 1.50 1.65 [m, 2H, -O-
CH2-CH2-CH2-], 2.97 [t, 2H, NH2-CH2-CH2-O-], 3.10 [brs, NH2-CH2-],
3.42 3.57 [m, 4H, -CH2-O-CH2-].
The secondary amine intermediates (IV, Scheme 1) in the syntheses of
amphiphiles 1 6 were not isolated as pure compounds. Instead, the crude
secondary amines were subjected to quaternization with alkaline 2-chlor-
oethanol. As a representative example, synthetic details for N,N-di-[2-n-
dodecyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium chloride (amphi-
phile 3, Scheme 1) is outlined below.
N,N-Di-[2-n-dodecyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium chlor-
1
99%). Rf ˆ 0.40 (hexane/ethyl acetate 95:5). H NMR (200 MHz, CDCl3):
ide (3, Scheme 1): In
a 10 mL round-bottomed flask, a mixture of
d ˆ 0.90 [t, 3H, CH3-(CH2)9-], 1.15 1.40 [m, 18H, -(CH2)9-], 1.50 1.65 [m,
-O-CH2-CH2-CH2-], 3.32 [t, 2H, N3-CH2-CH2-O-CH2-], 3.42 [t, 2H, N3-
CH2-CH2-], 3.55 [t, 2H, N3-CH2-CH2-O-].
2-aminoethyl n-dodecyl ether (240 mg, 1.05 mmol), 2-bromoethyl n-do-
decyl ether (307 mg, 1.05 mmol), and potassium carbonate (173.3 mg,
1.26 mmol) were taken in dimethyl sulfoxide (1 mL) and the reaction
mixture was heated to 658C. The reaction mixture was kept under stirring
for 42 h. Saturated brine solution (15 mL) was added to the reaction
mixture and extracted with ethyl acetate (3 Â 20 mL). The combined
organic phases was dried with anhydrous Na2SO4, filtered and the filtrate
was concentrated on a rotary evaporator. The secondary amine intermedi-
ate, N,N-di-[2-n-dodecyloxyethyl]amine (IV, Scheme 1) was purified fur-
ther from the residue by silica gel column chromatography using 60
120 mesh size silica and 4% methanol in chloroform (v/v) as the eluent.
The combined fractions eluted with 4% methanol in chloroform was
concentrated and the residue (401mg, pale yellow semisolid) was
quaternized using 2-chloroethanol and NaOH as described below.
2-Azidoethyl octyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 3H,
CH3-(CH2)5-], 1.15 1.45 [m, 10H, -(CH2)-], 1.50 1.65 [m, 2H, -O-CH2-
CH2-CH2-], 3.35 [t, 2H, N3-CH2-CH2-O-CH2-], 3.45 [t, 2H, N3-CH2-CH2-
O-], 3.60 [t, 2H, N3-CH2-CH2-O-].
2-Azidoethyl decyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 3H,
CH3-(CH2)7-], 1.20 1.50 [m, 14H, -(CH2)7], 1.50 1.70 [m, 2H, -O-CH2-
CH2-CH2-], 3.35 [t, 2H, N3-CH2-CH2-O-CH2-], 3.45 [t, 2H, N3-CH2-CH2-
O-], 3.60 [t, 2H, N3-CH2-CH2-O-].
2-Azidoethyl tetradecyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)11-], 1.15 1.40 [m, 22H, -(CH2)11-], 1.50 1.62 [m, 2H, -O-
CH2-CH2-CH2-], 3.32 [t, 2H, N3-CH2-CH2-O-CH2-], 3.45 [t, 2H, N3-CH2-
CH2-O-], 3.58 [t, 2H, N3-CH2-CH2-O-].
2-Azidoethyl hexadecyl ether: 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t,
3H, CH3-(CH2)13-], 1.201.40 [m, 26H, -(CH2)13-], 1.50 1.65 [m, 2H, -O-
A mixture of the residue obtained above (155 mg), chloroethanol (2 mL)
and NaOH (150 mg in 0.5 mL water) in a 5 mL round-bottomed flask was
stirred at 1108C for 38 h. The unreacted 2-chloroethanol was evaporated
from the reaction mixture as far as possible by raising the temperature to
906
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Chem. Eur. J. 2002, 8, No. 4

Cationic Lipofectins
900 909
1308C. The residual 2-chloroethanol was removed on a rotary evaporator
by repeated addition of methanol. Column chromatographic purification of
the resulting residue using 60 120 mesh size silica and 8 10% methanolic
chloroform (v/v) as the eluent afforded pure title cationic amphiphile (V,
Scheme 1) as a colorless semi solid (98.4 mg, 49.4%). Rf ˆ 0.30 (chloro-
form/methanol 90:10).
combined filtrate was cooled to 08C. NaBH4 (0.11 g, 3.0 mmol) was added
to the cold solution and the mixture was kept under stirring for 4 h at room
temperature. The reaction mixture was then diluted with chloroform
(25 mL), washed with water (3 Â 25 mL), the chloroform layer dried over
anhydrous Na2SO4 and filtered. Solvents from the filtrate was removed on a
rotary evaporator. Column chromatographic purification of the residue
using silica gel 60 120 mesh size and 4% (v/v) methanol in chloroforn as
eluent afforded the title secondary amine (Rf ˆ 0.5, chloroform/methanol
90:10) as a pale yellowish solid (0.18 g, 33.4%).
N,N-Di-[2-n-dodecyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium chlor-
ide (3): 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 6H, CH3-(CH2)9-],
1.20 1.45 [m, 36H, -(CH2)9-], 1.45 1.60 [m, 4H, -O-CH2-CH2-(CH2)9-],
3.45 [t, 4H, -O-CH2-(CH2)10-], 3.70 3.95 [m, 12H, (HO-CH2-
N,N-Di-n-dodecylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.8 [t, 6H, CH3-
‡
‡
CH2)2N (CH2-CH2-O-)2-], 4.05 4.15 [m, 4H, (HO-CH2-CH2)2N (CH2-
CH2-O-)2]; HRMS (LSIMS): m/z: calcd for C32H68NO4: 530.5148; found:
530.5160.
(CH2)9-], 1.05 1.4 [m, 18H, CH -(CH2)9], 1.45 1.7 [m, 4H, NH(CH2-CH2-
3
)2-], 2.8 [t, 4H, NH(CH2-CH2-)2], 4.0 4.7 [br, NH].
N,N-Di-n-octylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.8 [t, 6H, CH3-
N,N-Di-[2-n-octyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium chloride
(1): 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 6H, CH3-(CH2)5-], 1.15
1.40 [m, 20H, -(CH2)5-], 1.45 1.60 [m, 4H, -O-CH2-CH2-(CH2)5-], 3.45 [t,
(CH2)5-], 1.05 1.45 [m, 10H, CH -(CH2)5], 1.6 1.85 [m, 4H, NH(CH2-
3
CH2-)2-], 2.8 [t, 4H, NH(CH2-CH2-)2], 5.95 6.4 [br, NH].
N,N-Di-n-decylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, 6H, CH3-
‡
6H, -O-CH2-(CH2)6-], 3.70 3.90 [m, 12H, (HO-CH2-CH2)2N (CH2-CH2-
(CH2)7-], 1.1 1.45 [m, 14H, CH -(CH2)7], 1.45 1.75 [m, 4H, NH(CH2-
3
‡
O-)2-], 4.10 [brs, 4H, (HO-CH2-CH2)2N (CH2-CH2-O-)2-]; HRMS
CH2-)2-], 2.8 [t, 4H, NH(CH2-CH2-)2], 4.1 4.7 [br, N H].
(LSIMS): m/z: calcd for C24H52NO4: 418.3896; found: 418.3913.
N,N-Di-n-tetradecylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, 6H,
CH3-(CH2)9-], 1.15 1.45 [m, 18H, CH3-(CH2)11], 1.45 1.7 [m, 4H,
NH(CH2-CH2-)2-], 2.55 2.7 [t, 4H, NH(CH2-CH2-)2], 4.0 4.7 [br, NH].
N,N-Di-[2-n-decyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium chloride
(2): 1H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 6H, CH3-(CH2)7-], 1.15
1.35 [m, 28H, -(CH2)7-], 1.45 1.60 [m, 4H, -O-CH2-CH2-(CH2)7-], 3.42 [t,
N,N-Di-n-hexadecylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.85 [t, 6H,
‡
4H, -O-CH2-(CH2)8-], 3.65 3.90 [m, 12H, (HO-CH2-CH2)2N (CH2-CH2-
CH3-(CH2)14-], 1.15 1.60 [m, 28H, CH -(CH2)14], 2.6 [t, 4H, NH(CH2-
3
‡
O-)2-], 4.05 [brs, 4H, (HO-CH2-CH2)2N (CH2-CH2-O-)2-]; HRMS
CH2-)2].
(LSIMS): m/z: calcd for C28H60NO4: 474.4522; found: 474.4526.
N,N-Di-n-octadecylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.85 [t, 6H,
N,N-Di-[2-n-tetradecyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium
CH3-(CH2)15-], 1.15 1.35 [m, 30H, CH -(CH2)15], 1.4 1.7 [m, 4H,
3
1
chloride (4): H NMR (200 MHz, CDCl3‡CD3OD): d ˆ 0.90 [t, 6H, CH3-
NH(CH2-CH2-)2-], 2.6 [t, 4H, NH(CH2-CH2-)2].
(CH2)11-], 1.20 1.45 [m, 44H,-(CH2)11-], 1.50 1.65 [m, 4H, -O-CH2-CH2-
(CH2)11-], 3.45 [t, 4H,-O-CH2-(CH2)12-], 3.60 3.75 [m, 8H, (HO-CH2-
N,N-Oleyl-n-octadecylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.85 1.1
‡
‡
[t, 6H, CH3-(CH2)15-], 1.1 1.5 [m, 18H, CH-(CH2)15], 1.6 1.85 [m, 4H,
3
CH2)2N (CH2-CH2-O-)2-], 3.80 [brs, 4H, (HO-CH2-CH2)2N (CH2-CH2-O-
ˆ
‡
NH(CH2-CH2-)2-], 1.95 2.15 [m, 4H, -CH2-CH CH-CH2-], 2.9 [t, 4H,
)2-], 4.00 [brs, 4H, (HO-CH2-CH2)2N (CH2-CH2-O-)2-]; HRMS (LSIMS):
ˆ
NH(CH2-CH2-)2], 5.3 5.6 [t, 2H, -CH2-CH CH-CH2-].
m/z: calcd for C36H76NO4: 586.5774; found: 586.5798.
N,N-Di-oleylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, 6H, CH3-
N,N-Di-[2-n-hexadecyloxyethyl]-N,N-di-[2-hydroxyethyl]ammonium
1
(CH2)15-], 1.1 1.5 [m, 18H, CH-(CH2)15], 1.5 1.75 [m, 4H, NH(CH2-CH2-
3
chloride (5): H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 6H, CH3-(CH2)13-],
ˆ
)2-], 2.0 [m, 4H, -CH2-CH CH-CH2-], 2.8 [t, 4H, NH(CH2-CH2-)2], 5.3 [t,
1.15 1.35 [m, 52H, -(CH2)13-], 1.45 1.60 [m, 4H, -O-CH2-CH2-(CH2)13-],
3.45 [t, 4H, -O-CH2-(CH2)14-], 3.60 4.00 [m, 12H, (HO-CH2-
ˆ
2H, -CH2-CH CH-CH2-].
N,N-Di-n-archidylamine: 1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, 6H, CH3-
‡
‡
CH2)2N (CH2-CH2-O-)2-], 4.10 [brs, 4H, (HO-CH2-CH2)2N (CH2-CH2-
O-)2-]; HRMS (LSIMS): m/z: calcd for C40H84NO4: 642.6400; found:
642.6418.
(CH2)17-], 1.15 1.6 [m, 34H, CH -(CH2)17], 1.6 1.8 [m, 4H, NH(CH2-CH2-
3
)2-], 2.95 [t, 4H, NH(CH2-CH2-)2], 4.0 4.7 [br, NH].
All the new DHDEAB analogues were prepared by quaternizing the
appropriate hydrophobic secondary amines with alkaline 2-chloroethanol
as outlined in Scheme 2. As a representative example, the details of the
final quaternization step for the synthesis of N,N-di-n-dodecyl-N,N-di-n-
hydroxyethylammonium chloride (amphiphile 9, Scheme 2) is outlined
below.
N,N-Di-[2-n-octadecyloxyethyl]-N,N-di-[2-hydroxyethyl]
ammonium
1
chloride (6): H NMR (200 MHz, CDCl3): d ˆ 0.90 [t, 6H, CH3-(CH2)15-],
1.15 1.40 [m, 60H, -(CH2)15-], 1.45 1.65 [m, 4H, -O-CH2-CH2-(CH2)15-],
3.42 [t, 4H, -O-CH2-(CH2)16-], 3.60 3.87 [m, 12H, (HO-CH2-
‡
‡
CH2)2N (CH2-CH2-O-)2-], 4.07 [brs, 4H, (HO-CH2-CH2)2N (CH2-CH2-
O-)2-]; HRMS (LSIMS): m/z: calcd for C44H92NO4: 698.7026; found:
698.7050.
N,N-Di-n-dodecyl-N,N-dihydroxyethylammonium chloride (9, Scheme 2):
In a 25 mL round-bottomed flask a mixture of N,N-di-n-dodecylamine
(0.18 g, 0.50 mmol) and 2-chloroethanol (5 mL) was stirred at room
temperature for 15 min. NaOH (0.18 g, 0.46 mmol) dissolved in water
(1.5 mL) was added and the reaction mixture was heated for 36 h at a
temperature of 858C. Excess 2-chloroethanol was removed on a rotary
evaporator by repeated addition of methanol. Column chromatographic
purification of the residue using silica gel (60 120 mesh size) and 10%
methanol in chloroform (v/v) as the eluent afforded the title amphiphile
(118.6 mg, 44%) as a colorless liquid (Rf ˆ 0.4 10% methanol in chloro-
form).
All the dihydroxyethyl head group containing DHDEAB analogues 7 15
were prepared following the same protocol as outlined in Scheme 2. The
necessary starting primary amines I were conventionally synthesized in
three steps from the corresponding primary alcohols namely, conversion of
alcohol to primary azide through the intermediate mesylate followed by
reduction of the primary azides with triphenyl phosphine and water. As a
representative example, synthetic details for N,N-di-n-dodecylamine is
outlined below.
1-Dodecanal: Pyridinium chlorochromate (2.63 g, 12.3 mmol) and Celite
(2.63 g) was taken in dry dichloromethane (13 mL) in a two-necked round-
bottomed flask and to this was added n-dodecyl alcohol (1.52 g, 8.2 mmol)
dissolved in dry dichloromethane (6 mL). The reaction mixture was cooled
to 08C and stirring was continued at room temperature under nitrogen for
4 h. Dichloromethane was removed on a rotary evaporator. Column
chromatographic purification of the residue using 60 120 mesh size silica
gel and 10% ethyl acetate in petroleum ether (b.p. 60 808C) (v/v) as the
eluent afforded pure title compound as a colorless liquid (1.22 g, 81.5%).
N,N-Di-n-dodecylammonium chloride (9): 1H NMR (200 MHz, CDCl3):
d ˆ 0.9 [t, 6H, CH3-(CH2)n-], 1.55 1.9 [m, 4H, (HOCH2-CH2)2N(CH2-
CH2)2-], 3.3 3.5 [m, 4H, (HOCH2-CH2)2N(CH2-(CH2)2], 3.55 3.85 [m,
4H, (HOCH2-CH2)2N(CH2-CH2)2-], 3.95 4.2 [m, 4H, (HOCH2-
‡
CH2)2N (CH2-CH2-)2], 5.20 5.45 [m, 2H, OH]; FABMS (LSIMS): m/z:
‡
442 [M‡H] for C28H60NO2; HRMS(LSIMS): m/z: calcd for C28H60NO2:
442.4624; found: 442.4610.
N,N-Di-n-dodecylamine: In a 50 mL round-bottomed flask a mixture of n-
dodecanal (0.28 g, 1.5 mmol), n-dodecylamine (0.28 g, 1.5 mmol) was
dissolved in dry dichloromethane (10 mL) and the solution was cooled to
08C. Anhydrous MgSO4 (0.22 g, 1.8 mmol) was added to the cold solution
and the reaction mixture was stirred under N2 overnight during which
period the temperature gradually raised to room temperature. MgSO4 was
filtered off, the precipitate washed with methanol (3 Â 2 mL) and the
N,N-Di-n-octyl-N,N-dihydroxyethylammonium chloride (7): 1H NMR
(200 MHz, CDCl3): d ˆ 0.9 [t, 6H, (CH3-CH2)n-], 1.15 1.45 [m, 20H,
‡
-(CH2)5-], 1.5 1.8 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.3 3.5 [m,
‡
4H, (HO-CH2-CH2)2N (CH2-CH2-)2], 3.50 3.75 [m, 4H, (HOCH2-
‡
‡
CH2)2N (CH2-CH2-)2], 3.95 4.15 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2];
FABMS(LSIMS): m/z: 330 for C20H42NO2; HRMS(LSIMS): m/z: calcd for
C20H42NO2: 330.3372; found: 330.3358.
Chem. Eur. J. 2002, 8, No. 4
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0804-0907 $ 17.50+.50/0
907

A. Chaudhuri, N. M. Rao et al.
FULL PAPER
N,N-Di-n-decyl-N,N-dihydroxyethylammonium chloride (8): 1H NMR
(200 MHz, CDCl3): d ˆ 0.9 [t, 6H, CH3-(CH2)n-], 1.20 1.45 [m, 28H,
cells were lysed in 50 mL of lysis buffer (0.25m Tris ¥ HCl, pH 8.0 and 0.5%
NP40). The b-galactosidase activity per well was estimated by adding 50 mL
of 2 Â substrate solution (1.33 mgmLÀ1 ONPG, 0.2m sodium phosphate,
pH 7.15 and 2 mm magnesium chloride) to the lysate in a 96-well plate.
Absorption at 405 nm was converted to b-galactosidase units by using
calibration curve constructed with pure commercial b-galactosidase
enzyme. The values of b-galactosidase units in replicate plates assayed on
the same day varied by less than 30%. The transfection efficiency values
are the average values from four replicate transfection plates assayed on
the same day. Each transfection experiment was repeated three times on
different days and the day-to-day variation in average transfection
efficiency was found to vary within 20 30%. The transfection profiles
obtained on different days were identical.
‡
-(CH2)7-], 1.55 1.88 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.3 3.5 [m,
‡
4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.50 3.8 [m, 4H, (HOCH2-
‡
‡
CH2)2N (CH2-CH2)2], 3.95 4.15 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-
)2]; FABMS(LSIMS): m/z: 386 for C24H52NO2; HRMS(LSIMS): m/z: calcd
for C24H52NO2: 386.3998; found: 386.4002.
N,N-Di-n-tetradecylammonium chloride (10): (200 MHz, CDCl3): d ˆ 0.9
[t, 6H, CH3-(CH2)n-], 1.20 1.5 [m, 44H, -(CH2)11-], 1.55 1.9 [m, 4H,
‡
‡
(HOCH2-CH2)2N (CH2-CH2-)2], 3.3 3.5 [m, 4H, (OHCH2-CH2)2N (CH2-
‡
CH2-)2], 3.55 3.85 [m, 4H, (OHCH2-CH2)2N (CH2-CH2-)2], 3.95 4.2 [m,
4H, (OHCH2-CH2)2N (CH2-CH2-)2], 5.20 5.45 [m, 2H, -OH]; FABMS
‡
‡
(LSIMS): m/z: 498 [M‡H] for C32H68NO2; HRMS(LSIMS): m/z: calcd for
Dynamic light scattering measurements: Dynamic light scattering (DLS)
measurements were performed using a DLS-700 instrument from Otsuka
Electronics Co. Ltd., Japan, fitted with a 5 mW He/Ne laser operating at
632.8 nm by placing the sample tube in the thermostated chamber of the
goniometer. All measurements were taken at 908 angle. Each sample was
prepared using doubly distilled water filtered several times through a
Millipore 0.22 mm membrane filter. The DLS intensity data were processed
using the instrumental software to obtain the hydrodynamic diameter (dh).
C32H68NO2: 498.5250; found: 498.5274.
N,N-Di-n-hexadecyldihydroxyethylammonium chloride (11): 1H NMR
(200 MHz, CDCl3): d ˆ 0.9 [t, 6H, (CH3-CH2)n-], 1.20 1.5 [m, 52H,
‡
-(CH2)13-], 1.55 1.85 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.35 3.6
‡
[m, 4H, (HOCH2-CH2-)2N (CH2-CH2-)2], 3.60 3.80 [m, 4H, (HOCH2-
‡
‡
CH2)2N (CH2-CH2-)2], 3.95 4.2 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2];
‡
FABMS (LSIMS): m/z: 555 [M‡H] for C36H76NO2.
N,N-Di-n-octadecyl-N,N-dihydroxyethylammonium
chloride
(12):
Exclusion of ethidium bromide (EtBr) from DNA by the cationic lipids:
The extent of EtBr binding to the DNA was monitored by the changes in
the fluorescence. EtBr fluorescence was monitored in Hitachi 4500
fluorimeter by setting the excitation wavelength at 518 nm and emission
wavelength at 585 nm. To 1mL TE buffer (pH 8.0), 0.78 nmol DNA and
2.5 nmol EtBr were added. The change in fluorescence was monitored after
adding small volumes of lipids 1 15 to the EtBr/DNA complex. Arbitrary
fluorescence values were recorded after allowing sufficient time for
equilibration. The order of addition of EtBr or lipid to DNA did not alter
the final values indicating that the equilibrium does not depend on the
order of addition and reaches in minutes. Percent fluorescence was
calculated considering the fluorescence value in the absence of lipid as 100.
1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, H, CH3-(CH2)n-], 1.2 1.4 [m,
‡
60H, -(CH2)15-], 1.6 1.85 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.3
‡
3.5 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.6 [m, 4H, HOCH2-
‡
‡
CH2)2N (CH2-CH2-)2], 4.0 4.1[m, 4H, (HOC H2-CH2)2N (CH2-CH2-)2],
‡
5.2 [m, 2H, OH]; FABMS (LSIMS): m/z: 61 1 M[‡H] for C40H84NO2;
HRMS(LSIMS): m/z: calcd for C40H84NO2: 610.6502; found: 610.6525.
N-Oleyl-n-octadecyl-N,N-dihydroxyethylammonium
chloride
(13):
1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, 6H, CH3-(CH2)n], 1.20 1.45 [m,
‡
52H, -(CH2)13-], 1.6 1.8 [br, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 1.90
‡
2.10 (m, -CH2-CH ˆ CH-CH2-); 3.3 3.5 [m, 4H, (HOCH2-CH2)2N (CH2-
‡
CH2-)2], 3.55 3.8 [m, 4H, (HO-CH2-CH2)2N (CH2-CH2-)2], 3.95 4.15 [m,
‡
ˆ
4H, (HOCH2-CH2)2N (CH2-CH2-)2], 5.28 5.4 [m, 2H, -CH2-CH CH-
Toxicity assay: Cytotoxicity of lipids 1 15 was assessed using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction
assay as described earlier.[32] The cytotoxicity assay was performed in 96-
well plates by maintaining the ratio of number of cells to amount of cationic
lipid constant as in transfection experiments. MTT was added three hours
after adding the cationic lipid to the cells.
‡
CH2-], 5.45 5.55 [br, 2H, OH]; FABMS(LSIMS): m/z: 609 [M‡H] for
C40H82NO2; HRMS(LSIMS): m/z: calcd for C40H82NO2: 608.6346; found:
608.6354.
N,N-Di-n-oleyl-N,N-dihydroxyethylammonium chloride (14):
1H NMR (200 MHz, CDCl3): d ˆ 0.9 [t, 6H, CH3-(CH2)n], 1.20 1.5 [m,
ˆ
44H, (-CH2-(CH2)5-CH2-CH CH-CH2-(CH2)6-CH3)2], 1.60 1.80 [br, 4H,
Anisotropy measurements of cationic liposomes: Cationic lipids were
dissolved in chloroform along with DPH at a mol ratio of 300:1and dried
under N2 gas. The lipid was hydrated in buffer (TrisCl, pH 7.4, 100 mm) for
few hours and then sonicated in a Branson (Model B-50) sonifier at
temperatures above the transition temperatures till clarity. Anisotropy was
calculated by recording the fluorescence values (excitation at 354 nm;
emission at 427 nm) at parallel and perpendicular polariser positions in an
Hitachi F4010 fluorimeter. Anisotropy was calculated according to the
procedure given in Shinitzky and Barenholz (M. Shinitzky, Y. Barenholz,
Biochem. Biophys. Acta 1978, 515, 367 394).
‡
ˆ
CH3(HOCH2-CH2)N (CH2-CH2-)2], 1.90 2.10 [m, 8H, (-CH2-CH CH-
‡
CH2-)2], 3.3 3.5 [m, 4H, (HOCH2-CH2)2N (CH2-CH2-)2], 3.5 3.7 [m, 4H,
(HO-CH2-CH2)2N (CH2-CH2-)2], 3.95 4.15
CH2)2N (CH2-CH2-)2], 5.3 [m, 4H, -(-CH2CH CH-CH2-)2]; FABMS
‡
[m, 4H,
(HOCH2-
‡
ˆ
‡
(LSIMS): m/z: 607 [M‡H] for C40H80NO2.
N,N-Di-n-arachidyl-N,N-dihydroxyethylammonium
chloride
(15):
1H NMR (200 MHz,CDCl3): d ˆ 0.9 [t, 6H, CH3-(CH2)n-], 1.2 1.4 [m,
‡
68H,-(CH2)34-], 1.45 1.65 [m, 4H, (HOCH2-CH2)2N (CH2-CH2)2], 3.33.5
‡
[m,
4H,
(HOCH2-CH2)2N (CH2-CH2-)2],
(HOCH2CH2)2N (CH2CH2)2],
CH2)2N (CH2-CH2-)2]; FABMS (LSIMS): m/z: 667 [M‡H]
3.53 3.65
[m, 4H,
[m,
(HOCH2-
4H
‡
3.95 4.15
‡
‡
for
C44H92NO2.
Acknowledgement
Preparation of liposomes and plasmid DNA: Preparation of liposomes,
isolation of DNA and characterizaton of DNA were done, essentially, as
described in our previous work.[5] Briefly, cationic amphiphiles and
cholesterol were co-dried in chloroform under nitrogen gas and hydrated
in sterile water overnight. The vortexed liposomes were sonicated to clarity.
pCMV b-gal plasmid DNA was prepared by alkaline lysis procedure and
purified by PEG-8000 precipitation according to the published proce-
dures.[31] The plasmid preparations showing a value of A260/A280 more than
1.8 were used.
Financial support for this work from the Department of Biotechnology,
New Delhi, Government of India (to A.C.) is gratefully acknowledged.
Financial supports in the form of doctoral research fellowships from the
Council of Scientific and Industrial Research (CSIR), Government of
India, New Delhi (to R.S.S. and R.B.) and University Grant Commision
(UGC), Government of India, New Delhi (to K.M. and S.K.H.) are
gratefully acknowledged.
Transfection of cells: Transfection of the cells were done essentially as
described earlier.[5] Briefly, COS-1or CHO cells were seeded at a density of
15000 cells per well in
a
96-well plate eighteen hours before the
[1] S. Li, L. Huang, Gene Ther. 2000, 7, 31 34.
transfection. 0.3 mg of plasmid was complexed with varying amount of
lipid and cholesterol (0.05 4.3 nmol) in plain DMEM medium for 30 min.
The charge ratios were varied from 0.1:1to 9:1( ‡/ À ) over this range of
the lipid. The complex was diluted to 100 mL with plain DMEM and added
to the wells. After 3 h of incubation, 100 mL of DMEM with 10% FCS was
added to the cells. The medium was changed to 10% complete medium
after 24 h and the reporter gene activity was assayed after 48 h. Washed
[2] D. Luo, W. M. Saltzman, Nat. Biotechnol. 2000, 18, 33 37.
[3] A. D. Miller, Angew. Chem. 1998, 110, 1862 1880; Angew. Chem. Int.
Ed. 1998, 37, 1768 1785 and references therein.
[4] S. Kawakami, A. Sato, M. Nishikawa, F. Yamashita, M. Hashida, Gene
Ther. 2000, 7, 292 299.
[5] R. Banerjee, P. K. Das, G. V. Srilakshmi, A. Chaudhuri, N. M. Rao, J.
Med. Chem. 1999, 42, 4292 4299.
908
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0804-0908 $ 17.50+.50/0
Chem. Eur. J. 2002, 8, No. 4

Cationic Lipofectins
900 909
[6] J. P. Behr, B. Demeneix, J. P. Loeffler, J. Perez-Mutul, Proc. Natl.
Acad. Sci. USA 1989, 86, 6982 6986.
[20] I. Solodin, C. Brown, M. Bruno, C. Chow, E.-H. Jang, R. Debs, T. A.
Heath, Biochemistry 1995, 34, 13537 13544.
[7] J. Wang, X. Guo, Y. Xu, L. Barron, F. C. Szoka, Jr., J. Med. Chem.
1998, 41, 2207 2215.
[21] Y. Liu, D. Liggitt, G. Tu, W. Zhong, K. Gaensler, R. Debs, J. Biol.
Chem. 1995, 270, 24864 24870.
[8] G. Byk, C. Dubertret, V. Escriou, M. Frederic, G. Jaslin, R. Rangara,
B. Pitard, J. Crouzet, P. Wils, B. Schwartz, D. Scherman, J. Med. Chem.
1998, 41, 224 235.
[22] J. H. Felgner, R. Kumar, C. N. Sridhar, C. J. Wheeler, Y.-J. Tsai, R.
Border, P. Ramsey, M. Martin, P. L. Felgner, J. Biol. Chem. 1994, 269,
2550 2561.
[9] G. Byk, B. Wetzer, M. Frederic, C. Dubertret, B. Pitard, G. Jaslin, D.
Scherman, J. Med. Chem. 2000, 43, 4377 4387.
[10] M. J.Bennett, A. M. Aberle, R. P. Balasubramaniam, J. G. Malone,
R. W. Malone, M. H. Nantz, J. Med. Chem. 1997, 40, 4069 4078.
[11] V. Floch, G. Le Bolc×h, C. Gable-Guillaume, N. Le Bris, J.-J. Yaouanc,
[23] E. W. Alton, P. G. Middleton, N. J. Caplen, S. N. Smith, D. M. Steel,
F. M. Munkonge, P. K. Jeffery, B. J. Stevension, G. McLachlan, J. R.
Dorin, D. J. Porteous, Nat. Genet. 1993, 5, 135 142.
[24] M. A. W. Eaton, T. S. Baker, C. F. Catterall, K. Crook, G. S. Macaulay,
B. Mason, T. J. Norman, D. Parker, J. J. B. Perry, R, J. Taylor, A.
Turner, A. N. Weir, Angew. Chem. 2000, 112, 4229 4233; Angew.
Chem. Int. Ed. 2000, 39, 4063 4067.
¬
¬
H. des Abbayes, C. Ferec, J.-C. Clement, Eur. J. Med. Chem. 1998, 41,
923 934.
[12] J. P. Behr, Acc. Chem. Res. 1993, 26, 274 278.
[25] X. Gao, L. Huang, Biochem. Biophys. Res. Commun. 1991, 179, 280
285.
[26] P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, W. Chan, M. Wenz,
J. P. Northorp, G. M. Ringold, M. Danielsen, Proc. Natl. Acad. Sci.
USA 1987, 84, 7413 7417.
[27] Y. K. Ghosh, S. S. Visweswarial, S. Bhattacharya, FEBS Lett. 2000,
473, 341 344.
[28] T. Fichert, A. Regelin, U. Massing, Bioorg. Med. Chem. Lett. 2000, 10,
787 791.
[13] D. M. Lynn, R. Langer, J. Am. Chem. Soc. 2000, 122, 10761 10768.
[14] Y. Liu, L. C. Mounkes, H. D. Liggitt, C. S. Brown, I. Solodin, T. D.
Heath, R. J. Debs, Nat. Biotechnol. 1997, 15, 167 173.
[15] N. S. Templeton, D. D. Lasic, P. M. Frederik, H. H. Strey, D. D.
Roberts, G. N. Pavlakis, Nat. Biotechnol. 1997, 15, 647 652.
[16] N. J. Caplen, E. W. Alton, P. G. Middleton, J. R. Dorin, B. J. Steven-
sion, X. Gao, S. R. Durham, P. K. Jeffery, M. E. Hodson, C. Coutelle,
L. Huang, D. J. Porteous, R. Williamson, D. M. Geddes, Nat. Med.
1995, 1, 39 46.
[29] A. E. Regelin, S. Fankhaenel, L. Gurtesch, C. Prinz, G. v. Kiedrowski,
U. Massing, Biochim. Biophys. Acta 2000, 1464, 151 164.
[30] T. Ren, Y. K. Song, G. Zhang, D. Liu, Gene Ther. 2000, 7, 764 768.
¬
¬
[17] E. Guenin, A.-C. Herve, V. Floch, S. Loisel, J.-J. Yaouanc, J.-C.
¬
¬
Clement, C. Ferec, H. des Abbayes, Angew. Chem. 2000, 112, 643
645; Angew. Chem. Int. Ed. 2000, 39, 629 631.
[31] J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning:
A
[18] R. G. Cooper, C. J. Etheridge, L. Stewart, J. Marshall, S. Rudginsky,
S. H. Cheng, A. D. Miller, Chem. Eur. J. 1998, 4, 137 154.
[19] C. J. Wheeler, P. L. Felgner, Y. J. Tsai, J. Marshall, L. Sukhu, G. Doh, J.
Hartikka, J. Nietupski, M. Manthorpe, M. Nichols, M. Piewe, X. Liang,
J. Norman, A. Smith, S. H. Cheng, Proc. Natl. Acad. Sci. USA 1996, 93,
11454 11459.
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989.
[32] M.-B. Hansen, S. E. Neilson, K. Berg, J. Immunol. Methods 1989, 119,
203 210.
Received: May 22, 2001
Revised: September 10, 2001 [F3277]
Chem. Eur. J. 2002, 8, No. 4
¹ WILEY-VCH Verlag GmbH, 69451Weinheim, Germany, 2002
0947-6539/02/0804-0909 $ 17.50+.50/0
909