Novel series of non-glycerol-based cationic transfection lipids for use in liposomal gene delivery

Article abstract of DOI:10.1021/jm9806446

A novel series of nontoxic and non-glycerol-based simple monocationic transfection lipids containing one or two hydroxyethyl groups directly linked to the positively charged nitrogen atom were synthesized. The in vitro transfection efficiencies of these new liposomal gene delivery reagents were better than that of lipofectamine, a widely used transfection agent in cationic lipid-mediated gene transfer. The most efficient transfection formulation was observed to be a 1:1:0.3 mol ratio of DHDEAB (N,N-di-n- hexadecyl-N,N-dihydroxyethylammonium bromide): cholesterol:HDEAB (N-n- hexadecyl-N,N-dihydroxyethylammonium bromide) using a DHDEAB-to-DNA charge ratio (+/-) of 0.3:1. Observation of good transfection at charge ratios lower than i suggests that the amphiphile-DNA complex may have net negative charge. Our results reemphasize the important point that in cationic lipid-mediated gene delivery, the overall charge of the lipid-DNA complex need not always be positive. In addition, our transfection results also imply that favorable hydrogen-bonding interactions between the lipid headgroups and the cell surface of biological membranes may have some role for improving the transfection efficiency in cationic lipid-mediated gene delivery.

Full text of DOI:10.1021/jm9806446

4292
J . Med. Chem. 1999, 42, 4292-4299
Novel Ser ies of Non -Glycer ol-Ba sed Ca tion ic Tr a n sfection Lip id s for Use in
Lip osom a l Gen e Deliver y1,†
Rajkumar Banerjee, Prasanta Kumar Das, Gollapudi Venkata Srilakshmi, and Arabinda Chaudhuri*
Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Nalam Madhusudhana Rao*
Centre for Cellular & Molecular Biology, Hyderabad 500 007, India
Received November 13, 1998
A novel series of nontoxic and non-glycerol-based simple monocationic transfection lipids
containing one or two hydroxyethyl groups directly linked to the positively charged nitrogen
atom were synthesized. The in vitro transfection efficiencies of these new liposomal gene delivery
reagents were better than that of lipofectamine, a widely used transfection agent in cationic
lipid-mediated gene transfer. The most efficient transfection formulation was observed to be a
1:1:0.3 mol ratio of DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide):
cholesterol:HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide) using a DHDEAB-
to-DNA charge ratio (+/-) of 0.3:1. Observation of good transfection at charge ratios lower
than 1 suggests that the amphiphile-DNA complex may have net negative charge. Our results
reemphasize the important point that in cationic lipid-mediated gene delivery, the overall charge
of the lipid-DNA complex need not always be positive. In addition, our transfection results
also imply that favorable hydrogen-bonding interactions between the lipid headgroups and
the cell surface of biological membranes may have some role for improving the transfection
efficiency in cationic lipid-mediated gene delivery.
In tr od u ction
based liposomal transfection lipids has been reported,
e.g. DC-Chol synthesized by Huang and co-workers28
and the long chain alkyl acyl carnitine esters recently
designed by Szoka and colleagues.13 These non-glycerol-
based liposomal gene delivery reagents have no hy-
droxyethyl groups present in their polar headgroup
regions. Except for the patented report by Nantz et al.
on the development of a 1,4-diaminobutane-based di-
cationic transfection lipids,21 detailed investigations on
the transfection efficiencies of non-glycerol-based mono-
cationic liposomal transfection vectors containing hy-
droxyethyl groups directly attached to the positively
charged nitrogen atoms have not been reported. Herein,
we report on the synthesis and remarkably high in vitro
transfection efficiencies of a novel series of simple,
nontoxic and non-glycerol-based transfection lipids 1-5
(Chart 1) containing hydroxyethyl group(s) directly
attached to the positively charged quaternized nitrogen
atom.
In gene therapy, patients carrying identified defec-
tive genes are supplemented with the copies of the
corresponding normal genes.1,2 Many gene delivery
reagents (also known as transfection vectors) including
retrovirus,3 adenovirus,4 positively charged polymers
and peptides,5-7 and cationic amphiphilic compounds8,9
are currently being used as carriers of genes in combat-
ing the hereditary diseases by gene therapy. Repro-
ducibility, low cellular and immunological toxicities,
and ease in preparation and administration asso-
ciated with cationic transfection lipids are increasingly
making them the transfection vector of choice in gene
therapy.
Since the first reports8 on cationic liposome-mediated
gene delivery by Felgner et al. in 1987, an upsurge of
global interest has been witnessed in synthesizing
efficient cationic transfection lipids.10-29 Many of the
reported liposomal transfection vectors, e.g. DOTMA,8
DMDHP,18 DMRIE,25 and DOTAP,29 have a common
element in their molecular structures: namely, the
presence of a glycerol backbone. Interestingly, among
the glycerol-based cationic transfection lipids, the polar
headgroup domains of the most efficient lipids, such as
DMRIE25 and DMDHP,18 contain one or two hydroxy-
ethyl groups directly linked to the positively charged
nitrogen atoms. Development of efficient non-glycerol-
Resu lts a n d Discu ssion
Ch em istr y. The key structural elements common to
all the transfection lipids 1-5 (Chart 1) described in
the present investigation include: (a) the presence of a
hydrophobic group either directly linked to the positively
charged nitrogen atom or linked to the positively
charged nitrogen via an ester group, (b) the presence of
at least one hydroxyethyl group directly linked to the
positively charged nitrogen atom, and (c) the absence
of glycerol backbone in the molecular architecture of the
monocationic amphiphiles. The details of the synthetic
procedures for all the novel transfection lipids shown
in Chart 1 are described in the Experimental Section.
† IICT Communication No. 4179.
* Authors to whom correspondence should be addressed. E-mail:
arabinda@iict.ap.nic.in (A.C.) and madhu@ccmb.ap.nic.in (N.M.R.).
Fax: 91-40-7173757 (A.C.) and 91-40-7171195 (N.M.R.). Tel: 91-40-
7173370 (A.C.) and 91-40-7172241 (N.M.R.).
10.1021/jm9806446 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/01/1999

Non-Glycerol-Based Cationic Transfection Lipids
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4293
Sch em e 2. Synthesis of MOOHAC and DOMHACa
Ch a r t 1. Structures of New Transfection lipids
a
Reagents: (a) anhyd MgSO4 (1.0 equiv)/DCM, NaBH4 (2.0
equiv)/DCM/MeOH; (b) Br-(CH2)2-OTBDPS (1 equiv)/K2CO3 (1.1
equiv)/ethyl acetate; (c) TBAF (2.5 equiv)/THF, MeI (huge excess)/
CHCl3/MeOH, Amberlyst A-26 chloride ion-exchange resin.
Sch em e 3. Synthesis of DOHEMABa
Sch em e 1. Synthesis of DHDEAB and HDEAB
As delineated in Schemes 1-3, the chemistries involved
in preparing these new lipids are straightforward.
However, given the high transfection efficiencies of
these nontoxic hydroxyethyl-containing cationic trans-
fection lipids, the overall yields need to be improved in
the future.
Scheme 1 outlines the one-step synthetic procedure
for preparing DHDEAB and HDEAB. Diethanolamine
was initially refluxed with n-hexadecyl bromide in the
presence of potassium carbonate in methanol. The
resulting intermediate tertiary amine (N-n-hexadecyldi-
ethanolamine, not isolated) was then refluxed in a
mixed solvent containing 80:15:5 (v/v) acetonitrile:ethyl
acetate:methanol. Finally, column chromatographic pu-
rification of the product mixture afforded pure DHDEAB
and HDEAB.
The steps used in synthesizing MOOHAC and DOM-
HAC (Scheme 2) include: (a) coupling the appropriate
aliphatic saturated or unsaturated aldehyde with the
appropriate long chain aliphatic amine followed by
reduction of the resulting imine to obtain the corre-
sponding secondary amine, (b) conversion of the second-
ary amine obtained in step (a) to N-hydroxyethyl-N,N-
dialkylamine (tertiary amine) by reacting with the
a
Reagents: (a) n-hexadecanoyl chloride (2.2 equiv)/DMF; (b)
1.0 M aq NaOH/DCM (biphasic system); (c) 2-bromoethanol (1.5
equiv)/85 °C/4 h.
hydroxyl-protected 2-bromoethanol followed by removal
of the hydroxyl protecting group, and (c) quaternizing
the tertiary amine obtained in step (b) with excess of
methyl iodide followed by chloride ion-exchange chro-
matography of the resulting intermediate quaternary
amphiphilic iodide.
Synthesis of DOHEMAB (Scheme 3) essentially con-
sists of: (a) reacting n-hexadecanoyl chloride with
N-methyldiethanolamine to obtain the hydrochloride

4294 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Banerjee et al.
F igu r e 2. Transfection efficiencies of DHDEAB and DDAB
with cholesterol and DOPE as helper lipids on COS-1 cells.
The charge ratios and amount of DNA used are as in Figure
1. Lipofectamine (A) was used for comparision. DHDEAB (C,
E) and DDAB (B, D) were used in combination with DOPE
(B, C) and cholesterol (D, E) at a mole ratio of 1:1. The X-axis
is given in mole ratio ([cationic amphiphile]/[DNA]) instead
of charge ratio for comparing the lipofectamine with the
cationic amphiphiles on the same scale. The charge ratio and
the mole ratio are same for our cationic amphiphiles (since
they carry one charge per molecule) unlike lipofectamine
carrying five positive charges for one molecule.
F igu r e 1. Transfection efficiencies of (1) DOMHAC, (2)
MOOHAC, (3) DOHEMAB, and (4) DHDEAB on COS-1 cells.
The transfection efficiencies of the four lipids were tested by
varying both the charge ratio (X-axis) and cholesterol (Z-axis).
The following mole ratios of cholesterol:cationic lipids were
used in the Z-axis: 0.2:1 (A, E, I, M); 0.4:1 (B, F, J , N); 0.6:1
(C, G, K, O); 1:1 (D, H, L, P); 1.2:1 (Q); 1.5:1 (R). In each well
of a 24-well plate a fixed amount of plasmid DNA (0.3 µg) was
used to complex with 0.1-9 nmol of cationic lipid to vary the
charge ratio (+/-) from 0.1 to 9.
An interesting observation with these non-glycerol-
based hydroxyethyl headgroup amphiphiles was that
the optimal transfection efficiencies were in most cases
observed with formulations containing lipid-to-DNA
charge ratios (+/-) less than 1 (Figure 1). Amphiphiles
1 and 3 were most efficient at lipid-to-DNA charge ratios
of 0.3:1 and 0.1:1, respectively (Figure 1). Formulations
with less than 1 lipid-to-DNA charge ratios for optimal
transfection have previously been reported for cationic
lipids with hydroxyethyl groups directly attached to the
positively charged nitrogen atom.21,25 However, in lipo-
somal gene delivery it is generally believed that the
overall positive charge of the cationic lipid-DNA com-
plex plays a key role in their interaction with the
negatively charged biological membranes. Thus, the
remarkable efficiencies of the presently described trans-
fection complexes prepared using lipid-to-DNA charge
ratios significantly less than 1 (Figures 1 and 2)
convincingly indicate that the overall charge of the
lipid-DNA complex for efficient gene delivery need not
be necessarily positive. The positive charge may be
important for condensation of DNA or/and improve the
uptake of the complex by the cells. Since the plasmid
DNA has to interact with a variety of environments and
membranes before it is expressed in the nucleus, higher
expression of plasmid complexed with the novel hy-
droxyethyl group containing amphiphiles outlined here
suggests that the plasmid longevity is enhanced on its
route to nucleus. Thus, the function of the positive
charge on lipids in cationic lipid-mediated gene delivery
is still open to investigation. An important point de-
serves to be emphasized at this point of discussion. The
charge ratios of the present work refer to the charge
ratios of the lipid-to-DNA used in preparing the trans-
fection complex, and these may or may not be the net
charge of the resulting complexes.
salt of the di-O-acylated intermediate, (b) neutralizing
the hydrochloride salt obtained in step (a) with alkali,
and (c) quaternizing the resulting tertiary amine ob-
tained in step (b) with 2-bromoethanol.
Biology. The transfection efficiencies of the cationic
amphiphiles 1-5 (Chart 1) were tested in COS-1 cells
using pCH110 plasmid carrying a â-galactosidase re-
porter gene under the control of RSV promoter. Initially
we tested the transfection efficiencies of all the novel
transfection lipids using the widely used auxilliary lipid
DOPE. All the amphiphiles with DOPE showed very
poor transfection. Interestingly enough, the amphiphiles
1 and 3-5 showed remarkable transfection efficiencies
with varying amounts of cholesterol as helper lipid
(Figure 1). Amphiphile 2 did not show any transfection
even with cholesterol as helper lipid at any ratio,
probably because of the single acyl chain, which might
interfere with the proper formation of a bilayer. Am-
phiphiles 4 and 5 showed the highest transfection
efficiency in the presence of 60 mol % cholesterol (with
respect to the cationic lipid), whereas 3 showed highest
efficiencies in the presence of 40 mol % of cholesterol.
The transfection efficiencies of the amphiphiles 3-5
having a single hydroxyethyl group in the headgroup
regions were poorer than that of amphiphile 1. Am-
phiphile 1 with two hydroxyethyl functionalities directly
linked to the positively charged nitrogen atom in
combination with an equimolar amount of cholesterol
was clearly the most efficient transfection lipid among
all the lipids tested (Figure 1). Among amphiphiles 3-5
with single hydroxyethyl groups directly linked to the
positively charged nitrogen atom, amphiphile 3 was the
most efficient one (Figure 1). The transfection efficiency
of the most efficient amphiphile DHDEAB was observed
to be 2-3 times more in COS-1 cells than that of
lipofectamine, one of the most widely used commercially
available transfection lipids (Figure 2).
Amphiphile 2 with only one aliphatic chain in the
hydrophobic tail did not show any transfection either
in pure form or in combination with any helper lipids
(data not shown). However, at 0.3 charge ratio of
DHDEAB to DNA, amphiphile 2 when used at 30 mol
% with respect to DHDEAB modestly enhanced the

Non-Glycerol-Based Cationic Transfection Lipids
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4295
tion results shown in Figure 2 demonstrate that in the
presence of an equimolar amount of cholesterol as the
auxiliary lipid, DDAB was 2-3 times less efficient than
DHDEAB. Interestingly, both DHDEAB and DDAB
were also observed to show their optimal transfection
efficiencies at the lipid-to-DNA charge ratio of 0.3:1
(Figure 2). Such modestly improved transfection ef-
ficiency of DHDEAB compared to that of DDAB (Figure
2) implies that the presence of hydroxyethyl function-
alities in the headgroup regions of the monocationic non-
glycerol-based amphiphiles possibly may have some role
in enhancing the transfection efficiencies. Similar ob-
servations were made earlier with glycerol-based am-
phiphiles such as DMRIE, DORIE, DORI, and DMDHP,
with one or two hydroxyethyl groups linked to the
nitrogen atom,18,25 and in the case of 1,4-diaminobutane-
based dicationic transfection lipid.21 Perhaps, hydrogen-
bonding interactions with the cell surface of biological
membranes play some role for transfection lipids con-
taining hydroxyethyl groups in their headgroup struc-
tures. Previous reports have also indicated that the
enhanced transfection efficiencies of glycerol-based cat-
ionic lipids containing polar hydroxyethyl headgroups
may originate from improved interactions of such func-
tionalized cationic lipids or lipid-DNA complexes with
cellular membranes via hydrogen bonding.18,25 However,
given the modestly (2-3 fold) enhanced transfection
efficiency of DHDEAB compared to that of DDAB with
no hydroxyalkyl functionalities in the headgroup regions
(Figure 2), the role of hydrogen-bonding interactions
between the lipid headgroups and the cell surface of
biological membranes is not likely to be a key issue in
cationic lipid-mediated gene delivery. In sharp contrast
to most other reported transfection results, transfection
capabilities of both DHDEAB and DDAB were observed
to be virtually lost when used in combination with
DOPE as the helper lipid (Figure 2). It is worth
mentioning here that the commercially available DDAB-
containing transfection reagent lipofectamine also con-
tains DOPE as the auxiliary lipid. However, to our
knowledge, except for the present comparative study
(Figure 2), the relative transfection efficiencies of DDAB-
cholesterol and DDAB-DOPE combinations have not
been reported so far.
F igu r e 3. Transfection efficiency of DHDEAB:cholesterol (1:1
mol ratio) with HDEAB (2) on COS-1 cells. HDEAB, which
carries a single hydrophobic chain, was added at a mole ratio
of 0 (A), 0.15 (B), 0.20 (C), 0.30 (D), and 0.5 (E) with respect
to DHDEAB. The charge ratios on the X-axis are based on the
charge of DHDEAB only.
F igu r e 4. Cytotoxicities of cationic amphiphiles on COS-1
cells. The amphiphiles DOMHAC (filled circles); MOOHAC
(open circles), DOHEMAB (filled squares), and DHDEAB (open
squares) were tested in combination with cholesterol at a 1:1
mol ratio. DHDEAB:cholesterol:HDEAB (1:1:0.5) mole ratio
was also tested (filled triangles).
transfection efficiency of DHDEAB (Figure 3). Use of
higher mol % of 2 with respect to DHDEAB and higher
charge ratios of DHDEAB to DNA (i.e. >0.3) did not
improve the transfection further (Figure 3). Given that
the single-chain micelle-forming surfactants are known
to destroy the bilayer structures of liposomes, the
observed modest increase in the transfection efficiency
of DHDEAB in the presence of 30 mol % HDEAB is
intriguing. Clearly, detailed investigations using a host
of known transfection lipids and varying the mole
percent of HDEAB need to be carried out to ensure
possible future use of HDEAB as a new helper lipid.
The cytotoxicities (Figure 4) of the amphiphiles as 1:1
amphiphile:cholesterol preparations were tested in COS-1
cells by using reduction of MTT.30 The cytotoxicity
assays were performed in identical conditions as those
in transfection experiments. In most cases, the cell
viabilities were more than 80% up to 9 nmol of lipid,
which is the highest concentration of the lipid used in
transfections. Low cytotoxicities of the amphiphiles and
good transfection efficiencies indicate that the formula-
tions may be used in a variety of cell lines.
Nantz et al.33 have recently shown that the elec-
tron-withdrawing headgroup functionalities diminish
the transfection activities of glycerol-based cationic
lipids. This report implies that the mechanism for the
enhanced transfection efficiencies observed with the
presently described hydroxyethyl headgroup-containing
cationic amphiphiles is not based on inductive activa-
tion of the ammonium ions and indirectly emphasizes
the possible role of favorable hydrogen-bonding in-
teractions in the case of the present amphiphiles. The
in vivo transfection efficiencies and in vivo cytotoxicities
of the cationic amphiphiles 1-5 are currently being
evaluated.
Con clu sion s
Toward understanding any key role played by the
hydroxyethyl headgroups of the present transfection
lipids, we have compared the transfection efficiency of
the most efficient amphiphile DHDEAB with that of
DDAB having two methyl groups directly linked to
nitrogen instead of two hydroxyethyl groups. Transfec-
We have synthesized a novel series of nontoxic and
non-glycerol-based simple monocationic liposomal trans-
fection lipids containing one or two hydroxyethyl groups
directly linked to the positively charged nitrogen atom.
The in vitro transfection efficiency of DHDEAB, the

4296 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Banerjee et al.
The solvent was removed on a rotary evaporator and the
residue was extracted with chloroform (100 mL). Potassium
carbonate was filtered from the chloroform extract and the
chloroform was removed on a rotary evaporator. The residue
was dissolved in ethyl acetate (30 mL) with the help of little
methanol (0.3-0.5 mL) and the resulting solution of the
product mixture showed three spots (with Rf ) 0.9, 0.8, and
0.7) on thin layer chromatography (TLC) using 30:70 (v/v)
methanol:chloroform as the TLC developing solvent. The
solution was kept at 4 °C for 36 h. The precipitate that
appeared at this point was predominantly the compound with
Rf ) 0.9 which was further purified by silica gel chromatog-
raphy using finer than 200 mesh size silica and eluting with
2-5% methanolic (by volume) chloroform. The NMR and
HRMS of this column purified product (yield 470 mg) showed
it to be an O-n-hexadecyl derivative of DHDEAB that did not
show any gene transfection activity. The mother liquor was
concentrated and dry-packed with 60-120 mesh size silica.
The dry packed residue was loaded on a 60-120 mesh size
silica column and was eluted first with ethyl acetate and then
with chloroform to wash off unreacted n-hexadecyl bromide
and diethanolamine. The compounds with Rf ) 0.8 and 0.7
were then isolated as a mixture by changing the eluent to 90:
10 (v/v) chloroform:methanol. The isolated product mixture
was finally dry-packed with finer than 200 mesh size silica
and the dry-packed mixture was loaded on a finer than 200
mesh size silica column. The title amphiphiles 1 and 2 (with
Rf ) 0.8 and 0.7, respectively) were finally isolated in pure
forms by eluting the dry-packed column carefully with chlo-
roform containing 2-5% methanol (by volume). The yields of
the purified amphiphile 1 (DHDEAB) and amphiphile 2
(HDEAB) were respectively 8% (500 mg, 0.787 mmol, white
solid, mp 68-70 °C) and 12.7% (500 mg, 1.2 mmol, white solid,
mp 64-65 °C).
most efficient transfection lipid described herein, is
better than that of lipofectamine, one of the most widely
used transfection vectors in cationic lipid-mediated gene
transfer. Unlike most of the reported liposomal trans-
fection studies, cholesterol instead of DOPE is needed
to be used as the helper lipid with the presently
described amphiphiles. Interestingly, the most efficient
formulations contained cationic lipid-to-DNA charge
ratios of 0.3:1. Such improved transfection efficiencies
with lower lipid-to-DNA ratios have also been previously
observed for the 1,4-diaminobutane-based dicationic
amphiphile, N,N,N′,N′-tetramethyl-N,N′-bis(hydroxy-
ethyl)-2,3-bis(oleoyloxy)-1,4-butanediammonium iodide.21
Thus, our results reemphasize the important point that
in cationic lipid-mediated gene delivery, the overall
charge of the lipid-DNA complex need not always be
positive. In addition, our transfection results also imply
that favorable hydrogen-bonding interactions between
the lipid headgroups and the cell surface of biological
membranes may have some role for improving the
transfection efficiency in cationic lipid-mediated gene
delivery. However, the transfection results delineated
in the present investigation also indicate that such
hydrogen-bonding interactions are not likely to be a key
controlling parameter in liposomal transfection.
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Ma ter ia ls. The high-resolution
mass spectrometric (HRMS) analyses were performed on a
Micromass AUTOSPEC-M mass spectrometer (Manchester,
U.K.) with OPUS V3.1X data system. Data were acquired by
liquid secondary ion mass spectrometry (LSIMS) technique
using m-nitrobenzyl alcohol as the matrix. LSIMS analyses
were performed in the scan range 100-1000 amu at the rate
1H NMR of DHDEAB (200 MHz, CDCl3): δ/ppm ) 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]; 3.4-3.6 [m, 4H, (HOCH2-
CH2)2N+(CH2-CH2-)2]; 3.65-3.80 [m, 4H, (HOCH2-CH2)2N+-
(CH2-CH2-)2]; 4.05-4.2 [m, 4H, (HOCH2-CH2)2N+(CH2-CH2-)2];
1
of 3 scans/s. H NMR spectra were recorded on a Varian FT
4.75- 4.95(m, 2H, -OH). HRMS (LSIMS) m/z: calcd (for C36H76
-
200 MHz. Melting point determinations were performed using
a Fisher-J ohns appratus and are uncorrected. Elemental
analyses (C, H, N) were performed at the microanalytical
Laboratories of Central Salt and Marine Chemical Research
Institute, Bhavnagar, Gujarat, India, and National Chemical
Laboratory, Poona, India. Unless otherwise stated, all reagents
were purchased from local commercial suppliers and were used
without further purification. The progress of the reactions was
monitored by thin-layer chromatography using 0.25-mm silica
gel plates. Column chromatography was performed with silica
gel (Acme Synthetic Chemicals, India; finer than 200 and 60-
120 mesh). COS-1 cell line (SV 40 transformed African green
monkey kidney, ATCCCRL 1650) was obtained from ATCC,
MD. o-Nitrophenyl â-D-galactopyranoside, cholesterol, â-ga-
lactosidase, and MTT were purchased from Sigma Co. DOPE
was purchased from Avanti Polar Lipids. Lipofectamine was
purchased from Life Technologies Ltd.
Syn t h esis of N,N-Di-n -h exa d ecyl-N,N-d ih yd r oxyet h -
yla m m on iu m Br om id e (DHDEAB, a m p h ip h ile 1) a n d
N-n-Hexa d ecyl-N,N-d ih yd r oxyeth yla m m on iu m Br om id e
(HDEAB, a m p h ip h ile 2). A mixture of diethanolamine (1 g,
9.5 mmol), n-hexadecyl bromide (2.9 g, 9.5 mmol), and potas-
sium carbonate (1.44 g, 10.5 mmol) was refluxed in methanol
for 48 h. Methanol was removed on a rotatory evaporator and
the residue was extracted with chloroform (3 × 100 mL). The
combined chloroform extract was filtered repeatedly (3 times)
to remove potassium carbonate. Chloroform was removed from
the filtrate on a rotary evaporator and the residue was taken
in 50 mL 80:15:5 (v/v) acetonitrile:ethyl acetate:methanol. One
equivalent of n-hexadecyl bromide (2.9 g, 9.5 mmol) was added
and the mixture was refluxed for 56 h. At this point, an
additional equivalent of potassium carbonate (1.44 g, 10.5
mmol) was added and refluxing was continued for another 30
h. Finally, one more equivalent of n-hexadecyl bromide (2.9 g,
9.5 mmol) was added and the mixture was refluxed for 24 h.
NO2 the 4°-ammonium ion, 100%) 554.5876, found 554.5899.
Anal. (C36H76BrNO2) C, H, N.
1H NMR of HDEAB (200 MHz, CDCl3): δ/ppm ) 0.9 [t,
3H, CH3-(CH2)n-]; 1.15-1.5 [m, 26H, CH3-(CH2)13-]; 1.75-1.85
[m, 2H, (HOCH2-CH2)2N+(CH2-CH2-)2]; 3.2-3.35 [m, 2H,
(HOCH2-CH2)2N+(CH2-CH2-)2]; 3.35-3.5 [m, 4H, (HOCH2-
CH2)2N+(CH2-CH2-)2]; 4.0-4.15 [m, 4H, (HOCH2-CH2)2N+(CH2-
CH2-)2]. HRMS (LSIMS) m/z: calcd (for C20H44NO2 the 4°-
ammonium ion, 100%) 330.3372, found 330.3346. Anal.
(C20H44BrNO2) C, H, N.
Syn t h esis of N-Met h yl-N-n-oct a d ecyl-N-oleyl-N-h y-
d r oxyeth yla m m on iu m Ch lor id e (MOOHAC, a m p h ip h ile
3). Step (a ). 100 mL dry dichloromethane was cooled to 0 °C
and to the cold dichloromethane solution were added 1.9 g of
oleyl aldehyde (7.14 mmol), 1.92 g of stearylamine (7.14 mmol),
and 0.86 g of anhydride magnesium sulfate (7.14 mmol). The
mixture was kept under stirring for 3 h while the tempera-
ture of the stirred mixture raised gradually from 0 °C to
room temperature. The magnesium sulfate was filtered from
the reaction mixture and the filtrate was diluted with 50
mL of methanol. The diluted dichloromethane/methanol
solution was cooled to 0 °C and to the cold solution, 0.54 g
sodium borohydride (14.0 mmol) was added. The solution was
kept stirred for 4 h during which time the temperature of
the reaction mixture gradually raised to room temperature.
The reaction mixture was taken in 100 mL chloroform and
washed with water (2 × 100 mL); the chloroform layer was
dried over anhydride magnesium sulfate and filtered. Chlo-
roform was removed from the filtrate on a rotary evaporator
and column chromatographic (using 60-120 mesh size silica)
purification of the residue using 20-50% ethyl acetate in pet
ether as the eluent afforded 2.34 g (64% yield) of the desired
intermediate secondary amine, namely, N-oleyl-N-n-octade-
cylamine.

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J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4297
Step (b). A mixture of 0.95 g (1.83 mmol) of N-oleyl-N-n-
octadecylamine obtained in step (a) and 0.67 g (1.83 mmol) of
2-bromoethyl tert-butyldiphenylsilyl ether (prepared conven-
tionally by the reaction between 2-bromoethanol and tert-
butyldiphenylsilyl chloride in the presence of triethylamine
and N,N-dimethylaminopyridine) was refluxed in ethyl acetate
in presence of anhydrous potassium carbonate (0.28 g, 2.01
mmol) for 48 h. The reaction mixture was taken in 100 mL
chloroform, washed with water (2 × 100 mL), dried over
anhydrous magnesium sulfate, and filtered. Chloroform was
removed from the filtrate on a rotary evaporator. Silica gel
column chromatographic purification of the resulting residue
using 60-120 mesh size silica and 3-4% ethyl acetate (by
volume) in pet ether as the eluent afforded 0.69 g (47% yield)
of the intermediate tertiary amine, namely the O-tert-butyl-
diphenylsilyl derivative of N-2-hydroxyethyl-N-oleyl-N-n-oc-
tadecylamine. The tert-butyl-diphenylsilyl protecting group of
this intermediate tertiary amine (0.67 g, 0.84 mmol) was
conventionally removed by stirring with tetrabutylammonium
fluoride (2.1 mmol, 2.1 mL of 1 M tetrabutylammonium
fluoride solution in tetrahydrofuran) in dry tetrahydrofuran
(cooled to 0 °C) for 4 h during which the temperature of the
reaction mixture gradually raised to room temperature. The
usual workup and silica gel column purification (as described
above in detail for isolating pure protected intermediate except
that the eluent used in this case was 7-10% ethyl acetate in
pet ether) of the product mixture afforded 0.32 g (67.5% yield)
of pure deprotected tertiary amine, namely, N-oleyl-N-n-
octadecyl-N-2-hydroxyethylamine.
ammonium ion, 100%) 580.6388, found 580.6396. Anal.
(C39H82ClNO‚0.5H2O) C, H, N.
Syn th esis of N,N-Di[O-h exa d eca n oyl]h yd r oxyeth yl-N-
h yd r oxyeth yl-N-m eth yla m m on iu m Br om id e (DOHEM-
AB, a m p h ip h ile 5). St ep (a ). Di-O-palmitoylation of N-
methyldiethanolamine was effected following a published
protocol.31 Briefly, N-methyldiethanolamine (1 g, 8.39 mmol)
was reacted with palmitoyl chloride (5.075 g, 18.5 mmol) in
10 mL dry N,N-dimethylformamide at 0 °C and the temper-
ature was gradually raised to room temperature within a
period of 4 h. The resulting hydrochloride salt of N-methyl-
di-O-palmitoylethanolamine was filtered, crystallized from 20
mL of dry ether. Finally, recrystallization from 20 mL 5:15
(v/v) methanol:ethyl acetate afforded 3.8 g of the pure hydro-
chloride intermediate (Scheme 4) in 68% yield.
Step (b). The recrystallized hydrochloride salt (200 mg) was
stirred for 5 min in a dichloromethane (10 mL)/1.0 M aqueous
NaOH (10 mL) biphasic system. The top aqueous layer was
discarded and the lower organic layer was washed with water
(2 × 50 mL), dried over anhydrous sodium sulfate, and filtered
and dichloromethane was removed on a rotary evaporator.
Pure N-methyl-di-O-palmitoylethanolamine (0.14 g, 0.22 mmol,
74.1% yield) was purified from the residue by silica gel column
chromatography using 60-120 mesh size silica and 98:2 (v/v)
chloroform:methanol as the eluent.
Step (c). Quaternization of N-methyl-di-O-palmitoyletha-
nolamine was effected by reacting the purified tertiary amine
(0.14 g, 0.22 mmol) with 1.5 equiv of neat 2-bromoethanol
(0.063 g, 0.34 mmol) at 85 °C for 4 h. The quaternized title
amphiphile salt 5 was crystallized from the residue using 10
mL 2:8 (v/v) benzene:n-pentane. Silica gel column chromato-
graphic purification of the resulting crystal using 60-120 mesh
size silica and 4:96 (v/v) methanol:chloroform as the eluent
finally afforded 0.04 g of the pure title amphiphile 5 (white
solid, mp 85-87 °C) in 16.5% yield. All the isolated intermedi-
ates (shown in Scheme 4) gave spectroscopic data in agreement
with their assigned structures. Thus, the title amphiphile 5
was prepared in 3 steps with an overall yield of 8.3%.
1H NMR of DOHEMAB (200 MHz, CDCl3): δ/ppm ) 0.88
[t, 6H, CH3-CH2-C13H26-]; 1.20-1.40 [m, 48H, -(CH2)n-]; 1.50-
1.68 [m, 4H, CH3(HOCH2-CH2)N+(CH2-CH2-O-CO-CH2-CH2-
)2]; 2.21-2.40 [2t, 4H, CH3(HOCH2-CH2)N+(CH2-CH2-O-CO-
CH2-CH2-)2]; 3.43 [s, 3H, CH3(HOCH2-CH2)N+(CH2-CH2-O-CO-
CH2-CH2-)2]; 3.50-4.30 [m, 9H, CH3(HOCH2-CH2)N+(CH2-
CH2-O-CO-CH2-CH2-)2]; 4.51-4.58 [br, t, 4H, CH3(HOCH2-
CH2)N+(CH2-CH2-O-CO-CH2-CH2-)2]. HRMS (LSIMS) m/z: calcd
(for C39H79NO5, the 4°-ammonium ion, 100%) 641.5958, found
641.5907. Anal. (C39H79BrNO5) C, H, N.
Step (c). Quaternization of 0.12 g (0.21 mmol) of N-oleyl-
N-n-octadecyl-N-2-hydroxyethylamine obtained in step (b) was
carried out in 5 mL of 5:4 chloroform:methanol (v/v) at room
temperature for overnight using huge excess of methyl iodide
(4 mL). The solvents were removed on a rotary evaporator and
silica gel column chromatographic purification of the residue
using 60-120 mesh size silica and 3-4% methanol (by volume)
in chloroform afforded the quaternary iodide salt, 0.1 g (66.5%
yield), of the title amphiphile. Finally, 0.08 g of pure title
amphiphile 3 (waxy white solid) in 91.9% yield was obtained
by subjecting the quaternized ammonium iodide salt (0.1 g,
0.14 mmol) to repeated (4 times) chloride ion-exchange chro-
matography, each time using a freshly generated Amberlyst
A-26 chloride ion-exchange column and about 80 mL of
chloroform as the eluent. All the isolated intermediates gave
spectroscopic data in agreement with their assigned structures
shown in Scheme 3. Thus, the title amphiphile 3 was prepared
in 3 steps with an overall yield of 12.4%.
1H NMR of MOOHAC (200 MHz, CDCl3): δ/ppm ) 0.89
[t, 6H, CH3-(CH2)n-]; 1.20-1.45 [m, 52H, -(CH2)13-]; 1.60-1.80
[br, 4H, CH3(HOCH2-CH2)N+(CH2-CH2-)2]; 1.90-2.10 (m, 4H,
-CH2-CH)CH-CH2-); 3.35 [s, 3H, CH3(HOCH2-CH2)N+(CH2-
CH2-)2)]; 3.41-3.58 [br, 4H, CH3(HOCH2-CH2)N+(CH2-CH2-)2];
3.65-3.80 [br, 2H, CH3(HOCH2-CH2)N+(CH2-CH2-)2]; 4.07-
4.12 [br, 2H, CH3(HOCH2-CH2)N+(CH2-CH2-)2]; 5.32 (m, 2H,
Lip osom e P r ep a r a tion . The mixture of cationic am-
phiphiles and cholesterol in the appropriate ratio were dis-
solved in chloroform in a glass vial. The chloroform was
removed with a thin flow of moisture free nitrogen and the
dried film of lipid left in the vial was then kept under high
vacuum for 8 h. 1 mL of autoclaved sterile deionized water
was added to the vacuum dried lipid film and the mixture was
allowed to swell for 15 h (overnight). The vial was then
vortexed for 2-3 min at room temperature and occasionally
shaken in a 45 °C water bath to produce multilamellar vesicles
(MLV). Small unilamellar vesicles (SUV) were then prepared
by sonicating the MLV placed in an ice bath for 3-4 min until
clarity using a Branson 450 sonifier at 100% duty cycle and
25-W output power.
-CH2-CH)CH-CH2-). HRMS (LSIMS) m/z: calcd (for C39H80
-
NO, the 4°-ammonium ion, 100%) 578.6239, found 578.6233.
Anal. (C39H80ClNO‚1.5H2O) C, H, N.
Syn th esis of N-m eth yl-N,N-d i-n -octa d ecyl-N-h yd r oxy-
eth yla m m on iu m Ch lor id e (DOMHAC, a m p h ip h ile 4).
The title amphiphile 4 (white solid, mp 83-85 °C) was
synthesized from n-octadecyl aldehyde and stearylamine fol-
lowing the same synthetic procedure for preparing the am-
phiphile 3 (MOOHAC) as described above with an overall
yield of 7.1%. All the isolated intermediates gave spectroscopic
data in agreement with their assigned structures shown in
Scheme 3.
P r ep a r a tion of P la sm id DNA. pRSV-â-Gal plasmid DNA
was prepared by alkaline lysis procedure and purified by PEG-
8000 precipitation according to Maniatis and co-workers.32 The
plasmid preparations showing OD260/OD280 more than 1.8 were
used.
1H NMR of DOMHAC (200 MHz, CDCl3): δ/ppm ) 0.89
[t, 6H, CH3-(CH2)n-]; 1.20-1.45 [m, 60H, -(CH2)13-]; 1.60-1.80
[br, 4H, CH3(HOCH2-CH2)N+(CH2-CH2-)2]; 3.35 [s, 3H, CH3-
(HOCH2-CH2)N+(CH2-CH2-)2)]; 3.41-3.58 [br, 4H, CH3(HOCH2-
CH2)N+(CH2-CH2)2]; 3.70-3.80 [br, 2H, CH3(HOCH2-CH2)N+-
(CH2-CH2-)2]; 4.07-4.13 [br, 2H, CH3(HOCH2-CH2)N+(CH2-
CH2-)2]. HRMS (LSIMS) m/z: calcd (for C39H82NO, the 4°-
Tr a n sfection Assa y. COS-1 cells were seeded at a density
of 50 000 cells/well in a 24-well plate 18 h before the trans-
fection. 0.3 µg of plasmid was complexed with varying amount
of lipid (0.1-9.0 nmol) in 25 µL of plain DMEM medium for
30 min. The charge ratio would vary from 0.1:1 to 9:1 (+/-)
over this range of the lipid. The complex was diluted to 200
µL with plain DMEM and added to the wells. After 3 h of

4298 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Banerjee et al.
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incubation, 200 µL of DMEM with 10% FCS was added to the
cells. The medium was changed after 24 h and the reporter
gene activity was estimated after 48 h. The cells were washed
twice with PBS and lysed in 100 µL of lysis buffer (0.25 M
Tris‚HCl, pH 8.0, and 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 ONPG, 0.2 M sodium phosphate, pH 7.15, and 2 mM
magnesium chloride) to 50 µL of lysate in a 96-well plate.
Absorption at 405 nm was converted to â-galactosidase units
by using the calibration curve obtained each day using pure
commercial â-galactosidase enzyme. The values of â-galactosi-
dase units in replicate plates assayed on the same day varied
by less than 20%. The transfection efficiency values shown in
Figures 1-3 are the average values from two replicate trans-
fection plates assayed on the same day. Each transfection
experiment was repeated 3 times on 3 different days, and the
day-to-day variation in average transfection efficiency values
for identically treated two replicate transfection plates was
approximately 2-fold and was dependent on the cell density
and conditions of the cells.
Toxicity Assa y. Cytotoxicity of amphiphiles was assessed
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) reduction assay as described earlier.30 The assay
is performed in 96-well plates by maintaining the ratio of
number of cells to amount of cationic amphiphile constant in
cytotoxicity and transfection experiments. MTT was added 3
h after adding the cationic amphiphile to the cells. Results
were expressed as percent viability ) [OD540(treated cells) -
background]/[OD540(untreated cells) - background] × 100.
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Heath, T. D.; Debs, R. J . Factors influencing the Efficiency of
Cationic Liposome-Mediated Intravenous Gene Delivery. Nature
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Ack n ow led gm en t. Financial support in the form of
doctoral research fellowships from the Council of Sci-
entific and Industrial Research (CSIR), Government of
India, New Delhi (to R.K.B. and G.V.S.), and from the
University Grant Commision (UGC), Government of
India, New Delhi (to P.K.D.), is gratefully acknowledged.
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