Distance of hydroxyl functionality from the quaternized center influence DNA binding and in vitro gene delivery efficacies of cationic lipids with hydroxyalkyl headgroups

Article abstract of DOI:10.1021/jm049656j

In vitro gene delivery efficacies of cationic amphiphiles 1-7 (Scheme 1) were measured by both the reporter gene expression assays in CHO, COS-1, HepG2, and MCF7 cells and by the whole cell histochemical X-gal staining of representative Chinese hamster ovary cells. Our results demonstrated that in vitro gene delivery efficiencies of cationic lipids with hydroxyalkyl headgroups are adversely affected by increased covalent distances between the hydroxyl functionality and the cationic centers. Findings in the DNase I protection experiments and transmission electron microscopic study support the notion that such compromised gene delivery efficacies may originate from poor lipid - DNA binding interactions and significantly increased lipoplex nanosizes.

Full text of DOI:10.1021/jm049656j

J. Med. Chem. 2004, 47, 5721-5728
5721
Distance of Hydroxyl Functionality from the Quaternized Center Influence
DNA Binding and in Vitro Gene Delivery Efficacies of Cationic Lipids with
Hydroxyalkyl Headgroups
†
†
‡
Yenugonda Venkata Mahidhar, Mukthavaram Rajesh, Sunkara Sakunthala Madhavendra, and
Arabinda Chaudhuri*,†
Division of Lipid Science and Technology and Electron Microscopy Center, Indian Institute of Chemical Technology,
Hyderabad-500 007, India
Received May 6, 2004
In vitro gene delivery efficacies of cationic amphiphiles 1-7 (Scheme 1) were measured by
both the reporter gene expression assays in CHO, COS-1, HepG2, and MCF7 cells and by the
whole cell histochemical X-gal staining of representative Chinese hamster ovary cells. Our
results demonstrated that in vitro gene delivery efficiencies of cationic lipids with hydroxyalkyl
headgroups are adversely affected by increased covalent distances between the hydroxyl
functionality and the cationic centers. Findings in the DNase I protection experiments and
transmission electron microscopic study support the notion that such compromised gene delivery
efficacies may originate from poor lipid-DNA binding interactions and significantly increased
lipoplex nanosizes.
Introduction
and demonstrated that the in vitro gene transfer ef-
ficiencies of some of these non-glycerol-based cationic
lipids are superior to that of LipofectAmine, one of the
most extensively used commercially available liposomal
transfection kits.2
Safety and efficiency of vehicles for delivering thera-
peutic genes into body cells are the two most important
prerequisites toward ensuring clinical success of gene
therapy. Recombinant viral vectors, despite being re-
markably efficient in transfecting body cells, suffer from
numerous biosafety-related disadvantages including
generation of inflammatory and immunogenic responses
7-35
To probe the influence of the distance between the
hydroxyl functionality and the cationic centers in modu-
lating the in vitro gene delivery efficacies of the above-
mentioned non-glycerol-based cationic transfection lip-
ids containing hydroxyalkyl headgroups, in the present
investigation we have designed and synthesized seven
novel structural analogues (lipids 1-7, Scheme 1) of
DOMHAC (N,N-di-n-octadecyl-N-2-hydroxyethyl-N-meth-
ylammonium chloride), one of our previously designed
non-glycerol-based cationic lipids.35 The relative in vitro
gene transfer efficacies of lipids 1-7 were measured by
reporter gene expression assays in CHO, COS-1, HepG2,
and MCF7 cells as well as by whole cell histochemical
X-gal staining of the representative CHO cells. In
addition, the lipid-DNA binding interaction profiles for
lipids 1-7 were evaluated by DNase I sensitivity assays.
Results convincingly demonstrate that both in vitro gene
transfer properties and the strength of lipid-DNA
binding interactions get adversely affected with the
increasing distance between the hydroxyl functionality
and the quaternized center of cationic lipids with
hydroxyalkyl headgroups. Results of the DNase I pro-
tection experiments and transmission electron micro-
scopic study, taken together, support the notion that the
poor transfection efficacies of cationic lipids with hy-
droxyl headgroups separated from the positively charged
nitrogen atoms by more than three methylene units may
originate from weak lipid-DNA binding interactions
and increased lipoplex (lipid-DNA complex) sizes.
and production of replication-competent virus through
recombination events with host genome.1-8 Conversely,
cationic transfection lipids, because of their least im-
munogenic nature, robust manufacture, ability to de-
liver large pieces of DNA, and ease of handling and
preparation techniques, are finding increasing uses as
the nonviral vectors of choice in delivering genes into
9
-24
body cells.
Defining the key architectural elements necessary for
imparting enhanced transfection properties to cationic
amphiphiles through detailed structure-activity inves-
tigations using libraries of new cationic lipids is an
intensely pursued area of contemporary research in
cationic lipid mediated gene delivery. Ever since the
pioneering development of glycerol-backbone-based
cationic transfection lipid (N-[1-(2,3-dioleyloxy)propyl])-
N,N,N-trimethylammonium chloride (DOTMA) in 1987,
the design and synthesis of more efficacious cationic
2
5
9
-24
amphiphiles have been reported.
Interestingly, in
the molecular architectures of many of these subse-
quently developed cationic transfection lipids, the glyc-
erol backbone of DOTMA was retained. Towards un-
derstanding how mandatory is the presence of the
glycero -backbone in the molecular structure of cationic
transfection lipids, we have designed and synthesized
a series of novel non-glycerol-based cationic amphiphiles
Results and Discussion
*
To whom correspondence should be addressed. Phone: 91-40-
2
7193201. Fax: 91-40-27160757. E-mail: arabinda@iict.res.in.
†
Chemistry. Lipids 1-7 (Scheme 1) were synthesized
essentially following our previously described method
Division of Lipid Science and Technology.
Electron Microscopy Center.
‡
1
0.1021/jm049656j CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/12/2004

5
722 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Mahidhar et al.
Scheme 1. Synthesis of Lipids 1-7a
compared to that of lipid 1 with only two methylene
units as the headgroup spacer arm (Figure 2). For the
sake of comparison, the transfection profiles of the
widely used commercially available DMRIE-C were also
included in both the reporter gene expression assay
(
(
Figure 1) and the whole cell X-gal staining experiments
Figure 2). Taken together, the results summarized in
Figures 1 and 2 convincingly demonstrate that the
transfection efficacies of cationic lipids with hydroxy-
alkyl headgroups get progressively compromised with
increasing distance between the hydroxyl headgroup
and the quaternized center.
To gain insight into whether the observed transfection
profiles of lipids 1-7 were related to their varying
inherent toxicity profiles, MTT-based cell viability as-
says were performed in representative CHO and COS-1
cells across the entire range of lipid/DNA charge ratios
used in the actual transfection experiments. Percent cell
viabilities of all the cationic lipids 1-7 were found to
be remarkably high even at the higher end of the lipid/
DNA ratios (Figure 3). Thus, the cell viability results
summarized in Figure 3 ruled out the possibility of
inherent cellular cytotoxicities of the individual lipids
playing a role behind the observed transfection profiles
of lipids 1-7.
a
Reagents: (a) Br-(CH2)nOTBDMS, K2CO3, ethyl acetate; (b)
TBAF, THF; (c) CH3I, DCM, Amberlyst A-26 chloride ion-exchange
resin.
To understand whether the contrasting transfection
efficacies of the present cationic lipids could originate
from varying lipid/DNA electrostatic binding interac-
tions, we performed the DNase I protection experiments
across the entire range of the lipid/DNA charge ratios
by monitoring the sensitivities of the lipoplexes upon
treatment with DNase I. After the free DNA digestion
by DNase I, the total DNA (both digested and inacces-
sible DNA) was separated from the lipid and DNase I
for preparing DOMHAC.35 Briefly, the appropriate tert-
butyldimethylsilyl-protected bromo alcohols were coupled
to N,N,di-n-hexadecylamine (I, Scheme 1) in the pres-
ence of potassium carbonate. Tetra-n-butylammonium
fluoride mediated silyl deprotection of the resulting
tertiary amines (II, Scheme 1) afforded the tertiary
alcohol intermediates (III, Scheme 1). Quaternization
with excess methyl iodide followed by final ion exchange
over Amberlyst A-26 chloride ion-exchange resin af-
forded the target lipids. Structures of all the final lipids
(
1
by extracting with organic solvent) and loaded onto a
% agarose gel. Figure 4 summarizes the electrophoretic
1-7, Scheme 1) were confirmed by 1H NMR and
gel patterns observed for representative lipids 1 and 7.
Band intensities of inaccessible and therefore undi-
gested DNA associated with transfection incompetent
lipoplexes prepared from lipid 7 were significantly less
than those associated with the most transfection ef-
ficient lipoplexes made from lipid 1 essentially across
the range of lipid/DNA charge ratios of 9:1 to 0.3:1
(
LSIMS, and the purity of the target lipids were con-
firmed by C, H, N analysis.
Transfection Biology. Figure 1 summarizes the
relative efficacies of lipids 1-7 in transfecting CHO,
COS-1, HepG2, and MCF7 cells. The cationic liposomes
of lipids 1-7 were prepared in combination with equiva-
lent mole of cholesterol as colipid and using pCMV-
SPORT-â-gal plasmid as the reporter gene across the
lipid/DNA charge ratios of 9:1 to 0.1:1. In general, the
transfection efficacies of lipids 1-7 were found to be
progressively compromised in all four cells as the
distance between the headgroup hydroxyl functionality
and the cationic center increased (Figure 1). The most
dramatic fall of transfection property was observed with
lipid 7 (with 10 methylene units spacer group in its
headgroup region), which was found to be practically
incompetent in transfecting all the four cells across the
entire lipid/DNA charge ratios (Figure 1). The same
transfection profiles were further confirmed by whole
cell histochemical X-gal staining experiments. Figure
(Figure 4). Such gel patterns in DNase I sensitivity
assays indicate that the plasmid DNA associated with
lipid 7 is likely to be more susceptible to degradation
by cellular DNase I than the DNA complexed to lipid 1.
Similar gel characteristics in DNase I protection experi-
ments were also observed for lipoplexes prepared with
lipid 6 (data not shown). These findings are consistent
with the notion that the compromised in vitro trans-
fection efficacies of lipids 6 and 7 could, in part, originate
from poor lipid-DNA binding interactions, which in
turn renders the loosely associated DNA susceptible to
degredation by cellular DNase I.
Finally, to find a correlation, if any, between the
lipoplex sizes and their in vitro gene transfer efficacies,
transmission electron microscopic images of the repre-
sentative lipoplexes prepared from lipids 1, 4, and 7
using lipid/DNA charge ratios of 1:1 were taken (Figure
5). Contrastingly, while the aqueous lipoplexes prepared
from lipids 1 and 4 were observed to be much smaller
and well dispersed, those prepared with lipid 7 were
found to be remarkably large and aggregate-forming in
2
shows a representative pattern observed in such whole
cell X-gal staining experiments for transfected CHO
cells with lipids 1, 6, and 7 at a lipid/DNA charge ratio
of 1:1. Once again, the transfection efficacies for lipids
with long spacer arms between the hydroxyl functional-
ity and the cationic center in the headgroup region
(lipids 6 and 7) were found to be strikingly poor

Lipids with Hydroxyalkyl Headgroups
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5723
Figure 1. In vitro transfection efficiencies of lipids 1-7 in CHO (A), COS-1 (B), and HepG2 (C), and MCF-7 (D) cells using
cholesterol as colipid (at lipid/cholesterol mole ratio of 1:1). Units of â-galactosidase activity were plotted against the varying
lipid to DNA (() charge ratios. The o-nitrophenol formation (micromoles of o-nitrophenol produced per 10 min) was converted to
activity units using a standard curve obtained with pure (commercial) â-galactosidase. The transfection efficiencies of commercially
available DMRIE-C are shown for comparison. The transfection values shown are the average of triplicate experiments performed
on the same day.
nature (Figure 5). Such relatively big size and ag-
gregate-forming characteristics of the lipoplex prepared
with lipid 7 (Figure 5) are likely to play some inhibitory
role in the cellular uptake step of the transfection
pathway. In addition, we also measured the nanosizes
of the pure liposomes (in the presence of DMEM)
prepared from cationic lipids 1-7 by the dynamic laser
light scattering technique. While the average nanosizes
of the liposomes prepared from transfection efficient
lipids 1-5 were found to vary within the range 120-
In summary, the results of the present structure-
activity investigation involving the use of seven new
structural analogues (1-7) of our previously reported
cationic transfection lipid DOMHAC35 demonstrate that
the in vitro gene delivery efficacies of simple twin-chain
cationic lipids with hydroxyalkyl headgroups get ad-
versely affected with increasing covalent distance be-
tween the hydroxyl functionality and the quaternized
center. Results of our DNase I protection experiments
and transmission electron microscopic images of repre-
sentative lipoplexes, taken together, indicate that the
severely compromised transfection efficacies of lipid 7
may result from the poor lipid-DNA binding interac-
tions and its relatively large size and aggregate-forming
characteristics. In conclusion, caution should be exer-
cised in designing future-generation cationic transfec-
tion lipids by covalent grafting of hydroxyalkyl head-
groups with the hydroxyl functionality far away from
the cationic centers.
1
70 nm (data not shown), those of liposomes prepared
with transfection incompetent lipids 6 and 7 were
measured to be 565 ( 16 and 535 ( 11 nm, respectively.
Thus, the significantly large sizes might inhibit effective
cellular uptake process for liposomes prepared with
lipids 6 and 7 imparting thereby severely compromised
transfection efficacies to lipids 6 and 7. Moreover, the
presence of 8 and 10 methylene units between the
positively charged nitrogen atom and the hydroxyl
functionality of lipids 6 and 7 essentially makes them
Experimental Section
“
three-tailed” cationic amphiphiles. This, in turn, may
adversely affect the stability of the liposomes prepared
with lipids 6 and 7, causing them to form larger lipid/
DNA complexes and imparting to them poor DNA
binding characteristics. Clearly, further cell biology
experiments need to be carried out for more mechanistic
insights into the origin of lipid 7 being completely
transfection-incompetent.
General Procedures and Materials. The high-resolution
mass spectrometric (HRMS) experiments were performed on
a Micromass AUTOSPEC-M mass spectrometer (Manchester,
U.K.) with an OPUS V 3.1X data system. Data were acquired
by liquid secondary ion mass spectrometry (LSIMS) using
1
m-nitrobenzyl alcohol as the matrix. H NMR spectra were
recorded on a Varian FT 200 MHz or AV 300 MHz or Varian
Unity 500 MHz. Elemental analyses (C, H, N) were performed

5
724 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Mahidhar et al.
Figure 2. Histochemical whole cell X-gal staining of transfected CHO cells with lipids 1, 6, and 7 and DMRIE-C at lipid/DNA
charge ratios of 1:1. Cells expressing â-galactosidase were stained with X-gal as described in the text.
on a Perkin-Elmer 240c at our microanalytical facility. 1-Bro-
mohexadecane, n-hexadecylamine, tert-butyldimethylsilyl chlo-
ride, tetra-n-butylammonium fluoride (1 M solution in THF),
and Amberlyst A-26 were purchased from Lancaster (More-
cambe, U.K.). 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,
The reaction mixture was taken in ethyl acetate (50 mL) and
washed with water (2 × 50 mL), dried over anhydrous sodium
sulfate, and fitered, and the filtrate was concentrated on a
rotary evaporator. The residue upon purification by column
chromatography (using 60-120 mesh size silica gel and 2%
ethyl acetate in hexane as eluent) afforded the intermediate
tertiary amine, namely, O-tert-butyldimethylsilyl derivative
of N-2-hydroxyethyl-N,N-di-n-hexadecylamine (II, Scheme 1)
60-120 mesh). The p-CMV-SPORT-â-gal plasmid used was a
as a semisolid (0.62 g, 46% yield, R
f
) 0.6, 10:90 ethyl acetate/
hexane). H NMR (200 MHz, CDCl ) of intermediate II
(Scheme 1): δ (ppm) ) 0.00-0.10 (6H, Si(CH ), 0.90-1.00
(m, 15H, (CH C-Si(CH -O- and (CH (CH , 1.20-1.60
[m, 56H, -N(-CH -(CH ], 2.40-2.60 (m, 6H,
N(CH ), 3.60 (t, 2H, -CH -O-Si(CH C(CH ).
1
generous gift from Dr. Nalam Madhusudhana Rao of Centre
for Cellular and Molecular Biology, Hyderabad, India. Cell
culture media, fetal bovine serum, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), poly(ethylene gly-
col) 8000, o-nitrophenyl-â-D-galactopyranoside, and cholesterol
were purchased from Sigma, St. Louis, MO. NP-40, antibiotics,
and agarose were purchased from Hi-media, India. Unless
otherwise stated, all reagents were purchased from local
commercial suppliers and were used without further purifica-
tion. COS-1 (SV 40 transformed African green monkey kidney
cells), and CHO (Chinese hamster ovary), MCF-7 (human brest
adenocarcinoma cell), and HEPG2 (human hepatoblastoma
cell) cell lines were procured from the National Centre for Cell
Sciences (NCCS), Pune, India. Cells were grown at 37 °C in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS
3
3 2
)
3
)
3
)
3 2
3
2 14 2
) )
2
2 3 2
)14-CH )
2
2
)
3
3
)
2
3 3
)
Step b. Tetrabutylammonium fluoride (1.3 mmol, 0.4 mL
of 1.0 M tetrabutylammonium fluoride solution in tetrahydro-
furan) was added at 0 °C to a solution of compound II (0.40 g,
0.7 mmol) in dry tetrahydrofuran (15 mL), and the reaction
mixture was kept under stirring at 0 °C for 2 h. The
temperature was then raised to room temperature for 6 h. The
reaction mixture was then taken in 50 mL of chloroform and
washed with water (3 × 20 mL). The organic layer was
2 4
separated, dried over anhydrous Na SO , filtered, and con-
centrated on a rotary evaporator. The residue upon purification
by column chromatography (using 60-120 mesh size silica gel
and 16% ethyl acetate in hexane as eluent) afforded compound
N-2-hydroxyethyl-N,N-di-n-hexadecylamine (III, Scheme 1) as
2
in a humidified atmosphere containing 5% CO /95% air. The
purity of lipids 1-7 was confirmed by elemental analyses (C,
H, N).
Syntheses of Lipids 1-7. As representative details,
syntheses and spectral characterization of lipid 1 and all their
synthetic intermediates shown in Scheme 1 are provided
below. Lipids 2-7 were synthesized following essentially the
same protocols adopted for preparing lipid 1.
a liquid (0.17 g, 50% yield, R
f
) 0.4, 30:70 ethyl acetate/
hexane). H NMR (200 MHz, CDCl ) of intermediate III
(Scheme 1): δ (ppm) ) 0.90 (t, 6H, [(CH )-(CH ), 1.20-
1.60 [m, 56H, -N(-CH -(CH ], 2.40-2.60 (m, 6H,
N(CH ), 3.60 (t, 2H, -CH -OH). FABMS (LSIMS), m/z: 511
[M] + 2 for C34
1
3
3
2 14 2
) ]
2
2 3 2
)14-CH )
)
2 3
2
+
Synthesis of N,N-Di-n-hexadecyl-N-methyl-N-(2-hy-
droxyethyl)ammonium Chloride (Lipid 1, Scheme 1).
Step a. A mixture of N,N-di-n-hexadecylamine (1.00 g, 2.2
H71NO.
Step c. Methyl iodide (6 mL) was added to intermediate
III (prepared in step b, 0.14 g, 0.27 mmol) dissolved in 4 mL
of 5:4 chloroform/methanol (v/v), and the reaction mixture was
kept at room temperature for 3 h. The reaction mixture was
concentrated on a rotary evaporator, and the residue upon
purification by column chromatography (using 60-120 silica
gel mesh size and 2% methanol in chloroform as eluent)
followed by chloride ion exchange (using Amberlyst A-26 with
3
5
mmol, prepared as described previously ) and 2-bromoethyl
tert-butyldimethylsilyl ether (0.61 g, 2.6 mmol, prepared by
reacting 2-bromoethanol and tert-butyldimethylsilyl chloride
in the presence of imidazole followed by the usual workup)
was refluxed in ethyl acetate (25 mL) in the presence of
anhydrous potassium carbonate (0.74 g, 5.4 mmol) for 40 h.

Lipids with Hydroxyalkyl Headgroups
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5725
Figure 3. MTT-assay-based cellular cytotoxicities of lipids 1-7 against representative CHO and COS-I cells (details of assay
protocol are described in the main text). The percent cell viability values shown are the average of triplicate experiments performed
on the same day. Results were expressed as percent viability ) {[A550(treated cells) - background]/[A550(untreated cells) -
background]} × 100.
methanol as eluent) afforded the pure title lipid 1 (80 mg, 58%
yield, white solid, R ) 0.4 with 5:95 methanol/chloroform).
H NMR (200 MHz, CDCl
CH ), 1.20-1.60 [m, 52H, -N(-CH
CH ], 1.60-1.80 (brs, 4H, CH (HOCH -CH
(HOCH -CH
-CH -(CH
-N(-CH
2
-CH
2
-(CH
2
)
13-CH
3
)
2
), 1.60-1.80 (brs, 4H, CH
), 2.00-2.20 (brs, 2H, CH
)N ), 3.23 [s, 3H, CH (HOCH -CH
], 3.30-3.40 (brs, 4H, CH (HOCH -CH
), 3.70-3.80 (4H, CH (HOCH -CH
3
-
-
+
f
(HOCH
2
-CH
-CH
2 2 2
)N (CH2-CH )
3
1
+
3
): δ (ppm)) 0.90 (t, 6H, [CH
3
-
-
-
(HOCH
2
2
-CH
2
3
2
2
-
+
(
2
)
2
15
]
2
2
-CH
2
-(CH
2
)
13
CH2)N (CH
2
-CH
2
)
2
2
3
2
2
-
-
+
+
3
)
2
3
2
2
)N [CH
2
-CH
2
CH
2
)N (CH
2
-CH
)
2
3
2
2
+
+
(
CH
brs, 4H, CH
.70-3.85 (2H, CH
)13-CH
3
)]
(HOCH
(HOCH
2
, 3.39 [s, 3H, CH
3
+
2
2
)N ], 3.40-3.60
CH
2
)N ), 3.90-4.00 (m, 1H, OH). FABMS (LSIMS), m/z: 538
+
[
3
2
-CH
2
)N (-CH
2
2
2
)
13-CH
3
)
2
],
-
[M] for C36H76NOCl. Anal. (C36H76NOCl) C, H, N.
+
3
3
2
-CH
2
)N ), 4.10-4.30 (m, 3H, CH
3
Lipid 3 (N,N-Di-n-hexadecyl-N-methyl-N-(4-hydroxy-
+
+
1
(
HOCH
2
-CH
1 for C35 74NOCl. Anal. (C35
Synthesis of Lipids 2-7. Lipids 2-7 (white solids) were
2
)N ) and OH). FABMS (LSIMS), m/z: 525 [M]
n-butyl)ammonium chloride). H NMR (200 MHz, CDCl ):
3
+
H
H
74NOCl) C, H, N.
δ (ppm) ) 0.90 (t, 6H, [CH (CH ) ] ), 1.20-1.60 (m, 52H,
3
2 15 2
-N(-CH
(HOCH -CH
(HOCH -(CH
(CH -CH
+ 1 for C37
2
-CH
-CH
-CH
2
-(CH
2 3 2 3
)13-CH ) ), 1.60-2.10 (m, 8H, CH -
+
synthesized following the same synthetic procedure as de-
scribed above for preparing lipid 1 except using the appropriate
tert-butyldimethylsilyl protected bromoalkanols. All the iso-
lated intermediates gave spectroscopic data in agreement with
2
2
2
-CH
2
2
-)N (CH
2
-CH
)N ), 3.40-3.80 (m, 8H, CH
-CH . FABMS (LSIMS), m/z: 553 [M]
78NOCl. Anal. (C37 78NOCl) C, H, N.
2 2
)
), 3.35 (s, 3H, CH
3
-
+
2
2
)
2
3
(HOCH -
2
+
+
)
2 2
2
)N (CH
2
2 2
)
H
H
1
their structures shown in Scheme 1. The H NMR and LSIMS
Lipid 4 (N,N-Di-n-hexadecyl-N-methyl-N-(5-hydroxy-
n-pentyl)ammonium chloride). H NMR (200 MHz, CDCl ):
1
results for the new lipids 2-7 are provided below.
3
Lipid 2 (N,N-Di-n-hexadecyl-N-methyl-N-(3-hydroxy-
δ (ppm) ) 0.90 (t, 6H, [CH
(HOCH -(CH -CH )N(-CH
CH (HOCH -(CH -CH
3
(CH
2
)
2 15
]
2
), 1.20-1.98 (m, 62H,
), 3.30 (s, 3H,
)N ), 3.35-3.70 (m, 8H, CH
1
n-propyl)ammonium chloride). H NMR (300 MHz, CDCl
3
):
2
2
)
3
2
-(CH )14-CH )
2 3 2
+
δ (ppm)) 0.90 (t, 6H, [CH
3
(CH
2
)
15
]
2
), 1.20-1.60 (m, 52H,
3
2
2
)
3
2
3
-

5
726 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Mahidhar et al.
Figure 4. DNase I sensitivities of lipid/DNA complexes made from the representative cationic lipids 1 and 7 and pCMV-SPORT-
â-gal. Free pCMV-SPORT-â-gal and pCMV-SPORT-â-gal treated with DNase I were loaded in lanes 1 and 2 from the left,
respectively. DNase I treated lipoplexes of lipids 1 and 7 at the indicated lipid/DNA charge ratios were loaded in the next 10
lanes from the left, and the DNA markers were loaded in the extreme right lane. The details of the treatment are as described
in the text.
Figure 5. Transmission electron micrographs of representative lipoplexes prepared from pCMV-SPORT-â-gal and the
representative cationic lipids 1 (A), 4 (B), and 7 (C) at a lipid/DNA charge ratio of 1:1. Bars in parts A-C correspond to 140 nm.
Experimental details are as described in the text.
+
(LSIMS), m/z: 637 [M]+ + 1 for C43
H90NOCl. Anal. (C43H -
90
(
HOCH
2
-(CH
2
)
3
-CH
67 [M] + 1 for C38
Lipid 5 (N,N-Di-n-hexadecyl-N-methyl-N-(6-hydroxy-
2
)N (CH
2
-CH
2
)
2
). FABMS (LSIMS), m/z:
+
5
H
80NOCl. Anal. (C38
H
80NOCl) C, H, N.
NOCl) C, H, N.
Preparation of Liposomes. The cationic lipid and cho-
lesterol in 1:1 mole ratio were dissolved in a mixture of
chloroform and methanol (3:1, v/v) in a glass vial. The solvent
was removed with a thin flow of moisture-free nitrogen gas,
and the dried lipid film was then kept under high vacuum for
8 h. An amount of 5 mL of sterile deionized water was added
to the vacuum-dried lipid film, and the mixture was allowed
to swell overnight. The vial was then vortexed for 2-3 min at
room temperature to produce multilamellar vesicles (MLVs).
MLVs were then sonicated in an ice bath until clarity was
attained using a Branson 450 sonifier at 100% duty cycle and
25 W output power to produce small unilamellar vesicles
(SUVs).
1
n-hexyl)ammonium chloride). H NMR (300 MHz, CDCl
δ (ppm) ) 0.90 (t, 6H, [CH (CH ), 1.20-1.98 (m, 64H,
HOCH -(CH -CH )N(-CH -(CH ), 3.35 (s, 3H,
(HOCH -(CH -CH )N ), 3.40-3.70 (m, 8H, CH
HOCH -(CH -CH
3
):
3
2 15 2
) ]
(
2
2
)
4
2
2
2 3 2
)14-CH )
+
CH
3
2
2
)
4
2
3
-
+
(
5
2
2
)
4
2
)N (CH
2
-CH
2
)
2
). FABMS (LSIMS), m/z:
82NOCl) C, H, N.
+
81 [M] + 1 for C39
H
82NOCl. Anal. (C39H
Lipid 6 (N,N-Di-n-hexadecyl-N-methyl-N-(8-hydroxy-
1
n-octyl)ammonium chloride). H NMR (200 MHz, CDCl
δ (ppm) ) 0.90 (t, 6H, [CH (CH ), 1.10-2.00 (m, 68H,
HOCH -(CH -CH )N(-CH -(CH ), 3.35 (s, 3H,
(HOCH -(CH -CH )N ), 3.40-3.70 (m, 8H, CH
HOCH -(CH -CH
09 [M] + 1 for C41
Lipid 7 (N,N-Di-n-hexadecyl-N-methyl-N-(10-hydroxy-
3
):
3
2 15 2
) ]
(
2
2
)
6
2
2
2 3 2
)14-CH )
+
CH
3
2
2
)
6
2
3
-
+
(
6
2
+
2
)
6
2
)N (CH
2
-CH
2
)
2
. FABMS (LSIMS), m/z:
86NOCl) C, H, N.
H
86NOCl. Anal. (C41
H
Preparation of Plasmid DNA. pCMV-SPORT-â-gal plas-
mid DNA was prepared by alkaline lysis procedure and
purified by PEG-8000 precipitation according to the protocol
1
n-dodecyl)ammonium chloride). H NMR (200 MHz,
CDCl ): δ (ppm)) 0.90 (t, 6H, [CH (CH ), 1.20-1.80
m, 72H, (HOCH -(CH -CH )N(-CH -(CH ), 3.20
s, 3H, CH (HOCH -(CH -CH )N (CH -CH ), 3.20-3.70
m, 8H, CH (HOCH -(CH -CH )N (CH -CH . FABMS
36
described by Maniatis and co-workers. The plasmid prepara-
tions showing a value of OD260/OD280 more than 1.8 were used.
3
3
2 15 2
) ]
(
(
(
2
2
)
8
2
2
2 3 2
)14-CH )
+
3
2
2
)
8
2
2
2
)
2
Transfection Biology. Cells were seeded at a density of
20 000 cells (for CHO, MCF-7, HepG2) and 15 000 cells (for
+
3
2
2
)
8
2
2
2
)
2

Lipids with Hydroxyalkyl Headgroups
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5727
COS-1) per well in a 96-well plate 18-24 h before the
transfection. An amount of 0.3 µg of plasmid DNA was
complexed with varying amounts of lipids (0.09-8.1 nmol) in
plain DMEM medium (total volume made up to 100 µL) for
filtered uranyl acetate (1.33%). Once again, the excess fluid
was wicked away and the grid was air-dried.
Acknowledgment. Financial support received from
the Department of Biotechnology, Government of India
30 min. The lipid/DNA charge ratios were varied from 0.1:1
to 9:1 over these ranges of the lipids. The complexes were then
added to the cells. After 3 h of incubation, 100 µL of DMEM
with 20% FBS was added to the cells. The medium was
changed to 10% complete medium after 24 h, and the reporter
gene activity was estimated after 48 h. The cells were washed
twice with PBS (100 µL each) and lysed in 50 µL of lysis buffer
(
to A.C.) is gratefully acknowledged. Y.V.M. and M.R.
thank the Council of Scientific and Industrial Research
CSIR), Government of India, for their doctoral research
(
fellowship.
[
0.25 M Tris-HCl, pH 8.0, 0.5% NP40]. Care was taken to
Supporting Information Available: Elemental analysis
results (C, H, N) for lipids 1-7. This material is available free
of charge via the Internet at http://pubs.acs.org.
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 7.3), and 2 mM
magnesium chloride] to the lysate in a 96-well plate. Absorp-
tion at 405 nm was converted to â-galactosidase units by use
of a calibration curve constructed with pure commercial
â-galactosidase enzyme. The values of â-galactosidase units
in triplicate experiments assayed on the same day varied by
less than 20%. The transfection efficiency values shown in
Figure 1 are the average of triplicate experiments performed
on the same day. Each transfection experiment was repeated
twice, and the day-to-day variation in average transfection
efficiency was found to be within 2-fold. The transfection
profiles obtained on different days were identical.
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