M. Sakashita et al. / Bioorg. Med. Chem. 22 (2014) 5212–5219
5213
16),
to C12 in the meta-meta position exhibit the highest efficiencies.13
1.49–1.43 (4H, m, 2 ꢀ CH
0.91–0.88 (6H, t, J = 6.9 Hz, 2 ꢀ CH
for C40 Na 742.69; found 742.98. We obtained the glucose
CH
), 1.38–1.29 (32H, m, 2 ꢀ C
H
2
3
8
+
In addition, lipids can form stable micelles at low concentrations as
a result of hydrophobic interactions among aromatic rings and 2
3
). FAB-MS (m/z): [M+Na] calcd
69 3 8
H N O
alkyl chains as well as
p–p
stacking derived from aromatic rings.
modified lipid in the same way using 1-azido-1-deoxy-b-D-
The advantages of using the aromatic linker have been described
glucopyranoside.
in our previous papers.1
2–14
Furthermore, these lipids are easy to
synthesize with high yield by using click chemistry.
2.3. Preparation of the lipoplex composed of sugar-modified
lipid/cationic lipid micelle and plasmid DNA
In this study, we synthesized galactose-modified lipids with an
aromatic ring with C12 in the meta-meta position. The galactose-
modified lipids were mixed with cationic lipids to form complexes
with DNA, which are termed ‘lipoplexes’. We subsequently investi-
gated their transfection efficiencies into cells expressing ASGPR.
We mixed the sugar-modified lipid and dioleoyltrimethylammo-
niumpropane (DOTAP; Sigma–Aldrich) at the same molar ratio and
dissolved them in chloroform and vacuum dried. Each mixture
was dissolved in water and added the pDNA encoding luciferase
(
pGL3-Control Vector; Promega, Madison, WI) at the indicated
2
2
. Materials and methods
.1. Materials
N/P ratios (i.e., the cation/anion charge ratio, [cationic amino
group]DOTAP/[anionic phosphate group]nucleic acid) and incubated for
1
1
bromide and the image was obtained using a PharosFX (Bio-Rad,
Richmond, CA).
h. To confirm the complexations, the mixtures were separated by
% agarose gel electrophoresis. DNA was stained with ethidium
3
,5-Dihydroxybenzaldehyde, potassium carbonate, propa-
rgylbromide, sodium -ascorbic acid, Dulbecco’s modified Eagle’s
L
medium (DMEM) were purchased from Wako Pure Chemical
Industries, Ltd, (Osaka, Japan). 1-Bromododecane, sodium boron-
hydride, sodium hydride were purchased from Tokyo Chemical
Industry Co., (Tokyo, Japan). N-N-dimethlformamide, sodium
methoxide were purchased from Kanto Chemical Co., (Tokyo,
2
.4. f Potential and size measurements
We prepared the lipoplexes at indicated N/P ratios in 150 mM
NaClaq, where we fixed DOTAP concentration at 0.3 mM. The zeta
potentials and hydrodynamic radiuses were measured with a Mal-
vern Zetasizer nano (Malvern Instruments, Malvern, UK) at room
temperature.
Japan). 1-azido-1-deoxy-b-D-galactopyranoside acetate, copper(II)
sulfate pentahydrate were purchased from Sigma–Aldrich (St.
Louis, MO).
2
.2. Synthesis of the sugar-modified lipid (Scheme 1)
,5-Dihydroxybenzaldehyde, 1-bromododecane and potassium
2
.5. Small-angle X-ray scattering (SAXS) measurement
3
SAXS measurements from the D/Gal and D/Glc lipoplex were
carbonate was mixed in N,N-dimethylformamide at molar ratio
of 1:2:5 and stirred at 80 °C for 6 h. The reaction mixture was
extracted with ethyl acetate and purified compound I by silica
gel chromatography using a mixture of hexane and dichlorometh-
ane (hexane/dichloromethane = 1:1) as a mobile phase. The com-
pound I and sodium boronhydride were mixed in solution of
methanol and tetrahydrofuran (methanol/tetrahydrofuran = 5: 1)
at molar ratio of 1: 2 and stirred at rt for 2 h under a nitrogen
atmosphere. The reactant was added dichloromethane and 1 N
hydrogen chloride for neutralization. We extracted the organic
phase and purified compound II by silica gel chromatography using
dichloromethane. The compound II, propargylbromide and sodium
hydride were mixed in N,N-dimethylformamide at molar ratio of
carried out at BL40B2 SPring-8 with a 0.7 m camera using a Rigaku
imaging plate (30 ꢀ 30 cm, 3000 ꢀ 3000 pixels) as a detector. The
wavelength of the beam was 0.71 Å, and the exposure time was
3
00 s. The obtained two dimensional image was circularly aver-
aged to give an intensity I(q) versus q plots, where q is the magni-
tude of the scattering vector defined by q = 4 sinh/k with the
p
scattering angle of 2h. The concentration of sugar-modified lipids
was 6 mM.
2
.6. Interaction between ASGPR and lipoplexes
The anti-His6 antibody (Thermo SCIENTIFIC, Waltham, MA) was
immobilized on gold substrate in the quartz-crystal microbalance
QCM) sensor cell (AFFINIX QN ; INITIUM, Inc., Tokyo, Japan) for
h at rt After blocking with 1% BSA in PBS for 1 h, ASGPR (R&D Sys-
tems, Inc., Minneapolis, MN) in PBS was added into the sensor cell
at 25 g/ml and incubated for 1 h at rt After washing with PBS
three times, the sensor cell was filled with 500 l of 10 mM Tris–
1
:3:11 and stirred at rt for 19 h under a nitrogen atmosphere.
(
l
The reactant was extracted with ethyl acetate and purified com-
pound III by silica gel chromatography using a mixture of hexane
and dichloromethane (hexane/dichloromethane = 1:1). The com-
1
l
pound III, 1-azido-1-deoxy-b-D-galactopyranoside acetate, sodium
l
L-ascorbic acid and copper(II) sulfate pentahydrate were added in
HCl containing 2 mM CaCl
at 4
2
and 0.1% BSA and added the samples
g/ml and measured the frequency changes at 25 °C.
N,N-dimethylformamide at molar ratio of 1:1:1:0.07 and stirred
at 90 °C for 16 h. The reactant was extracted with ethyl acetate
and purified compound IV by silica gel chromatography using a
mixture of ethyl acetate and hexane (ethyl acetate/hexane = 2:3).
The compound IV and sodium methoxide was mixed in methanol
at molar ratio of 1:0.6 and stirred at rt for 4 h under a nitrogen
atmosphere. The reactant was added 6 N hydrogen chloride for
neutralization and centrifuged at 300ꢀg for 20 min. We collected
the organic phase and obtained compound V with evaporation.
l
2
.7. Gene transfection
4
HepG2 or A549 cells were seeded at 1.0 ꢀ 10 cells in a colla-
gen-coated 96-well microplate and incubated at 37 °C under 5%
CO . The cells were cultured in DMEM containing 10% FBS and
00 U/ml penicillin, and 0.1 mg/ml streptomycin. After 24 h, the
cells were transfected with the pDNA at 0.2 g/ml using the lipo-
2
1
l
1
The obtained galactose modified lipid was identified by H NMR.
plexes or Lipofectamine 2000 (invitrogen, Carlsbad, CA). In brief,
on the day of transfection, the wells were replaced with fresh med-
ium without serum and added the lipoplexes at the indicated N/P
ratio. After 6 h, the wells were replaced with fresh medium con-
taining serum. After 48 h, the cells were washed with PBS twice
adequately and then lysed with a lysis buffer from the luciferase
d
6
H
(500 MHz, methanol-d
.36 (1H, s, ArH), 5.58 (1H, d, J = 9.4 Hz, 1-CH), 4.64 (2H, s,
OCH ), 4.50 (2H, s, OCH ), 4.14 (1H, t, J = 9.4 Hz, 3-CH), 3.98 (1H,
d, J = 3.0 Hz, 2-CH), 3.93 (5H, t, 4-CH and 2 ꢀ OCH ), 3.85–3.68
3H, m, 5-CH and 6-CH CH ),
), 1.77–1.71 (4H, m, 2 ꢀ OCH
4
): d = 8.23 (1H, s, CH), 6.50 (2H, s, ArH),
2
2
2
(
2
2
2