Synthesis and transfection activity of novel galactosylated polycationic lipid

Article abstract of DOI:10.1016/j.bmcl.2011.03.056

In this study, we synthesized a new galactosylated cationic lipid and investigated its biological activity. The structure of lipid combines both spermine residue for DNA compaction and galactose moiety for the improvement of aggregation behavior of lipopl

Full text of DOI:10.1016/j.bmcl.2011.03.056

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2937–2940
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and transfection activity of novel galactosylated polycationic lipid
M. A. Maslov a, , D. A. Medvedeva b, D. A. Rapoport b, R. N. Serikov b, N. G. Morozova a, G. A. Serebrennikova a,
⇑
V. V. Vlassov b, M. A. Zenkova b
a Lomonosov Moscow State Academy of Fine Chemical Technology (MITHT), 86 Vernadskogo Ave., Moscow 119571, Russian Federation
b Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Lavrentiev Ave., Novosibirsk 630090, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized a new galactosylated cationic lipid and investigated its biological activity.
The structure of lipid combines both spermine residue for DNA compaction and galactose moiety for the
improvement of aggregation behavior of lipoplexes. Lipid was low toxic for different mammalian cells,
and was able both to compact plasmid DNA and to mediate cellular accumulation of various nucleic acids
(ODN, pDNA and siRNA) exhibiting biological activity (transgene expression, gene silencing).
Ó 2011 Elsevier Ltd. All rights reserved.
Received 31 January 2011
Revised 15 March 2011
Accepted 16 March 2011
Available online 21 March 2011
Keywords:
Cationic lipids
Transfection efficiency
Oligonucleotides
siRNA
EGFP expression
Gene and antisense oligonucleotides based therapies have been
developed for the treatment of both acquired and inherited dis-
eases.1 These approaches are based on the correction of the basis
of diseases by therapeutic nucleic acids, namely DNA, oligonucleo-
tides, small interfering RNA etc. Naked nucleic acids degrade easily
within the organism, therefore special transport systems were
developed to protect and to deliver nucleic acids into the targeted
cells. Cationic lipids are widely used as non-viral delivery systems
for the improvement of nucleic acid delivery into eukaryotic cells.2
However, lipids used for the gene delivery often display both the
high cytotoxicity and insufficient transfection efficiency. Hence,
intensive development and study of the new non-toxic and
efficient cationic lipids for the gene delivery are currently in pro-
gress.3,4 Cationic lipids have common structural features such as
the presence of positively charged and hydrophobic domains con-
nected by a spacer. Numerous chemical modifications have been
made to augment the delivering activity of cationic lipids.2,5,6
One promising strategy for the improvement of nucleic acids
delivery is the design of cationic agents for the cell specific gene
targeting, with the aid of covalently bounded ligands for specific
cell receptors. Recently, cationic lipids with incorporated carbohy-
drate residues were under intensive investigation as targeted sys-
tems for the delivery of nucleic acids to hepatocytes, macrophages,
and into the nucleus.7–9 Several groups have reported that
carbohydrate fragments increase the colloid stability of nucleic
acid–lipid complexes (lipoplexes) in a blood serum and decrease
the toxicity of cationic lipids.10,11 Some polycationic single and
double long-alkyl chain galactospermine bolaamphiphile have
been synthesized and appeared as a promising non-viral synthetic
vector for specific gene delivery.12,13 Here we describe the new and
convenient route to obtain a galactosylated polycationic lipid 1
(Fig. 1) and assess its biological activity in terms of cytotoxicity
and transfection activity, in respect to different nucleic acids.
The structure of lipid 1 combines both spermine residue for
DNA compaction and galactose moiety for improvement of aggre-
gation behavior of lipoplexes. Galactosyl residue may then serve as
a molecular signal for hepatocytes targeting. 1,2-di-O-tetradecyl-
rac-glycerol was used as the hydrophobic anchor for incorporation
into a lipid bilayer. It is known that cationic lipids containing tet-
radecyl substituents possess higher in vitro transfection efficacy
OH
HO
HO
OC14H29
OC14H29
O
( )4
O
O
O
N
S
HO
O
H
N
NH2
NH
N
H
H
N
O
1
⇑
Corresponding author. Tel.: +7 495 9368903; fax: +7 495 9368901.
E-mail address: mamaslov@mail.ru (M.A. Maslov).
Figure 1. Galactose-containing polycationic lipid.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.056

2938
M. A. Maslov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2937–2940
compared to other long-chain hydrocarbon analogues.14,15 Natural
polyamines such as spermine or spermidine are capable of binding
and condensing DNA molecules into small, dense particles. Lipo-
philic derivatives of polyamines condense DNA more efficiently
than polyamines themselves.16 Among the cationic lipids tested
lipophilic spermines are the most efficient synthetic gene delivery
agents.17 Therefore, for the creation of new polycationic lipid nat-
ural polyamine—spermine have to be covalently attached to the
hydrophobic anchor by means of an amide bond. On the other
hand it was necessary to link to the same anchor galactosyl resi-
due. The attachment of hydrophobic, cationic, and carbohydrate
domains was performed by using a Fukuyama amine synthesis—
the reaction of 6-hydroxyhexyl 4-nitrobenzenesulfonamide with
1,2-ditetradecyl-3-bromo-rac-glyceride. This convenient approach
permits to obtain a bifunctional molecule for the assembly of both
spermine and galactose residues into a single structure of polycat-
ionic glycolipid.
According to the developed synthetic route the sulfonylation of
6-aminohexanol (2) with 2-nitrobenzenesulfonyl chloride in the
presence of Et3N afforded amide 3 in 92% yield (Scheme 1). Bro-
mide 4 was prepared from 1,2-di-O-tetradecyl-rac-glycerol by
treatment with CBr4 in the presence of Ph3P as described previ-
ously.18 The alkylation of amide 3 with bromide 4 was carried
out with a Fukuyama amine synthesis19 and gave 5 in 64% yield.
The glycosylation of compound 5 with 2,3,4,6-tetra-O-acetyl-
-galactosyl bromide under the modified Königs–Knorr method in
a-
D
a Soxlet apparatus using CdCO3 as the promoter gave galactoside
6a. It is known that the ratio of the anomeric glycosides obtained
is strongly dependent upon the reaction conditions. Upon optimis-
ing glycosylation conditions the best results were obtained by re-
flux of compound 5 (1 equiv) with galactosylbromide (3 equiv) in
benzene for 1.5 h. Under these conditions b-glycoside 6 was
formed in 65% yield accompanied by only 2% of
a-galactoside.
The preferable formation of b-glycoside 6 was a result of neighbor-
ing-group participation of a 2-O-acyl functionality in the glycosyl
donor.20 The reduction of nitro group by catalytic hydrogenolysis
on Pd/C in the presence of ammonium formate as a hydrogen
source afforded amine 7 with 90% yield. Acylation of lipid 7 with
succinic anhydride in the presence of N,N-dimethyl-4-aminopyri-
dine (DMAP) (2 equiv) and N-hydroxybenzotriazole (HOBT) at
70 °C for 24 h gave the corresponding succinate 8 in the very low
yield (29%). The main product obtained in this reaction was cyclic
imide 8a (70%). In order to minimize the amount of undesirable 8a
we optimized ratios of reagents and temperature conditions and
found that the treatment of compound 7 with succinic anhydride
in the presence of Et3N (2 equiv) and catalytic amount of DMAP
(0.2 equiv) for 36 h at the room temperature gave succinate 8 in
73% yield.
OC14H29
OC14H29
OC14H29
OC14H29
( )4
ii,
HO
NH
Br
( )4
iii
4
HO
N
S
i
O
S
O
( )4
HO
NH2
O
O
2
3
5
NO2
NO2
AcO
AcO
OAc
OC14H29
OC14H29
AcO
AcO
OAc
OC14H29
OC14H29
O
O
( )4
O
N
S
v
( )4
O
N
S
AcO
AcO
O
O
O
O
O
R
O
R
OH
N
H
8, R =
6, R = NO2
7, R = NH2
iv
N
8a, R =
O
O
Boc
N
Boc
NH
OAc
O
AcO
AcO
OC14H29
OC14H29
N
Boc
9
NH2
, vi
( )4
O
O
O
N
S O
AcO
vii
1
Boc
N
Boc
NH
NH
N
Boc
H
N
O
10
Scheme 1. Synthesis of polycationic lipid 1. Reagents and conditions: (i) 2-NO2-C6H4SO2Cl, TEA, CH2Cl2, 24 °C, 36 h, 73%; (ii) Cs2CO3, TBAI, DMF, 90 °C, 8 h, 65%; (iii) 2,3,4,6-
tetra-O-acetyl–
-galactopyranosyl bromide, CdCO3, C6H6, reflux, 1.5 h, 65%; (iv) NH4ꢀHCO2, Pd/C, MeOH/THF (2:1), 60 °C, 30 min, 90%; (v) succinic anhydride, TEA, DMAP,
CH2Cl2, 24 °C, 36 h, 73%; (vi) tri-Boc-spermine (9), HBTU, DIEA, DMF, 4 °C, 40 min, 85%; (vii) (a) TFA, CH2Cl2, 24 °C, 3 h, 86%; (b) 0.04 N MeONa, MeOH, 24 °C, 4 h, 53%.
D

M. A. Maslov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2937–2940
2939
The key step of the synthesis involved the condensation of pro-
tected spermine 921 with succinate 8. The use of both 1,3-diisopro-
pylcarbodiimide (DIC)/HOBT/DMAP and 1,10-carbonyldiimidazole
as condensing agents was unsuccessful and provided large
amounts of by-products (N-acyl diisopropylurea in the case of
DIC or cyclic imide 8a for both approaches). The condensation of
amino 8 and carboxy 7 derivatives in the presence of 2-(1H-benzo-
ror. The transfection of the nucleic acids mediated by Lipofect-
amine 2000 was used as a positive control.
The cationic lipid 1 (10
lM) enhanced significantly the accumu-
lation of FITC-ON (10 M) in the cells (HEK 293, and BHK) both in
l
the absence and in the presence of serum (Table 1). The accumula-
tion efficiency (number of FITC-positive cells in a population) was
similar for HEK 293 and BHK cells. A comparison of 1 with Lipofect-
amine 2000 shows that 1 stimulates an accumulation of FITC-ON
with an efficiency that is close to or exceeds these parameters
for Lipofectamine 2000.
triazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HBTU) at 4 °C for 40 min afforded lipopolyamine 10 in 85% yield.
At the final stage, the unblocking of amino groups in the spermine
moiety was carried out by treatment of 10 with trifluoroacetic acid.
The removal of acetyl groups under Zemplen reaction using 0.04 N
sodium metoxide in methanol and subsequent dialysis against
water provided the key compound 1 in 46% yield in two stages.
The cytotoxicity of the polycationic lipid 1 was estimated by the
MTT22 test using several cell lines: HeLa (human cervical carci-
noma), HEK 293 (human embryo kidney), BHK (baby hamster kid-
ney) and transgenic BHK IR-780 cell lines. The cells were incubated
with either the lipid 1 or with the complexes of 1 with plasmid
Analysis of the cellular delivery of plasmid DNA (2
lg/mL) med-
iated by 1 (20 M) was performed 24 h post incubation of HEK 293
l
cells with pDNA/lipid 1 complexes to achieve high levels of EGFP
expression (Table 1). In the absence of serum 1 mediates the accu-
mulation of pEGFP-C2 in the cells with the efficiency close to that of
Lipofectamine. However, in the presence of serum the number of
EGFP-positive cells is decreased noticeably, indicating that cellular
delivery of pDNA mediated by 1 is strongly affected in the presence
of serum.
DNA (2
l
g/mL) at lipid concentrations ranging from 1 to 90
l
M
Small interfering RNAs are widely used for gene silencing. Anal-
ysis of the delivery of siRNA (50 nM) targeted EGFP mediated by 1
(10 lM) was performed using transgenic BHK IR-780 cells. siRNA
for 24 h in the presence of 10% fetal bovine serum (FBS) in the cell
medium or under serum-free conditions. IC50 values of 1 (a lipid
concentration corresponding to the viability of 50% of the cells
after incubation in the presence of lipid for 24 h) for different cells
used in the study delivered in the complex with Lipofectamine (po-
sitive control) efficiently inhibits expression of EGFP both in the
absence and in the presence of serum in the cell medium, while
scrambled siRNA (50 nM) under similar conditions causes insignif-
icant changes of the EGFP expression (primary data not shown).
Under serum-free conditions the efficiency of siRNA delivery med-
iated by 1 is distinctly higher than with Lipofectamine both in
terms of the number of non-fluorescent cells in population and
an average cell fluorescence (Table 1), however in the presence
of serum efficiency of siRNA delivery is decreased 2.5 times. The
inhibitory effect of serum on the delivery mediated by 1 of both
plasmid DNA and siRNA allow us to suppose that additional stabil-
ization of the complexes nucleic acid/1 possibly is required.
The ability of the lipid to compact nucleic acids (plasmid DNA)
was studied using atomic force microscopy (Fig. 2). The topo-
were around 20
lM (18–21 lM) in the absence and around 40 lM
(36–42 M) in the presence of 10% FBS. Note that cytotoxicity of
DNA/lipid 1 complexes was lower than that of 1 alone both in
the absence and in the presence of serum. For comparison, IC50 va-
lue for Lipofectamine2000 was 20 lM for HEK 293 cells.
l
The cationic lipid 1 was used for the delivery of a 25-mer 50-
fluorescein isothiocyanate-labeled oligonucleotide (hereinafter,
FITC-ON); pEGFP-C2 plasmid, encoding enhanced green fluorescent
protein (EGFP); and small interfering RNA (siRNA) targeted EGFP
gene into the HEK 293, BHK, and transgenic BHK IR-780 cells
expressing EGFP. The results of these experiments monitored by
a flow cytometry are summarized in Table 1. The data are pre-
sented as a percentage of either the fluorescence-positive cells
(FITC-ODN, pDNA) or non-fluorescent cells (when silencing of EGFP
gene caused by the delivered siRNA was achieved) in a sample and
as an average fluorescence of the cells population in a sample. The
cell population incubated with nucleic acids in the absence of 1
were used as a negative control: in these samples the percentage
of both the fluorescence-positive cells (FITC-ODN, pDNA) and the
non-fluorescent cells (siRNA) did not exceed the experimental er-
graphic AFM images show that particles formed by 1 (20
in the experiments with the plasmid DNA delivery) with plasmid
DNA (5.5 g/mL) have predominately spherical shape 50–
lM, as
l
a
100 nm in a diameter (Fig. 2B). The ability of the polycationic lipid
to compact the large plasmid DNA (Fig. 2A) to the small granules,
having the size lower than 100 nm, was in accordance with the
effective uptake and delivery of the nucleic acids by the lipid.23
Table 1
Analysis of cellular accumulation of various nucleic acids mediated by polycationic lipid 1
Nucleic acid
10% FBS
Control
Lf
Lipid 1
% of positive cells in population
RFU
% of positive cells in population
RFU
% of positive cells in population
RFU
FITC-ODNa
pDNAb
ꢁ
+
0.2
0.2
1.1
1.8
1.2d
1.3d
95
97
236
250
13988
16522
75.4
72.7
29
2714
1654
7512
8295
7122
6065
63.3
94.6
31.2
2.9
4198
13,243
5108
406
6872
14,246
ꢁ
+
27
siRNAc
ꢁ
+
39.8d
45.4d
49.4d
19.6d
Flow cytometry data.
a
An accumulation of the complexes FITC–ODN (5
l
M)/1 (10
l
M) in the BHK cells in the absence (ꢁ) or in the presence (+) of 10% FBS. The percentage of FITC-positive cells
and the average fluorescence of the cells in a population were determined by FACS analysis after 4 h of cell incubation with the FITC–ODN/1 complexes.
b
An expression of EGFP-transgene in the HEK 293 cells. The cells were incubated with the complexes composed of plasmid pEGFP-C2 (2 g/mL) and 1 (20 M) in the
l
l
absence (ꢁ) or in the presence (+) of 10% FBS. The percentage of EGFP-positive cells and the average fluorescence of a cell population were estimated by FACS analysis after
24 h of cell incubation with the cationic lipid/pEGFP-C2 complex.
c
An inhibition of the EGFP expression in the transgenic TGFP expressing BHK IR-780 cells. The cells were incubated with the complexes composed of 1 (10 lM) and siRNA-
EGFP (50 nM) in the absence (ꢁ) or in the presence (+) of 10% FBS. The levels of EGFP expression in the cells were evaluated using flow cytometry after 72 h of incubation with
these complexes.
d
The percentage of non-fluorescent BHK IR-780 cells, which means siRNA mediated the silencing of the EGFP gene.

2940
M. A. Maslov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2937–2940
Figure 2. The atomic force microscopy images of the free plasmid DNA (A) and the complexes formed by the plasmid DNA (5.5 lg/mL) and the cationic lipid 1 (20 lM) (B).
The AFM analysis was performed in 20 mM Tris–HCl buffer (pH 7.0) containing 5 mM MgCl2. The bar size is 100 nm.
In conclusion, we have elaborated the synthetic route for prep-
aration of the new galactose-containing polycationic lipid, which
was shown to be a promising transfection agent in terms of both
the low cytotoxicity and the efficient delivery of various nucleic
acids in vitro. Lipid 1 stimulates an accumulation of FITC-ON, plas-
mid DNA and siRNA with an efficiency that is close to or exceeds
these parameters for Lipofectamine 2000. The inhibitory effect of
serum on the delivery mediated by lipid 1 of both plasmid DNA
and siRNA allow us to suppose that additional studies, including
the formation of liposomal compositions, nucleic acid-to-carrier
ratio, and delivery conditions are required.
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Authors greatly acknowledge Dr. Dmitry V. Sheglov and Dr.
Alexander V. Latyshev (Institute of Semiconductor Physics SB
RAS, Novisibirsk State University) for their assistance in atomic
force microscopy, Mrs. Albina V. Vladimirova (Institute of Chemical
Biology and Fundamental Medicine SB RAS) for the cell mainte-
nance and Dr. Yaroslav R. Efremov for his assistance in FACS
analysis.
This research was supported by the Russian Academy of Science
under the programs ‘Molecular and Cell Biology’ and ‘Science for
Medicine’; Russian Foundation for Basic Research (grants no. 08-
04-00753 and 09-03-00874), Ministry of Science and Education
of the Russian Federation (state contract P438 and P715), Presi-
dent’s program in support of leading scientific schools SS-
7101.2010.4.
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Supplementary data
23. Medvedeva, D. A.; Maslov, M. A.; Serikov, R. N.; Morozova, N. G.; Serebrenikova,
G. A.; Sheglov, D. V.; Latyshev, A. V.; Vlassov, V. V.; Zenkova, M. A. J. Med. Chem.
2009, 52, 6558.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.056.