The synthesis of cholesterol-based cationic lipids with trimethylamine head and the effect of spacer structures on transfection efficiency

Article abstract of DOI:10.1016/j.bmcl.2011.04.071

Five cholesterol-based cationic lipids were newly synthesized based on cholest-5-en-3β-oxyethane-N,N,N-trimethylammonium bromide (Chol-ETA) structure where the cholesterol backbone is linked to cationic head via various lengths of ether-linked carbon spacer. The transfection efficiency of these compounds was increased in order of three (Chol-PRO) < four (Chol-BTA) < two (Chol-ETA) methylene unit in their spacer, and was decreased by an addition of isomethyl group to Chol-PRO spacer. In case of the presence of multiple bonds in the spacer, it required the more cationic lipids in liposome formulation than single bond in the spacer to present similar transfection efficiency. Crown Copyright

Full text of DOI:10.1016/j.bmcl.2011.04.071

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3734–3737
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis of cholesterol-based cationic lipids with trimethylamine head
and the effect of spacer structures on transfection efficiency
Bieong-Kil Kim a, , Yun-Ui Bae b, , Kyung-Oh Doh b, , Guen-Bae Hwang a, Sung-Hye Lee b, Hyungu Kang a,
Young-Bae Seu a,
⇑
a School of Life Science & Biotechnology, Kyungpook National University, Sankyukdong, Bookgu, Daegu 702-701, Republic of Korea
b Department of Physiology, Yeungnam University College of Medicine, 317-1 Daemyung-dong, Daegu 705-717, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Five cholesterol-based cationic lipids were newly synthesized based on cholest-5-en-3b-oxyethane-
N,N,N-trimethylammonium bromide (Chol-ETA) structure where the cholesterol backbone is linked to
cationic head via various lengths of ether-linked carbon spacer. The transfection efficiency of these com-
pounds was increased in order of three (Chol-PRO) < four (Chol-BTA) < two (Chol-ETA) methylene unit in
their spacer, and was decreased by an addition of isomethyl group to Chol-PRO spacer. In case of the pres-
ence of multiple bonds in the spacer, it required the more cationic lipids in liposome formulation than
single bond in the spacer to present similar transfection efficiency.
Received 11 February 2011
Revised 14 April 2011
Accepted 18 April 2011
Available online 24 April 2011
Keywords:
Cholesterol
Cationic liposome
Transfection
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Gene transfer technique
By means of gene therapy, gene transfer technique using cat-
ionic liposomes has been continuously researched in the view of
the fact that these non-viral vectors can avoid the in vivo problems
such as immune response caused by viral vectors.1–3 These cationic
liposomes have been also synthesized in order to deliver various
genetic materials including plasmid DNA and siRNA into cells
in vitro.4,5 For the effective delivery of anionic genetic materials,
amphipathic cationic lipids generally consist of cationic head
group, linker, and hydrophobic domain. Cationic head groups and
polar linkers vary quite substantially, while hydrophobic regions
are mostly composed of long chain fatty acids or cholesterol deriv-
atives.6 This molecular structure of cationic lipids is important fac-
tor influencing gene delivery. The relationship between structure
of cationic lipid and transfection efficiency has been actively
researched from various angles. However, it was very hard to
understand structure-transfection activity relationship (SAR) for
transfection due to involving numerous steps related to cellular
delivery of DNA.7
Chol-ETA was generally superior to complicated structures.5,8–10
Not only the structure of hydrophobic domain and cationic head
but also the linker between them is one of the important factor re-
lated to successful gene delivery according to the kind of bond9,11
and spacer8,12–14 in linker (Fig. 1). Therefore, we newly synthesized
five compounds based on Chol-ETA, and investigated the effect of
spacer structure on transfection efficiency (Scheme 1).
Six cholesterol-based cationic lipids have been synthesized
based on Chol-ETA structure where the cholesterol backbone is
linked to cationic head via a various types of ether-linked carbon
spacer (Scheme 1). Compound 2 and 3 were synthesized by modi-
fying the methods of Bajaj et al.10 To an ice-cooled solution of cho-
lesterol 1 in pyridine-chloroform (v/v, 1/1), p-toluenesulfonyl
chloride was added and stirred for 12 h at room temperature. Cho-
lesterol tosylate 2 tosylated from cholesterol 1 in practical yield
was used in the next reaction without a purification process be-
cause it was sufficiently purified by precipitating as a result of
thin-layer chromatography (TLC) and 1H NMR analysis, and was
unstable in a silica gel column. Cholesterol tosylate 2 and 7–
10 equiv of appropriate diol in anhydrous dioxane were refluxed
for 7 h to afford compound 3 in 70–87% yield. To link cationic head
of trimethylamine, compound 3 was bromized by reacting with
1 equiv of carbon tetrabromide and 1.5 equiv of triphenylphos-
phine in methylene chloride in 91–100% yield. The last compounds
were synthesized by reacting compound 4 with trimethylamine
(ca. 4.3 mol/L in water) in dimethylsulfoxide/chloroform (v/v, 7/
3) at room temperature for 24 h in 40–90% yield. Structures
of all the synthetic intermediates and last compounds shown in
Recently, Ghosh’s group synthesized cholest-5-en-3b-
oxyethane-N,N,N-trimethylammonium bromide (here called as
Chol-ETA) where cholesterol lipid domain and trimethylamine
head are linked to two methylene units via ether linker (Fig. 1).
Although this structure is very simple, transfection efficiency of
⇑
Corresponding author. Tel.: +82 53 950 5380; fax: +82 53 955 5522.
E-mail address: ybseu@knu.ac.kr (Y.-B. Seu).
Three authors contributed equally to this work.
0960-894X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.071

B.-K. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3734–3737
3735
Transfection optimizing medium (TOM), pCMVTnT-GFP, and
pcDNA-Luc were obtained from Welgene (Korea) to test the ability
of liposomes delivering plasmid DNA in COS-7 cell (african green
monkey kidney cell). The day before transfection, COS-7 cells
(4 ꢀ 104 per well) were seeded into 48-well plates. Thirty minutes
before transfection, each well was briefly washed and added with
100
mixed with pCMVTnT-GFP or pcDNA-Luc plasmid DNA (0.3
in 50 l of TOM. The lipoplexes, consisting of liposomes and
ll of TOM. Each liposome was diluted to 50
ll of TOM and
lg)
l
DNA, were incubated for 10 min in room temperature, and then
added to each well. The culture medium containing lipoplex was
removed 4 h after the transfection, and fresh medium with 10% fe-
tal bovine serum (FBS) was added to each well. After 18 h of further
incubation, the medium was removed, and cells were washed
twice with sterile phosphate buffered saline (PBS). The transfection
efficiency was determined by means of a LMax II 384 luminometer
(Molecular devices corp., USA) using a luciferase assay kit from
Promega (USA). The protein content was quantified using a
bicinchoninic acid (BCA) assay (PIERCE, USA). The luciferase effi-
ciency was normalized by the protein content and expressed as rel-
ative light unit/lg of protein (RLU/lg protein). To observe GFP
expression, fluorescence protein was observed on a Nikon ECLIPSE
TE300 fluorescence microscope (Japan).
This study describes the synthesis and SAR of six cholesterol-
based cationic lipids including Chol-ETA. These compounds were
synthesized to observe the effect of change of a spacer structure
Figure 1. The common structure of cholesterol-based cationic lipids linked to
cationic head via a various linker.
Scheme 1. Synthesis of Chol-ETA series. Reagents and conditions: (a) Cholesterol,
p-toluenesulfonyl chloride, pyridine/CHCl3 = 1/1 (yield: 100%); (b) X-diol/anhy-
drous dioxane, reflux (yield: 70–87%); (c) Carbon tetrabromide, triphenylphos-
phine/CH2Cl2 (yield: 91–100%); (d) Trimethylamine, DMSO/CHCl3 = 7/3 (yield:
40–90%).
Scheme 1 were confirmed by 1H NMR. The final compounds were
characterized by FAB-MS to confirm the identity of the molecular
ions.
The optimum transfection efficiency was shown with the addi-
tion of appropriate amount of helper lipids such as DOPE (1,2-diol-
eoyl-L-a-glycero-3-phosphatidyl-ethanolamine) and cholesterol in
Figure 2. Transfection optimization of Chol-ETA series. (A) Transfection efficiencies
of Chol-ETA series with various weight ratio of DOPE. The concentration of pcDNA-
many cationic liposomes.3,6,12 To form liposome formulation, lipid
film made from appropriate weight ratio of cationic lipids and
DOPE was briefly dispersed in an aqueous solution. The lipid film
was hydrated at 4 °C overnight, followed by sonication at 60 °C
for 20 min. Next, cationic liposome was passed 10 times through
200 nm polycarbonate membrane to remove overlarge liposomes.
Luc was 0.3
Transfection efficiencies of Chol-ETA series using optimized lipid/DOPE formula-
tions at various N/P weight ratios. Data are expressed as relative light unit (RLU)/
lg/well, and liposoems were used at 15 N/P weight ratio. (B)
l
g
total protein content as obtained from luciferase gene expression assay. Each bar
value represents the mean SD of duplicate experiments performed on the same
day.

3736
B.-K. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3734–3737
connecting cationic head and hydrophobic domain on transfection
efficiency. We used DOPE as co-lipid to enhance transfection effi-
ciency, and sought the optimum transfection condition by varying
the weight ratio of lipid:DOPE. We also measured the transfection
efficiency at various liposome/DNA (N/P) weight ratios to find
the optimum condition. In 15 N/P weight ratio, the optimum
lipid:DOPE weight ratio was 1:1 for newly synthesized five com-
pounds and 1:2 for Chol-ETA. In the optimum lipid:DOPE ratio,
15 N/P weight ratio showed the highest transfection efficiency in
all liposomes except Chol-BTA (Fig. 2). Their transfection efficiency
was compared with commercial cationic liposomes at optimal con-
dition. These liposomes were as good as or better than commercial
cationic liposomes for in vitro transfection in COS-7 cell (Fig. 3).
The transfection efficiency of Chol-ETA having two methylene
unit in their spacer was higher than others and there are no signif-
icant difference between three (Chol-PRO) and four (Chol-BTA)
methylene units (Fig. 3). These results were also confirmed by
green fluorescent protein (GFP) expression (Fig. 4). In aminoglycer-
ol-based cationic lipids with diverse length of a spacer structure,
transfection efficiency of the spacer length exceeding 6 carbons
was lower than short counterparts, and binding with DNA was also
weaker.12 In cholesterol-based lipids, the spacer length presenting
the highest transfection efficiency was three carbons in carbar-
mate-linked cationic lipid but was two carbons in amide and
ester-linked cationic lipids.13,14 Elongation of the distance between
cationic head and lipid domain from one oxyethylene units to four
oxyethylene units represented to have negative effect on the trans-
fection efficiency.8 In this study, we found that two methylene unit
in ether-linked carbon spacer of cationic lipid having cholesterol
domain and trimethylamine cationic head is more feasible struc-
ture for in vitro transfection.
We added the isomethyl group in the spacer to know whether
spatial factor causes steric inhibition in lipoplex conformation.
The addition of isomethyl group in the spacer, Chol-MPRO,
decreased transfection efficiency when compared with Chol-PRO.
In addition, the presence of multiple bonds in the spacer did not
make much difference in the transfection efficiencies at the opti-
mum condition (Fig. 3: Chol-BTA, Chol-BTE, Chol-BTY). The trans-
fection efficiency in 2:1 cationic lipid:DOPE weight ratio (as in a
large amount of cationic lipid) was increased in order of single
bond (Chol-BTA) < double bond (Chol-BTE) < triple bond (Chol-
BTY). The transfection efficiency in 1:2 cationic lipid:DOPE weight
ratio (as in a small amount of cationic lipid) was increased in order
of single bond > double bond > triple bond (Fig. 2A). In other words,
although the best transfection efficiencies were similar, the more
cationic lipids in the liposome formulation were required for the
optimal transfection condition according to the increase of
from single bond to triple bond (Fig. 2B). It can be attributed to the
fact that the face of an electron-rich system (e.g. benzene, ethyl-
p-bond
p
Figure 3. Comparision of transfection efficiencies using optimized Chol-ETA series
ene) noncovalently interact with an adjacent cation including sim-
ple inorganic ions (e.g. Li+, Na+ and K+), protonated amines (RNHþ3 ),
quaternary ammoniums, sulfoniums and carbocations, known as
with commercial cationic liposomes (LFA: Lipofectamine, DT: DOTAP, DM-C:
DMRIE-C). Data are expressed as relative light unit (RLU)/lg total protein content
as obtained from luciferase gene expression assay. Each bar value represents the
mean SD of duplicate experiments performed on the same day.
cation-p
interaction15,16 which may inhibit charge interaction be-
tween a cationic liposome with plasmid DNA.
In summary, five compounds were newly synthesized based on
Chol-ETA to investigate the effect of various spacer structures on
the transfection efficiency. These compounds showed sufficient
transfection efficiency when compared with commercial cationic
liposomes in COS-7 cell. The transfection efficiency of cationic lipid
with two methylene unit in their spacer was highest than the oth-
ers. The addition of isomethyl group in the spacer decreased trans-
fection efficiency when compared with its counterpart, Chol-PRO.
Also, the structure including multiple bonds in their spacer re-
quired the more cationic lipids in the liposome preparation than
single bond to present similar transfection efficiency.
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (No.2010-
0024376).
References and notes
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