Synthesis and gene transfection activity of cyclen-based cationic lipids with asymmetric acyl-cholesteryl hydrophobic tails

Article abstract of DOI:10.1039/c4ob00384e

A series of novel 1,4,7,10-tetraazacyclododecane (cyclen)-based cationic lipids with asymmetric double hydrophobic tails (cholesteryl and long aliphatic chains) were designed and synthesized. Lysine was chosen as a linking moiety in the molecular backbone. The liposomes formed from 8 and dioleoylphosphatidylethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles under a low N/P ratio. These nano-scaled lipoplexes have low cytotoxicity, and might efficiently transfect A549 cells. In vitro transfection results revealed that all cationic lipids showed a comparable or better transfection efficiency (TE) than commercially available Lipofectamine 2000. The length and saturation degree of the aliphatic chain would affect their gene transfection performance, and the linoleic acid-containing 8e could give the best TE. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00384e

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Synthesis and gene transfection activity of
cyclen-based cationic lipids with asymmetric
acyl-cholesteryl hydrophobic tails
Cite this: Org. Biomol. Chem., 2014,
12, 3484
Bao-Quan Liu,†a,b Wen-Jing Yi,†a Ji Zhang,*a Qiang Liu,a Yan-Hong Liu,a
Sheng-Di Fanb and Xiao-Qi Yu*a
A series of novel 1,4,7,10-tetraazacyclododecane (cyclen)-based cationic lipids with asymmetric double
hydrophobic tails (cholesteryl and long aliphatic chains) were designed and synthesized. Lysine was
chosen as a linking moiety in the molecular backbone. The liposomes formed from 8 and dioleoylphos-
phatidylethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles under a low N/P
ratio. These nano-scaled lipoplexes have low cytotoxicity, and might efficiently transfect A549 cells.
In vitro transfection results revealed that all cationic lipids showed a comparable or better transfection
efficiency (TE) than commercially available Lipofectamine 2000. The length and saturation degree of the
aliphatic chain would affect their gene transfection performance, and the linoleic acid-containing 8e
could give the best TE.
Received 19th February 2014,
Accepted 25th March 2014
DOI: 10.1039/c4ob00384e
www.rsc.org/obc
linkage (including a backbone domain), and a hydrophobic
moiety.9 The cationic hydrophilic domain generally has one or
Introduction
Gene therapy is a promising approach for treating both genetic more positively charged groups usually formed from the proto-
and acquired diseases. The most challenging issue for the suc- nation of amino groups (monocationic and polycationic lipids,
cessful application of gene therapy is to develop safe and respectively).10 Polyamine-containing lipids are expected to
highly efficient vectors for the delivery of the transgene into form liposomes with a greater surface charge density than
host cells.1 Traditionally, gene delivery systems are broadly monocationic lipids. In our ongoing studies of novel polycatio-
based on either viral or non-viral mediated vectors. Despite the nic lipids for gene transfection, we recently demonstrated the
high transfection efficiency (TE) of viral vectors, safety con- potential of 1,4,7,10-tetraazacyclododecane (cyclen) based gene
cerns have been raised in clinical trials. The inherent draw- delivery systems.11,12 Besides easy modification, the unique
backs of viral vectors include immunogenicity, restricted cyclic structure and wide-ranging pKa (ref. 13) of the amino
targeting of specific cell types, size limitation on DNA, poten- groups on cyclen may facilitate the binding toward negatively
tial mutagenesis, etc.2 Non-viral vectors, such as cationic lipo- charged DNA and other processes in the transfection.
some/polymer-based systems2,3 and some naturally existing or
artificially synthesized cell-penetrating peptides4–7 are con- its cytotoxicity and biodegradability in
The gene delivery performance of a cationic lipid, including
physiological
a
sidered to be less toxic, less immunogenic, and easier to environment, is largely dependent upon the structure of the
prepare than viral vectors and are potentially more attractive linking group between the hydrophobic and the hydrophilic
for clinical applications.
domains.14,15 The common linkage bonds are ether, ester,
Appropriate modification of cationic lipid structures and amide, carbamate groups, etc. Although some commercially
the study of the structure–activity relationships (SAR) are of available transfection agents such as Lipofectamine 2000
great importance for the rational design of efficient lipidic contain ether bridges, other linkages are considered to have
gene vectors.8 The molecular architectures of cationic lipids lower cytotoxicity and the potential for degradation under
include three functional units: a hydrophilic head group, a special conditions. Carbamate and amide linkers are expected
to serve as a reasonable balance between the TE and biocom-
patibility.3,16,17 Besides the linkage bonds, the backbone
aKey Laboratory of Green Chemistry and Technology (Ministry of Education), College
domain also acts as a scaffold on which the cationic lipid is
built.9 The most common backbone is a glycerol-based struc-
of Chemistry, Sichuan University, Chengdu, 610064, China.
E-mail: jzhang@scu.edu.cn, xqyu@scu.edu.cn; Tel: +86 2885415886
bKey Laboratory of Biochemical Engineering (Ministry of Education), Dalian
ture. However, as a natural and biocompatible unit, amino
acids were used as building blocks in the design of cationic
Nationalities University, Dalian, 116600, China
†These authors contributed equally to this work.
lipids, and high TEs were achieved in several cases.18,19
3484 | Org. Biomol. Chem., 2014, 12, 3484–3492
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
The hydrophobic tails of synthesized lipids usually contain intensities significantly decreased with the rise of the N/P ratio
a steroid skeleton or a double-chain hydrocarbon with either (0–5). At the N/P ratio of 5, only about 20% of the original fluo-
saturated or unsaturated chains.9 Among the sterol-containing rescent intensity was found, indicating that these lipids could
cationic lipids, cholesterol derivatives are most widely used efficiently displace EB, which previously intercalated into DNA
due to their importance in human metabolic pathways.20–22 base pairs. The parameters of CE50 which represent the N/P
Although many cationic lipids with cholesterol or double- charge ratios for quenching 50% fluorescence intensity of EB
chain hydrocarbon have been extensively studied, lipids with were evaluated to be 2.8, 2.5, 2.3, 1.9 and 2.1 for lipids 8a–8e,
asymmetric hydrophobic tails have received less attention.23–25 respectively. On the other hand, the agarose gel retardation
For example, Yingyongnarongkul et al. synthesized a series of assay was also applied to evaluate the interaction between 8
novel cationic lipids with asymmetric acyl-cholesteryl hydro- and pDNA. Results of Fig. 2 show that complete DNA retar-
phobic tails with high TE and low cytotoxicity.26 In this report, dation could be achieved at the N/P of 4 for the liposomes
we develop a new series of cyclen-based polycationic lipids formed from all five lipids. These results further demonstrate
with asymmetric acyl-cholesteryl hydrophobic tails, which were the strong interaction of 8 with plasmid DNA.
connected by the lysine moiety. These materials were proved to
The average particle size, surface charge and morphology of
have the potential to be efficient gene vectors with relatively the lipoplexes may largely influence the apparent cytotoxicity,
low cytotoxicity.
cellular uptake/trafficking, and release of the encapsulated
gene.27 Fig. 3A depicts the N/P ratio dependence of the mean
particle sizes of the lipoplexes formed from 8a to 8e. The
average diameters were observed in the range of 100–380 nm,
which strongly depended on the N/P ratio. In a general view,
the average diameters gradually dropped with the increase of
N/P, and then became stable around 120–150 nm at the N/P
ratio of 4, indicating that full DNA condensation was achieved.
Meanwhile, surface potentials of the lipoplex nanoparticles
were also measured by DLS, and the results are shown in
Fig. 3B. Zeta potentials of the five lipoplexes showed a similar
trend along with the change of the N/P ratio. The values
increased from −12 mV to 38 mV with the rise of the N/P ratio
from 2 to 8. The difference of zeta-potentials between these
lipoplexes under the N/P of 2 was much larger than those
under the N/P of ≥4. This might be attributed to the different
binding situation at such a lower N/P ratio (incomplete
condensation).
Transmission electron microscopy (TEM) was further per-
formed to get information about the shape and morphology of
lipoplexes. A representative electron micrograph of lipoplexes
formed from 8e is shown in Fig. 4. The lipoplexes showed
homogeneous spherical particles with sizes in the range of
40–50 nm. From these results we considered that lipoplexes
formed from 8 may condense DNA to form nanoparticles with
proper sizes for gene delivery. It is worth mentioning that the
discrepancy of particle size measured by DLS (158 6 nm)
Results and discussion
Synthesis of target cationic lipids
The building blocks of target lipids include a cyclen head-
group, a lysine-containing backbone, a hydrophobic choles-
teryl tail and an aliphatic tail with different chain lengths and
saturation degrees, which are conducive to the SAR study. The
preparation route is shown in Scheme 1. Firstly, commercially
available α-N-boc-ε-N-(2-chlorobenzyloxycarbonyl)-L-lysine (1)
was coupled with different aliphatic amines (2a–2e) in the
presence of (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride) (EDC·HCl) and N-hydroxybenzotriazole (HOBt)
to yield 3a–e. After removal of the boc group by trifluoroacetic
acid, the compounds were subsequently reacted with tri-boc-
cyclen-acetic acid 4 to give the intermediates 5a–e. The benzyl-
oxycarbonyl group was then removed by Pd/C under a H2
atmosphere. The coupling between the product and cholesteryl
chloroformate 6 gave the precursors 7a–e. Finally deprotection
led to target lipids 8a–e. The structures of all new compounds
were confirmed by NMR and HRMS.
Interaction with DNA and characterization of liposome/DNA
complexes (lipoplexes)
Cationic liposomes are formed from either individual cationic and TEM (40–50 nm) could be attributed to the fact that
lipids or more frequently from a combination of cationic lipids DLS determines the hydrodynamic diameter of micelles in
and neutral lipids such as dioleoyl phosphatidylethanolamine water, whereas TEM reveals the morphology of the micelles in
(DOPE), which might increase the transfection efficiency sig- the dehydrated state.28
nificantly.19 Each cationic lipid was mixed in different molar
ratios (1 : 0, 1 : 1, 1 : 2, and 1 : 3) with DOPE to determine the
Cytotoxicity
optimal combination. According to the best behavior in the The cytotoxicity of the prepared lipoplexes was examined in
transfection experiment (data not shown), the 8/DOPE ratio of A549 cells by the MTT assay. As shown in Fig. 5, the percentage
1 : 2 was used herein. Ethidium bromide (EB) dye replacement of viable cells decreased with the rise of the N/P ratio. Under
and agarose gel electrophoresis assays were first employed to the N/P of 4, the cytotoxicity of these lipids is slightly lower
evaluate their binding abilities toward plasmid DNA. Fig. 1 than that of the commercially available transfection agent
shows the EB replacement assay results of relative fluorescence Lipofectamine 2000. In addition, the results also revealed that
intensity as a function of the N/P charge ratio in 10 mM of the variation of hydrophobic chains would influence the cyto-
HEPES solution (pH 7.4). It was shown that the fluorescent toxicity. Lipoplexes with longer chains led to higher cell viabi-
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 3484–3492 | 3485

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 1 Synthetic routes of title lipids 8a–8e.
lity, while unsaturated chains seemed to be harmful to the
cells. This might come from the greater membrane fluidity
caused by the bended unsaturated long chain.
In vitro gene transfection
Gene transfection experiments were then conducted to assess
the suitability of 8 as non-viral vectors for nucleic acid delivery.
To directly visualize the infected cells expressing pEGFP-Nl,
lipids 8 (prepared at N/P ratios of 4, 6 and 8) mediated eGFP
expression in A549 tumor cells was firstly studied and observed
using an inverted fluorescence microscope. Fig. 6 shows the
eGFP fluorescence microscope images obtained after 8-pro-
moted transfection. It was found that the transfection
mediated by lipoplexes 8a–8d showed the highest green fluo-
rescence density at the N/P of 4 (Fig. 6A, D, G and J), indicating
the largest amount of transfected cells. Meanwhile, lipoplexes
8e gave the best transfection result at the N/P of 6 (Fig. 6N).
Among the five lipids, the linoleic acid-containing 8e is
Fig. 1 Fluorescence quenching of EB by 8/DOPE liposomes under
various N/P ratios in 10 mM of HEPES buffer. The molar ratio of 8/DOPE
was 1 : 2.
3486 | Org. Biomol. Chem., 2014, 12, 3484–3492
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 2 Electrophoretic gel retardation assays of 8/DOPE/pDNA complexes at different N/P ratios. The molar ratio of 8/DOPE was 1 : 2.
Fig. 3 Mean particle sizes (A) and zeta-potentials (B) of the lipoplexes formed from 8a to 8e under various N/P ratios (DLS at room temperature).
Fig. 4 TEM image of 8e/DOPE/DNA complexes at the N/P ratio of 6.
Fig. 5 Cell toxicity of the lipoplexes prepared at various N/P ratios, with
Lipofectamine 2000 as the control.
distinctly the best choice for high TE. Subsequently, a luciferase
assay was conducted to quantitatively study the TE of 8 in the
same cell line. As shown in Fig. 7, the results were quite con-
sistent with those obtained in eGFP experiments. 8e gave the
best TE at the N/P ratio of 6, and the TE was about 1.6 times
higher than that of Lipofectamine 2000. Except 8e, the other
four lipids gave their best TE at the N/P of 4. Among the lipids
containing saturated long chains, palmitic (hexadecanoic)
acid-containing 8b seemed to be the best transfection reagent,
and its TE was close to that of Lipofectamine 2000. It was also
reported that linoleic acid modified lipids29,30 or lipopoly-
mers28 might show a higher gene delivery efficiency than their
analogs. We speculate that the relatively higher DNA-binding
ability and better fluidic character may benefit their transfec-
tion behavior.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 3484–3492 | 3487

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 6 Fluorescent microscope images of A549 cells transfected with 8a (A–C), 8b (D–F), 8c (G–I), 8d (J–L), and 8e (M–O). The lipid/DOPE ratio
was 1 : 2, and N/P ratios were 4 (top row), 6 (middle row) and 8 (bottom row). The cells were observed by fluorescence microscopy after 24 h
transfection.
fication of such types of lipids and relative mechanism studies
are now in progress.
Experimental section
Materials and methods
All chemicals and reagents were obtained commercially and
were used as received. Anhydrous chloroform and dichloro-
methane were dried and purified under nitrogen by using
standard methods and were distilled immediately before
use. [4,7,10-Tris(tert-butoxy-carbonyl)-1,4,7,10-tetraaza-cyclo-
dodecan-1-yl] acetic acid (tri-boc-cyclen-acetic acid, compound
4),31 and cholesteryl chloroformate (compound 6)16 were
synthesized according to the literature. MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
purchased from Sigma-Aldrich (St. Louis, MO, USA). A micro-
BCA protein assay kit was obtained from Pierce (Rockford, IL,
USA). A luciferase assay kit was purchased from Promega
(Madison, WI, USA). A endotoxin-free plasmid purification kit
was purchased from TIANGEN (Beijing, China). The plasmids
used in the gene transfection assay were pGL3 (Promega)
coding for luciferase DNA and pEGFP-N1 (Clontech, Palo Alto,
CA, USA) coding for enhanced green fluorescent protein
(eGFP) DNA. The supercoiled plasmid DNA (pUC-19) used in
the agarose-gel assay was purchased from Takara (Dalian,
China). Dulbecco’s modified Eagle’s medium (DMEM), RPMI
1640 medium, and fetal bovine serum (FBS) were purchased
from Invitrogen Corp. A549 (human lung adenocarcinoma epi-
thelial cells) was purchased from the American Type Culture
Collection (ATCC). The NMR spectra were measured on a
Varian INOVA-400 spectrometer and the d scale in parts per
Fig. 7 Transfection efficiencies of lipids 8a–8e in A549 cells. The molar
ratio of lipid/dioleoylphosphatidylethanolamine (DOPE) was 1 : 2.
Conclusion
In summary, a series of cyclen-based cationic lipids with asym-
metric double hydrophobic tails were prepared. Lysine was
used as a linking group in the molecular backbone. The lipo-
somes formed from the lipids and DOPE could efficiently con-
dense DNA into nanoparticles with proper size and zeta-
potential. The length and saturation degree of the aliphatic
chain would affect their gene transfection performance.
Results showed that the linoleic acid-containing 8e could give
1.6 times higher TE than the commercially available transfec-
tion reagent Lipofectamine 2000. The cytotoxicity of these
lipids was similar to that of Lipofectamine 2000. Further modi-
3488 | Org. Biomol. Chem., 2014, 12, 3484–3492
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
million was referenced to residual solvent peaks or internal 2H), 4.31 (s, 1H), 3.73–2.74 (m, 22H), 1.67–1.20 (m, 53H), 0.87
tetramethylsilane (TMS). MS-ESI spectra data were recorded on (t, J = 6.8 Hz, 3H). HR-MS (ESI): C51H88ClN7NaO10 [M + Na]+,
a Bruker Daltonics BioTOF mass spectrometer.
1016.6173, found: 1016.6183.
5b: (736 mg, 0.7 mmol, yield 70%). 1H NMR (400 MHz,
CDCl3) δ 7.47–7.29 (m, 2H), 7.26–7.17 (m, 2H), 5.35–5.08 (m,
Preparation of compounds 3a–e
A solution of compound 1 (830 mg, 2 mmol), long chain alkyl 2H), 4.31 (s, 1H), 3.50–2.62 (m, 22H), 1.59–1.21 (m, 61H),
amines 2a–e (2.2 mmol), and HOBt (324 mg, 2.4 mmol) in 0.97–0.74 (m, 3H). HR-MS (ESI): C55H96ClN7NaO10 [M + Na]+,
CH2Cl2 (50 mL) was cooled to 0 °C. EDC·HCl (460 mg, 1072.6799, found: 1072.6806.
2.4 mmol) and N,N-diisopropylethylamine (DIEA, 776 mg,
5c: (788 mg, 0.73 mmol, yield 73%). 1H NMR (400 MHz,
3 mmol) were gradually added, and the reaction mixture was CDCl3) δ 7.40–7.20 (m, 4H), 5.12 (s, 2H), 4.28 (s, 1H), 3.6–2.50
stirred for 1 h at 0 °C and at room temperature overnight. The (m, 22H), 1.67–1.0 (m, 65H), 0.83–0.80 (m, 3H). HR-MS (ESI):
mixture was washed with saturated aqueous NaHCO3 solution C57H100ClN7NaO10 [M + Na]+, 1100.7112, found: 1100.7118.
(2 × 20 mL) and saturated brine (20 mL). The organic phase
5d: (754 mg, 0.7 mmol, yield 70%). 1H NMR (400 MHz,
was dried over Na2SO4, filtered, and concentrated to afford a CDCl3) δ 7.49–7.34 (m, 2H), 7.26–7.24 (m, 2H), 5.50–5.12 (m,
white solid, which was further purified by column chromato- 4H), 4.32 (s, 1H), 3.70–2.75 (m, 22H), 2.10–1.94 (m, 4H),
graphy on silica gel (EtOAc–PE = 1 : 2, Rf = 0.3) to yield 3 as a 1.82–1.20 (m, 57H), 0.89 (t, J = 6.8 Hz, 3H). HR-MS (ESI):
white solid.
3a: (1.06 g, 1.82 mmol, yield 91%). 1H NMR (400 MHz,
C57H98ClN7NaO10 [M + Na]+, 1098.6956, found: 1098.6962.
5e: (774 mg, 0.72 mmol, yield 72%). 1H NMR (400 MHz,
CDCl3) δ 7.60–7.39 (m, 2H), 7.33–7.19 (m, 2H), 5.08 (s, 2H), CDCl3) δ 7.55–7.32 (m, 2H), 7.28–7.25 (m, 2H), 5.57–5.11 (m,
4.05 (s, 1H), 3.45–3.01 (m, 4H), 2.18–1.11 (m, 35H), 1.00–0.76 6H), 4.33 (s, 1H), 3.94–2.58 (m, 24H), 2.21–1.93 (m, 4H),
(m, 3H). HR-MS (ESI): C31H52ClN3NaO5 [M + Na]+, 604.3488, 1.76–1.17 (m, 55H), 0.91 (t, J = 6.7 Hz, 3H). HR-MS (ESI):
found: 604.3495.
C57H96ClN7NaO10 [M + Na]+, 1096.6799, found: 1096.6797.
3b: (1.15 g, 1.8 mmol, yield 90%). 1H NMR (400 MHz,
CDCl3) δ 7.50–7.34 (m, 2H), 7.30–7.24 (m, 2H), 5.06 (s, 2H),
4.19 (s, 1H), 3.40–3.10 (m, 4H), 1.96–1.75 (m, 2H), 1.71–1.22
Preparation of compounds 7a–e
(m, 41H), 0.89 (t,
J
=
6.8 Hz, 3H). HR-MS (ESI): Compound 5 (1.0 mmol) was dissolved in methanol (20 mL),
and then 10% palladium on carbon (0.5 g, containing 50%
C35H60ClN3NaO5 [M + Na]+, 660.4114, found: 660.4115.
3c: (1.15 mg, 1.72 mmol, yield 86%). 1H NMR (400 MHz, water) was added. The 2-chlorobenzyl group was removed from
CDCl3) δ 7.47–7.30 (m, 2H), 7.26–7.15 (m, 2H), 5.17 (s, 2H), 5 using a hydrogen atmosphere (balloon pressure) at room
4.07 (m, 1H), 3.16 (d, J = 2.6 Hz, 4H), 1.77–1.23 (m, 47H), 0.86 temperature for 24 h. The catalyst was removed by filtration,
(t, J = 6.8 Hz, 3H). HR-MS (ESI): C35H60ClN3NaO5 [M + Na]+, and the filtrate was concentrated. The residue was dissolved in
688.4427, found: 688.4427.
CH2Cl2 (30 mL). Triethylamine (101 mg, 2.0 mmol) and choles-
3d: (1.13 g, 1.7 mmol, yield 85%). 1H NMR (400 MHz, teryl chloroformate 6 (460 mg, 1.0 mmol) were gradually
CDCl3) δ 7.46–7.33 (m, 2H), 7.27–7.21 (m, 2H), 5.40–5.30 (m, added, and the reaction mixture was stirred at room tempera-
2H), 5.20 (s, 2H), 4.05 (s, 1H), 3.23–3.17 (m, 4H), 2.12–1.91 (m, ture overnight. The mixture was washed with saturated
4H), 1.78–1.12 (m, 39H), 0.88 (t, J = 6.7 Hz, 3H). HR-MS (ESI): aqueous NaHCO3 solution (20 mL) and saturated brine
C37H62ClN3NaO5 [M + Na]+, 686.4270, found: 686.4272.
(20 mL). The organic phase was dried over Na2SO4, filtered,
3e: (1.19 g, 1.8 mmol, yield 90%). 1H NMR (400 MHz, and concentrated to afford a white solid, which was further
CDCl3) δ 7.60–7.30 (m, 2H), 7.36–7.00 (m, 2H), 5.51–5.18 (m, purified by column chromatography on silica gel (EtOAc–PE =
6H), 4.05 (s, 1H), 3.16 (s, 4H), 2.75 − 1.25 (m, 39H), 0.86 (s, 2 : 1, Rf = 0.3) to yield 7 as a white solid.
3H). HR-MS (ESI): C37H60ClN3NaO5 [M + Na]+, 684.4114,
found: 684.4115.
7a: (1.10 g, 0.89 mmol, yield 89%). 1H NMR (400 MHz,
DMSO-d6) δ 5.33 (s, 1H), 4.29 (s, 1H), 3.53–2.61 (m, 22H),
2.33–0.57 (m, 100H). HR-MS (ESI): C71H127ClN7NaO10
[M + Na]+, 1260.9537, found: 1260.9542.
Preparation of compounds 5a–e
Compound 3 (1.0 mmol) was dissolved in anhydrous CH2Cl2
7b: (1.04 g, 0.8 mmol, yield 80%). 1H NMR (400 MHz,
(4 mL), and then 2 mL CF3COOH was added at 0 °C. After stir- DMSO-d6) δ 5.33 (s, 1H), 4.21 (s, 1H), 3.66–2.60 (m, 22H),
ring for 6 h, the solvent was removed under reduced pressure 2.37–0.46 (m, 108H). HR-MS (ESI): C75H135ClN7NaO10
to obtain an oily product which was directly coupled with tri- [M + Na]+, 1317.0163, found: 1317.0172.
boc-cyclen-acetic acid 4 (531 mg, 1.0 mmol) in the presence of
7c: (953 mg, 0.72 mmol, yield 72%). 1H NMR (400 MHz,
EDC·HCl (230 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), and DMSO-d6) δ 5.33 (s, 1H), 4.19 (s, 1H), 3.32–2.67 (m, 22H),
DIEA (259 mg, 2 mmol) in CH2Cl2 by the same synthetic 2.38–0.65 (m, 112H). HR-MS (ESI): C77H139ClN7NaO10
method as that for compound 3. After column chromatography [M + Na]+, 1345.0476, found: 1345.0479.
on silica gel (EtOAc–PE = 2 : 1), 5 was obtained as a white
powder.
7d: (990 mg, 0.75 mmol, yield 75%). 1H NMR (400 MHz,
DMSO-d6) 5.76 (s, 2H), 5.33 (s, 1H), 4.21 (s, 1H),
δ
5a: (706 mg, 0.71 mmol, yield 71%). 1H NMR (400 MHz, 3.32–2.67 (m, 22H), 2.38–0.53 (m, 110H). HR-MS (ESI):
CDCl3) δ 7.52–7.32 (m, 2H), 7.29–7.20 (m, 2H), 5.38–5.06 (m, C77H137ClN7NaO10 [M + Na]+, 1343.0319, found: 1343.0324.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 3484–3492 | 3489

View Article Online
Paper
Organic & Biomolecular Chemistry
7e: (1.12 g, 0.85 mmol, yield 85%). 1H NMR (400 MHz, concentrations of plasmids were determined by ultraviolet
DMSO-d6) δ 5.70 (s, 4H), 5.33 (s, 1H), 4.19 (s, 1H), 3.30–2.65 (UV) absorbance at 260 nm.
(m, 22H), 2.38–0.53 (m, 108H). HR-MS (ESI): C77H135ClN7-
Preparation of lipid/DOPE/DNA complexes (lipoplexes)
NaO10 [M + Na]+, 1341.0163, found: 1341.0162.
To prepare the lipid/DOPE/pDNA complexes (lipoplexes),
various amounts of cationic lipids were mixed with a constant
Preparation of title lipids 8a–8e
Compound 7 (0.5 mmol) was suspended in anhydrous CH2Cl2 amount of DNA by pipetting thoroughly at various N/P ratios,
(4 mL), and then a solution of CF3COOH (2 mL) in anhydrous and the mixture was incubated for 30 min at room tempera-
dichloromethane (2 mL) was added dropwise under an ice ture. The theoretical N/P ratio represents the charge ratio of
bath and a N2 atmosphere. The obtained mixture was stirred at cationic lipid to nucleotide base (mole ratios) and was calcu-
room temperature for 6 h. After the solvent and CF3COOH lated by considering the average nucleotide mass of 350.
were removed, the residue was washed with anhydrous ether
Ethidium bromide replacement assay
twice to afford the title lipids 8 as an oily solid.
1
8a: Yield 80%. H NMR (400 MHz, DMSO-d6) δ 5.36 (s, 1H), The ability of lipid 8 to condense DNA was studied using ethi-
4.22 (s, 1H), 3.73–2.73 (m, 22H), 2.49–0.61 (m, 74H). HR-MS dium bromide (EB) exclusion assays. Fluorescence spectra
(ESI): C56H104ClN7NaO4 [M + H]+, 938.8144, found: 938.8145.
were measured at room temperature in air by a Horiba Jobin
8b: Yield 82%. 1H NMR (400 MHz, DMSO-d6) δ 5.36 (s, 1H), Yvon Fluoromax-4 spectrofluorometer and corrected for the
4.20 (s, 1H), 3.80–2.71 (m, 22H), 2.49–0.61 (m, 82H). HR-MS system response. EB (5 μL, 1.0 mg mL−1) was put into a quartz
(ESI): C60H112ClN7NaO4 [M + H]+, 994.8770, found: 994.8770.
cuvette containing 2.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piper-
1
8c: Yield 80%. H NMR (400 MHz, DMSO-d6) δ 5.37 (s, 1H), azineethanesulfonic acid (HEPES) solution (pH 7.4). After
4.21 (s, 1H), 3.74–2.75 (m, 22H), 2.45–0.51 (m, 86H). HR-MS shaking, the fluorescence intensity of EB was measured. Then,
(ESI): C62H116ClN7NaO4 [M
1022.9087.
+
H]+, 1022.9083, found: CT DNA (10 μL, 1.0 mg mL−1) was added to the solution with
mixing. The measured fluorescence intensity is the result of
8d: Yield 74%. 1H NMR (400 MHz, DMSO-d6) δ 5.44–5.32 the interaction between DNA and EB. Subsequently, the solu-
(m, 3H), 4.20 (s, 1H), 3.47–2.63 (m, 22H), 2.38–0.51 (m, 81H). tions of lipid 8 (1.0 mM, 2 μL for each addition) were added to
HR-MS (ESI): C62H114ClN7NaO4 [M + H]+, 1020.8927, found: the above solution for further measurement. All the samples
1020.8929.
were excited at 520 nm, and the emission was measured at
8e: Yield 70%. 1H NMR (400 MHz, DMSO-d6) δ 5.45–5.32 600 nm.
(m, 5H), 4.20 (s, 1H), 3.48–2.61 (m, 22H), 2.31–0.56 (m, 77H).
HR-MS (ESI): C62H112ClN7NaO4 [M + H]+, 1018.8770, found:
1018.8774.
Agarose-gel retardation assay
Lipid 8/DOPE/pDNA complexes at different N/P ratios (the
amino groups of lipids to phosphate groups of DNA) ranging
from 0.5 to 4 were prepared by adding an appropriate volume
Preparation of cationic liposomes
The neutral lipid dioleoylphosphatidylethanolamine (DOPE; of lipids (in PBS solution) to 0.125 μg pUC-19 DNA. The com-
0.0025 mmol) was combined with 8 (0.0025 mmol) in chloro- plexes were incubated at 37 °C for 30 min. Thereafter, the com-
form and dried with nitrogen gas under reduced pressure to plexes were electrophoresed on the 1.0% (W/V) agarose-
remove the chloroform solvent. The lipid film was hydrated gel-containing gelred and with Tris-acetate running buffer at
with 2.5 mL of MilliQ water to the final lipid concentration 110 V for 30 min. DNA was visualized with a UV lamp at a
of 1 mM. The samples were sonicated in a bath sonicator to wavelength of 312 nm using a Bio-Rad Universal Hood II
generate small unilamellar vesicles according to previously (Bio-Rad, Hercules, CA, USA).
described procedures.17 After being cooled to room tempera-
ture, the liposome solution was filtered through a 0.22 μm
Lipoplex particle sizes and zeta potentials
filter to generate homogeneous size of lipid vesicles.
Sizes and zeta potentials of the lipid/pDNA lipoplex at various
N/P ratios were analyzed at room temperature on a dynamic
light scattering (DLS) system (Zetasizer Nano ZS; Malvern
Amplification and purification of plasmid DNA
pGL3 and pEGFP-N1 plasmids were used in the gene transfec- Instruments Led, Malvern, UK) at 25 °C. The lipoplex particle
tion assay in vitro. The former one as the luciferase reporter solutions were first prepared by mixing 8/DOPE lipids and
gene was transformed in Escherichia coli JM109, and the latter pDNA (1 μg mL−1) under diverse N/P charge ratios in 1 mL
one as the green fluorescent protein reporter gene was trans- deionized water.
formed in E. coli DH5a. Both plasmids were amplified in terri-
fic broth media at 37 °C overnight. The plasmids were purified
Transmission electron microscopy (TEM)
by an EndoFree Tiangen TM Plasmid Kit (Tiangen Biotech, TEM images were obtained on a JEM-100CX (JEOL) trans-
Beijing, China). Then the purified plasmids were dissolved in mission electron microscope at an acceleration voltage of
TE buffer solution and stored at 20 °C. The integrity of plas- 100 kV. The TEM samples were prepared by dipping a copper
mids was confirmed by agarose-gel electrophoresis. The purity grid with Formvar film into the freshly prepared nanoparticle
of plasmid was checked by the A260/A280 ratio (∼1.9). The solution (10 μL). A few minutes after the deposition, the
3490 | Org. Biomol. Chem., 2014, 12, 3484–3492
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
aqueous solution was blotted away with a strip of filter paper J. Z. thanks the Program for New Century Excellent Talents in
and then the samples were dried for 2 min at room tempera- University (NCET-11-0354). We also thank Analytical and
ture. The samples were stained with phosphotungstic acid Testing Center of Sichuan University for structural analysis of
(ATP) aqueous solution and dried in air.
the compounds.
Cell culture
Human non-small-cell lung carcinoma A549 cells were incu-
bated in DMEM containing 10% FBS and 1% antibiotics (peni-
cillin 10 000 U mL−1 and streptomycin 10 000 μg mL−1) at
37 °C under a humidified atmosphere containing 5% CO2.
References
1 X. Guo and L. Huang, Acc. Chem. Res., 2012, 45, 971.
2 M. A. Mintzer and E. E. Simanek, Chem. Rev., 2009, 109,
259.
MTT cytotoxicity assay
3 C. H. Jones, C. K. Chen, A. Ravikrishnan, S. Rane and
B. A. Pfeifer, Mol. Pharmaceutics, 2013, 10, 4082.
4 Y. C. Su, T. Doherty, A. J. Waring, P. Ruchala and M. Hong,
Biochemistry, 2009, 48, 4587.
5 Y. C. Su, A. J. Waring, P. Ruchala and M. Hong, Biochemis-
try, 2010, 49, 6009.
6 Y. C. Su, S. H. Li and M. Hong, Amino Acids, 2013, 44, 821.
7 Y. N. Zhao, S. B. Zhang, S. H. Cui, B. Wang and S. F. Zhang,
Expert Opin. Drug Deliv., 2012, 9, 127.
The toxicity of lipoplexes toward A549 cells was determined by
the MTT assay following literature procedures.7 About 7000
cells per well were seeded into 96-well plates. After 24 h, opti-
mized lipid/DOPE formulations were complexed with 0.2 μg of
DNA at various N/P ratios. Lipoplexes prepared from Lipofecta-
mine 2000 were used as the control.
In vitro transfection assays
8 S. Bhattacharya and A. Bajaj, Chem. Commun., 2009, 4632.
9 D. Zhi, S. Zhang, B. Wang, Y. Zhao, B. Yang and S. Yu, Bio-
conjugate Chem., 2010, 21, 563.
10 D. Zhi, S. Zhang, S. Cui, Y. Zhao, Y. Wang and D. Zhao, Bio-
conjugate Chem., 2013, 24, 487.
11 Q.-D. Huang, G.-X. Zhong, Y. Zhang, J. Ren, J. Zhang, Y. Fu,
W. Zhu and X.-Q. Yu, PLoS One, 2011, 6, e23134.
12 Q.-D. Huang, J. Ren, H. Chen, W.-J. Ou, J. Zhang, Y. Fu,
W. Zhu and X.-Q. Yu, ChemPlusChem, 2012, 584.
13 R. M. Izalt, K. Pawlak and J. S. Bradshaw, Chem. Rev., 1991,
91, 1721.
A549 cells were seeded into 24-well plates at a density of 80 000
cells per well in 0.5 mL of complete medium. Twenty-four
hours prior to transfection experiments, the medium was
replaced with 1 mL fresh culture medium with or without
FBS.13 Lipid 8/DNA complexes equivalent to 1 μg of plasmid
DNA at the desired N/P ratios were added to each well and
incubated with cells for 4 h. The medium was then replaced
with 0.5 mL of fresh complete medium and incubated for a
further 24 h at 37 °C. For fluorescence microscopy assays, cells
were transfected with complexes containing pEGFP-N1. After
24 h of incubation, the microscopy images were obtained at
the magnification of 100× and recorded using the VIEWFIN-
DER LITE (1.0) software (Olympus, Tokyo, Japan). For lucifer-
ase assays, cells were transfected with lipoplexes containing
pGL3 plasmid DNA. The luciferase assay was performed using
the Picagene luciferase assay kit (Toyo Ink, Tokyo, Japan). The
transfected cells were washed three times with PBS and lysed
in a cell lysis buffer. The lysate was centrifuged at 10 000g
at 4 °C for 2 min, and the supernatant was subjected to the
luciferase assay. The relative light unit (RLU) of chemilumines-
cence was measured using a luminometer (Turner designs,
20/20; Promega), and the luminescent RLU values were nor-
malized to the protein content as determined by a BCA assay.
Transfection with Lipofectamine 2000/DNA complexes was
performed as a positive control. All transfection experiments
were performed in triplicate.
14 H. T. Lv, S. B. Zhang, B. Wang, S. H. Cui and J. Yan, J. Con-
trolled Release, 2006, 114, 100.
15 M. A. Maslov, T. O. Kabilova, I. A. Petukhov,
N. G. Morozova, G. A. Serebrennikova, V. V. Vlassov and
M. A. Zenkova, J. Controlled Release, 2012, 160, 182.
16 J.-L. Liu, Q.-P. Ma, Q.-D. Huang, W.-H. Yang, J. Zhang,
J.-Y. Wang, W. Zhu and X.-Q. Yu, Eur. J. Med. Chem., 2011,
46, 4133.
17 Q. Liu, W.-J. Yi, Y.-M. Zhang, J. Zhang, L. Guo and X.-Q. Yu,
Chem. Biol. Drug Des., 2013, 82, 376.
18 Y. Obata, D. Suzuki and S. Takeoka, Bioconjugate Chem.,
2008, 19, 1055.
19 Q. Liu, Q.-Q. Jiang, W.-J. Yi, J. Zhang, X.-C. Zhang,
M.-B. Wu, Y.-M. Zhang, W. Zhu and X.-Q. Yu, Bioorg. Med.
Chem., 2013, 21, 3105.
20 R. L. Sheng, T. Luo, Y. D. Zhu, H. Li, J. J. Sun, S. D. Chen,
W. Y. Sun and A. M. Cao, Biomaterials, 2011, 32, 3507.
21 Q.-D. Huang, W.-J. Ou, H. Chen, Z.-H. Feng, J.-Y. Wang,
J. Zhang, W. Zhu and X.-Q. Yu, Eur. J. Pharm. Biopharm.,
2011, 78, 326.
Acknowledgements
This work was financially supported by the National Program
on Key Basic Research Project of China (973 Program, 22 Q.-D. Huang, J. Ren, W.-J. Ou, Y. Fu, M.-Q. Cai, J. Zhang,
2012CB720603 and 2013CB328900), the National Science W. Zhu and X.-Q. Yu, Chem. Biol. Drug Des., 2012, 79, 879.
Foundation of China (no. 21232005, 51133004, J1310008 and 23 W. K. Tao, J. P. Davide, M. M. Cai, G. J. Zhang, V. J. South,
J1103315) and the Specialized Research Fund for the Doctoral
Program of Higher Education (20120181130006) in China.
A. Matter, B. Ng, Y. Zhang and L. Sepp-Lorenzino, Mol.
Ther., 2010, 18, 1657.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 3484–3492 | 3491

View Article Online
Paper
Organic & Biomolecular Chemistry
24 S. S. Le Corre, M. Berchel, N. Belmadi, C. Denis, 28 W.-J. Yi, Q.-F. Zhang, J. Zhang, Q. Liu, L.-F. Ren, Q.-M.
J. P. Haelters, T. Le Gall, P. Lehn, T. Montier and
P. A. Jaffrès, Org. Biomol. Chem., 2014, 12, 1463.
25 J. T. Zhang, H. H. Fan, D. A. Levorse and L. S. Crocker,
Langmuir, 2011, 27, 1907.
Chen, L. Guo and X.-Q. Yu, Acta Biomater., 2014, 10, 1412.
29 B. Yu, S. H. Hsu, C. G. Zhou, X. M. Wang, M. C. Terp,
Y. Wu, L. S. Teng, Y. C. Mao, F. Wang, W. M. Xue,
S. T. Jacob, K. Ghoshal, R. J. Lee and L. J. Lee, Biomaterials,
2012, 33, 5924.
26 W. Radchatawedchakoon, A. Krajarng, N. Niyomtham,
R. Watanapokasin and B. Yingyongnarongkul, Chem. – Eur. 30 T. L. Gall, D. Loizeau, E. Picquet, N. Carmoy, J.-J. Yaouanc,
J., 2011, 17, 3287.
27 S. Gratton, P. A. Ropp, P. D. Pohlhaus, J. C. Luft,
L. Burel-Deschamps, P. D. epine, P. Giamarchi, P. A. Jaffres,
P. Lehnand and T. Montier, J. Med. Chem., 2010, 53, 1496.
V. J. Madden, M. E. Napier and J. M. Desimone, Proc. Natl. 31 J. W. Jeon, S. J. Son, C. E. Yoo, I. S. Hong, J. B. Song and
Acad. Sci. U. S. A., 2008, 105, 11613.
J. Suh, Org. Lett., 2002, 4, 4155.
3492 | Org. Biomol. Chem., 2014, 12, 3484–3492
This journal is © The Royal Society of Chemistry 2014