Rigid aromatic linking moiety in cationic lipids for enhanced gene transfection efficiency

Article abstract of DOI:10.1016/j.ejmech.2017.05.038

Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a series of lysine-derived cationic lipids contain

Full text of DOI:10.1016/j.ejmech.2017.05.038

European Journal of Medicinal Chemistry 136 (2017) 585e595
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Rigid aromatic linking moiety in cationic lipids for enhanced gene
transfection efficiency
Bing Wang, Rui-Mo Zhao, Ji Zhang*, Yan-Hong Liu, Zheng Huang, Qing-Ying Yu,
Xiao-Qi Yu**
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity
relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a
series of lysine-derived cationic lipids containing linkages with different rigidity were designed and
synthesized. SAR studies showed that lipids with rigid aromatic linkage could promote the formation of
tight liposomes and enhance DNA condensation, which is essential for the gene delivery process. These
lipids could give much higher transfection efficiency than those containing more flexible aliphatic
linkage in various cell lines. Moreover, the rigid aromatic linkage also affords the material higher serum
tolerance ability. Flow cytometry assay revealed that the target lipids have good cellular uptake, while
confocal microscopy observation showed weaker endosome escape than Lipofectamine 2000. To solve
such problem and further increase the transfection efficiency, some lysosomotropic reagents were used
to improve the endosome escape of lipoplex. As expected, higher transfection efficiency than Lipofect-
amine 2000 could be obtained via this strategy. Cytotoxicity assay showed that these lipids have lower
toxicity in various cell lines than Lipofectamine 2000, suggesting their potential for further application.
This work demonstrates that a rigid aromatic linkage might distinctly improve the gene transfection
abilities of cationic lipids and affords information to construct safe and efficient gene vector towards
practical application.
Received 11 April 2017
Received in revised form
10 May 2017
Accepted 14 May 2017
Available online 17 May 2017
Keywords:
Non-viral gene vector
Cationic lipid
Structure-activity relationship
Gene delivery
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
intra-cellular delivery [8]. The early research efforts focused on
using viral gene vectors such as retrovirus and adenovirus since
Gene therapy is a promising treatment method for genetic dis-
orders and acquired diseases, as well as an alternative means of
cancer treatment to traditional chemotherapy [1e4]. However, the
intrinsic physicochemical properties of nucleic acid make itself
grossly unsatisfactory as an independent therapeutic pharmaceu-
tical, and a series of barriers exist in their transfection process [5].
Generally, free nucleic acids will be rapidly degraded by nucleases
and/or esterases in the fluid circulatory system. On the other hand,
naked genes cannot easily pass through the negatively charged cell
membranes because of electrostatic repulsion [6,7]. Therefore, a
great amount of research works have concentrated on the devel-
opment and utilization of gene vectors with the ability to condense
and protect gene cargos from degradation during the extra- and
these vectors revealed excellent transfection efficiency (TE) in
numerous cell lines [9]. However, many issues such as toxicity,
immunogenic, inflammatory, and mutagenic effects may lead to
safety risks and limit their application, prompting the extensive
studies of non-viral vectors.
In the past decades, many kinds of non-viral vectors, such as
lipids, organic polymers or inorganic materials, have been designed
for safer gene delivery [10e13]. Among these materials, cationic
lipids, a family of amphipathic small molecules usually containing a
hydrophilic cationic headgroup, hydrophobic tails and a linking
moiety, have been realized as promising gene delivery platforms
because of their exact structure, good biocompatibility, high effi-
ciency, and potential for clinical application [14,15]. Since the
pioneer work from Felgner and coworkers utilizing DOTMA as
cationic gene delivery vector [16], a wide range of lipids were
investigated with the hope to overcome in vitro and in vivo trans-
fection barriers [17]. In order to obtain ideal lipid materials with
* Corresponding author.
** Corresponding author.
E-mail addresses: jzhang@scu.edu.cn (J. Zhang), xqyu@scu.edu.cn (X.-Q. Yu).
http://dx.doi.org/10.1016/j.ejmech.2017.05.038
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

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B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
high efficiency and biocompatibility, many works have focused on
the clarification of the structure-activity relationship (SAR)
[18e20]. Various kinds of cationic groups, such as amines, amino-
acids, guanidiniums and peptides, were used as polar head for
nucleic acid condensation and further balancing the TE and cyto-
toxicity [21]. Meanwhile, as another one of the important structural
domain of the lipids, the hydrophobic tails were also considered to
play significant roles in the liposome formation and gene delivery.
Efforts were carried out on discussing the influence of the number,
length, unsaturation, branching and asymmetry of hydrophobic
chains on the TE [22]. Compared to the headgroup and hydrophobic
tails, the studies on linking moiety were rare. It is known that
biodegradable linkage (such as disulfide, ester, acetal, etc.) may
benefit the lipoplex dissociation and thus to improve TE [23].
Recently, it was found that the integration of rigid elements might
benefit the gene vectors with enhanced self-assembly, biocom-
patibility and efficiency [24e27].
To obtain instructional information about the SAR of linking
moiety of cationic lipids, we herein designed and synthesized a
series of lipids containing the linking groups with different rigidity.
Besides the linking moiety, these lipids contain a bio-essential
lysine group as cationic headgroup, a hydrophobic tail formed by
C18 oleyl group, which has been used in many efficient lipidic gene
vectors [21,28]. Transfection experiment results revealed that lipids
with rigid aromatic linkage could promote the formation of tight
liposomes and enhance DNA condensation, which is essential for
the gene delivery process. Moreover, these lipids exhibited higher
serum tolerance and TE than those containing more flexible
aliphatic linkage. Mechanism studies revealed that the target lipids
have good cellular uptake but weak endosome escape, which is the
main reason for their lower TE than Lipofectamine 2000. Thus, with
the help of lysosomotropic reagents, the TE of the lipids could be
effectively promoted as expected.
2.2. The properties of lipids, liposomes and lipoplexes
It is widely recognized that amphiphilic lipids can self-assemble
in aqueous solution when the lipid concentration surpasses a
critical value, which is defined as the critical aggregation concen-
tration (CAC) value [29]. Herein, two representative lipids without
(LBO) or with (LPO) rigid aromatic group were chosen to measure
the CAC. The results in Fig. S1 show that LPO holds a distinctly
lower CAC value (0.04 mg/mL) than LBO (0.18 mg/mL), demon-
strating that the aromatic ring in the linkage would promote the
aggregation. The five lipids were then mixed with DOPE (1: 2, mol/
mol) to form relative liposomes, of which the particle sizes and zeta
potentials were measured via dynamic light scattering (DLS) assay.
It was found that with the increase of rigidity, the particle size
decreased gradually from LO to LNO (Fig. S2A). This also indicates
that rigid structure might facilitate the formation of tight aggre-
gates. On the other hand, the zeta potentials of the five liposomes
were similar around þ55 mV (Fig. S2B), indicating their remarkable
cationic property. Subsequently, agarose gel electrophoresis assay
was performed to evaluate the DNA binding ability of the liposomes
at different N/P ratios. As shown in Fig. 1, complete DNA retardation
could be observed at N/P ratio of 4 in the case involving LO. The
aliphatic linkage seemed not to have any effect on the binding,
meanwhile, the aromatic linkage might increase the binding, and
complete retardation was found at lower N/P ratio (N/P ¼ 2). Such
results suggest that the rigid aromatic structure in the lipid may not
only promote the formation of tight liposomes, but also enhance
the DNA condensation, which is essential for the gene delivery
process [30,31].
The physical properties including particle size and zeta potential
of the liposome/DNA complexes (lipoplexes) were also studied for
their significance in cellular uptake, cytotoxicity and release of gene
cargo. The DLS results shown in Fig. 2A reveal that at N/P ratio of 1,
the liposomes and DNA formed particles with the sizes larger than
500 nm, indicating an incomplete DNA condensation. With the
increase of N/P ratio, the sizes of all the lipoplexes reduced grad-
ually and finally became steady at 100e150 nm, suggesting the full
condensation. On the other hand, the zeta potentials of the lip-
oplexes were negative at the N/P of 1, and charge reverse occurred
from N/P of 2 and remained stable at 30e45 mV (Fig. 2B). The
positive surface charge may facilitate their interaction with nega-
tively charged cell membrane, leading to better cellular uptake. In
addition, a visual image of LPO/DNA complexes at N/P of 4 by TEM
(Fig. S3) revealed an irregular spherical morphology features with
diameter of ~50 nm, which was smaller than that measured by DLS.
This might be due to the different measure conditions of the two
assays [20].
2. Results and discussion
2.1. Synthesis of target lipids
Cationic lipids designed for gene therapy usually utilize
biocompatible amino acids as functional headgroup for nucleic acid
binding while using oleyl chain as hydrophobic tail for lipid bilayer
formation and cell/endosome membrane fusion. Lysine, which has
two primary amines in the structure, was chosen as headgroup of
the target lipids, and several linking structures were constructed to
study their SAR in gene delivery. The synthetic route of the lipids
was shown in Scheme 1. The target lipids were named according to
their chemical structures, e.g. LBO represents the lysine-
butylidene-oleyl architecture, while LPO represents lysine-
phenyl-oleyl structure. Two types of rigid aromatic ring (phenyl
in LPO and naphthyl in LNO) were introduced into the structure of
cationic lipids as linking moiety. For comparison, aliphatic linkage
with similar carbon atoms (cyclohexyl in LHO and butyl in LBO)
were also designed and prepared. Further, a lipid with only an
amide bond as linking group (LO) was also prepared as control
lipid. For the preparation of LBO and LHO containing aliphatic
linkage, the linkage was first reacted to the oleyl tail, and then
coupled with lysine headgroup. However, the aromatic ring-
contained lipids LPO and LNO could not be obtained via similar
method. In their preparation, the aromatic linkage was first reacted
to the lysine headgroup in the presence of IBCF, a stronger
condensation reagent, and then coupled with oleyl amine. The
structures of all target lipids were confirmed by 1H NMR, 13C NMR
and HRMS.
2.3. In vitro gene transfection
The gene transfection activity of the lipoplexes was first
assessed by using enhanced green fluorescent protein (eGFP) as
reporter gene in HeLa cells for direct visualization and preliminary
investigation. Fig. 3 shows the eGFP gene expression mediated by
lipoplexes at various N/P ratios, and Lipofectamine 2000 (Lipo) was
used as control. Compared to naked DNA transfection, all the
complexes induced large area of green florescent dots and the
florescence density reached the maximum at N/P of 2. It was
observed that by comparison with LO, the aromatic ring-contained
lipids LPO and LNO (especially the phenyl-derived LPO) gave
higher transfection efficiency (TE), while alkyl linkage-contained
LBO and LHO did not lead to enhanced TE.
To quantitatively study the TE of the lipids and to confirm the
superiority of LPO, luciferase reporter gene was also applied for the
transfection. As shown in Fig. 4A, in HeLa cells, the five lipids gave

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587
Scheme 1. Synthetic routes of target lipids.
similar TE trend to the EGFP assay, and LPO could mediate the
transfection with the highest efficiency. However, the TE of the
lipids were still lower than Lipo. The transfection in the presence of
serum was also studied. It is known that the anionic components in

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B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
Fig. 1. Agarose gel electrophoresis retardation assays of lipoplexes at different N/P ratios.
Fig. 2. Mean particle sizes (A) and zeta-potentials (B) of the lipoplexes at various N/P ratios.
serum may interfere with the transfection process by complexation
with positively charged cationic vectors, thus the development of
novel vectors with high serum tolerance is of great importance
[32,33]. Results in Fig. 4B reveal that with the presence of 10%
serum, the TEs of LO, LBO and LHO decreased dramatically. On the
contrary, to our delight, the aromatic ring-contained LPO and LNO
kept high gene expression level, and the gap between the two lipids
and Lipo narrowed. Moreover, same assay was also carried out in
some other cell lines such as A549 and HL 7702 cells by using LO
and LPO for comparison. As shown in Fig. S4, LPO still reflected a
significant better TE than LO in the two cell lines. Thus, it was
believed that the rigid aromatic moiety in the lipid may play
important role in the gene transfection and may effectively
enhance the serum tolerance of the vector material.
Generally, effective gene transfection associates with many
factors [34]. To figure out the large TE difference among the lipids in
the presence of serum, flow cytometry was applied to evaluate the
cell-penetrating efficiency of the lipoplexes by using Cy5-labeled
DNA as fluorescent source. As shown in Fig. 5, the columns reflect
that LO induced the lowest percentage of positive cells, indicating
that a linkage is necessary for efficient cellular uptake. Lipoplexes
formed from LPO and LNO with aromatic ring in the structure
might enter more cells than other lipoplexes, even including Lipo.
More significantly, the fluorescence intensity, which reflects the
total amount of internalized DNA, showed huge difference between
those with and without aromatic ring. As we expected, LPO and
LNO delivered much more (almost 10 fold) DNA cargo into the cells.
This might be the main reason for their higher TE than those
without a rigid linkage.
derived lipids as non-viral gene vectors, their TEs were still lower
than Lipo. Since the cellular uptake of LPO or LNO was not lower
than that of Lipo, we speculate that our lipids would encounter
more difficult intracellular trafficking such as endosomal escape
and nuclear entry. Therefore, confocal laser scanning microscopy
(CLSM) was performed to visually investigate their endosome/
lysosome escape ability by using HeLa cells with the presence of
10% FBS. The nuclei were stained by DAPI (blue) and acidic com-
partments such as late endosome or lysosome were stained by
LysoTracker green (green), while pDNA was labeled by Cy5 (red). As
shown in Fig. 6, after incubation for 4 h, LPO could deliver large
amount of pDNA into cytosol, and the red fluorescence density was
even higher than that mediated by Lipo. However, in the merge
image, it could be found that many of the DNA was trapped in late
endosome or lysosome, which was reflected by the yellow signals.
After 12 h incubation, there were still obvious yellow signals. On
the contrary, much less yellow dots were found in the case
involving Lipo. Such results indicate that compared to Lipo, LPO/
DNA complex has much weaker endosome escape ability, leading to
its lower TE. With these findings, some corresponding strategy may
be used to further improve the TE of the lipids.
It is reported that lysosomotropic reagents such as chloroquine
(CQ), ammonium chloride (AC) and methylamine (MA) possess the
function of promoting endo/lysosome escape [35,36]. Hence, the
luciferase assay using LPO was subsequently performed in HeLa
and HepG2 cells with the addition of such three reagents at various
concentrations. Results show that the TE of LPO could be enhanced
by CQ and AC in HeLa cells. With 50 mM of CQ, LPO could achieve
similar efficiency to Lipo (Fig. 7A). In HepG2 cells, AC could largely
improve the performance of LPO, and the result TE was distinctly
Although the aromatic ring exhibited certain advantage in Lys-

B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
589
Fig. 3. Fluorescence microscopy images of eGFP expression HeLa cells at various N/P ratios. Lipo was used as control.
higher than Lipo (Fig. 7B). These results demonstrate that the
lysosome trapping of LPO lipoplexes was indeed a considerable
barrier in the transfection process. This may give us clues for further
design of novel lipid vector with improved gene transfection
efficiency.

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B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
Fig. 4. Luciferase expression in HeLa cells transfected by lipoplexes at various N/P ratios without (A) or with (B) 10% serum. Lipo served as control.
from these lipids and helper lipid DOPE could condense DNA to
form nanosized lipoplexes which showed good transfection effi-
ciency and low cytotoxicity. SAR studies showed that lipids with
rigid aromatic linkage could promote the formation of tight lipo-
somes and enhance DNA condensation, which is essential for the
gene delivery process. These lipids could give much higher trans-
fection efficiency than those containing more flexible aliphatic
linkage in various cell lines. Moreover, the rigid aromatic linkage
also affords the material higher serum tolerance ability. Mechanism
studies revealed that the target lipids have good cellular uptake but
weak endosome escape, which is the main reason for their lower
transfection efficiency than Lipo. Therefore corresponding strategy
was applied to further improve the transfection performance of the
lipids by using lysosomotropic reagents, and higher efficiency than
Lipo was obtained as expected. To sum up, this work demonstrates
that a rigid aromatic linkage might distinctly improve the gene
Fig. 5. Cellular uptake of lipoplexes with 10% serum at N/P ratio of 4 in HeLa cells
quantified by flow cytometry. Columns: percentage of Cy5-positive cells; Dots and
lines: fluorescence intensity of internalized Cy-5 labeled DNA. Lipo was used as control.
Data represent mean SD (n ¼ 3).
transfection abilities of cationic lipids. Further studies including
aromatic modification of current efficient lipid vectors are now in
progress.
4. Experimental section
2.4. Cytotoxicity
4.1. Materials and methods
Besides high TE, a relatively low toxicity is necessary for suc-
cessful gene vector. Cell viability caused by lipoplexes prepared at
different N/P ratios (1, 2, 4 and 6) was evaluated by MTS assay
(Fig. 8). All the materials showed a dose-dependent cytotoxicity. LO
exhibited lower cell viability than the others, indicating that a
linkage may not only enhance the TE but also reduce the toxicity of
the lipid. The introduction of rigid segments did not increase more
severe cell death, the toxicity of LPO is similar to its aliphatic an-
alogs LBO and LHO. LNO with larger aromatic ring induced higher
N,N-Diisopropylethylamine (DIEA), N-Methyl morphofine
(NMM), Isobutyl chloroformate (IBCF), Benzocaine, 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-
hydroxybenzotriazole hydrate (HOBt), 4-Bromo-1-naphthoic acid
were purchased from Aladdin® (Shanghai, China). Boc-Lys(Boc)-
OH, 4-Aminocyclohexanecarboxylic acid,
g-aminobutyric acid
(GABA), oleyl amine, Di-tert-butyl pyrocarbonate (Boc2O) were
purchased from Asta Tech Pharmaceutical (Chengdu, China).
Compounds 5 and 6 were prepared according to literature pro-
cedures [37,38]. Trifluoroacetic acid (TFA), dichloromethane (DCM),
methanol were purified before use. Column chromatography was
performed using 200e300 mesh silica gel.
1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was
purchased from Avanti Polar Lipids, Inc. pUC19 DNA was purchased
from TIANTAI (Chengdu, China). MicroBCA protein assay kit was
obtained from Pierce (Rockford, IL, USA). Luciferase assay kit and
MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H tetrazolium, inner salt) were purchased from
Promega (Madison, WI, USA). Endotoxin-free plasmid purification
kit was purchased from TIANGEN (Beijing, China). The plasmids
used in the study were pGL-3 (Promega, Madison, WI, USA) coding
for luciferase and pEGFP-N1 (Clontech, Palo Alto, CA, USA) coding
for EGFP. The Dulbecco's modified Eagle's medium (DMEM), fetal
toxicity, this may be attributed to the stronger
p-p interaction
between the material and DNA base pairs. Under optimal trans-
fection N/P ratio, all of the Lys-derived lipids had lower cytotoxicity
than Lipo, suggesting their potential for further application. Be-
sides, the cell viability of cationic lipids in HeLa cells and selected
lipoplexes in other cell lines were also investigated (Fig. S5), and the
results were in accordance with those in Fig. 8. It was confirmed
that LPO is more biocompatible than LO, and also induced less cell
death than Lipo.
3. Conclusions
In summary, a series of lysine-derived cationic lipids containing
linkages with different rigidity were designed and synthesized to
investigate their SAR as non-viral gene vectors. Liposomes formed

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591
Fig. 6. CLSM observation of the intracellular distribution of LPO/pDNA or Lipo/pDNA complexes (Cy5-labeled, red), late endosomes and lysosomes (LysoTracker Green-labeled,
green), nuclei (DAPI-labled, blue) at different incubation time. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
Fig. 7. Relative luciferase expression in HeLa (A) and HepG2 cells (B) transfected by LPO complexes at N/P ratios of 4 (10% serum) in the presence of chloroquine (CQ), ammonium
chloride (AC) and methylamine (MA) at varying concentrations. Lipo was used as control.

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B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
reduced pressure to give brown residues. Finally, under stirring,
anhydrous diethyl ether (3 ꢀ 20 mL) was added into the residue to
carry off redundant impurities and finally compound 3 or 4 was
obtained as yellow oil solid in 93% and 92% yields, respectively.
4.2.3. Preparation of compounds 7e9
The preparation method of compounds 7e9 was similar to that
of compounds 1 and 2 (step 2) by using starting materials RNH2, 3
and 4, respectively. Boc-Lys(Boc)-OH was used as raw material. It's
worth mentioning that compounds 3 and 4 contained a TFA
molecule, thus one more equiv. of DIEA should be used. The yield of
products 7e9 were 82%, 77% and 64%, respectively.
4.2.4. Preparation of compounds 10 and 11
NMM (2 equiv.) was added to a solution of Boc-Lys(Boc)-OH (1
equiv.) in anhydrous DCM at 0 ꢁC, followed by dropwise addition of
IBCF (1.5 equiv.). The mixture was stirred for 30 min, then benzo-
caine or compound 6 (1.5 equiv.) was added, and the resulting
mixture was stirred at 0 ꢁC for 1 h and then at room temperature for
12 h. The precipitate was filtered off and the filtrate was washed
with H2O (2 ꢀ 40 mL), brine (40 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (EA/DCM ¼ 1/2, v/v) to yield
pure compound 10 (37%) or 11 (25%).
Fig. 8. Cytotoxicity of the liposome/pDNA complexes at varying N/P ratios against
HeLa cells. The percentage of cell viability was expressed relative to blank control cells.
Naked DNA and Lipo/DNA complex were used as controls.
bovine serum (FBS) and Lipofectamine 2000 were purchased from
Invitrogen Corp.
The 1H NMR and 13C NMR spectra were recorded on a Bruker
AM400 NMR spectrometer. Proton Chemical shifts of NMR spectra
were given in ppm relative to internals reference TMS (1H,
0.00 ppm). HRMS spectral data was measured on a Bruker Daltonics
Bio TOF mass spectrometer. The average particle sizes and zeta
potentials were detected by dynamic light scattering (DLS) on a
Malvern Zetasizer Nano ZS. Transmission electron microscopy
(TEM), amplification and purification of plasmid DNA, cell culture
and other procedures were performed according to our reported
literature [20,39].
4.2.5. Preparation of compounds 12 and 13
Step 1: To the solution of 10 or 11 in CH3OH (50 mL) was added
aq. NaOH (5 equiv. 50 mL), and the reaction mixture was stirred for
2 h. The organic solvent was evaporated off, the residue was dis-
solved in 10% aq. hydrochloric acid, and pH was adjusted to 3. After
the solution was extracted with EA (3 ꢀ 50 mL) and the organic
layer was dried over Na2SO4 and evaporated, an amorphous solid
was obtained with desired yield (95% and 94% for compound 10 and
11 respectively).
4.2. Synthesis of lipids (characterization data for the intermediates,
see supplementary information)
Step 2: Compounds 12 and 13 were synthesized via the same
procedure for compound 7 with yields of 79% and 72% respectively.
4.2.1. Preparation of compounds 1 and 2
4.2.6. Preparation of target lipids
Step
1:
g-aminobutyric
acid
(GABA)
or
4-
The final lipids LO, LBO, LHO, LPO and LNO were synthesized by
Boc deprotection procedure described in preparation of compound
3 with yields of >90%.
Aminocyclohexanecarboxylic acid (ACBA) (1 equiv.) was dissolved
in the solvent (H2O/THF ¼ 1/1, v/v), then Et3N (2 equiv.) and (Boc)2O
(1.2 equiv.) were added. The resulting mixture was stirred for
24 h at room temperature. THF was evaporated off, and the residue
was adjusted to pH ¼ 3 by 2N HCl, and the solution was then
extracted by ethyl acetate (EA, 3 ꢀ 50 mL). The organic layer was
combined and dried over anhydrous magnesium sulfate and then
filtered. The solvent was evaporated under reduced pressure to give
the products which were used directly.
Step 2: Oleylamine (1.2 equiv.) was added to the anhydrous
dichloromethane solution of the product obtained in last step (1
equiv.), HOBt (1.2 equiv.), EDCI (1.2 equiv.) and DIEA (2 equiv.) in
ice-salt-bath at 0 ꢁC. The mixture was stirred at 0 ꢁC for 1 h and then
at room temperature overnight. The reaction solution was washed
with saturated aqueous NaHCO3 solution (2 ꢀ 50 mL) and brine
(50 mL). The organic solvent was dried over Na2SO4, filtered, and
concentrated to afford an oil solid, which was further purified by
column chromatography on silica gel (EA/PE ¼ 1/2, v/v) to yield
pure compound 1 (85%) or 2 (66%).
LO: 1H NMR (CDCl3, 400 MHz)
d
0.87 (t, 3H, J ¼ 6.4 Hz, -CH3),
1.16e1.37 (m, 22H, -(CH2)5, -(CH2)6), 1.48e1.56 (m, 4H,
-NHCH2CH2CH2CH2-, -NHCH2CH2), 1.60e1.76 (m, 2H,
-NHCH2CH2CH2CH2-), 1.77e1.90 (m, 2H, -NHCH2CH2CH2CH2-),
1.92e2.06 (m, 4H, -CH2CH¼CHCH2-), 2,90e3.07 (m, 2H, -NHCH2-),
3.08e3.25 (m, 2H, -NHCH2CH2CH2CH2-), 3.97e4.12 (m,1H, -CH(NH)
CO-), 5.27e5.43 (m, 2H, -CH¼CH-). 13C NMR (CDCl3, 100 MHz): 14.0,
22.6, 26.8, 27.2, 28.7, 29.2, 29.3, 29.4, 29.5, 29.7, 31.9, 32.6, 39.7, 41.0,
129.7, 129.9, 176.6. HR-MS (ESI): Calcd for
C24H49N3O
[MþH]þ396.3954, found 396.3956.
LBO: 1H NMR (CDCl3, 400 MHz)
d
0.87 (t, 3H, J ¼ 6.4 Hz, -CH3),
1.20e1.40 (m, 24H, -NHCH2CH2CH2CH2-, -(CH2)5, -(CH2)6),
1.41e1.54 (m, 2H,, -NHCH2CH2), 1.58e1.71 (m, 2H,
-NHCH2CH2CH2CH2-), 1.76e1.89 (m, 4H, -NHCH2CH2CH2CO-,
-NHCH2CH2CH2CH2-), 1.92e2.07 (m, 4H, -CH2CH¼CHCH2-),
2.15e2.24 (m, 2H, -NHCH2CH2CH2CO-), 2.06e3.15 (m, 2H, -NHCH2-
), 3.18e3.26 (m, 2H, -NHCH2CH2CH2CH2-), 3.26e3.36 (m, 2H,
-NHCH2CH2CH2CO-), 3.95e4.04 (m, 1H, -CH(NH)CO-), 5.29e5.43
(m, 2H, -CH¼CH-). 13C NMR (CDCl3, 100 MHz): 14.1, 22.5, 26.4, 26.8,
27.2, 28.6, 29.1, 29.3, 29.4, 29.3, 29.8, 31.9, 32.6, 33.6, 39.4, 39.7, 41.1,
129.8, 129.9, 172.6, 176.5. HR-MS (ESI): Calcd for C28H56N4O2
[MþH]þ481.4482, found 481.4478.
4.2.2. Preparation of compounds 3 and 4
A solution of trifluoroacetic acid (3 mL) in anhydrous dichloro-
methane (3 mL) was added dropwise to a solution of compound 1
or 2 in anhydrous dichloromethane (3 mL) under ice bath. The
resulting mixture was stirred at room temperature for 6 h. Then
solvent and excess trifluoroacetic acid (TFA) was removed under
LHO: 1H NMR (CDCl3, 400 MHz)
d
0.87 (t, 3H, J ¼ 6.8 Hz, -CH3),
1.20e1.39 (m, 24H, -NHCH2CH2CH2CH2-, -(CH2)5, -(CH2)6),

B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
593
1.42e1.54
(m,
2H,
-NHCH2CH2), 1.55e1.93
(m, 10H,
with 1
mg DNA/mL sample.
-NHCH2CH2CH2CH2-, -NHCH(CH2CH2)2CHCO-), 1.93e2.05 (m, 4H,
-CH2CH¼CHCH2-), 2.17 (s, 1H, -NHCH(CH2CH2)2CHCO-), 3.02e3.27
(m, 4H, -NHCH2CH2CH2CH2-, -NHCH2-), 4.19e4.31 (m, 1H,
-NHCH(CH2CH2)2CHCO-), 5.30e5.41 (m, 2H, -CH¼CH-). 13C NMR
(CDCl3, 100 MHz): 14.1, 22.7, 24.9, 26.8, 27.2, 29.1, 29.2, 29.3, 29.4,
29.5, 29.6, 29.8, 31.9, 32.3, 32.6, 39.5, 39.8, 43.1, 44.5, 55.3, 54.6,
60.4, 79.0, 79.9, 129.7, 129.9, 171.5, 175.1. HR-MS (ESI): Calcd for
4.6. Determination of the critical aggregation concentration
Pyrene was used as the fluorescence probe in the critical ag-
gregation concentration (CAC) determination. The lipid concen-
tration varied from 1.0 ꢀ 10ꢂ6 mg mLꢂ1 to 1 mg mLꢂ1, and the
pyrene concentration was fixed at 6.0 ꢀ 10ꢂ7 M. The fluorescence
spectra were recorded using a HITACHI F-7000 Fluorescence
Spectrophotometer. Both the emission and excitation slit widths
were 5 nm. The samples were excited at 335 nm and the emission
spectra were recorded from 350 to 500 nm. The emission fluores-
cence values, I373 and I383 at 373 nm and 383 nm, respectively, were
used for the subsequent calculations. The CAC was determined
from the plots of the I383/I373 ratio versus the logarithm of the
polymer concentration using the intersection of the linear regres-
sion lines as the CAC value.
C
31H60N4O2 [MþH]þ521.4789, found 521.4791.
LPO: 1H NMR (DMSO-D6, 400 MHz): 0.89 (t, 3H, J ¼ 6.4 Hz,
-CH3), 1.16e1.34 (m, 22H, -(CH2)5, -(CH2)6), 1.34e1.44 (m, 2H,
-NHCH2CH2CH2CH2-),
1.45e1.60
(m,
4H,
-NHCH2CH2,
-NHCH2CH2CH2CH2-), 1.76e1.89 (m, 2H, -NHCH2CH2CH2CH2-),
1.90e2.03 (m, 4H, -CH2CH¼CHCH2-), 2.70e2.83 (m, 2H, -NHCH2-),
3.18e3.27 (m, 2H, -NHCH2CH2CH2CH2-), 3.93e4.02 (s, 1H, -CH(NH)
CO-), 5.29e5.39 (m, 2H, -CH¼CH-), 7.63e7.70 (d, 2H, J ¼ 5.4 Hz, Ar-
H), 7.81e7.88 (d, 2H, J ¼ 5.4 Hz, Ar-H). 13C NMR (DMSO-D6,
100 MHz): 14.1, 22.7, 26.8, 27.2, 28.4, 29.0, 29.3, 29.4, 29.6, 29.7,
29.8, 31.5, 31.9, 33.6, 39.6, 40.5, 55.7, 119.1, 121.8, 129.8, 130.1, 130.2,
141.7, 167.2, 172.0. HR-MS (ESI): Calcd for C31H54N4O2 [MþH]þ
515.4325, found 515.4323.
4.7. Cytotoxicity assays
Toxicity of lipoplexes toward HeLa cells was determined by
using MTS following the instruction method. About 1.0 ꢀ 104 cells/
well were seeded into 96-well plates. After 24 h, optimized lipo-
LNO: 1H NMR (CDCl3, 400 MHz)
1.18e1.60 (m, 26H, -NHCH2CH2CH2CH2-, -(CH2)5, -(CH2)6,
-NHCH2CH2), 1.63e1.98 (m, 4H, -NHCH2CH2CH2CH2-,
-NHCH2CH2CH2CH2-), 2.00e2.10 (m, 4H, -CH2CH¼CHCH2-),
3.07e3.22 (m, 2H, -NHCH2-), 3.46e3.56 (m, 2H,
d
0.87 (t, 3H, J ¼ 6.8 Hz, -CH3),
somes were complexed with 0.2
30 min and then 100 L of lipoplexes were added to the cells in the
presence of serum (10%). After 4 h of incubation, lipoplex solutions
were removed, and 100 L of media with 10% FBS was added. After
24 h, 20 L MTS mixed with 100 L PBS was added to each well and
mg of DNA at various N/P ratios for
m
-NHCH2CH2CH2CH2-), 4.70 (s, 1H, -CH(NH)CO-), 5.30e5.42 (m, 2H,
-CH¼CH-), 7.33e7.52 (m, 2H, Ar-H), 7.71 (s,1H, Ar-H), 7.85 (s,1H, Ar-
H), 8.23 (d, 1H, J ¼ 8.0 Hz, Ar-H), 9.13 (s, 1H, Ar-H). 13C NMR (CDCl3,
100 MHz): 14.1, 22.7, 27.1, 27.2, 29.2, 29.4, 29,5, 29.6, 29.7, 31.8, 32.7,
39.8, 40.1, 79.2, 80.6, 118.7, 121.1, 124.8, 125.9, 126.5, 126.8, 129.8,
129.9, 130.5, 131,7, 134.2, 169.6, 171.2. HR-MS (ESI): Calcd for
m
m
m
the plates were incubated at 37 ꢁC for another 1 h. Then, the
absorbance of each sample was measured using an ELISA plate
reader (model 680, BioRad) at a wavelength of 490 nm. The cell
viability (%) was obtained according to the manufacturer's in-
struction. Lipoplexes prepared from Lipofectamine 2000 were used
as control.
C
35H46N4O2 [MþH]þ 565.4476, found 565.4481.
4.3. Preparation of cationic liposomes
4.8. In vitro transfection procedure
Individual cationic lipid (0.005 mmol) or its mixture with DOPE
in the desired mole ratio of 2 (lipid/DOPE ¼ 1/2, mol/mol) was
dissolved in anhydrous chloroform (2.5 mL) in autoclaved glass
vials. Thin films were made by slowly rotary-evaporating the sol-
vent at room temperature. Last trace of organic solvent was
removed by keeping these films under vacuum above 8 h. Then
2.5 mL H2O was added to the glass vials, vortexed vigorously until
the films were completely resuspended and then kept at 70 ꢁC for
30 min in water bath. Sonication of these suspensions for 5 min
with an ice bath on the Noise Isolating Tamber (Ningbo Scientz
Biotechnology CO., LTD) afforded the corresponding cationic lipo-
somes that were stored at 4 ꢁC.
In order to obtain about 80% confluent cultures at the time of
transfection, 24-well plates were seeded with 100,000 cell/well in
500 mL of antibiotic-free media 24 h before transfection. For the
preparation of lipoplexes applied to cells, various amounts of li-
posomes and DNA were serially diluted separately in anti-biotic-
free DMEM culture medium; then, the DNA solutions were added
into liposome solutions and mixed briefly by pipetting up and
down several times, after which the mixtures were incubated at
room temperature for about 30 min to obtain lipoplexes of desired
N/P ratios, the final lipoplexes volume was 100
used at a concentration of 0.8 g/well. After 30 min of complexa-
tion, old cell culture medium was removed from the wells and
400 L fresh DMEM (with/without 10% FBS) was added, then the
above 100
mL, and the DNA was
m
4.4. Gel retardation assay
m
m
L lipoplexes was added to each well. The plates were
To determine the formation of liposome/pDNA complex (lip-
olexes), lipoplexes of various N/P ratios ranging from 0 to 6 were
prepared as mentioned in the literature [20]. Constant amount of
incubated for 4 h at 37 ꢁC in a humidified atmosphere containing 5%
CO2. At the end of incubation period, medium was removed, and
500 mL of fresh DMEM medium containing 10% FBS was added to
0.125
m
g DNA was used here; 20
m
L (containing 2
m
L loading buffer)
each well. Plates were further incubated for a period of 24 h before
checking the reporter gene expression.
of each lipoplexes solution was electrophoresed on the 1% (WVꢂ1
)
agarose gel containing GelRed and Triseaceate (TAE) running
buffer at 140 V for 30 min. DNA was visualized with a UV lamp
using a BioRad Universal Hood II.
For fluorescent microscopy assays, cells were transfected by
complexes containing pEGFP-N1. After 24 h incubation, the mi-
croscopy images were obtained at the magnification of 100 and
recorded using Viewfinder Lite (1.0) software. Control transfection
was performed in each case using a commercially available trans-
fection reagent Lipofectamine 2000 based on the standard condi-
tions specified by the manufacture.
4.5. Dynamic light scattering (DLS)
Particle size and zeta potential of liposomes or lipoplexes at
various N/P ratios were measured by a dynamic light scattering
system at 25 ꢁC. Lipoplexes were prepared by the same procedure
For luciferase assays, cells were transfected by complexes con-
taining pGL-3. For a typical assay in a 24-well plate, 24 h post

594
B. Wang et al. / European Journal of Medicinal Chemistry 136 (2017) 585e595
transfection as described above, the old medium was removed from
the wells, and the cells were washed twice with 500 L of pre-
chilled PBS. According to Luciferase assay kit (promega) manufac-
ture, 100
to each well, and the cells were lysed for 30 min in a horizontal
rocker at room temperature. The cell lysate was transferred
completely to Eppendorf tubes and centrifuged (4000 rpm, RT) for
2 min; the supernatant was transferred to another Eppendorf tubes
(chloroquine, methylamine and ammonium chloride) were added
to the 500 L fresh serum containing DMEM medium (10%).
m
m
m
L of 1 ꢀ cell lysis buffer diluted with PBS was then added
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Nos. 21232005, 21672155, J1310008).
We also thank the Comprehensive Training Platform of Specialized
Laboratory, College of Chemistry, and the Analytical & Testing
Center of Sichuan University for sample analysis.
and stored in ice. For the assay, 20 mL of this supernatant and 100 mL
of luciferase assay substrate (Promega) were used. The lysate and
the substrate were both thawed to RT before performing the assay.
The substrate was added to the lysate, and the luciferase activity
was measured in a luminometer (Turner designs, 20/20, Promega,
USA) in standard single-luminescence mode. The integration time
of measurement was 10,000 ms. A delay of 2 s was given before
each measurement. The protein concentration in the cell lysate
supernatant was estimated in each case with Lowry protein assay
kit (PIERCE, Rockford, IL, USA). Comparison of the transfection ef-
ficiencies of the individual liposomes was made based on the data
for luciferase expressed as relative light units (RLU)/mg of protein.
All the experiments were done in triplicates, and results presented
are the average of at least two such independent experiments done
on the same days.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.038.
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