[12]aneN3-based lipid with naphthalimide moiety for enhanced gene transfection efficiency

Article abstract of DOI:10.1016/j.bioorg.2018.04.018

Three cationic lipids derived from [12]aneN₃ modified with naphthalimide (1a), oleic acid (1b) and octadecylamine (1c) were designed and synthesized. In vitro transfection showed that all these liposomes can deliver plasmid DNA into the tested

Full text of DOI:10.1016/j.bioorg.2018.04.018

Accepted Manuscript
[
12]aneN -based lipid with naphthalimide moiety for enhanced gene transfec-
3
tion efficiency
Yong-Guang Gao, Uzair Alam, Ai-Xiang Ding, QuanTang, Zheng-Li Tan, You-
Di Shi, Zhong-Lin Lu, Ai-Rong Qian
PII:
S0045-2068(18)30256-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.018
YBIOO 2341
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
6 March 2018
16 April 2018
24 April 2018
Please cite this article as: Y-G. Gao, U. Alam, A-X. Ding, QuanTang, Z-L. Tan, Y-D. Shi, Z-L. Lu, A-R. Qian,
[
12]aneN -based lipid with naphthalimide moiety for enhanced gene transfection efficiency, Bioorganic
3
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.04.018
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*
**[12]aneN -based lipid with naphthalimide moiety for enhanced gene transfection efficiency
3
a,b
b
c
b
b
b
Yong-Guang Gao , Uzair Alam , Ai-Xiang Ding , QuanTang , Zheng-Li Tan , You-Di Shi , Zhong-Lin
Lu
b,*,luzl@bnu.edu.cn
a,*,qianair@nwpu.edu.cn
, Ai-Rong Qian
a
Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Biotechnology, School of Life
Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi 710072, China.
b
KeyLaboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry,
Beijing Normal University, Xinjiekouwai Street 19, Beijing 100875, China.
c
Xinyang Normal University, Xinyang, Henan, China, 464000
*
Corresponding authors.
Graphical abstract
Highlights
•
The effect of the rigid naphthalimide moiety and flexible aliphatic chain on gene
transfection efficiency was investigated.
•
The lipid with rigid naphthalimide 1a exhibited strong DNA binding ability and could
effectively condense DNA into homogeneous square shaped nanoparticles.
•
Lipid 1a showed excellent gene transfection efficiency and serum tolerance.
Abstract
Three cationic lipids derived from ***[12]aneN
3
modified with naphthalimide (1a), oleic acid (1b) and
octadecylamine (1c) were designed and synthesized. In vitro transfection showed that all these liposomes can deliver
plasmid DNA into the tested cell lines. Among these liposomes, 1a gave the best transfection efficiency (TE) in
A549 cells, which was higher than that of lipofectamine 2000. More importantly, the TE of 1a was dramatically
increased in the presence of 10% serum. These results suggested that 1a might be a promising non-viral gene vector,
and also give further insight for developing novel high performance gene delivery agents.
Keywords:
non-viral vectors, cationic lipids, transfection efficiency, ***[12]aneN3, naphthalimide
1
. Introduction
Gene therapy is a promising strategy in the treatment of genetic diseases and has attracted significant interest in
clinical trials over the past two decades[1-8]. However, it is greatly limited due to the lack of nontoxic and high
efficient gene delivery vectors[9]. Previously, viral vectors such as adenoviruses, lentiviruses and retroviruses have
been successfully applied in several clinical applications because of their effective gene delivery ability[10-14], but
their inherent drawbacks such as carcinogenicity, immunogenicity and difficulty of industrial production restrained
them from therapeutic applications in humanp[15]. In contrast, non-viral vectors such as cationic lipids, cationic
polymers and nanoparticles have received increased attention due to their low immunogenicity, good
biocompatibility and easy production as well[16-26]. Among these non-viral vectors, cationic polymers show high
transfection efficiency. However the usage of cationic polymers in applications in gene delivery is generally limited
because of their high toxicity and non-degradable nature. In comparison, cationic lipids hold great potential for
clinical gene therapy.
Cationic lipids as a type of important and potential non-viral gene vectors possess more advantages such as
biodegradability, low cytotoxicity, structure variety and easy production etc[27, 28]. Though these advantages of

cationic lipids appear attractive, still low transfection efficiency and serum stability cannot satisfy the requirements
of gene therapy. To improve the performance of cationic lipids, different kinds of aliphatic chains were used as
hydrophobic domain of cationic lipids. The structure−activity correlations of cationic lipids were also studied. But
these studies usually focus on changing the length and number of aliphatic chains[29]. The effects of big rigid
aromatic units and flexible aliphatic chains on transfection efficiency have been seldom studied.
Recently, we developed a series of ***[12]aneN
naphthalimide, tetraphenylethene and coumarin[30-33]. It was found that the transfection efficiency was related to the
number of rigid moieties and macrocyclic polyamine ***[12]aneN units[31, 33], as well as to the distance between
naphthalimide moiety and ***[12]aneN units[32]. Moreover, rigid naphthalimide unit, possessing strong green
3
cationic lipids modified with different rigid units such as
3
3
fluorescence and exceptional photo-stability, can be applied for monitoring the process of cellular uptake, DNA
translocation and release[30]. However, these works have just focused on rigid units, while the flexible aliphatic
chains had been ignored. To compare the difference between long flexible aliphatic chains and big rigid aromatic unit,
we designed and synthesized three cationic lipids containing rigid naphthalimide unit (1a), unsaturated aliphatic
chain (1b) and saturated aliphatic chain (1c). Due to good fluorescence properties of 1a, the distribution of 1a/DNA
complexes and cellular uptake mechanism studies can be easily carried out through fluorescence microscopy. On the
other hand, non-fluorescent compounds 1b and 1c can be applied in green fluorescent protein (eGFP) experiments.
Among these lipids, 1a gave the best transfection efficiency (TE) in A549 cells and exhibited good serum-tolerance
ability, indicating that 1a may serve as a promising non-viral gene delivery vector.
2
. Results and discussion
2.1 Synthesis of target cationic lipids 1a-1c
Target lipids 1a, 1b and 1c were synthesized as shown in Scheme 1. The key intermediate Boc-protected
*
**[12]aneN 5 was obtained through two steps. 1,3-bis(bromomethyl)-5-nitrobenzene 2[34] was reacted with
3
pre***-[12]aneN
3
3 in anhydrous acetonitrile, followed by acidification with 3M HCl solution and protection with
Boc
2
O to give compound 4. Subsequently, the reaction of 4 with hydrazine hydrate in presence of 10% Pd/C yielded
the key intermediate 5. Compounds 5 and 6 were easily linked by triphosgene under basic conditions and further de-
protection of Boc under acidic conditions resulted target compound 1a. Compound 1b was obtained by reaction of
oleic acid 7 with compound 5 in the presence of (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride)
(
EDC·HCl) and N-hydroxybenzotriazole (HOBt). 3,5-bis(azidomethyl)benzoic acid 8[30] was reacted with
octadecylamine in dichloromethane to produce the key intermediate N-(3,5-bis(azidomethyl)phenyl)stearamide.
Subsequently, the click reaction of the key intermediate with propargyl ***[12]aneN 9[35] and de-protection of Boc
under acidic conditions resulted in target cationic lipid 1c. All new compounds were fully characterized by H NMR,
3
1
13
C NMR, IR and MS.
2
.2 Gel retardation assay
Cationic liposomes were formed from the combination of title lipids with 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) in the molar ratio of 1:2. To investigate the interaction between liposomes and DNA,
agarose gel electrophoresis assays were carried out in HEPES buffer (50 mM, pH 7.4) at 37 °C (Fig. 1). The results
indicated that liposomes 1a (containing rigid naphthalimide unit) and 1c had stronger DNA retardation activity than
1
b, with complete retardation observed at concentrations of 20 μM, 20 μM and 50 μM, respectively. The weaker
retardation activity of 1b can be attributed to the presence of electron-rich unsaturated alkyl chain, which could
shield the positive charge of the liposome[36].
2
.3 Dynamic light scattering
It is well known that gene delivery ability is greatly affected by particle size of the liposome/DNA complexes.
Dynamic light scattering (DLS) was used to characterize the size distribution of lipoplexes. As shown in Fig. 2, the
liposomes of 1a-1c could efficiently condense DNA into nanoparticles with diameters in the range of 50–160 nm at
the concentrations ranging from 5 to 40 μM. The particle size seemed to be suitable for cell endocytosis and further
gene transfection experiments[37, 38]. It was also observed that the size of condensed DNA particles increased
significantly with increasing concentrations of compound 1a. As we all know, the formation of nanoparticles can be
attributed to the combination effects caused by electrostatic forces, intercalation between the adjacent DNA base
pairs, as well as to the π-stacking interactions between rigid units and nucleotides. In 1a/DNA complexes, π-π
stacking interaction between compound 1a and DNA might play a dominant role as compared to electrostatic forces,

which was solely the reason for the increased size of 1a/DNA complexes with increasing concentrations of
compound 1a.
2
.4 Scanning electron microscopy
The morphology of DNA condensates also played an important role in gene delivery. Scanning electron
microscope (SEM) was applied to get the information about morphology of liposome 1a/DNA complexes at different
concentrations of 5-40 μM. As shown in Fig. 3, liposome 1a can effectively condense DNA into homogeneous
square shaped nanoparticles, and the size of nanoparticles was gradually increased with increasing concentration of
compound 1a, which was consistent with the results of DLS assay. According to the literature report, the colipid
DOPE could increase the stability of the lipoplexes, and help lipid to form various structural phases, such as the
micellar, lamellar, cubic, and inverted hexagonal phase, and arrange back-to-back in bilayers[29]. It is possible that
π-π stacking interactions between rigid naphthalimide and nucleotides may have a strong tendency to form a
homogeneous square nanostructure. Furthermore, nanoparticle sizes measured by SEM (30-120 nm) were smaller
than those obtained by DLS (50-160 nm), which could be attributed to different methods for the preparation of
liposome/DNA complexes[39, 40]. The hydrated complex was examined by DLS, while dehydrated was measured
by SEM.
2
.5 Cytotoxicity assay
The cytotoxicity of gene delivery agents is a key index for their potential clinical applications. Thus the
cytotoxicity of liposomes 1a, 1b and 1c towards A549, HeLa, U2Os and SKOV-3 cells was investigated by using
MTT assays, and lipofectamine 2000 was used as control. The results (Fig. 4) revealed that percentage of viable cells
decreased with increasing concentrations of all the three liposomes. However, even at higher concentration, all three
liposomes exhibited acceptable biocompatibility with 60 % viable cells were still present at 40 μM in all cell lines,
which was suitable for further gene transfection.
2
.6 In vitro transfection by liposomes
To further investigate the transfection ability of the liposomes as non-viral gene vectors, firstly liposomes 1a, 1b
and 1c mediated luciferase assay was conducted at different concentrations in A549 cells. As shown in Fig. 5A, these
three liposomes exhibited the best transfection efficiency at the concentration of 30 μM, but at higher concentrations
of liposomes, transfection efficiency was decreased because of the increased cytotoxicity. Among the liposomes, 1a
modified with rigid naphthalimide exhibited the best transfection efficiency, which was slightly higher than that of
lipofectamine 2000. Compared to 1c, the liposome of 1b with an unsaturated aliphatic chain exhibited higher
transfection efficiency, showing that the suitable DNA release ability was resulted from unsaturated aliphatic chain
which might promote gene transfection[29]. Furthermore, the gene transfections mediated by 1a, 1b and 1c were also
performed at the concentration of 30 μM in other cell lines, and the results are shown in Fig. 5B. The liposome of 1a
showed the highest transfection efficiency in A549 cells. In contrast, other liposomes (1b and 1c) had lower
transfection efficiencies as compared with lipofectamine 2000. Subsequently, the gene transfection of liposome 1a in
the presence of fetal bovine serum (FBS) was investigated (Fig. 5C). As it can be seen from the figure, in the
presence of 10% serum, the transfection efficiency of 1a was dramatically increased 3 times as compared with the TE
of 1a in absence of serum. The good serum tolerance of liposome 1a is probably attributed to the rigid aromatic
moiety, which may enhance the resistance to serum-induced aggregation or premature DNA release[41-43].
Moreover, increased concentration of FBS resulted in decreased transfection efficiency of liposome 1a. In order to
study the stability of liposome 1a/DNA complexes in the presence of fetal bovine serum, gel electrophoresis was
carried out. 1a/DNA complexes were incubated with 10%, 30% and 50% serum for 0.5 h. As shown in Fig. S1, DNA
was found to be gradually released with the increase of serum. It was also observed that 1a/DNA complexes could
remain stable after 24 h incubation (Fig. S2), indicating good serum tolerance of liposome 1a. To directly visualize
the infected cells expressing pEGFP-Nl reporter gene, the liposomes 1b and 1c (30 μM) mediated enhanced green
fluorescent protein (eGFP) expression in A549 cells was observed. As shown in Fig. 6, 1b-promoted transfection
produced higher fluorescence emission than 1c at 30 μM concentration, which was consistent with the luciferase
gene expression experiments.

2
.7 Cellular uptake assays
To further examine the gene delivery ability of liposome 1a in serum, cellular uptake assays were carried out in
A549 cells using Cy5-labeled dsDNA. The gene delivery agent 1a exhibited strong fluorescent emission at 551 nm
(
Fig.S3), while Cy5-labeled dsDNA emitted red fluorescence under fluorescence microscope. A common blue
fluorescent dye DAPI was used to stain the nuclei of cells. As shown in Fig. 7, Cy5-DNA could be delivered into
A549 cells with or without serum, but concentrations of the serum had an obvious influence on cellular uptake. It is
obvious that the strongest fluorescence of 1a and Cy5-DNA was observed simultaneously in the presence of 10%
serum and the fluorescence intensity was gradually decreased with increasing concentrations of serum. When 50%
serum was used as medium, almost no red fluorescent spot was observed, and the green fluorescence was also much
weaker than that observed in 10% serum. These above results were in accordance with gene transfection experiments,
indicating that serum had a significant effect both in the cellular uptake and gene transfection.
2
.8 Mechanism Studies
It is a well-known fact that gene transfection is affected by several factors, such as temperature, cellular uptake
level and internalization pathway. Endocytosis as a general entry mechanism is an energy-dependent uptake which
can be blocked at low temperature (4 °C instead of 37 °C)[44, 45]. Cellular incubations with 1a/DNA complexes
were carried out at 4 °C and 37 °C. As shown in Fig. 8B, the fluorescence of 1a was almost completely quenched
after incubation for 2h at 4 °C, suggesting that 1a-mediated endocytosis as the internalization mechanism is an
energy-dependent uptake. Furthermore, the cellular uptake pathways of clathrin, macropinocytosis, caveolae, and
microtubule mediated endocytosis could effectively be inhibited by chlorpromazine, cytochalasin D, genistein and
nocodazole, respectively[46]. Therefore, we studied the influence of different chemical inhibitors on cellular uptake
pathways. Results revealed that chlorpromazine, cytochalasin D and genistein had no obvious inhibiting effect on
cellular uptake (Figs. 8C, 8D and 8F), suggesting that 1a-mediated transfection was not dependent on clathrin,
macropinocytosis and caveolae mediated endocytosis. The cellular uptake level was significantly inhibited by
nocodazole (Fig. 8E), indicating that the cellular uptake proceeded via a microtubule-mediated endocytosis.
3
. Conclusion
In conclusion, three cationic lipids 1a-1c bearing different kinds of hydrophobic moieties were designed and
synthesized through multistep reactions. These lipids were mixed with DOPE in the ratio of 1:2 to form
corresponding liposomes, which could efficiently condense DNA into square shaped nanoparticles with the size in
the range of 50–160 nm. In vitro gene transfection experiments indicated that all these liposomes could effectively
deliver gene into different cell lines. Amongst them, 1a with big rigid naphthalimide moiety exhibited the highest
transfection efficiency, which was higher than that of lipofectamine 2000 in A549 cells. More importantly, 1a
showed excellent serum tolerance, as its transfection efficiency was increased 3 folds in the presence of 10% fetal
bovine serum, indicating its good potential as promising gene vector in vivo application.
4
. Experimental section
.1 Physical Measurements
4
1
13
o
H and C NMR spectra were obtained on a Bruker Avance III 400 MHz spectrometer at 25 C. Chemical shifts
were referenced on residual solvents peaks. The infrared spectra were taken on a Nicolet 380 spectrometer in the
-
1
range of 4000-400 cm . Mass spectra were acquired on a Waters Quattro Mocro spectrometer and high resolution
mass spectra were acquired on a Waters LCT Premier XE spectrometer. Electrophoresis apparatus was a BG-
subMIDI sub marine system (Baygene Biotech Company Limited, Beijing, China). Bands were visualized by UV
light and recorded on a UVP EC3 visible imaging system. Hydrodynamic diameters were determined using a Brook
Haven Zeta Plus Partical Size and Zeta Potential Analyzer. The morphologies of the lipoplexes were observed by
SEM (aHitachi, X 650).
4
.2 Chemicals
Anhydrous dichloromethane (DCM) was purified and dried under nitrogen by using standard methods and was
distilled immediately before use. All other reagents were of analytical grade and were used as received. Pre***-
[
12]aneN (compound 3), 3,5-bis(azidomethyl) benzoic acid (compound 8) and di-tert-butyl 9-(prop-2-ynyl)-1,5,9-
3
triazacyclododecane-1,5-dicarboxylate (compound 9) were prepared according to the literature[34, 35, 47].
Electrophoresis grade agarose, 6 × loading buffer, Goldview II, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bormide (MTT), plasmid DNA (pUC 18) were purchased from Beijing Solarbio Science﹠ Technology Co. Ltd.
(
Beijing, China), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was bought from Santa Cruz
TM
Biotechnology, Inc. (Texas, USA). Lipofectamine 2000 and plasmid PGL-3 vector were purchased from Clontech
Laboratories, Inc. (Beijing, China). Ultrapure milli-Q water (18.25 MΩ) was used in all DNA condensation assays.
4
.3 Synthesis
.3.1 Preparation of compound 4
4

To a solution of 1,3-bis(bromomethyl)-5-nitrobenzene 2 (3.30 g, 10.68 mmol) in anhydrous acetonitrile (50 mL)
was added pre***-[12]aneN 3 (4.00 g, 22.07 mmol). The mixture was heated at 80 °C for 12 h. After cooling to
3
room temperature, blue precipitates were filtered and washed with diethyl ether (30 mL) to obtain white solid. The
above crude intermediate was added to 3 M hydrogen chloride solution (60 mL) and then refluxed for 12 h. The
solvent was removed by reduced pressure to give white solid, which was added to a solution of Et
mmol) in dichloromethane (30 mL). Boc
druing half an hour and stirred for 24 h. The mixture was concentrated to dryness and extracted with dichloromethane
3 × 30 mL). The combined organic layers were washed with water (3 × 10 mL) and dried over anhydrous Na SO
After filtration and concentration to dryness, the residue was purified by chromatography on silica gel (ethyl
3
N (5.11 g, 50.50
2
O（4.00 g, 18.33 mmol）was added in above mixture in small portions
(
2
4
.
1
acetate/petroleum ether = 1/1) to give compound 4 (3.60 g, 38%) as yellow solid. H NMR (400 MHz, CDCl
3
) δ 8.05
(
s, 2H), 7.49 (s, 1H), 3.61 (s, 4H), 3.40-3.31 (m, 16H), 2.51-2.35 (m, 8H), 1.97-1.86 (m, 4H), 1.85-1.74 (m, 8H), 1.44
13
(
s, 36H). C NMR (101 MHz, CDCl
3
) δ 155.27, 147.53, 140.78, 133.96, 121.24, 78.51, 57.36, 48.96, 43.96, 42.69,
-
1
2
8
7.55, 26.69, 25.68. IR (KBr, cm ): 3433, 2971, 2938, 2797, 1691, 1532, 1472, 1411, 1349, 1250, 1202, 1159, 1068,
+
54, 564. ESI-MS Cacld. for C46
.3.2 Preparation of compound 5
To a solution of compound 4 (2.40 g, 2.70 mmol) in methanol (25 mL) was added Pd/C (0.24 g，10% w/w), and
H N
80 7
O10(M+H) : 890.6, found: 890.9.
4
hydrazine hydrate (20 mL，330.2 mmol). The mixture was heated to 75 °C for 10 h under N . After cooling to room
2
temperature, the mixture was filtered, and the filtrate was distilled under vacuum to remove the methanol. The
residue was extracted with dichloromethane (3 × 20 mL), dried over Na
(
2
SO
4
, and concentrated to give the product 5
2.12 g, 91%) as white solid, which was stored under nitrogen condition. H NMR (400 MHz, CD SOCD ) δ 6.39 (s,
H), 6.36 (s, 2H), 4.97 (s, 2H), 3.34 (s, 4H), 3.29-3.21 (m, 16H), 2.34-2.29 (m, 8H), 1.85-1.76 (m, 4H), 1.75-1.63 (m,
1
3
3
1
8
4
1
13
H), 1.38 (s, 36H). C NMR (101 MHz, CDCl ) δ 155.41, 145.48, 139.55, 119.26, 113.46, 78.46, 57.84, 49.21,
3
-1
4.15, 42.83, 27.64, 26.55, 25.84. IR (KBr, cm ): 3450, 2974, 2932, 2792, 1692, 1477, 1416, 1366, 1305, 1253,
+
+
170, 1142, 1065, 868, 773. HR-MS (ES ) Cacld. for C46
.3.3 Preparation of compound 1a[48]
To a mixture of triethylamine (0.12 g, 1.16 mmol) and triphosgene (0.69 g, 0.23 mmo) in 10 mL of
H
82
N
7
O
8
(M+H) : 860.6225, found: 860.6229.
4
dichloromethane was added compound 5 (0.50 g, 0.58 mmol) under ice-bath condition. A solution of compound 6
0.25 g, 0.58 mmol) in 2 mL of dichloromethane was added to the mixture. The reaction mixture was stirred for 10 h
at room temperature. The solvent was removed by reduced pressure, and the residue was extracted with
(
dichloromethane and dried over anhydrous Na
2
SO . After removal of the solvent, the crude product was purified over
4
silica gel using dichloromethane/methanol (10/1) as the eluent to give the inermediate as yellow solid (0.1 g), which
was added to a saturated HCl/ethyl acetate solution (5 mL). After 0.5 h of stirring, the resulting suspension was
filtered, washed with ethyl acetate, and dried in vacuum at 60 °C for 24 h to give compound 1a (80 mg, 17%) as
1
yellow solid. H NMR (400 MHz, D
2
O) δ 8.27 (s, 1H), 8.13 (s, 1H), 7.95 (s, 1H), 7.48 (s, 2H), 7.38 (s, 1H), 7.19 (s,
1
2
1
4
2
H), 6.22 (s, 1H), 4.22 (s, 2H), 3.77 (s, 2H), 3.75-3.30 (m, 20H), 3.02 (s, 4H), 2.38-2.13 (m, 12H), 1.84-0.58 (m,
13
4H), 0.50 (s, 3H); C NMR (101 MHz, D O) δ 164.61, 163.77, 156.69, 156.27, 150.52, 140.95, 139.81, 130.55,
2
30.25, 129.17, 128.31, 126.96, 123.53, 122.66, 121.22, 119.73, 107.52, 103.59, 58.26, 52.88, 46.76, 42.21, 42.10,
-1
1.06, 40.96, 31.26, 29.47, 28.35, 27.10, 26.71, 22.26, 20.58, 20.47, 17.54, 17.39, 13.70; IR (KBr, cm ): 3425.58,
+
927.94, 2852.72, 1676.14, 1633.71, 1573.91, 1467.83, 1388.75, 1246.02, 1112.93, 773.46; HR-MS (ES ) calcd. For
+
C
53
H
84
N
10
O
3
(M+H) : 909.6728, found 909.6807.
4
.3.4 Preparation of compound 1b[30]
Oleic acid (0.28 g, 1.00 mmol), EDCI (0.23 g, 1.20 mmol), BtOH (0.17 g, 1.20 mmol) and DIEA (0.25 g, 2.00
mmol) were added into dichloromethane (10 mL). After stirring for 0.5 h, compound 5 (0.86 g, 1.00 mmol) was
added to the reaction mixture and stirred for 12 h. The mixture was extracted with dichloromethane (3 × 15 mL). The
organic phase was dried (Na
2
SO ), filtered, and the solvent was evaporated under reduced pressure. The crude
4
material was purified by column chromatography on silica gel (dichloromethane/methanol = 10/1) to give a white
solid, which was reacted with a saturated HCl/ethyl acetate solution to give compound 1b (0.53 g, 56%) as white
1
solid. H NMR (400 MHz, D
2
O) δ 7.80 (s, 2H), 7.45 (s, 1H), 5.29 (s, 2H), 4.31 (s, 4H), 3.37 (s, 24H), 2.44-2.18 (m,
13
1
1
2
1
7
4H), 2.05-1.85 (m, 4H), 1.61 (s, 2H), 1.29-1.19 (m, 20H), 0.82 (t, J = 6.6 Hz, 3H); C NMR (101 MHz, D
2
O) δ
74.74, 139.60, 129.96, 129.67, 124.12, 57.69, 47.16, 42.33, 41.17, 36.89, 31.85, 29.69, 29.49, 29.29, 29.24, 29.15,
-
1
7.19, 27.10, 25.45, 22.60, 20.46, 17.93, 13.98; IR (KBr, cm ): 3425.58, 2926.01, 2852.72, 1606.43, 1562.34,
+
+
463.97, 1361.03, 1209.37, 1070.49, 742.59, 547.76; HR-MS (ES ) calcd. For C44
81
H N
7
O (M+H) : 724.6503, found
3
24.6577.
4
.3.5 Preparation of compound 1c
Compound 1c was synthesized according to a similar method with earlier report[30]. Briefly, compound 8 (0.23 g,
1
.00 mmol) was reacted with octadecan-1-amine (0.27 g, 1.00 mmol) in the presence of EDCI (0.23 g, 1.20 mmol),
BtOH (0.17 g, 1.20 mmol) and DIEA (0.25 g, 2.00 mmol). After reaction finished, the crude material was obtained
by simple purification, which was directly reacted with compound 9 (0.82 g, 2.00 mmol) in THF-H O (10 mL/5 mL).
2
The mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the
residue was extracted with dichloromethane (3 × 20 mL). The Boc-protected compound was purified by column
chromatography to give white solid, which was reacted with a saturated HCl/ethyl acetate solution to give compound
1
1
c (0.32 g, 27%) as yellow solid. H NMR (400 MHz, D
2
O) δ 8.23 (s, 2H), 7.75 (s, 2H), 7.27 (s, 1H), 5.53 (s, 4H),

1
3
4
.11 (s, 4H), 3.32 (s, 18H), 3.00 (s, 8H), 2.24 (s, 4H), 2.07 (s, 8H), 1.50 (s, 2H), 1.19 (s, 30H), 0.80 (s, 3H); C NMR
(
101 MHz, D O) δ 167.47, 138.61, 136.33, 135.47, 130.65, 127.30, 127.10, 53.28, 49.01, 47.00, 43.24, 42.02, 40.22,
2
-1
3
1
9
1.94, 29.88, 29.43, 29.20, 27.14, 22.65, 20.09, 19.38, 14.01; IR (KBr, cm ): 3423.65, 2924.09, 1637.56, 1602.85,
+
+
460.11, 1362.96, 1238.30, 1116.78, 1058.92, 744.52, 582.50; HR-MS (ES ) calcd. For C51
91
H N
13O (M+H) :
02.7470, found 902.7542.
4
.4 Agarose gel retardation assays
Lipid/DOPE/pDNA complexes at different concentrations were prepared by adding appropriate volume of the
liposome solution to 0.9 μL of pUC18 DNA (200 μg/mL) and 4 μL of HEPES (50 mM，pH 7.4). The obtained
complex solution was then diluted to the total volume of 20 μL. After incubation at 37 °C for 5 min, 10 μL of each
lipoplex solution was electrophoresed on a 0.7 % (w/v) agarose gel containing Gold view in Tris-acetate (TAE)
running buffer at 120 V for 30 min. Then DNA was visualized under an ultraviolet lamp using a Vilber Lourmat
imaging system.
4
.5 Dynamic light scattering (DLS)
The liposome/DNA complexes were prepared by adding pUC18 DNA (4 μg/mL) to the appropriate volume of the
liposome solution. The complex solution was then vortexed for 30 s before being incubated at 37 °C for 5 min and
was measured at 25 °C. Data were shown as average of four times measurements.
4
.6 Scanning electron microscope (SEM) images
.9 μL of pUC18 DNA (200 μg/mL) was mixed with the appropriate volume of liposomes solution to form
0
complexes, diluted by water to a total volume of 20 μL, and incubated at 37 °C for 5 min. The lipoplexes were added
dropwise to the silicon slice. The slice was then dried at room temperature at atmospheric pressure for several hours
before observation.
4
.7 Cytotoxicity assays
The cytotoxicity of lipoplexes towards A549, HeLa, U2Os and SKOV-3 cell lines were tested by MTT assays[43].
The cells were seeded in 96-well plates at 7000 cells and 100 μL medium per well and cultured for 24h. The cells
were then treated with different N/P ratios of liposomes 1a-1c in 100 μL DMEM. After a 4h co-incubation, 100 μL
DMEM with 10% FBS was added to each well and cells were further cultured for 24h. After that the medium was
removed and 10 µL of MTT (5 mg/mL) was added to wells. Finally, MTT was replaced with 150 µL of DMSO. The
optical density (OD) of the medium was measured by a microplate reader at 490 nm.
4
.8 In vitro gene transfection
Luciferase assay[36]. The in vitro transfections mediated by the plasmid pGL-3-containing complexes were
conducted A549, HeLa, U2Os, SKOV-3 and E1 cells. Briefly, cells were seeded in 24-well plates and cultured until
reach 70-80% cell confluence. After a 4 h co-incubation and subsequent removal of the medium, the cells were
further cultured in fresh DMEM medium containing 10% FBS for 20 h. The gene transfection efficiency of each
sample was represented by firefly luciferase expression and calculated as relative light units per milligram of the total
protein.
Green fluorescent protein assay[36]. To examine the expression of the internalized DNA, A549 cells were
transfected by the condensates containing pEGFP-N1. Cells were seeded in glass bottom cell culture dishes and
cultured until 80% cell confluence. Before transfection, the medium was washed three times with DMEM, and
treated with freshly prepared pEGFP-DNA condensates and the controls (500 µL). After 4 h under standard culture
conditions, the medium was replaced with 500 μL of fresh DMEM medium containing 10% FBS. After 20h
incubation, the cells were washed for 3 times with PBS, and observed under Zeiss Inverted Fluorescence Microscope
with a 10 × objective to examine the expression of the intracellular EGFP.
4
.9 Cellular uptake assays
The cellular uptake of 1a/Cy5-DNA condensates was observed with or without FBS by fluorescence
microscope[30]. A549 cells were cultured in DMEM medium with 10% FBS in at 37 °C. The cells were seeded at a
3
density of 10 cells per dish and cultured for 24 h. After washed three times with DMEM, the cells were treated with
1
a/Cy5-DNA condensates (1a: 30 µM, Cy5-DNA: 9 µg/mL). The blue fluorescence dye DAPI (5 µg/mL) was also
added to each dish for nuclear staining, after that the cells were cultured for 2 h. Finally, the cells were washed for 6
times with PBS buffer, observed using a Zeiss Inverted Fluorescence Microscope with a 40 × objective and DAPI
filter for DAPI (blue), GFP filter for lipid 1a (green), and Rhodamine filter for Cy5 (red), respectively.
4
.10 Mechanism Studies
To probe the internalization mechanism of the lipoplexe 1a, the cellular uptake study was performed at 4 °C or in
the presence of various endocytic inhibitors[44, 45]. Briefly, cells were incubated with 1a/pUC18 DNA complexes
(
10 μM) at 4 °C for 4 h, the energy-dependent endocytosis was completely blocked. Otherwise, cells were
preincubated with various endocytic inhibitors including chlorpromazine (20 μg/mL), cytochalasin D (10 μg/mL),
genistein (200 μM), and nocodazole (33 μM). Following pretreatment for 2h, the inhibitor solutions were replaced by
the 1a/pUC18 DNA complexes (10 μM). After further incubation for 2 h, the cells were washed 6 times with PBS
buffer and were observed using a Zeiss Inverted Fluorescence Microscope with a 10 × objective.

Acknowledgments
The authors gratefully acknowledge the financial assistance provided by the National Natural Science Foundation
of China (31570940, 31400725, 81700784 and 21372032).
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Fig. 1. Agarose gel electrophoresis of liposomes 1a-1c complexed with DNA to form lipoplexes at different
concentrations; Condition: pUC18 DNA (9 μg/mL), HEPES (50 mM, pH 7.4), 37 °C, 5 minutes.
Fig. 2. The effective diameter of liposomes of 1a-1c/DNA complexes at different concentrations, [DNA] = 9 μg/mL,
HEPES buffer (50 mM, pH 7.4), 25 °C.
Fig. 3. SEM Images of pUC18 DNA (9 μg/mL) and its condensation induced complexes of 1a with DOPE (1:2) at
different concentrations in HEPES buffer (50 mM, pH 7.4).
Fig. 4. Cytotoxicity of liposomes 1a (A), 1b (B) and 1c (C) at different concentrations toward A549, HeLa, U2Os
and SKOV-3 cell lines, with lipofectamine 2000 (10 μg/mL) as the control.
Fig. 5. Luciferase gene expression transfected by (A) 1a, 1b and 1c at different concentrations toward A549 cells, (B)
1
a, 1b and 1c at a concentration of 30 μM toward different cell lines, (C) 1a at a concentration of 30 μM in the
absence or presence of serum toward A549 cells.
Fig. 6. Fluorescence microscope image of pEGFP-transfected A549 cells of liposomes 1b (30 μM), 1c (30 μM) and
Lipo2000 (10 μg/mL)
Fig. 7. CLSM images of A549 cells transfected with Cy5-labaled DNA (9 μg/mL) by 1a/DOPE (1:2) at 30 μM in the
absence (A) and presence of 10%, 30% and 50% serum. A: BF; B: cell stained by 1a (green); C: Cy5-labeled DNA
(red); D: cell nuclei stained by DAPI (blue); E: merged images.
Fig. 8. CLSM images of A549 cells at 37 °C, 4 °C and in the presence of various endocytic inhibitors. 1: BF; 2: cell
stained by 1a (10 μM); 3: merged images.
Scheme 1. The synthesis of cationic lipids 1a, 1b and 1c