Cyclen-based double-tailed lipids for DNA delivery: Synthesis and the effect of linking group structures

Article abstract of DOI:10.1016/j.bmc.2015.07.005

The gene transfection efficiency (TE) of cationic lipids is largely influenced by the lipid structure. Six novel 1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids L1-L6, which contain double oleyl as hydrophobic tails, were designed and synthesized. The difference between these lipids is their diverse backbone. Liposomes prepared by the lipids and DOPE showed good DNA affinity, and full DNA condensation could be achieved at N/P of 4 to form lipoplexes with proper size and zeta-potentials for gene transfection. Structure-activity relationship of these lipids as non-viral gene delivery vectors was investigated. It was found that minor backbone structural variations, including linking group and the structural symmetry would affect the TE. The diethylenetriamine derived lipid L4 containing amide linking bonds gave the best TE, which was several times higher than commercially available transfection reagent lipofectamine 2000. Besides, these lipids exhibited low cytotoxicity, suggesting their good biocompatibility. Results reveal that such type of cationic lipids might be promising non-viral gene vectors, and also afford us clues for the design of novel vectors with higher TE and biocompatibility.

Full text of DOI:10.1016/j.bmc.2015.07.005

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cyclen-based double-tailed lipids for DNA delivery: Synthesis
and the effect of linking group structures
⇑
⇑
Yi-Mei Zhang, De-Chun Chang, Ji Zhang , Yan-Hong Liu, 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:
The gene transfection efficiency (TE) of cationic lipids is largely influenced by the lipid structure. Six novel
1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids L1–L6, which contain double oleyl as
hydrophobic tails, were designed and synthesized. The difference between these lipids is their diverse
backbone. Liposomes prepared by the lipids and DOPE showed good DNA affinity, and full DNA conden-
sation could be achieved at N/P of 4 to form lipoplexes with proper size and zeta-potentials for gene
transfection. Structure–activity relationship of these lipids as non-viral gene delivery vectors was inves-
tigated. It was found that minor backbone structural variations, including linking group and the struc-
tural symmetry would affect the TE. The diethylenetriamine derived lipid L4 containing amide linking
bonds gave the best TE, which was several times higher than commercially available transfection reagent
lipofectamine 2000. Besides, these lipids exhibited low cytotoxicity, suggesting their good biocompatibil-
ity. Results reveal that such type of cationic lipids might be promising non-viral gene vectors, and also
afford us clues for the design of novel vectors with higher TE and biocompatibility.
Received 22 May 2015
Revised 7 July 2015
Accepted 8 July 2015
Available online xxxx
Keywords:
Cationic lipids
Gene delivery
Non-viral vector
Cyclen
Structure–activity relationship
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
number of advantages including their simple structures, good
repeatability, ease of production, biocompatibility, and potentially
Gene therapy has been expected to be a powerful approach to
the treatment of genetic and acquired diseases.1 Generally speak-
ing, gene transfection vectors commonly used in gene therapy
mainly fall into two categories: viral and non-viral.2 In early days,
viral vectors such as adenovirus and retrovirus have achieved some
success particularly in cancer gene therapy for their relatively high
transfection efficiency (TE). However, their disadvantages such as
adverse immunogenic responses, oncogenic effects, potential
mutagenesis and high cost limited their applications.3 People thus
try to find a more efficient and safe delivery system.2 For their easy
preparation and modification, safety, and reproducibility, non-viral
vectors attracted the attentions of scientists.1 Cationic lipids and
polymers4 are two main types of non-viral vectors for nucleic
acids. Compared to polymers, cationic lipids brought about a
commercial value.3 Since Felgner and co-workers in 1987 first
reported cationic lipid DOTMA for gene delivery,5 an upsurge of
global interest has been witnessed in the design and synthesis of
novel efficient cationic transfection lipids.6 A number of struc-
ture–activity relationship studies have demonstrated that each
building block of the lipids, including positive charged polar head
group,7 hydrophobic tails,8–12 linking group,13–16 would largely
influence the TEs of the delivery materials.
As a polyamine compounds, 1, 4, 7, 10-tetraazacyclododecane
(cyclen) has unique cyclic structure. The amino groups, which are
easy to be modified, have wide range of pKa values.17 In our previ-
ous studies, we have demonstrated the potential of cyclen-based
gene delivery systems.11,12,16 On the other hand, lipids with
monounsaturated hydrophobic chain(s) (e.g., oleyl group) might
facilitate the transfection, since they could facilitate membrane
disruption and DNA escape from endosome by increasing the
membrane fluidity.8 Many oleyl-contained lipids have been proved
to be efficient delivery materials,8,12 and some commercially avail-
able transfection reagents also have oleyl moiety in their struc-
tures. Besides the cationic headgroup and hydrophobic tails, the
linking group also plays important role in the vector structure.
Bajaj et al. showed that the length of polymethylene18 or oxyethy-
lene spacers19 would influence the gene delivery properties of
Abbreviations: Boc, tert-butoxycarbonyl; cyclen, 1, 4, 7, 10-tetraazacyclodode-
cane; lipoplexes, liposome/DNA complexes; TE, gene transfection efficiency; EDC
HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt, 1-hy-
droxybenzotriazole;
TFA,
trifluoroacetic
acid;
DOPE,
dioleoylphos-
phatidylethanolamine; N/P, numbers of protonable amines to phosphates on
DNA; EB, ethidium bromide; DLS, dynamic light scattering; DCC,
N,N0-Dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine.
⇑
Corresponding authors. Fax: +86 28 85415886 (X.-Q.Y.).
E-mail addresses: ji_zhang@163.com (J. Zhang), xqyu@scu.edu.cn (X.-Q. Yu).
http://dx.doi.org/10.1016/j.bmc.2015.07.005
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang, Y.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.005

2
Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
L1
4CF3COOH
O
L3
L5
4CF3COOH
O
O
O
O
R
NH HN
NH
NH HN
NH
NH HN
NH
O
O
N
H
R
R
O
O
O
R
R
O
O
N
N
H
N
N
N
H
N
N
H
4CF3COOH
O
R
R
(C17H34
)
R =
O
L2
H
N
L4
4CF3COOH
O
L6
4CF3COOH
O
NH HN
NH HN
NH
NH HN
NH
N
R
R
O
O
O
O
H
2
NH
N
N
H
N
N
N
H
O
R
R
N
N
H
O
4CF3COOH
O
N
H
R
O
Figure 1. Chemical structures of the title lipids L1–L6.
aromatic backbone based gemini lipids. Kedika and Patri found
that the lipid with symmetrical backbone might give much higher
TE than those with asymmetrical backbone structure.9 More
recently, Niyomtham et al. revealed that the spermine-based catio-
nic lipids with diverse central core structures had much different
TEs.20
hydrophobic chains were connected via different bonds (e.g., ester
bonds for L3, L5 and L6; amide bonds for L1, L2 and L4). These
structure varieties would help us to study the structure–activity
relationship of the cyclen-based double-tailed cationic lipid vec-
tors. All novel compounds were purified and characterized by
NMR and HRMS.
Considering this, herein we design and synthesize a series of
cationic lipids with cyclen as cationic headgroup and oleyl as
hydrophobic tails. These lipids have different linking group struc-
tures, which would help us to study their influence on the transfec-
tion behavior of the liposome/DNA complexes (lipoplexes). The
structures of lipids studied here are listed in Figure 1: Aspartic
acid,21 glutamic acid,21 diethanolamine,20 diethylenetriamine, seri-
nol and 3-amino-1, 2-propanediol9,22,23 were used as linking group
for lipids L1–L6, respectively. Their interactions with plasmid DNA
together with their structure–activity relationship as non-viral
gene delivery vectors were investigated.
2.2. Interactions between the liposomes and plasmid DNA
Cationic liposomes were formed from a combination of cationic
lipid and a neutral lipid dioleoylphosphatidylethanolamine (DOPE)
in different molar ratios (1:1, 1:2, and 1:3). According to the trans-
fection behavior of liposomes from different lipid combinations,
the molar ratio of 1:2 was chosen for further investigation. Since
effective DNA package is prerequisite for cationic lipids serving
as gene vectors, agarose gel retardation assay was first applied to
evaluate the condensation capabilities of lipids L1–L6 to pDNA.
As shown in Figure 2, all of the lipids showed good DNA binding
abilities. Completely DNA retardation could be achieved at the
charge ratio (numbers of protonable amines to phosphates on
DNA, N/P) of 2 for all liposomes except that formed from L3, which
needed an N/P ratio of 4 for full condensation.
2. Results and discussion
2.1. Synthesis of target cationic lipids
Furthermore, ethidium bromide (EB) dye replacement assay16
was also carried out to study the strength of the binding between
liposomes and DNA. EB that bound to DNA in advance would be
replaced by another binding reagent and become free molecules,
leading to obvious fluorescence decrease. As shown in Figure 3,
the fluorescent intensities were significantly decreased with the
rise of N/P ratio (0–3). The N/P ratios for quenching 50% of fluores-
cence intensity were evaluated to be 0.94 (L4), 1.10 (L1, L5), 1.16
(L2, L6) and 1.35 (L3), respectively. Further increase of N/P ratio
(3 –7) led to a plateau of the fluorescence, indicating a saturate
DNA binding. It is revealed that among the liposomes, L1 has the
best affinity toward DNA, while L3 is on the opposite. Such results
are consistent with the gel electrophoresis results.
Mean particle size, surface charge of the lipoplex may largely
influence its activity during gene transfection. These parameters
of free liposomes derived from L1 to L6 were first studied by
dynamic light scattering (DLS, Fig. S1). It was found that these lipo-
somes had similar size with diameters about 120 nm and gave the
particles with surface charges about +55 mV. The stability of these
liposomes was also studied. As Table S1 shows, the sizes of those
particles after being stored at 4 °C for 2 months seldom changed
compared to the freshly prepared liposomes, indicating their good
stability. Subsequently, the lipoplexes prepared from L1 to L6 with
DNA were measured at the N/P of 1, 2, 4, 6 and 8 via same method,
To study the influence of linking group structure in the cyclen-
based cationic lipids, target compounds L1–L6 were designed and
synthesized, and the preparation routes are listed in Scheme 1.
Several small molecules with three functional groups (1–6) were
chosen as the linking group between cyclen headgroup and double
oleyl tails. In the synthesis of each lipid, the long tails were first
connected to the linking molecule, and the obtained intermediate
was then reacted with triBoc-cyclen-acetic acid 7 to form the lipid
precursor. For the amino acid (aspartic acid 1 and glutamic acid 2)
contained lipids L1 and L2, the amino group was first protected
with Boc group, and the two carboxyls were then condensed with
oleyl amine to form 1a and 2a, respectively. After deprotection and
coupling with 7, the target lipids were obtained by deprotection
with trifluoroacetic acid. For the other four lipids L3–L6, the amino
group on the linking molecule 3–6 was first protected by (Boc)2O
except 4, in which the two primary amines must be previously pro-
tected by trifluoroacetylation with CF3COOEt (after reacted with
(Boc)2O, the trifluoroacetyls were removed by potassium carbon-
ate in methanol). The protected molecules 3a–6a were then trea-
ted with oleic acid and then trifluoroacetic acid to form the
intermediates 3b–6b. After deprotection of Boc group, the target
lipids L3–L6 could be obtained by similar procedure including
the reaction with 7, and subsequent deprotection with trifluo-
roacetic acid. Except the structure of linking moiety, the
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Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
O
O
1) (Boc)2O
OH
OH
NHR CF3COOH
NHR
NHR
NHR
DIEA/HOBt/EDC
n
n
n
Boc
2) oleyl amine
DIEA/HOBt/EDC
Boc
Boc
H2N
N
H
H2N
N
N
N
N
O
1,2
O
1a,2a
O
1b,2b
7
O
Boc
OH
4CF3COOH
O
Boc
O
Boc
O
NH HN
NH
N
N
N
CF3COOH
CH2Cl2
NHR
NHR
O
NHR
NHR
n
n
N
N
n=1 for 1 series
n=2 for 2 series
N
H
Boc
N
H
1c,2c
O
O
L1-L2
Boc
N
Boc
N
H
N
H
N
HO
OH
H2N
NH2
HO
OH
H2N
Boc
NH2
3
4
3a
4a
OH
3 5 6
(Boc)2O for , ,
OH
OH
1) CF3COOEt
2) (Boc)2O
3) K2CO3 for 4
OH
OH
H2N
H
N
HN
Boc
H2N
OH
O
OH
OH
6
5
6a
5a
O
Boc
N
O
Boc
N
O
R
O
O
R
R
N
H
N
H
R
3b
4b
1) CF3COOH
oleic acid
O
O
O
O
L3 L6
-
7
2)
R
O
3) CF3COOH
H
N
O
R
O
H
Boc
N
R
Boc
R
O
5b
6b
Scheme 1. Synthesis of title lipids L1–L6.
Figure 2. Electrophoretic gel retardation assays of lipids L/DOPE/pDNA complexes at different N/P ratios.
and the results are shown in Figure 4. The lipoplexes gave the par-
ticles with diameters about 150 nm at low N/P ratio of 1, and for
each example, the size was largely increased at N/P ratio of 2.
Then the particle sizes gradually reduced and plateaued at
120–250 nm, depending on the different structures of the lipids.
The data suggest the formation of stable lipoplex particles. On
the other hand, the zeta-potentials of the lipoplexes rose along
with the increase of N/P ratio, and the charge reverse was observed
at the N/P of 2 except L3, which happened at N/P ratio of 4 (Fig. 4B).
The unique performance of L3 might be due to its relatively lower
DNA binding ability, which was indicated by the gel retardation
and fluorescence quenching essays.
2.3. Cytotoxicity
The cytotoxicity of the lipoplexes prepared from L1 to L6 at var-
ious N/P ratios was examined in HEK 293 cells and A549 cells by
CCK-8 assay after culturing cells with lipoplexes for 24 h. As shown
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Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
significantly higher than that of lipofectamine 2000, indicating
the low cytotoxicity of these lipoplexes.
2.4. In vitro gene transfection
Luciferase reporter gene expression assay was used in evaluat-
ing the in vitro gene delivery efficacies of lipids L1–L6 at N/P ratios
of 3, 4, 6 and 8 in HEK 293 human and A549 cells, while lipofec-
tamine 2000 used as positive control. Figure 6 shows that the lipid
structure largely influenced their TE in each cell line. Generally,
lipids containing amide linking bonds (L1, L2 and L4) showed bet-
ter TE than those with ester bonds (L3, L5 and L6), which was sim-
ilar to our previous results.21 Among the six lipidic materials, L1
gave the best TE in HEK 293 cells (Fig. 6A), while L4 exhibited
the best performance in A549 cells (Fig. 6B). The optimal TE was
obviously higher than lipofectamine 2000, especially in A549 cells.
Interestingly, although L3 and L4 had almost the same structure
except the linking bonds, L4 gave much higher than L3 in both cell
lines. Except the linking bond, the symmetry of the hydrophobic
domain of cationic lipids is also an important factor which might
affect the transfection. In both cell lines, lipid L6 with asymmetric
hydrophobic structure exhibited lower TE than L5, which contains
symmetric hydrophobic domain. Such advantage of symmetric
structures was also confirmed by some other studies.9,22 It’s known
that TE was influenced not only by the chemical structure of the
Figure 3. Ethidium bromide displacement assay of plasmid DNA binding abilities
for the lipid L1–L6/DOPE liposomes under various N/P ratios in Hepes buffer
solution (pH = 7.4, 10 mM). The molar ratio of lipid/DOPE was 1:2.
in Figure 5, survival rate of cells was in a dose-dependent manner,
which decreased along with the rise of N/P ratio. Even at high N/P
ratio of 12, which was much higher than the optimal N/P of 4 for
transfection, the relative cell viabilities were more than 60% and
Figure 4. Mean particle sizes (A) and zeta-potentials (B) of the lipoplexes formed from L1 to L6 at N/P ratio of 1, 2, 4, 6 and 8.
Figure 5. Cytotoxicity of the lipoplexes prepared at various N/P ratios (2, 4, 6, 8 and 12) in HEK293 cells (A) and A549 cells (B). Data represent mean SD (n = 3).
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Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 6. Transfection efficiencies of lipoplexes in HEK 293 cells (A) and A549 cells (B) at various N/P ratios (3, 4, 6, 8) under lipid/DOPE ratio of 1:2.
carrier, but also by the cell type. Thus we applied L1 to the gene
delivery toward various other cell lines (7402, U-2OS, and HepG-
2 cells), the results are shown in Figure S2. Unfortunately, the
TEs of our material were much less than those of lipofectamine
2000 in these cell lines. We hope that the structure–activity rela-
tionship studies may afford us clues for further optimization of
cationic lipid gene delivery materials.
purchased from Promega (Madison, WI, USA). Endotoxin free plas-
mid purification kit was purchased from TIANGEN (Beijing, China).
HEK 293 (normal human embryonic kidney cells) and A549
(human lung carcinoma cells) were purchased from Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences.
4.2. Synthesis (Detailed yields and analysis data of novel
compounds are shown in Supporting Information)
3. Conclusion
4.2.1. Preparation of compound 1a and 2a
In summary, six cyclen-based cationic lipids L1–L6 were
designed and synthesized. These amphiphilic compounds contain
the same hydrophobic tails and headgroup, only differing in the
linking group structure. Liposomes formed by these lipids and
helper lipid DOPE could efficiently bind DNA and condense it into
nano-sized particles with positive zeta-potentials. These lipoplexes
were applied as non-viral gene delivery vectors, and their struc-
ture–activity relationship was investigated. Subtle changes in the
backbone structure of the lipids such as the linking bonds and
structural symmetry would lead to large effect on the TE. The
diethylenetriamine-derived lipid L4 gave the best TE, which was
several times higher than the commercially available transfection
reagent lipofectamine 2000. Besides, compared to the same
reagent, these lipids exhibited much less cytotoxicity. Results
reveal the potential of such type of cationic lipids as non-viral gene
vectors.
N-Boc-D D
-Aspartic acid (5 mmol)25 or N-Boc- -glutamic acid,26
which was prepared according to literature procedure, was dis-
solved in CH2Cl2 (50 mL), cooled to 0 °C. 1-Hydroxybenzotriazole
(HOBt) (1.83 g, 12 mmol), 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDCꢀHCl) (2.3 g, 12 mmol) and DIEA
(2.58 g, 2 mmol) were gradually added for activation of carboxylic
acid. After 0.5 h stirring, oleyl amine (3.57 g, 90%, 12 mmol) was
added, and resulting mixture was stirred at room temperature
overnight. The solvent was then removed under reduced pressure.
EtOAc was added to dissolve the residue, and the solution was
washed with water, saturated aqueous NaHCO3 solution and satu-
rated brine. The organic layer was dried over anhydrous sodium
sulfate and concentrated to give a white solid. The crude product
was purified by silica gel column chromatography (PE/EA = 1/1,
v/v).
4.2.2. Preparation of lipids L1 and L2
4. Experimental section
Compound 1a or 2a (2 mmol) was dissolved in anhydrous
CH2Cl2 (20 mL). Then CF3COOH (20 mmol) in anhydrous CH2Cl2
(10 mL) was added dropwise. The resulting mixture was stirred
at 0 °C overnight. The solvent was then removed under reduced
pressure. The residue was washed with anhydrous ether twice to
give pure compounds 1b or 2b. After similar coupling procedure
depicted in the preparation of 1a, 1c or 2c could be obtained and
further purified by silica gel column chromatography
(PE/EA = 1/2, v/v).
Compound 1c (or 2c) (300 mg) was dissolved in anhydrous
CH2Cl2 (3.0 mL), then trifluoroacetic acid (CF3COOH) (3.0 mL) in
anhydrous CH2Cl2 (3.0 mL) was added at 0 °C. After stirring for
6 h, the solvent was removed under reduced pressure. The residue
was washed with anhydrous ether twice to give pure compounds
L1 or L2.
4.1. Materials and methods
All chemicals and reagents were obtained commercially and
were used as received. Anhydrous acetonitrile, dichloromethane
(DCM) and chloroform (CHCl3) were dried and purified under
nitrogen by using standard methods and were distilled immedi-
ately before use. [4, 7, 10-Tris(tert-butoxycarbonyl)-1, 4, 7, 10-te-
traazacyclododecan-1-yl] acetic acid (tri-boc-cyclen-acetic acid,
compound 7, Scheme 1) was prepared according to the literature.24
The plasmids used in the study were pEGFP-N1 (Clontech, Palo
Alto, CA, USA). A Cell Counting Kit-8 (CCK-8) was purchased from
Dojindo Laboratories (Kumamoto, Japan). The Dulbecco’s
Modified Eagle’s Medium (DMEM) and fetal bovine serum were
purchased from Invitrogen Corp. MicroBCA protein assay kit was
obtained from Pierce (Rockford, IL, USA). Luciferase assay kit was
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Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.2.3. Preparation of compounds 3b, 5b and 6b
(10 mM, pH 7.4) to the lipid film, and then keep them in 70 °C
for 30 min to hydrate. The samples were sonicated for 5 min in a
bath sonicator to generate small unilamellar vesicles at a concen-
tration of 1 mM.15
Diethanolamine 3 (or threoninol 5, 3-amino-1,2-propanediol 6)
(0.047 mol) was dissolved in CH2Cl2 and triethylamine (9.60 g,
0.095 mol) was slowly added. The solution was cooled to 0 °C,
and (Boc)2O (12.45 g, 0.057 mol) was added dropwise. Then the
mixed solution was left for stirring at room temperature overnight.
The solvent was removed under reduce pressure to afford crude
product, which was further purified by silica gel column chro-
matography (CH2Cl2/CH3OH = 10/1, v/v) to give compound 3a (or
5a, 6a).
4.4. Amplification and purification of plasmid DNA
PGL-3 was seed as the luciferase reporter gene, which was
transformed in M109 Escherichia coli, and pEGFP-N1 plasmids
was used as the enhanced green fluorescent protein reporter gene,
To the solution of oleic acid (3.75 g, 13.2 mmol) and 4-dimethy-
laminopyridine (DMAP) (270 mg) in CH2Cl2 at 0 °C,
N,N0-Dicyclohexylcarbodiimide (DCC) (2.75 g, 13.3 mmol) in
CH2Cl2 were added dropwise. After 30 min, compound 3a (or 5a,
or 6a) (11 mmol) was added, then the mixture was left for stirring
at room temperature overnight. The reacting solution was cooled
to 0 °C, and the formed precipitate was filtered off. The filtrate
was evaporated to give the residue, which was purified by silica
gel column chromatography (PE/EA = 3/1, v/v) to give compound
3b (or 5b, 6b).
which was transformed in E. coli DH5a. Both plasmids were ampli-
fied in E. coli grown in LB medium at 37 °C and 220 rpm overnight.
The plasmids were purified by an EndoFree Tiangen™ Plasmid Kit.
Then, the purified plasmids were dissolved in TE (Tris + EDTA) buf-
fer solution and stored at ꢁ80 °C. The integrity of plasmids was
confirmed by agarose gel electrophoresis. The purity and concen-
tration of plasmids were determined by the ratio of ultraviolet
(UV) absorbances at 260 nm/280 nm (ꢂ1.9). The concentrations
of plasmids were determined by ultraviolet (UV) absorbance at
260 nm.
4.2.4. Preparation of compound 4b
4.5. Preparation of lipid/DOPE/DNA complexes (lipoplexes)
Diethylenetriamine
4 (3.35 g, 0.032 mol) was dissolved in
CH2Cl2 and cooled to 0 °C. Ethyl trifluoroacetate (9.69 g,
0.068 mol) in CH2Cl2 was slowly added. After 1 h, triethylamine
(9.10 mL, 68.8 mmol) was added to the mixed solution and
(Boc)2O (8.48 g, 38.9 mmol) was added dropwise. The mixture
was stirred at room temperature overnight and then washed with
saturated aqueous NaHCO3 solution twice and saturated brine
twice. The organic phase was dried over Na2SO4, filtered, and con-
centrated to afford crude product, which was crystallized from a
mixture of CH2Cl2/hexane to give 6.00 g needlelike solid. These
needles and K2CO3 were suspended in a mixture of CH3OH:H2O
(20:1, 40 mL). The reaction mixture was refluxed for 4 h. Then
the solvent was removed under reduced pressure to get 2.50 g
crude product 4a, which can be used without further purification.
A solution of oleic acid (4.60 g, 16.3 mmol) in CH2Cl2 was cooled
to 0 °C. HOBt (2.72 g, 16.3 mmol), EDCꢀHCl (3.40 g, 16.3 mmol),
DIEA (3.76 g, 29.1 mmol) were gradually added for activation of
carboxyl. After 0.5 h, compound 4a (1.48 g, 7.28 mmol) was added.
Then the mixture was stirred at room temperature overnight. The
reacting solution was washed with water, saturated aqueous
NaHCO3 solution and then saturated brine. The organic phase
was dried over Na2SO4, filtered, and concentrated to afford a white
solid, which was further purified by silica gel column chromatogra-
phy (PE/EA = 1/1, v/v).
To prepare the lipid/DOPE/pDNA complexes (lipoplexes), vari-
ous amounts of cationic lipids and a constant amount of DNA were
mixed together by pipetting thoroughly at various N/P ratios, and
then incubated for 30 min at room temperature. The theoretical
N/P ratio represents the charge ratio of cationic lipid to nucleotide
base (mole ratios) and was calculated by considering the average
nucleotide mass of 309.
4.6. Agarose-gel retardation assay
Lipid L1–L6/DOPE/pDNA complexes at different N/P ratios (the
amino groups of lipids to phosphate groups of DNA) ranging from
0 to 8 were prepared by adding an appropriate volume of lipids to
0.125 lg pUC-19 DNA. The complexes were incubated at 37 °C for
30 min. Then the complexes were electrophoresed on the 1.0%
(W/V) agarose gel containing gelred and with Tris-acetate (TAE)
running buffer at 110 V for 30 min. DNA was visualized with a
UV lamp at wavelength of 312 nm by using BioRad Universal
Hood II.
4.7. Ethidium bromide replacement assay
The ability of lipids L1–L6 to condense DNA was studied using
ethidium bromide (EB) exclusion assays. Fluorescence spectra
4.2.5. Preparation of lipids L3–L6
were measured at room temperature in air by
JobinYvon Flu-oromax-4 spectrofluorometer and corrected for the
system response. EB (5 L, 1.0 mg/mL) was put into quartz cuvette
a Horiba
Compound 3b (or 4b, 5b, 6b, 2 mmol) was dissolved in anhy-
drous CH2Cl2 (20 mL). Then CF3COOH (20 mmol) in anhydrous
CH2Cl2 (10 mL) was added dropwise, and the resulting mixture
was stirred at 0 °C overnight. The solvent was removed under
reduced pressure. The residue was washed with anhydrous ether
twice to get the deprotection product. Then L3–L6 could be
obtained by the same coupling and deprotection procedures
described in the preparation of L1 and L2.
l
containing 2.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES) solution (pH 7.4). After shaking 1 min,
the fluorescence intensity of EB was measured. Then CT-DNA
(10 lL, 1.0 mg/mL) was added to the solution and mixed symmet-
rically, and the measured fluorescence intensity is the result of the
interaction between DNA and EB. Subsequently, the solutions of
lipid L1–L6 (1 mmol/L, 4 lL for each addition) were added to the
4.3. Preparation of cationic liposome
above solution for further measurement. All the samples were
excited at 520 nm and the emission was measured at 600 nm.
The pure EB solution and DNA/EB solution without cationic lipo-
some were used as negative and positive controls, respectively.
The percent relative fluorescence (%F) was determined using the
equation %F = (F ꢁ FEB)/(F0 ꢁ FEB), wherein FEB and F0 denote the
fluorescence intensities of pure EB solution and DNA/EB solution,
respectively.
The cationic lipid L1–L6 was combined with the neutral lipid
dioleoylphosphatidylethanolamine (DOPE) in 1:2 mole ratio in
2.5 mL anhydrous chloroform in a glass vial. The lipid films were
got by slowly rotary-evaporating the solvent with a thin flow of
moisture-free nitrogen gas. Then, leave them under high vacuum
overnight to get dried films. Add 2.5 mL of Tris–HCl buffer
Please cite this article in press as: Zhang, Y.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.005

Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.8. Lipoplex particle sizes and zeta potentials
from the wells, and the cells were washed twice with 500
lL of
prechilled PBS. According to Luciferase assay kit (promega) manu-
facture, 100
added to each well, and the cells were lysed for 30 min in a hori-
zontal 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 Eppendorf tubes and
Sizes and zeta potentials of the lipid/pDNA lipoplexes at various
N/P ratios were analyzed at room temperature on a dynamic light
scattering system (Zetasizer Nano ZS, Malvern instruments Led) at
25 °C. The lipoplexes particle solutions were first prepared by mix-
l
L of 1ꢃ cell lysis buffer diluted with PBS was then
ing L/DOPE lipids and pUC-19 (2 lg/mL) under diverse N/P ratios in
1 mL deionized water.
stored in ice. For the assay, 20 lL of this supernatant and 100 lL
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
4.9. Cell culture
HEK293 cells, A549 cells, 7402 cells, HepG-2 cells and U-2OS
cells were incubated in DMEM or 1640 containing 10% fetal bovine
serum (FBS) and 1% antibiotics (penicillin-streptomycin,
10,000 U mLꢁ1) at 37 °C in a humidified atmosphere containing
5% CO2.
was measured in a luminometer (Turner designs, 20/20,
Promega, USA) in standard single-luminescence mode. The integra-
tion 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 TEs of the
individual lipids was made based on the data for luciferase
expressed as relative light units (RLU)/mg of protein. All the exper-
iments were done in triplicates, and results presented are the aver-
age of at least two such independent experiments done on the
same days.
4.10. CCK-8 cytotoxicity assay
Toxicity of lipoplexes toward HEK 293 cells and A549 cells
determined by using
a Cell Counting Kit-8 (CCK-8). About
1.2 ꢃ 104 cells/well were seeded into 96-well plates.27 After 24 h,
optimized lipid/DOPE formulations were complexed with 0.2 lg
of DNA at various N/P ratios for 30 min; 50
50 L of media with 10% FBS were added to the cells, after the used
media were removed. Then, the cells were incubated with those
lipoplexes for 24 h. 10 L CCK-8 and 90 L PBS were added to each
well and the plates were incubated at 37 °C for another 1 h, after
removed the mixed solution in cells. Then, the absorbance of each
sample was measured using an ELISA plate reader (model 680,
BioRad) at a wavelength of 450 nm. The cell viability (%) was
obtained according to the manufacturer’s instruction. Blank con-
trol and Lipoplex prepared from Lipofectamine 2000 were used
as comparisons.
lL of lipoplexes and
Acknowledgements
l
This work was financially supported by the National Program
on Key Basic Research Project of China (973 Program,
2012CB720603), National Science Foundation of China (Nos.
21472131, 21232005), and Specialized Research Fund for the
Doctoral Program of Higher Education (20120181130006).
l
l
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.005.
4.11. In vitro transfection procedure
Gene transfection of a series of complexes was investigated in
HEK293 cells and A549 cells. In order to obtain about 80% conflu-
ent cultures at the time of transfection, 24-well plates were seeded
References and notes
1. Guo, X.; Huang, L. Acc. Chem. Res. 2011, 45, 971.
2. Liu, F.; Huang, L. J. Controlled Release 2002, 78, 259.
3. El-Aneed, A. J. Controlled Release 2004, 94, 1.
4. Luo, K.; Li, C.; Wang, G.; Nie, Y.; He, B.; Wu, Y.; Gu, Z. J. Controlled Release 2011,
155, 77.
5. Felgner, T. R. G. P. L.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J.
P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 1987, 7413.
6. Bhattacharya, S.; Bajaj, A. Chem. Commun. 2009, 4632.
7. Zhi, D.; Zhang, S.; Cui, S.; Zhao, Y.; Wang, Y.; Zhao, D. Bioconjugate Chem. 2013,
24, 487.
8. Zhi, D.; Zhang, S.; Wang, B.; Zhao, Y.; Yang, B.; Yu, S. Bioconjugate Chem. 2010,
21, 563.
9. Kedika, B.; Patri, S. V. Bioconjugate Chem. 2011, 22, 2581.
10. Rajesh, M.; Sen, J.; Srujan, M.; Mukherjee, K.; Sreedhar, B.; Chaudhuri, A. J. Am.
Chem. Soc. 2007, 129, 11408.
11. Wang, H.-J.; Liu, Y.-H.; Zhang, J.; Zhang, Y.; Xia, Y.; Yu, X.-Q. Biomater. Sci. 2014,
2, 1460.
12. Zhang, Y.-M.; Liu, Y.-H.; Zhang, J.; Liu, Q.; Huang, Z.; Yu, X.-Q. RSC Adv. 2014, 4,
44261.
13. Mukthavaram, R.; Marepally, S.; Venkata, M. Y.; Vegi, G. N.; Sistla, R.;
Chaudhuri, A. Biomaterials 2009, 30, 2369.
14. Liu, Q.; Jiang, Q.-Q.; Yi, W.-J.; Zhang, J.; Zhang, X.-C.; Wu, M.-B.; Zhang, Y.-M.;
Zhu, W.; Yu, X.-Q. Bioorg. Med. Chem. 2013, 21, 3105.
15. Liu, Q.; Yi, W.-J.; Zhang, Y.-M.; Zhang, J.; Guo, L.; Yu, X.-Q. Chem. Biol. Drug Des.
2013, 82, 376.
16. Huang, Q.-D.; Zhong, G.-X.; Zhang, Y.; Ren, J.; Fu, Y.; Zhang, J.; Zhu, W.; Yu, X.-Q.
PLoS One 2011, 6, e23134.
17. Izatt, R. M.; Pawlak, K.; Bradshaw, J. S.; Bruening, R. L. Chem. Rev. 1991, 91,
1721.
with 85,000 cell/well in 500 lL of media with 10% FBS for 24 h
before transfection. For the preparation of lipoplexes applied to
cells, various amounts of liposomes and DNA were serially diluted
separately in antibiotic-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
to readd 400
100 L lipoplexes. The plates were then incubated for 4 h at
37 °C in a humidified atmosphere containing 5% CO2. At the end
of incubation period, mixed medium was removed, and 500 L of
l
L. After that, the used medium was removed from the wells,
lL fresh medium without FBS cells and the above
l
l
fresh DMEM medium containing 10% FBS was added to each well.
Plates were further incubated for a period of 24 h before checking
the reporter gene expression.
For fluorescent microscopy assays, cells were transfected by
complexes containing pEGFP-N1. After 24 h incubation, themi-
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.
18. Bajaj, A.; Paul, B.; Indi, S. S.; Kondaiah, P.; Bhattacharya, S. Bioconjugate Chem.
2007, 18, 2144.
19. Bajaj, A.; Kondaiah, P.; Bhattacharya, S. Biomacromolecules 2008, 9, 991.
20. Niyomtham, N.; Apiratikul, N.; Suksen, K.; Opanasopit, P.; Yingyongnarongkul,
B.-E. Bioorg. Med. Chem. Lett. 2015, 25, 496.
For luciferase assays, cells were transfected by complexes con-
taining pGL-3. For a typical assay in a 24-well plate, 24 h post-
transfection as described above, the old medium was removed
Please cite this article in press as: Zhang, Y.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.005

8
Y.-M. Zhang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
21. Wang, B.; Yi, W.-J.; Zhang, J.; Zhang, Q.-F.; Xun, M.-M.; Yu, X.-Q. Bioorg. Med.
Chem. Lett. 2014, 24, 1771.
22. Obika, S.; Yu, W.; Shimoyama, A.; Uneda, T.; Miyashita, K.; Doi, T.; Imanishi, T.
Bioorg. Med. Chem. 2001, 9, 245.
23. Heyes, J. A.; Niculescu-Duvaz, D.; Cooper, R. G.; Springer, C. J. J. Med. Chem.
2002, 45, 99.
25. Kiessling, F.; Heilmann, M.; Lammers, T.; Ulbrich, K.; Subr, V.; Peschke, P.;
Waengler, B.; Mier, W.; Schrenk, H.-H.; Bock, M.; Schad, L.; Semmler, W.
Bioconjugate Chem. 2006, 17, 42.
26. De Cat, I.; Gobbo, C.; Van Averbeke, B.; Lazzaroni, R.; De Feyter, S.; van Esch, J. J.
Am. Chem. Soc. 2011, 133, 20942.
27. Luo, K.; Li, C.; Li, L.; She, W.; Wang, G.; Gu, Z. Biomaterials 2012, 33, 4917.
24. Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Song, J. B.; Suh, J. Org. Lett. 2002, 4,
4155.
Please cite this article in press as: Zhang, Y.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.005