Novel cationic lipids possessing protonated cyclen and imidazolium salt for gene delivery

Article abstract of DOI:10.1016/j.ejpb.2011.03.017

In this study, two novel protonated cyclen and imidazolium salt-containing cationic lipids differing only in their hydrophobic region (cholesterol or diosgenin) have been designed and synthesized for gene delivery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including DLS, gel retardation assay, ethidium bromide intercalation assay, and TEM demonstrated that these amphiphilic molecules are able to bind and compact DNA into nanometer particles that could be used as non-viral gene delivery agents. Our results from in vitro transfection showed that in association with DOPE, two cationic lipids can induce effective gene transfection in HEK293 cells. Furthermore, the gene transfection efficiencies of two cationic lipids were dramatically increased in the presence of calcium ion (Ca²⁺). It is notable that the gene transfection abilities of two cationic lipids were maintained in the presence of 10% serum. Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vector.

Full text of DOI:10.1016/j.ejpb.2011.03.017

European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Research paper
Novel cationic lipids possessing protonated cyclen and imidazolium salt
for gene delivery
Qing-Dong Huang a,1, Wen-Jing Ou a,1, Hong Chen a,b, Zhi-Hua Feng a, Jing-Yi Wang a, Ji Zhang a,
,
⇑
a,
Wen Zhu a, , Xiao-Qi Yu
⇑
⇑
a Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, and State Key Laboratory of Biotherapy,
West China Hospital, Sichuan University, Chengdu, PR China
b Department of Basic Science , Rongchang Campus, Southwest China University, Chongqing, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, two novel protonated cyclen and imidazolium salt-containing cationic lipids differing only
in their hydrophobic region (cholesterol or diosgenin) have been designed and synthesized for gene deliv-
ery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures
of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including DLS, gel
retardation assay, ethidium bromide intercalation assay, and TEM demonstrated that these amphiphilic
molecules are able to bind and compact DNA into nanometer particles that could be used as non-viral
gene delivery agents. Our results from in vitro transfection showed that in association with DOPE, two
cationic lipids can induce effective gene transfection in HEK293 cells. Furthermore, the gene transfection
efficiencies of two cationic lipids were dramatically increased in the presence of calcium ion (Ca2+). It is
notable that the gene transfection abilities of two cationic lipids were maintained in the presence of 10%
serum. Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title
cationic lipids have large potential to be efficient non-viral gene vector.
Received 27 November 2010
Accepted in revised form 15 March 2011
Available online 23 March 2011
Keywords:
Gene therapy
Gene delivery
Cationic liposome
Cyclen
Imidazolium salt
Calcium ion
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
large pieces of DNA, and ease of handling and preparation tech-
niques [9,10]. Since Felgner et al. first reported the utilization of
Gene therapy has tremendous potential for both inherited and
acquired diseases [1,2]. The clinical success of gene therapy re-
quires a safe and effective gene delivery system, which involves
viral or non-viral vectors [3]. The gene delivery efficacies of viral
vectors are, in general, superior to those of their non-viral counter-
parts. However, as viral vectors suffer from numerous biosafety-re-
lated disadvantages such as immunogenicity, restricted targeting
of specific cell types, size limitation on DNA, and potential for
mutagenesis [4], their use in clinic is limited. Compared to viral
vectors, non-viral vectors including cationic lipids [2,5], cationic
polymers [6,7], dendrimers [8], etc. are highly attractive because
of their excellent safety profile despite their low transfection effi-
ciency. Among non-viral vectors, cationic lipids have particularly
excellent potential for gene delivery applications because of their
lesser immunogenic nature, robust manufacture ability to deliver
unnatural diether-linked cationic lipid, N-[1-(2,3-dioleyloxy)pro-
pyl]-N,N,N-trimethylammonium chloride (DOTMA), as a synthetic
carrier to deliver gene into cells in 1987 [11], hundreds of cationic
lipids have been synthesized as candidates for non-viral gene
delivery [2,5]. However, all those cationic lipids were still far from
the requirement of in vivo gene therapy because of their potential
toxicity and relative low transfection efficiency. Consequently, the
development of novel nontoxic cationic structures and high effec-
tive cationic lipids is of great importance.
Previous reports have proved that cationic lipids using poly-
amine as the headgroups generally have much superior perfor-
mance in terms of gene delivery than cationic lipids of
a
quaternary amine or a single protonated amine [12–14]. However,
cationic lipids with long linear polyamine chains as headgroups
may also decrease gene delivery efficiency because of their relative
low binding ability toward DNA, which is the result of self-folding
of the linear lipopolyamine chains in cationic lipid structure [15].
Since cyclic polyamines and branched polyamines are hard to
self-fold, we postulated that using macrocyclic polyamines or
branched polyamines as the hydrophilic headgroups of cationic
lipids may solve this problem. Although many cationic amphi-
philes of linear polyamines [16,17] or branched polyamines [18]
have been widely studied, it seems that nearly no example using
⇑
Corresponding authors. Key Laboratory of Green Chemistry and Technology
(Ministry of Education), College of Chemistry, and State Key Laboratory of
Biotherapy, West China Hospital, Sichuan University, Chengdu 610064, PR China.
Tel.: +86 28 8546 0576, +86 28 8516 4063; fax: +86 28 8541 5886.
E-mail addresses: jzhang@scu.edu.cn (J. Zhang), zwjulia@163.com (W. Zhu),
xqyu@scu.edu.cn (X.-Q. Yu).
1
These authors contributed equally to this work.
0939-6411/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejpb.2011.03.017

Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
327
macrocyclic polyamines as hydrophilic headgroup on a cationic li-
pid formulation have been investigated. In addition, because of the
dissociation of positive charge on imidazolium salt group, cationic
lipids containing imidazolium polar heads [19,20] have been re-
ported to display higher transfection efficiency and reduced cyto-
toxicity when compared with classical quaternary ammonium
cationic lipids. For this reason, we have designed and synthesized
a novel 1,4,7,10-tetraazacyclododecane (cyclen)-based and imi-
dazolium-containing cationic lipid L (Fig. 1) by linking N-dodecy-
limidazole (NDI) group and cyclen ring via a p-xylyl bridge.
Unfortunately, results showed that although the liposome pre-
pared from L could bind to DNA and transfer it into cell without
any other extraneous agent such as cholesterol or dioleoylphos-
phatidylethanolamine (DOPE), it need high N/P ratio (39:1) to com-
pletely bind DNA, and only weak gene express was observed. This
might be attributed to its less protonated nitrogen atom and low
water solubility [21].
The hydrophobic structures cationic lipids have large effects on
both gene transfection efficiency and cytotoxicity. Steroid com-
pounds such as vitamin D [22], bile acids [23], antibiotic steroid
[24], cholestane, and litocholic acid [15] were used as hydrophobic
region of cationic lipids, which showed promising gene delivery
performance and lower cytotoxicity. In this study, two trifluoro-
acetic acid salts L1 and L2, in which the hydrophilic cyclen head-
group and steroid tails (cholesterol for L1 and diosgenin for L2,
Fig. 1) were bridged by imdazolium and an ester group, were de-
signed and synthesized. Stable liposomes could be prepared from
the two lipids themselves or in the presence of DOPE. Studies re-
vealed that the liposomes could bind and compact DNA into nano-
particles (lipoplexes). Their cytotoxicities and in vitro transfection
efficiencies were systematically investigated. We hope that the
study could broaden the applications of macrocyclic polyamines,
especially of cyclen.
Absolute chloroform (CHCl3) and dichloromethane (CH2Cl2) were
distilled after being dried with calcium hydride (CaH2). Anhy-
drous acetonitrile (CH3CN) was distilled after being dried with
P2O5. Chloroacetyl chloride and trifluoroacetic acid were dried
and purified under nitrogen by using standard methods. Column
chromatography was performed using 200–300 mesh silica gel.
All aqueous solutions were prepared from deionized or distilled
water. Cholesteryl chloroacetate (2a) [25] and the precursor 1-
(p-bromomethyl
benzyl)-4,7,10
tris(tertbutyloxycarbonyl)-
1,4,7,10-tetraazacyclododecane 4 [26] were prepared according
to reported procedures. IR spectra were recorded on a Shimadzu
FTIR-4200 spectrometer as KBr pellets or thin films on KBr
plates. The 1H NMR and 13C NMR spectra were measured on a
Varian INOVA-400 spectrometer, and the d scale in parts per
million was referenced to residual solvent peaks or internal
tetramethylsilane (TMS). MS-ESI spectra data were recorded
on a Finnigan LCQDECA and a Bruker Daltonics BioTOF mass
spectrometer, respectively. Fluorescence spectra were measured
by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
was purchased from Sigma–Aldrich (St. Louis, MO, USA). Micro-
BCA protein assay kit was obtained from Pierce (Rockford, IL,
USA). Luciferase assay kit was purchased from Promega (Madi-
son, WI, USA). Endotoxin-free plasmid purification kit was pur-
chased from TIANGEN (Beijing, China). The plasmids used in
the study were pGL-3 (Promega, Madison, WI, USA) coding for
luciferase DNA and pEGFP-N1 (Clontech, Palo Alto, CA, USA) cod-
ing for EGFP DNA. The Dulbecco’s modified Eagle’s medium
(DMEM), RPMI 1640 Medium, and fetal bovine serum were
purchased from Invitrogen Corp.
2.2. Preparation of diosgenin chloroacetate (2b)
Chloroacetyl chloride (19.8 g, 180 mmol) in anhydrous
dichloromethane (30 mL) was added dropwise to a stirred solu-
tion of diosgenin (14.9 g, 36 mmol) and triethylamine (5.8 mL,
4.2 g, 41.4 mmol) in anhydrous dichloromethane (120 mL). The
resulting reaction mixture was stirred for 4 h at room tempera-
ture. Excess chloroacetyl chloride was removed by washing the
2. Experimental section
2.1. Materials and methods
Unless otherwise stated, all chemicals and reagents were
obtained commercially and used without further purification.
reaction mixture with
a 5% solution of aqueous NaHCO3
(3 ꢀ 50 mL) and brine (50 mL). The organic phase was collected
and dried over anhydrous magnesium sulfate and then filtered.
The solvent was evaporated under reduced pressure to give the
product as a white solid. IR (cmꢁ1): 2953, 2832, 1737, 1452,
1378, 1313, 1164, 1049, 1003, 900. 1H NMR (CDCl3, 600 MHz):
d 0.80 (t, J = 6.0 Hz, 6H, diosgenin-H), 0.98–2.01 (br, 28H, dios-
genin-H), 2.37 (d, 2H, diosgenin-H), 3.39 (d, 1H, diosgenin-H),
3.48 (t, J = 54.6 Hz, 1H, diosgenin-H), 4.05 (s, 2H, ClCH2CO),
4.42 (m, 1H, diosgenin-H), 4.70 (m, 1H, diosgenin-H), 5.40 (s,
1H, C@CHA). HR MS (C29H44ClO4 + H): Calcd. 491.2928; Found.
491.2934.
2.3. General procedure for the preparation of imidazole derivatives 3
Chloroacetate derivative 2 (27.1 mmol) was added to a solution
of imidazole (22.15 g, 325.2 mmol), sodium carbonate(2.9 g,
28 mmol), potassium iodine (0.24 g, 1.4 mmol) in anhydrous chlo-
roform (36 mL), and dimethyl sufloxide (22 mL). The reaction mix-
ture was heated at 90 °C for 10 h, chloroform (180 mL) was added,
and the resulted solution was washed with a 5% solution of aque-
ous NaHCO3 (3 ꢀ 100 mL) and brine (100 mL). The organic phase
was collected and dried over anhydrous magnesium sulfate and
then filtered. The solvent was evaporated under reduced pressure
to give crude products, which were purified by flash chromatogra-
phy over silica (ethyl acetate) to obtain the corresponding
imidazole derivatives 3.
Fig. 1. Molecular structures of the studied cationic lipids.

328
Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
Compound 3a: IR (cmꢁ1): 3427, 3113, 2930, 2855, 1746, 1509,
2.5. General procedure for the preparation of title lipids L1 and L2
1464, 1377, 1291, 1217, 1110, 1078, 1003. 1H NMR (CDCl3,
400 MHz): d 0.67 (s, 3H, cholesterol-H), 0.85–1.99 (br, 38H, choles-
terol-H), 2.32 (d, 2H, cholesterol-H), 4.67 (s, 3H, NCH2CO and cho-
lesterol-H), 5.35 (s, 1H, C@CHA), 6.96 (s, 1H, imidazole-H), 7.10 (s,
1H, imidazole-H), 7.51 (s, 1H, imidazole-H). 13C NMR (CDCl3,
100 MHz): 19.27, 21.02, 22.57, 22.82, 23.83, 24.27, 27.64, 28.00,
28.21, 31.81, 31.88, 35.78, 36.17, 36.52, 36.83, 37.91, 39.50,
39.68, 42.30, 48.27, 49.95, 56.12, 56.65, 76.08, 119.94, 123.22,
129.66, 137.93, 139.00, 166.80. HR MS (C32H50N2O2 + H):
Calcd.495.3951; Found. 495.3954.
Compound 5 (1.2 mmol) was suspended in anhydrous dichloro-
methane (15 mL), and then, a solution of trifluoroacetic acid (5 mL)
in anhydrous dichloromethane (5 mL) was added dropwise under
ice bath and N2 atmosphere. And then, the obtained mixture was
stirred at room temperature for 6 h. The solvent and excess triflu-
oroacetic acid were removed under reduced pressure to give yel-
low residue. Then, under stirring, anhydrous ethyl ether was
added dropwise into the residue to precipitate the desired lipids
as white solid in 98–100% yields.
Compound 3b: IR (cmꢁ1): 3117, 2932, 1751, 1508, 1457, 1379,
1219, 1178, 1052, 984, 901, 816, 752, 663, 617. 1H NMR (CDCl3,
600 MHz): d 0.80 (m, 6H, diosgenin-H), 0.98–2.00 (br, 32H, dios-
genin-H), 2.35 (d, 2H, diosgenin-H), 3.36 (d, 1H, diosgenin-H), 3.40
(d, 1H, diosgenin-H), 4.41 (m, 1H, diosgenin-H), 4.68 (m, 3H,
ClCH2CO and diosgenin-H), 5.39 (s, 1H, C@CHA), 6.97 (s, 1H, imidaz-
ole-H), 7.11 (s, 1H, imidazole-H), 7.52 (s, 1H, imidazole-H). 13C NMR
(CDCl3, 100 MHz): 16.27, 17.13, 19.29, 20.80, 27.60, 28.79, 30.28,
31.35, 31.37, 31.81, 32.01, 36.66, 36.79, 37.88, 39.67, 40.24, 41.60,
48.28, 49.86, 56.39, 62.06, 66.83, 75.99, 80.76, 109.27, 119.93,
122.95, 129.68, 137.92, 139.04, 166.79. HR MS (C32H46N2O4 + H):
Calcd. 523.3536; Found. 523.3532.
Lipid L1: IR (cmꢁ1): 3436, 3101, 2951, 2867, 1746, 1681, 1565,
1515, 1457, 1414, 1375, 1203, 1174, 1131, 832, 800, 722. 1H NMR
(DMSO-d6, 400 MHz): d 0.60 (s, 3H, cholesterol-H), 0.80–2.00 (br,
41H, cholesterol-H), 2.36 (d, 2H, cholesterol-H), 2.60–2.90 (m, 8H,
cyclen-H), 3.00–3.20 (m, 8H, cyclen-H), 3.75 (s, 2H, benzene-CH2-
cyclen), 4.55 (m, 1H, cholesterol-H), 5.20 (m, 2H, benzene-CH2-imi-
dazolium) 5.35 (s, 1H, C@CHA), 5.51 (s, 2H, imidazolium-CH2–COO),
7.40 (m, 4H, benzene-H), 7.78 (s, 1H, imidazole-H), 7.87 (s, 1H,
imidazole-H), 9.36 (s, 1H, imidazole-H). 13C NMR (DMSO-d6,
100 MHz): 27.86, 28.24, 31.80, 35.67, 36.13, 36.52, 36.81, 37.97,
39.35, 39.42, 39.56, 39.77, 39.97, 42.32, 42.53, 42.64, 45.17, 47.61,
49.85, 50.22, 52.17, 55.48, 56.05, 56.58, 75.96, 79.71, 116.14,
119.12, 122.71, 122.96, 124.70, 128.82, 131.05, 134.28, 136.49,
137.93, 139.55, 158.78, 159.09, 159.40, 166.73. HR MS
(C48H77N6O2): Calcd. 769.6108; Found. 769.6102.
2.4. General procedure for the preparation of compounds 5
Lipid L2: IR (cmꢁ1): 3430, 3101, 2949, 1747, 1682, 1566, 1455,
1203, 1133, 1055, 984, 834, 800, 722. 1H NMR (DMSO-d6,
400 MHz): d 0.72 (m, 6H, diosgenin-H), 0.89–1.92 (br, 31H, dios-
genin-H), 2.31 (d, 2H, diosgenin-H), 2.71–2.83 (br, 8H, cyclen-H),
3.04–3.19 (br, 9H, cyclen-H and diosgenein-H), 3.38 (d, 1H, diosgen-
ein-H), 3.74 (s, 2H, benzene-CH2-cyclen), 4.26–4.28 (m, 1H,
diosgenin-H), 4.52 (m, 1H, diosgenin-H), 5.26 (s, 2H, benzene-CH2-
imidazolium), 5.35 (s, 1H, C@CHA), 5.51 (s, 2H, imidazolium-CH2–
COO), 7.38–7.42 (br, 4H, benzene-H), 7.79 (s, 1H, imidazolium-H),
7.87 (s, 1H, imidazolium-H), 9.36 (s, 1H, imidazolium-H). 13C NMR
(DMSO-d6, 100 MHz): 28.92, 30.25, 31.37, 31.89, 31.94, 36.67,
36.77, 37.94, 39.28, 39.49, 39.70, 39.91, 41.54, 42.49, 42.61, 45.15,
47.60, 49.79, 50.22, 52.19, 55.50, 56.16, 62.26, 66.38, 75.97, 80.63,
108.89, 122.69, 122.79, 124.71, 128.82, 131.06, 134.26, 136.46,
137.90, 139.59, 166.72. HR MS (C48H73N6O4): Calcd. 797.5693;
Found. 797.5691.
A solution of imidazole derivatives 3 (6 mmol) and 1-(p-bromo-
methyl benzyl)-4,7,10 tris(tertbutyloxycarbonyl)-1,4,7,10-tetraaz-
acyclododecane
4 (7.9 g, 12 mmol) in anhydrous acetonitrile
(30 mL) was refluxed at 90 °C over a period of 48–96 h until TLC
indicated the disappearance of the starting materials. Then, reac-
tion mixture was cooled, and the acetonitrile was rotary-evapo-
rated to give a yellow residue, which could be purified by
chromatography over silica (dichloromethane/methanol, 12:1) as
white foamy solid in 55–65% yields.
Compound 5a: IR (cmꢁ1): 3424, 2941, 2860, 1749, 1691, 1564,
1461, 1415, 1366, 1249, 1165, 1107, 1027, 978, 859, 773. 1H NMR
(CDCl3, 400 MHz): d 0.68 (s, 3H, cholesterol-H), 0.85–2.03 (br,
63H, cholesterol-H and Boc-H), 2.36 (d, 2H, cholesterol-H), 2.37–
2.38 (br, 4H, cyclen-H), 3.31–3.71 (br, 12H, cyclen-H), 3.73–3.76
(t, J = 11.6, 2H, benzene-CH2-cyclen), 4.69 (m, 1H, cholesterol-H),
5.37–5.38 (m, 3H, C@CHA and benzene-CH2-imidazolium), 5.49
(s, 2H, imidazolium-CH2–COO), 7.27 (s, 1H, imidazole-H), 7.32–
7.38 (br, 5H, imidazole-H and benzene-H), 10.70 (s, 1H, imidaz-
ole-H). 13C NMR (CDCl3, 100 MHz): 22.53, 22.79, 23.80, 24.24,
27.60, 27.97, 28.18, 28.45, 28.66, 30.91, 31.76, 31.86, 35.75, 36.14,
36.49, 36.80, 37.86, 39.47, 39.66, 42.27, 49.94, 50.46, 56.11, 56.65,
79.47, 121.30, 123.28, 123.84, 128.87, 138.93, 165.38, 206.98. HR
MS (C63H101N6O8): Calcd. 1069.7675; Found. 1069.7616.
2.6. Preparation of cationic liposome
Individual cationic lipid (0.005 mmol) or its mixture with DOPE
in the desired mole ratio was dissolved in anhydrous chloroform
(5 mL) in autoclaved glass vials. Thin films were made by slowly
rotary-evaporating the solvent at room temperature. Last trace of
organic solvent was removed by keeping these films under vacuum
above 8 h. The dried films and Tris–HCl buffer (10 mM, pH 7.4)
were preheated to 70 °C, and then the buffer was added to the
films to the final lipid concentration of 1 mM. The mixtures were
vortexed vigorously until the films were completely resuspended.
Sonication of these suspensions for 20 min in a bath sonicator at
60 °C afforded the corresponding cationic liposomes that were
stored at 4 °C.
Compound 5b: IR (cmꢁ1): 3423, 3145, 3951, 1748, 1690, 1564,
1459, 1415, 1367, 1248, 1163, 1105, 1052, 1006, 982, 920, 900,
862, 774. 1H NMR (CDCl3, 400 MHz): d 0.79 (m, 6H, diosgenin-H),
0.96–2.03 (br, 55H, diosgenin-H and Boc-H), 2.36 (d, 2H, dios-
genin-H), 2.51–2.70 (br, 4H, cyclen-H), 3.30–3.71 (br, 14H, cyclen-
H and diosgenein-H), 3.72 (t, J = 8, 2H, benzene-CH2-cyclen), 4.42
(m, 1H, diosgenin-H), 4.68 (m, 1H, diosgenin-H), 5.35–5.39 (m, 3H,
C@CHA and benzene-CH2-imidazolium), 5.46 (s, 2H, imidazolium-
CH2–COO), 7.20 (s, 1H, imidazolium-H), 7.30–7.39 (br, 4H,
benzene-H), 7.44 (s, 1H, imidazolium-H), 10.52 (s, 1H, imidazoli-
um-H). 13C NMR (CDCl3, 100 MHz): 20.77, 27.56, 28.44, 28.65,
28.75, 30.24, 31.30, 31.34, 31.78, 31.99, 36.62, 36.76, 37.83, 39.65,
40.21, 41.56, 49.85, 50.46, 53.23, 56.39, 62.05, 66.79, 76.79, 77.06,
79.47, 80.72, 109.23, 121.38, 122.97, 123.87, 128.86, 137.93,
138.99, 165.45. HR MS (C63H97N6O10): Calcd. 1097.7266; Found.
1097.7002.
2.7. Amplification and purification of plasmid DNA
pGL-3 and pEGFP-N1 plasmids were used. The former one was
used as the luciferase reporter gene, which was transformed in
JM109 Escherichia coli, and the latter one was used as the enhanced
green fluorescent protein reporter gene, which was transformed in
E. coli DH5a. Both plasmids were amplified in E. coli grown in LB
media at 37 °C and 220 rpm overnight. The plasmids were purified

Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
329
by an EndoFree TiangenTM Plasmid Kit. Then, the purified plas-
mids were dissolved in TE buffer solution and stored at ꢁ20 °C.
The integrity of plasmids was confirmed by agarose gel electropho-
resis. The purity and concentration of plasmids were determined
by the ratio of ultraviolet (UV) absorbances at 260 nm to 280 nm.
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 de-
sired N/P ratios, the final lipoplexes volume was 200
lL, and the
DNA was used at a concentration of 2 g/well unless otherwise
l
2.8. Preparation of lipid/DOPE/DNA complexes (lipoplexes)
noted. After 30 min of complexation, old cell culture medium
was removed from the wells, cells were washed once with ser-
To prepare the liposome/pDNA complexes (lipoplexes), various
amounts of cationic lipids were mixed with a constant amount of
DNA by pipetting thoroughly at various N/P ratio, and the mixture
was incubated for 30 min at room temperature. The theoretical N/P
ratio represents the charge ratio of cationic lipid to nucleotide base
(in mole) and was calculated by considering the average nucleotide
mass of 350.
um-free DMEM, and the above 200 lL lipoplexes was added to
each well. The plates were then incubated for 4 h at 37 °C in a
humidified atmosphere containing 5% CO2. At the end of incuba-
tion period, medium was removed, and 500 lL of 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 calcium ion-promoted transfection studies, lipoplexes ap-
plied to cells were prepared as follows: appropriate amounts of lip-
osomes, DNA, and CaCl2 were serially diluted, respectively, in both
serum- and antibiotic-free DMEM or serum-free RPMI 1640 (or
RPMI 1640 containing 10% FBS); then, the DNA solutions were
added into liposome solutions and mixed briefly by pipetting up
and down several times, after which the mixtures were immedi-
ately added to CaCl2 solution and incubated at room temperature
for about 30 min to obtain lipoplexes that were applied to cells.
2.9. Gel retardation assay
To determine the formation of liposome/pDNA complex (lipo-
plexes), lipoplexes of various N/P ratios ranging from 1 to 12 were
prepared as described above. Constant amount of 5 lg DNA was
used here; 10 lL of each lipoplexes solution was electrophoresed
on the 1% (W Vꢁ1) agarose gel containing Gold view and Tris–ace-
tate (TAE) running buffer at 110 V for 20 min. DNA was visualized
with a UV lamp using a BioRad Universal Hood II.
The final lipoplex volume was 200
lL, the DNA was used at a con-
centration of 2 g/well unless otherwise noted, and the CaCl2
l
2.10. Ethidium bromide intercalation assay
(100 mM) stock solution was prepared in dH2O and sterilized by
filtering.
Fluorescence emission due to ethidium bromide (EB) at 605 nm
was monitored in a fluoromax-4 spectrofluorimeter (excitation
wavelength was 497 nm with 3 nm slit widths for both excitation
and emission). Typically fluorescence emission was measured for
For fluorescent microscopy assays, cells were transfected by
complexes containing pEGFP-N1. After 24 h incubation, the
microscopy 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 transfection reagent Lipofectamine 2000™ based on the
standard conditions specified by the manufacture.
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
EB (5
(11 M) was added, and the fluorescence emission due to EB upon
intercalative complexation with DNA was measured again at 25 °C.
Then, aliquots of a given cationic liposome (0.68 M) were added
lM) in 10 mM Tris, pH 7.4 buffer. To this solution, CT-DNA
l
l
into the EB/CT-DNA solution for further measurement. If F0 is the
fluorescence intensity (FI) of unintercalated, Fmax is the FI of fully
intercalated EB, and Fx is the FI for a given concentration of lipo-
some, then %FI was calculated as %FI = (Fx ꢁ F0)/(Fmax ꢁ F0).
from the wells, and the cells were washed twice with 500
pre-chilled PBS. According to Luciferase assay kit (promega) man-
ufacture, 100
lL of
l
L of 1 ꢀ cell lysis buffer diluted with PBS was then
2.11. Dynamic light scattering (DLS)
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
Particle size and zeta potential of liposomes or lipoplexes at var-
ious N/P ratio were measured by a dynamic light scattering system
(Zetasizer Nano ZS, Malvern Instruments Led) at 25 °C. Then,
stored in ice. For the assay, 20 lL of this supernatant and 100 lL
100
l
L of liposomes or lipoplexes prepared as aforementioned
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 effi-
ciencies of the individual lipids 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.
was diluted to 1 mL with deionized water, RPMI 1640 (or contain-
ing 10% serum), or DMEM (or containing 10% serum) prior to
measurement.
2.12. Cell culture
HEK (human embryonic kidney) 293 cells were incubated in
Dulbecco’s modified Eagle’s medium (DMEM) 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.
2.13. Transfection procedure
In order to obtain about 80% confluent cultures at the time of
transfection, 24-well plates were seeded with 75,000 cell/well in
2.14. Cytotoxicity assays
500
l
L antibiotic-free media 24 h before transfection. For the prep-
Toxicity of lipoplexes toward HEK293 cells was determined by
using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) reduction assay following literature procedures [25].
aration of lipoplexes applied to cells, various amounts of liposomes
and DNA were serially diluted separately in both serum- and anti-

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Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
About 7000 cells/well were seeded into 96-well plates. After 24 h,
optimized lipid/DOPE formulations were complexed with 0.2 g of
DNA at various N/P ratios for 30 min; 100 L of lipoplexes were
added to the cells in the absence of serum. After 4 h of incubation,
lipoplex solutions were removed, and 200 L of media with 10%
FBS was added. After 24 h, 20 L of MTT solution was added, and
the cells were incubated further for 4 h. Blue formazan crystals
were seen at well when checked under microscope. Media were re-
moved, 150 lL of DMSO was added per well, and then, plates were
incubated on a shaker for 10 min at room temperature to dissolve
blue crystal. The absorbance was measured using a microtiter plate
reader. The % viability was then calculated as [[A490(treated cells)-
background]/[A490(Untreated cells)-background]] ꢀ 100. Lipo-
plexes prepared from Lipofectamine 2000 were used as control.
first time as the hydrophobic region of cationic lipid to formulate
the diosgenin-based cationic lipid L2. As shown in the scheme,
chloroacetate derivatives 2 were first prepared through esterifica-
tion between corresponding steroids (cholesterol 1a or diosgenin
1b) and chloroacetyl chloride. With potassium iodide as catalyst
and sodium carbonate as base, chloroacetate derivatives 2 react
with imidazole in chloroform and dimethyl sulfoxide at 90 °C for
about 10 h to give the imidazole derivatives 3, which could further
react with precursor 4 in acetonitrile to form tri-Boc-protected lip-
ids 5. The final products L1 and L2 were obtained by removing the
Boc groups by trifluoroacetic acid in CH2Cl2. To improve the water
solubility, the trifluoroacetic acid salt was directly used for further
l
l
l
l
studies. All new compounds were characterized by IR, 1H NMR, 1
NMR, and HRMS.
C
2.15. Transmission electron microscopy
3.2. Formation of liposomes and lipid/DOPE/DNA complexes
(lipoplexes)
Transmission electron microscopy was carried out to determine
the morphology and particle size distribution of the optimized
Cationic liposomes are formed from either individual cationic li-
pid or more frequently from a combination of cationic lipid and
neutral lipids, such as DOPE, which usually dramatically enhance
the transfection efficiency. Each of cationic lipids was mixed in dif-
ferent molar ratios (1:0, 1:1, 1:2, 1:3, and 1:4) with DOPE to deter-
mine the most auspicious combination. Under these mole ratios,
the particle size of formed liposomes ranged from 40 nm to
180 nm with zeta potentials in the range of 20–75 mV. Under the
optimized mole ratio of 1:3 obtained in transfection experiments
(see Fig. 7), the mean particle sizes of the liposome vesicles were
47.6 and 68.3 nm for L1/DOPE and L2/DOPE, respectively. Mean-
while, the zeta potentials for these two nanoparticles were 50.9
(L1/DOPE) and 24.9 mV (L2/DOPE). L1-based liposome has dis-
tinctly smaller size and higher zeta potential, and these differences
were undoubtedly originated from the different hydrophobic moi-
eties of cationic lipids.
lipoplexes by negative staining using 1% uranyl acetate. A 10 lL
sample of freshly prepared lipoplexes was loaded onto Formvar-
coated, 400 mesh copper grids and allowed to remain for 1 min.
Excess fluid was wicked off the grids by touching their edges to fil-
ter paper, and 10 lL of 1% uranyl acetate was applied on the same
grid after which the excess stain was similarly wicked off. The grid
was air dried for 20 min, and the specimens were observed under
TEM (JEM 100CX II) operating at an acceleration voltage of 80 keV.
3. Results and discussion
3.1. Synthesis of cationic lipids
The title lipids, whose protonated cyclen headgroup and a ste-
roid tails were bridged by imdazolium and an ester group, were de-
signed and synthesized along the route shown in Scheme 1.
Considering the influence of hydrophobic moiety of cationic lipids
on their gene transfection efficiency [27] and the unique structure
of diosgenin that has an acetal group, we selected diosgenin for the
Liposome/DNA complexes (lipoplexes) were prepared by com-
bining constant amount of DNA with varying amounts of lipo-
somes. Conventional electrophoretic gel retardation assay was
first used to study the electrostatic interactions between plasmid
Scheme 1. Synthesis of target lipids L1 and L2. Reaction conditions: (a) chloroacetyl chloride/Et3N/CH2Cl2, 4 h, rt; (b) imidazole/sodium carbonate/potassium iodine/CHCl3/
DMSO, 10 h, 90 °C; (c) CH3CN, reflux; (d) trifluoroacetic acid/CH2Cl2, 6 h, rt.

Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
331
cence quench results were also quite consistent with the following
classical Stern–Volmer equation [28]:
F
max=Fx ¼ 1 þ KsvðQÞ
where Fmax and Fx are as aforementioned in experimental section, Q
is the mole concentration (mol/L) of lipid, and Ksv is the quenching
constant (namely binding constant), which reflect the binding abil-
ity of lipid to DNA. After linear fitting (Fig. 3B), the Ksv values
(namely binding constant) of the L1/DOPE and L2/DOPE were ob-
tained as 1.02 ꢀ 105 Mꢁ1 (R2 = 0.99788) and 1.69 ꢀ 105 Mꢁ1
(R2 = 0.99288), respectively, showing that L2 has better DNA-bind-
ing ability than L1.
Fig. 2. Electrophoretic gel retardation assays of liposomes/DNA complexes (lipo-
plexes) at different N/P ratios. The molar ratio of lipid/DOPE was 1:3.
DNA and cationic lipids under different N/P ratios. As shown in
Fig. 2, the liposomes formed from L1 and L2 could partly retard
plasmid DNA at N/P ratio of 3. Further, both liposomes completely
inhibit the electrophoretic mobility of plasmid DNA at N/P P 6,
indicating that they have similar DNA-binding abilities. Compared
with lipid L (Fig. 1) that need high N/P ratio (39/1) to completely
retard DNA [21], L1 and L2 showed dramatically increased DNA-
binding abilities.
The binding ability of the liposomes (lipid/DOPE = 1:3) to CT
(calf thymus) DNA was further studied by fluorescence spectros-
copy with the use of ethidium bromide (EB). As shown in Fig. 3A,
the addition of liposomes to EB pretreated with DNA caused appre-
ciable decrease in the emission intensity, indicating that EB that
bound to DNA was partially replaced by the lipids. The fluores-
The appropriate size and zeta potential of lipoplexes are of crit-
ical important for cationic lipids used as gene vectors. For easier
deposition on cell membrane and subsequent uptake, the lipoplex-
es with moderate diameters (from 0.4 to 1.4 lm) were most effec-
tive for in vitro gene transfection [29]. The mean particle size and
zeta potential of lipoplexes at various N/P ratio (as in gel retarda-
tion assays) in water were shown in Fig. 4. The two liposomes
could bind and compact DNA into nanoparticles at various N/P ra-
tios (1–12, Fig. 4A). To our surprise, the sizes of both lipoplexes at
N/P ratio of 3 suddenly changed to above 700 nm compared with
about 100 nm at other N/P ratios. The zeta potentials shown in
Fig. 4B give an increased trend along with the increase in N/P ra-
tios. Generally, the lipoplex formed from L1 showed relatively
higher zeta potential (about ꢁ4 to 40 mV) than that formed from
Fig. 3. (A) Change of relative fluorescence of EB bound to DNA by the addition of L1/DOPE or L2/DOPE to different N/P ratios. The lipid/DOPE ratio was 1:3. (B) Calculation of
DNA-binding constant for the two liposomes according to the Stern–Volmer equation.
Fig. 4. Particle sizes (A) and zeta potentials (B) of lipid/DOPE/DNA (mole ratio of lipid/DOPE: 1:3) complexes at different N/P ratios by DLS.

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Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
Fig. 5. Particle sizes (A) and zeta potentials (B) of L2/DOPE/DNA lipoplexes (N/P = 6) at different times by DLS.
L2 (about ꢁ10 to 20 mV). Both lipoplexes have low particle sizes
(100–200 nm) and moderate positive zeta potentials (20–30 mV)
at the N/P ratio of 6, indicating that DNA could be tightly com-
pacted into nanoparticles with proper size and electric charge un-
der such N/P ratio. These results were consistent with those
obtained from gel retardation assays.
To determine the stability of the formed liposomes and lipo-
plexes in different media, the particle size and zeta potential of
L2-based liposomes (L2/DOPE = 1:3) and lipoplexes at different
times have been measured in water, RPMI 1640 medium, and
DMEM medium with or without 10% serum. The L2/DOPE liposome
was stable in water, and no evident change of particle size was ob-
served even after 4 days (data not shown). For the lipoplex formed
from L2/DOPE/DNA, the results in Fig. 5 showed that in the absence
of serum, the lipoplex is stable in water with relatively small par-
ticle size (100–200 nm) and positive zeta potential. However, after
addition of 10% serum, the particle size increased dramatically to
Transfection efficiency of cationic lipids has been linked to not
only the physicochemical characteristics but also the morphology
of lipoplexes. To get further insights into the lipid–DNA complexa-
tion, we examined the lipoplexes formed at the lipid/DOPE ratio of
1:3 and N/P ratio of 1:6 by transmission electron microscopy. Rep-
resentative electron micrographs of the lipoplexes are shown in
Fig. 6. The images showed that L1/DOPE/DNA lipoplex gave homo-
geneous spherical particles, while L2/DOPE/DNA lipoplex gave
non-homogeneous oval-shaped particles. The sizes of these nano-
particles were found to be in the range of 100–250 nm, which
was in accord with the results described in DLS analysis.
3.3. In vitro transfection
The gene delivery efficiencies of many of the cationic lipid-based
formulations depend on the lipid/DOPE ratio in liposomes and on
the molecular structure of the cationic lipids. To find out the opti-
mized lipid/DOPE ratio, we carried out the fluorescent microscopy
assay by using pEGFP-N1 plasmid DNA as reporter gene in
HEK293 cells. The mole ratio of cationic lipids against DOPE was
varied (lipid/DOPE ratio of 1:0, 1:1, 1:2, 1:3, and 1:4) under the
N/P ratio of 6. The pictures listed in Fig. 7 showed that two neat lip-
ids were found to have no transfection ability (Fig. 7A and F). Dis-
tinct results could be obtained as that L1/DOPE/DNA lipoplexes
have less transfection efficiency (Fig. 7B–E) than L2/DOPE/DNA
(Fig. 7G–J). For both lipids, the lipid/DOPE ratio of 1:3 (Fig. 7D and
I) was found to be the best combination for efficient transfection.
Therefore, this ratio was used in our subsequent investigations.
Calcium ion was used to improve transfection efficiency of cat-
ionic liposomes by facilitating the cell uptake of nucleic acids
through endocytosis and the entrance toward nucleus after endo-
somal escape [30,31]. To investigate the effect of calcium ions on
the gene transfection performance of the title lipids, keeping the li-
pid/DOPE ratio at 1:3 and N/P ratio at 6, we carried out fluorescent
1–5 lm, while zeta potential was reversed to negative. These re-
sults indicated that the lipoplex is prone to bind with the serum
proteins, promoting aggregation and possibly phagocytosis. On
the other hand, the positive surface charge was neutralized by
the negatively charged proteins, leading to the charge reverse.
microscopy assay by varying the concentration (0–20 lM) of cal-
cium ions in transfection medium and using pEGFP-N1 plasmid
DNA as reporter gene in HEK293 cells. The results shown that sig-
nificant transfection enhancement was achieved in the presence of
4–6 lM calcium ions (data not shown). To further verify the obser-
vations, luciferase assays were also processed by using pGL-3 as re-
porter gene in the same cell type. As shown in Fig. 8A, the
transfection efficiency of L1/DOPE/DNA lipoplex increased with
the addition of Ca2+ to 4 mM, while further addition of Ca2+ led
to less transfection efficiency. To our delight, dramatic increase
in transfection efficiency was found in the experiments involving
L2 as cationic lipid. In the presence of 6 mM of Ca2+, the luciferase
expression in the transfection was found as about seven times as
Fig. 6. Representative transmission electron micrographs of the lipoplexes
prepared from L1/DOPE/DNA (A) and L2/DOPE/DNA (B).

Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
333
Fig. 7. Fluorescent microscope images of HEK293 cells transfected by L1-based (A–E) and L2-based (F–J) lipoplexes at the lipid/DOPE ratio of 1:0 (A, F), 1:1 (B, G), 1:2 (C, H),
1:3 (D, I), and 1:4 (E, J). N/P ratios in all experiments were 6. The cells were observed by fluorescence microscopy after 24 h transfection. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. (A) Luciferase expression in HEK293 cells transfected by Lipid/DOPE/DNA complexes (lipoplexes) at N/P ratio of 6 in the absence (control) and in the presence of 4 and
6 l l .
M of Ca2+. (B) Luciferase expression in HEK293 cells transfected by Lipid/DOPE/DNA complexes (lipoplexes) at various N/P ratios (3–15) in the presence of 6 M of Ca2+
pGL-3 was used as reporter gene in each case, and the concentration of Ca2+ was calculated with respect to the final volume (200
cells.
lL) of the transfection solution applied to
more than that without the using of Ca2+. According to the results,
Ca2+ was employed at the concentration of 6 mM in subsequent
experiments.
different changes of transfection efficiency were found for the two
lipoplexes. For L2-based lipoplex, transfection efficiency also de-
creased, and the amount of expressed luciferase was found as half
of those obtained in the experiment without the use of serum.
Meanwhile, for L1-participated transfection, increased luciferase
expression was found in the experiments using RPMI 1640 as cul-
ture in the presence of 10% serum, but the transfection efficiency
was still lower than that obtained in L2-participated transfection.
The lipoplexes were then subjected to luciferase assays at dif-
ferent N/P ratios (3–15) using optimized conditions studied above.
As shown in Fig. 8B, the lipoplexes formed from L1 and L2 showed
maximum transfection efficiencies at N/P ratio of 6. Furthermore,
at optimized N/P ratios studied in our experiments, L2 obviously
yielded much higher transfection than L1, indicating that minor
structure variation in the hydrophobic moiety might lead to large
difference in transfection efficiency. Cholesterol, as a major compo-
nent of cell membrane, was most commonly used as hydrophobic
moiety of cationic amphiphiles because of its easy fusion with cell
membranes. Cholesterol-based cationic lipids have been widely
studied for gene delivery and shown excellent gene transfection
[32,33]. However, results herein suggested that in the studied type
of cationic lipids, diosgenin is much suitable to be the hydrophobic
group for high in vitro gene transfection than cholesterol.
Serum has been reported to be the major hurdle in liposome-
mediated gene delivery, and many studies showed that the
transfection efficiency of cationic lipoplexes would decrease in
the presence of serum. The negatively charged serum proteins
might cause aggregation of cationic lipoplex by electrostatic inter-
action, leading to increased toxicity. Luciferase expression assays
were carried out to estimate the effect of serum in the transfection
system studied herein. As shown in Fig. 9, the transfection efficien-
cies dramatically decreased in the experiments using 10% serum in
DMEM medium. However, by using RPMI 1640 as culture medium,
Fig. 9. Luciferase expression in HEK293 cells transfected by Lipid/DOPE/DNA
complexes (lipoplexes) under optimized conditions. Cells were transfected in
DMEM culture medium without (ꢂ) or with (ꢂꢂ) 10% serum or in RPMI 1640 culture
medium with 10% serum (ꢂꢂꢂ). Lipofectamine 2000™ (Lip) was used as control.

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Q.-D. Huang et al. / European Journal of Pharmaceutics and Biopharmaceutics 78 (2011) 326–335
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In this study, we designed and synthesized two novel proton-
ated cyclen and imidazolium salt-based cationic lipids, which dif-
fer only in their hydrophobic region (cholesterol or diosgenin). In
association with DOPE, two cationic lipids can easily form lipo-
somes and effectively bind and compact DNA into nanoparticles,
which were characterized by size and zeta potential measurements
and TEM. The gene transfection efficiencies of two cationic lipids
could be dramatically increased in the presence of calcium ion
(Ca2+). Further, although DLS showed that aggregation might occur
in the presence of 10% serum, the gene transfection abilities of two
cationic lipids were maintained with serum. Different cytotoxici-
ties were found for two lipoplexes. Although the transfection
efficiencies of title lipids were lower than that of commercially
available lipofectamine 2000, we believe that this type of cationic
lipids has large potential to be efficient non-viral gene vectors. Fur-
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lipids are now in progress.
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This work was financially supported by the National Science
Foundation of China (Nos. 20972104, 20902062, and 20725206),
the Program for Changjiang Scholars and Innovative Research
Team in University, the Key Project of Chinese Ministry of Educa-
tion in China, and the Scientific Fund of Sichuan Province for Out-
standing Young Scientist. We also thank Analytical & Testing
Centre of Sichuan University for NMR analysis.
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