Synthesis and characterization of novel cationic lipids derived from thio galactose

Article abstract of DOI:10.1007/s11743-013-1474-0

Two double chain cationic lipids QAS C_n -2-S (n = 12, 14) derived from thio galactose and carbamate-linkage tertiary amine were synthesized and their structures were confirmed by MS, TOF-MS, ¹H NMR and ¹³C NMR. The QAS C₁₂-2-S revealed superior surface activity compared with QAS C₁₄-2-S with lower CMC and γ_CMC. Though Lipo C₁₂-2-S displayed large average particle-size with high polydispersity, positive charged Lipo C_n -2-S can be combined with the negative charged DNA, also negatively stained TEM images confirmed the formation of vesicles. All the above prove that the Lipo C_n -2-S is helpful for gene transfection.

Full text of DOI:10.1007/s11743-013-1474-0

J Surfact Deterg
DOI 10.1007/s11743-013-1474-0
ORIGINAL ARTICLE
Synthesis and Characterization of Novel Cationic Lipids
Derived from Thio Galactose
•
Weihong Qiao Min Zhou Limei Luo
•
Received: 22 October 2012 / Accepted: 8 April 2013
Ó AOCS 2013
Abstract Two double chain cationic lipids QAS Cn-2-S
(n = 12, 14) derived from thio galactose and carbamate-
linkage tertiary amine were synthesized and their structures
were confirmed by MS, TOF-MS, 1H NMR and 13C NMR.
The QAS C12-2-S revealed superior surface activity com-
human chromosomes [4]. However, cationic liposomes
have low transfection efficiency in comparison with vital
carriers. The transfection efficiency could be enhanced
owing to the specific binding of ligands and receptors. As
we all know, there is rich asialoglycoprotein on the surface
of the liver organ, which as a receptor can specially rec-
ognize the compounds whose terminal part has non-
reductive galactose and GalNAc [5]. It has been confirmed
that the galactose receptor-mediated pathway is the best for
the hepatocytes in the all receptor-mediated pathways [6].
The liver targeting and transfection efficiency would be
significantly improved by the galactose-modified drug [7]
or polymer carriers [8] in this way.
pared with QAS C14-2-S with lower CMC and cCMC
.
Though Lipo C12-2-S displayed large average particle-size
with high polydispersity, positive charged Lipo Cn-2-S can
be combined with the negative charged DNA, also nega-
tively stained TEM images confirmed the formation of
vesicles. All the above prove that the Lipo Cn-2-S is helpful
for gene transfection.
Keywords Synthesis ꢀ Thio galactose ꢀ Cationic lipids ꢀ
In 1998, Kawakami et al. [8] reported three novel ga-
lactosylated cholesterol derivatives, cholesten-5-yloxy-N-
(4-((1-imino-c-b-D-thiogalactosyl-ethyl) amino) alkyl)
formamide (Gal-C2-Chol, Gal-C4-Chol, Gal-C6-Chol).
Liposome/DNA complexes prepared with these lipids
showed low cytotoxicity in human hepatoma HepG2 cells.
Gal-C4-Chol (in Fig. 1)/DC-Chol/DOPE (3:3:4) liposomes
showed higher transfection activity. In 2007, Shigeta et al.
[9] proposed a novel pH-sensitive histidine-modified ga-
lactosylated cholesterol derivative (Gal-His-C4-Chol,
shown in Fig. 1), for a more efficient gene delivery to
hepatocytes. These researches show that thiogalactosyl
group may have a good influence on gene transfection
efficiency. Additionally, carbamate as a linker has chemi-
cal stability in a certain degree, but it can be decomposed
by variation of pH. So the transfection efficiency of the
cationic galactolipid could be enhanced and the cytotox-
icity could be reduced in theory.
Vesicle
Introduction
As one of the significant means of cancer treatment, gene
therapy has received much attention for decades. It is
crucial to find a carrier to enhance transfection efficiency
with the in-depth research of this technology.
Cationic liposome–DNA complexes are attracting con-
siderable attention as gene vectors due to their safety and
other inherent advantages over viral delivery methods
[1–3]. These advantages include ease and variability of
preparation, lack of immunogenicity, and a capacity for
DNA of unlimited size, allowing for delivery of artificial
W. Qiao (&) ꢀ M. Zhou ꢀ L. Luo
In this paper, we synthesized two novel double chains
cationic lipids Cn-2-S derived from thio galactose and car-
bamate-linkage tertiary amine and investigated their phys-
ico-chemical properties such as critical micelle
State Key Laboratory of Fine Chemicals, School of Chemical
Engineering, Dalian University of Technology, Dalian,
Liaoning 116024, People’s Republic of China
e-mail: qiaoweihong@dlut.edu.cn
123

J Surfact Deterg
Fig. 1 Chemical structures of
Gal-C4-Chol a and Gal-His-C4-
Chol b
(A) Gal-C4-Chol
CH2OH
OH
OH
O
NH
O
S
N
H
N
H
O
4
OH
(B) Gal-His-C4-Chol
H
N
N
H
OH
OH
HO
O
NH2
H
N
H
H
O
S
N
N
H
O
OH
O
The mass spectrum was obtained with Liquid chromato-
graphy/quadrupole time of flight tandem mass spectrometry
(Micromass Britain, LC/Q-TOF-MS) and High Perfor-
mance Liquid Chromatography/Mass selective Detector
(HP, America, Model HP 1100LC/MSD). 1H-NMR and
13C-NMR spectra were recorded on a Bruker 400-MHz
instrument (Bruker, Switzerland, Model AVANCE II
400 MHz). Melting points were tested with a micro-melting
point apparatus (Ningbo Yongxin Optical Co., Ltd., China,
Model X-4). The surface tension was determined with a
processor tensiometer (Bowing Industry Corporation,
America, Model TX-500C) by the spinning drop principle.
Particle sizes and Zeta potentials were measured by nano
particle size and zeta potential measurement (Zetasizer nano
series Nano-ZS90, Malvern, UK). Vesicles were observed
with a transmission electron microscope (JEOL, Japan,
Model JEM-1200EX).
OH OH
O
O
Br
N
R
S
HO
O
N
OH
H
H
O
N
R
O
R=C12H25, C14H29
Fig. 2 General structure of the cationic thio galactolipid
concentrations (CMC), surface tension at CMC (cCMC),
critical packing parameter (P). An ingenious combination
of thio galactose and carbamate was represented on the
structure of this new galactolipid (Fig. 2). The vesicle for-
mation was determined by transmission electron micros-
copy (TEM) as well as particle-size and zeta-potential.
Experimental Procedures
Synthesis
General
The thiogalactopyranoside was synthesized in four steps, as
shown in Scheme 1. The cationic galactolipid was obtained
during the quarterisation process. The general procedure is
described in Scheme 2.
D-Galactose was purchased from the Sinopharm Chemical
Reagent Co. Ltd., China. Thiourea and Potassium metabi-
sulfite were purchased from Guangdong Shantou Xilong
Chemical Factory, China. 1,2-Dibromoethane was obtained
from Chengdu Kelong Chemical Reagent Factory, China.
Potassium carbonate, sodium sulfate and sodium methox-
ide were purchased from Tianjin Fuchen Chemical Plant,
China. D113 cation exchange resin was purchased from
Shenyang Xinxing Reagent Factory, China. 1,2-Dioleoyl-
sn-glycero-3-phosphoethanolamine (DOPE) was purchased
from Sigma-Aldrich (USA). Dichloromethane, chloroform,
methanol, ethanol, hexane, ethyl acetate, and acetone were
of analytical grade. Deionized water was used in all
measurements.
Preparation of 2,3,4,6-Tetra-O-acetyl-a-D-
galactopyranosyl bromide (1)
2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl bromide was
prepared according to Ref. [10].
Preparation of 2,3,4,6-Tetra-O-acetyl-b-D-
galactopyranosylthiol (2)
Compound 1 (6.00 g, 14.63 mmol) was dissolved in
anhydrous acetone (7 mL), thiourea (1.17 g, 14.63 mmol)
123

J Surfact Deterg
Scheme 1 Synthesis route of
thiogalactopyranoside
OH
OH
O
OAc OAc
O
OAc OAc
O
Ac2O
HBr-HOAc
OAc
OH
HO
AcO
S
AcO
OH
OAc
OAc
Br
OAc OAc
O
OAc OAc
O
NH2CSNH2
K2S2O5
NH2
SH
AcO
AcO
AcO
OAc
OAc
Br
NH2
OAc OAc
O
BrCH2CH2Br
S
Br
OAc
13C NMR (400 MHz, MeOD, d ppm):169.84,169.97,
170.16,170.38 (C=O(Gal)), 79.19 (C-1(Gal)), 76.73–77.37
(CDCl3), 67.24,70.84,71.57,74.95 (C-2,3,4,5(Gal)), 61.47
(C-6(Gal)), 20.56,20.67,20.69,20.83 (CH3COO(Gal));
MS (positive): m/z 387.1420 [M ? Na]?.
O
OAc OAc
O
R
O
N
S
N
AcO
+
H
Br
NH
O
OAc
R
O
O
OAc OAc
Br
O
R
R
S
AcO
O
N
Preparation of 2-Bromoethyl-2,3,4,6-tetra-O-acetyl-b-D-
thiogalactopyranoside (3)
N
H
OAc
NH
O
R
O
O
OH
OH
O
O
Br
N
A mixture of compound 2 (2.00 g, 5.49 mmol) and
potassium carbonate (800 mg, 5.80 mmol) in acetone–
water (4:3, 14 mL) was stirred under nitrogen. Then, 1,2-
dibromoethane (3.6 mL, 7.85 mmol) was added and the
reaction was stirred at room temperature. The reaction was
monitored by thin layer chromatography with hexane: ethyl
acetate (2:1, v/v) as eluent. The mixture was extracted
twice with CH2Cl2 (50 mL). The combined organic layer
was dried with anhydrous sodium sulfate and concentrated
to give a yellow oil. The residue was purified by silica gel
chromatography using hexane and ethyl acetate (2:1, v/v)
as the eluent to give the bromide intermediate as white
crystals m.p. 71.8–74.5 °C, in a 44.6 % yield.
S
HO
O
N
H
OH
NH
O
R
R=C12H25, C14H29
Scheme 2 Synthesis route of cationic galactolipid
was added and the mixture was refluxed for 1 h. After a
rapid removal of acetone, the residue was dissolved in
water (10 mL) and CH2Cl2 (10 mL). Potassium metabi-
sulfite (2.59 g, 11.88 mmol) was now added and the mix-
ture was refluxed for another 15 h. The reaction was
monitored by thin layer chromatography (hexane: ethyl
acetate = 5:2, v/v). The solution was then cooled down to
r.t., and the mixture was extracted with CH2Cl2 twice. The
dichloromethane extracts were combined, dried over
anhydrous sodium sulfate, evaporated in vacuo and the
resulting residue was subjected to column chromatography
(hexane: ethyl acetate, 5:2, v/v) to obtain the white crystals
m.p. 81.6–84.5 °C, in a 49.6 % yield.
1H NMR (400 MHz, MeOD, d ppm): 1.99,2.05,2.09,
2.17 (12H, 4 9 s, 4 9 CH3COO(Gal)), 2.97–3.23 (2H, m,
S-CH2, in which 2.97–3.05 (1H, m), 3.17–3.25 (1H, m)),
3.52–3.61 (2H, m, CH2Br), 3.94–3.97(1H, t, H-5(Gal)),
4.08–4.17 (2H, m, H-6,60(Gal)), 4.54–4.56 (1H, d, J = 8.0,
H-1(Gal)), 5.03–5.07 (1H, dd, J1 = 12.0, J2 = 4.0, H-2
(Gal)), 5.22–5.27 (1H, t, H-3(Gal)), 5.44–5.45 (1H, d, J =
2.4, H-4(Gal)), 7.28 (s, CDCl3);
13C NMR (400 MHz, MeOD, d ppm): 169.56,169.98,
170.13,170.41 (C=O(Gal)), 84.51 (C-1(Gal)), 76.73–77.36
(CDCl3), 67.00,67.24,71.70,74.72 (C-2,3,4,5(Gal)), 61.69
(C-6(Gal)), 32.81(CH2Br), 30.64(S-CH2), 20.57,20.67,20.
70,20.77 (CH3COO(Gal));
MS (positive): m/z [M ? Na]? 495.0378 (1Br)
[2 M ? Na]? 967.0661.
1H NMR (400 MHz, MeOD, d ppm): 1.99,2.07,2.08,2.17
(12H, 4 9 s, 4 9 CH3COO (Gal)), 2.36,2.39 (1H, d,
J = 12.0, Gal-SH), 3.94–3.97 (1H, m, H-5(Gal)), 4.12–4.14
(2H, d, J = 6.8, H-6,60(Gal)), 4.52–4.56 (1H, t, H-1(Gal)),
5.01–5.04 (1H, dd, J1 = 10.0, J2 = 3.2, H-2(Gal)),
5.16–5.21 (1H, t, H-3(Gal)), 5.43–5.44 (1H, dd, J1 = 3.2,
J2 = 0.8, H-4(Gal)), 7.28 (s, CDCl3);
123

J Surfact Deterg
Preparation of 3-(Dimethylamino)-propane-1,
2-di-alkylcarbamate (4)
filtration. Compound 6 was obtained as a light yellow solid
with a 100 % yield.
1H NMR (400 MHz, MeOD, d ppm): 0.88,0.90,0.92
(6H, t, 2 9 CH2CH3), 1.29 (36H, s, 2 9 (CH2)9), 1.49,1.51
(4H, 2 9 s, 2 9 NHCH2CH2), 3.02–3.26 (12H, m,
2 9 NHCH2 2 9 N?CH3, OCH2 (Et-Linker)), 3.31 (s,
MeOD), 3.46–3.87 (10H, m, 2 9 N?CH2, H-2,3,4,5,6,60
(Gal)), 4.06–4.11 (2H, m, NHCOOCH2) 4.42–4.46 (1H, dd,
J1 = 12.0, J2 = 4.0, H-1(Gal)), 4.89 (s, H2O), 5.47–5.57
(1H, m, NHCOOCH), 8.55 (6H, s, 2 9 NH, 6 9 OH);
13C NMR (400 MHz, MeOD, d ppm): 155.44,156.58
(NHC=O), 85.66,86.36 (C-1, a/b(Gal)), 69.38,69.84,74.66,
79.60 (C-2,3,4,5(Gal)), 66.30 (NHCOOCH), 65.51,65.75
(N?CH2), 63.72 (NHCOOCH2), 61.83 (C-6(Gal)),
50.89,51.13 (N?(CH3)2), 49.00 (MeOD), 40.55,40.67
(NHCH2), 22.37–31.70 ((CH2)10, SCH2(Et-Linker)), 13.10
(CH3CH2);
Tertiary amines with carbamate as the important interme-
diates were synthesized according to the literature proce-
dures [11].
Preparation of 2,3-Bis(dodecylcarbamoyloxy)-N,N-
dimethyl-N-[2-(2,3,4,6-tetra-O-acetyl-1-thio-b-D-
galactopyranosyl) Ethyl] Propanyl Ammonium
Bromide (5)
Anhydrous compound 3 (1.25 g, 2.66 mmol) and 3-
(dimethylamino) propanyl-1,2-didodecylcarbamate (1.5 g,
2.67 mmol) were dissolved in anhydrous ethanol (15 mL).
The mixture was refluxed for 48 h under a stream of
nitrogen. The solvent was evaporated in vacuo and the
resulting residue was subjected to silica gel column chro-
matography ([CHCl3/NH3 = 5/1]/CH3OH = 10/1, v/v).
Compound 5 was obtained as a light yellow viscous fluid
with a 30 % yield.
1H NMR (400 MHz, MeOD, d ppm): 0.88,0.90,0.92
(6H, t, 2 9 CH2CH3), 1.29 (36H, s, 2 9 (CH2)9), 1.49,1.51
(4H, 2 9 s, 2 9 NHCH2CH2), 1.96,2.05,2.06,2.16 (12H,
4 9 s, 4 9 CH3COO(Gal)), 3.08–3.26 (12H, m,
2 9 NHCH2, SCH2(Et-Linker), 2 9 N?CH3), 3.31 (s,
MeOD), 3.69–3.79 (4H, m, 2 9 N?CH2), 4.10–4.42 (5H,
m, H-5,6,60(Gal), NHCOOCH2), 4.88 (s, H2O), 4.92–4.94
(1H, d, J = 8.0, H-1(Gal)), 5.15–5.23 (2H, m, H-2,3(Gal)),
5.48–5.52 (2H, m, H-4(Gal), NHCOOCH);
MS (positive): m/z 764.7 [M-Br]?.
Preparation of 2,3-Bis(tetradecylcarbamoyloxy)-N,N-
dimethyl-N-[2-(2,3,4,6-tetra-O-acetyl-1-thio-b-D-
galactopyranosyl) Ethyl] Propanyl Ammonium
Bromide (7)
Compound 7 was synthesized as the same procedure as
compound 5 with a 33.3 % yield.
1H NMR (400 MHz, MeOD, d ppm): 0.88,0.90,0.92 (6H,
t, 2 9 CH2CH3), 1.29 (44H, s, 2 9 (CH2)11), 1.49,1.51 (4H,
2 9 s, 2 9 NHCH2CH2), 1.96,2.05,2.06,2.16 (12H, 4 9 s,
4 9 CH3COO(Gal)), 3.08–3.26 (12H, m, 2 9 NHCH2,
SCH2(Et-Linker), 2 9 N?CH3), 3.30 (s, MeOD), 3.67–3.81
(4H, m, 2 9 N?CH2), 4.06–4.41 (5H, m, H-5,6,60(Gal),
NHCOOCH2), 4.88 (s, H2O), 4.91–4.94 (1H, d, J = 8.8,
H-1(Gal)), 5.15–5.23 (2H, m, H-2,3(Gal)), 5.48 (1H, s,
H-4(Gal)), 5.51–5.52 (1H, d, J = 4.8, NHCOOCH), 7.90 (s,
CHCl3);
13C NMR (400 MHz, MeOD, d ppm): 171.28,171.53,
171.80,172.22 (C=O(Gal)), 156.84,157.87 (NHC=O),
83.96,84.86 (C-1, a/b(Gal)), 68.37,69.17,72.95,76.43 (C-
2,3,4,5(Gal)), 67.58 (NHCOOCH), 65.21,66.45 (N?
(CH2)2), 64.93 (NHCOOCH2), 62.86 (C-6(Gal)), 52.61,52.
87 (N?(CH3)2), 49.00 (MeOD), 41.99,42.11 (NHCH2),
22.95–33.08 ((CH2)10, SCH2(Et-Linker)), 20.48,20.57,20.
69,20.80 (CH3COO(Gal)), 14.44 (CH3CH2);
13C NMR (400 MHz, MeOD, d ppm): 171.27,171.44,
171.52,171.79 (C=O(Gal)), 157.95,156.84 (NHC=O), 83.
94,84.86 (C-1, a/b(Gal)), 79.47 (CHCl3) 68.35,69.15,72.
94,76.42 (C-2,3,4,5(Gal)), 67.56 (NHCOOCH), 65.21,66.43
(N?(CH2)2), 64.87 (NHCOOCH2), 62.86 (C-6(Gal)),
52.59,52.87 (N?(CH3)2), 49.00 (MeOD), 41.98,42.10
(NHCH2), 22.94-33.08 ((CH2)12, SCH2(Et-Linker)), 20.57,
20.70,20.72,20.81 (CH3COO(Gal)), 14.45 (CH3CH2);
MS (positive): m/z 988.7 [M-Br]?.
MS (positive): m/z 932.4318 [M-Br]?.
Preparation of 2,3-Bis(dodecylcarbamoyloxy)-N,N-
dimethyl-N-[2-(1-thio-b-D-galactopyranosyl) Ethyl]
Propanyl Ammonium Bromide (6, denoted as QAS C12-2-S)
Compound 5 (0.83 g, 0.82 mmol) was dissolved in anhy-
drous ethanol (15 mL). Sodium methoxide (0.31 g,
5.74 mmol) was added and the mixture was stirred for 1 h.
D113 cation exchange resins were added when TLC
(CHCl3/CH3OH = 4/1, v/v) showed the above reaction
was completed. Until the pH of the solution had changed to
neutral, chloroform (5 mL) was added to distill the mix-
ture. The solvent was removed by rotary evaporation after
Preparation of 2,3-Bis(tetradecylcarbamoyloxy)-N,N-
dimethyl-N-[2-(1-thio-b-D-galactopyranosyl) Ethyl]
Propanyl Ammonium Bromide (8, denoted as QAS C14-2-S)
Compound 8 was synthesized as the same procedure as
compound 6 with a 100 % yield.
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J Surfact Deterg
1H NMR (400 MHz, MeOD, d ppm): 0.88,0.89,0.91
(6H, t, 2 9 CH2CH3), 1.28 (44H, s, 2 9 (CH2)11), 1.49,
1.50 (4H, 2 9 s, 2 9 NHCH2CH2), 1.96,2.15 (2 9 s, OH),
3.04–3.17 (6H, m, 2 9 NHCH2, SCH2(Et-Linker)), 3.20
(6H, s, N?(CH3)2) 3.30 (s, MeOD), 3.47–3.88 (10H, m,
2 9 N?CH2, H-2,3,4,5,6,60(Gal)), 4.07–4.11,4.25–4.27
(2H, m, NHCOOCH2) 4.45–4.48 (1H, dd, J1 = 9.6,
J2 = 5.6, H-1(Gal)), 4.84 (s, H2O), 5.48–5.56 (1H, m,
NHCOOCH), 7.89 (s, CHCl3), 8.48 (s, NH);
V
P ¼
ð3Þ
ð4Þ
a0Lc
3
˚
A
V ¼ 54:30 ꢂ C3 þ 27:05 ꢂ C2
3
˚
Lc ¼ 1:50 þ 1:265 ꢂ C1 A
a0 ¼ Amin
where, C1, C2 and C3 are the amount of carbon, methylene
and methyl, respectively.
ð5Þ
ð6Þ
13C NMR (400 MHz, MeOD, d ppm): 156.84,157.96
(NHC=O), 87.01,87.69 (C-1, a/b(Gal)), 81.03(CHCl3),
67.71,70.76,71.28,76.09 (C-2,3,4,5(Gal)), 67.22 (NHCOO
CH), 65.16,66.98 (N?CH2), 63.25 (NHCOOCH2), 63.18
(C-6(Gal)), 52.29,52.53 (N?(CH3)2), 49.00 (MeOD), 41.
95,42.08 (NHCH2), 23.29–33.07 ((CH2)12, SCH2 (Et-Lin-
ker)), 14.45 (CH3CH2);
Size and Zeta Potential Measurements
In the formulation of cationic liposomes containing DOPE
as a helper lipid, cationic lipid/DOPE at a molar ratio of 1:1
was prepared by conventional thin film evaporation and an
ultrasonic method [12, 13]. The aqueous solutions of the
cationic liposomes were sonicated in the ultrasonic bath.
The liposomes were obtained by filtration through the
200 nm membrane, final concentration of which was 1 mg/
mL. Particle sizes and zeta potentials were measured at r.t.
by Nanoparticle Size and Zeta Potential Measurement.
MS (positive): m/z 820.4453 [M-Br]?.
Equilibrium Surface Tension
The surface tensions of the galactolipid solutions of dif-
ferent concentrations were measured twice using a tensi-
ometer at 25 0.1 °C until the experimental error was
within 0.2 mNꢀm-1. The curves of surface tension versus
logarithm of surfactant concentrations (c-log C) were
plotted and the critical micelle concentration (CMC) values
were determined from the break points. The surface ten-
sions at CMC (cCMC) were also obtained from the curves of
c-log C. The maximum Gibbs surface excess, Cmax, at the
air–water was calculated by Gibbs adsorption isotherm
equation (Eq. 1), where the value of n (a constant deter-
mined by the number of species constituting the surfactant
and adsorbed at the interface) is taken as 1, R is the gas
constant (8.314 J mol-1K-1), T is the absolute temperature
(K), and dc/dlog C is determined by the slope of the curve
of dc-log C when the concentration is near the CMC.
Transmission Electron Microscopy
Observation by transmission electron microscope (TEM)
was performed with a negative staining method. A drop of
solution was placed on a Formvar covered TEM grid
(copper grid, 3.02 mm, 200 mesh) and stained with a drop
of 2 wt% phosphotungstic acid aqueous solution. The
excess solution was removed by blotting with a filter paper.
TEM, operating at 80.5 kV, was used to investigate the
samples.
Results and Discussion
ꢀ
ꢁ
ꢁ1
dc
2:303 nRT d log C
Cmax
¼
mol ꢂ cmꢁ2
ð1Þ
CMC, cCMC, Cm, Am and P
T
The minimum average area occupied per surfactant
molecule Amin (nm2) at the air/water interface when the
surface adsorption was saturated was calculated by Eq. 2,
where N is Avogadro’s number.
The surface tensions of aqueous solutions of QAS Cn-2-S
were measured at 25 0.1 °C. The results of surface
tension measurements are shown in Fig. 2. The surface
tension decreases with increasing concentration and then
reaches a clear break point, which is taken as the CMC.
CMC as one of the main parameters for surfactants is the
concentration at which micelles are formed initially, which
will be useful for the formation of molecular self-organized
assemblies. The values of CMC, cCMC, Cm, Am and P are
given in Tables 1 and 2.
1014
Amin
¼
nm2
ð2Þ
NACmax
The critical packing parameter, P, which is widely used
to elaborate the formation and transformation of the system
of the surfactant congeries was calculated by Eq. 3, where
V is the volume of hydrophobic portion of surfactant, and
a0 is the area of the molecular head or the hydrophilic part.
Lc is the maximum extended length of the hydrophobic
chain. Their values were estimated from Eqs. 4–6.
It is well known that the CMCs of conventional ionic
surfactants decrease with the increase of the carbon number
of the hydrophobic chain up to 16 [14]. In this research,
CMCs of QAS Cn-2-S increased from 4.5 9 10-6 to
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J Surfact Deterg
Amin/(nm2)
Table 1 Physicochemical properties of the surfactants QAS Cn-2-S(O)
Surfactants
c
CMC/(mNꢀm-1
)
CMC/(molꢀL-1
)
C
max/(molꢀcm-2
)
QAS C12-2-O
QAS C12-2-S
QAS C14-2-S
26.1
25.7
51.0
2.4 9 10-4
4.5 9 10-6
4.0 9 10-5
7.07 9 10-10
8.28 9 10-10
5.21 9 10-10
0.235
0.201
0.319
Note: Data of QAS C12-2-O is from Ref. [11]
process of the surfactant molecules and the formation of
micelles, thus giving higher CMC [15].
Table 2 Critical packing parameter of the surfactants QAS Cn-2-S
3
˚
V/A
˚
Lc/A
Surfactants
P
As expected, the surfactants QAS Cn-2-S exhibited
remarkable physicochemical properties with low CMC and
great efficiency at lowering surface tension. As shown in
Fig. 3, it is clearly seen that QAS C12-2-S displayed
smaller CMC than that of the conventional surfactant QAS
C12-2-O [11]. Of note, the CMC of the surfactant was
smaller by approximately two orders of magnitude, while
cCMC decreased by about 0.5 mN m-1. This indicates that
the surfactant QAS C12-2-S with sulfur bond absorbs
strongly at the air–water interface compared to QAS C12-2-
O, orienting itself to cause superior surface activity [16].
The water solubility of QAS Cn-2-S at 25 °C was markedly
dependent on the chain length, which affected the regu-
larity of the equilibrium surface tension of QAS C12-2-S.
The critical surface tension (cCMC) of QAS Cn-2-S was
thought to decrease with the increase of the chain length.
However, the cCMC of QAS C12-2-S increased from 25.7 to
51.04 mNꢀm-1 while n ranged from 12 to 14, which may
be ascribed to the low water solubility of QAS C14-2-S.
Furthermore, considering the small steric hindrance among
the long hydrocarbon chains of QAS Cn-2-S, the alkyl
chains can rotate freely and have the possibility to cover
each other. This may result in a decrease in methyl groups
and an increase in methylene groups at the outer surface.
As the surface energy of the methylene group is larger than
that of the methyl group, the more methylene groups at the
outer surface, the larger the value of cCMC of QAS Cn-2-S.
The surface energy of the methylene group may also
contribute to the fact that the cCMC of QAS C14-2-S was
larger than that of QAS C12-2-S.
QAS C12-2-S
QAS C14-2-S
1,001.65
1,109.31
50.835
55.755
0.98
0.62
80
70
60
50
40
30
QAS C12-2-S
QAS C14-2-S
QAS C12-2-O
20
-7
-6
-5
-4
-3
Log C(mol/L)
Fig. 3 Surface tension (c) versus log C of QAS Cn-2-S in H2O at
25 0.1 °C
Table 3 Size and Zeta-potential of liposomes
Liposomes
Average size/nm
PDI
Zeta potential/mV
Lipo C12-2-S
Lipo C14-2-S
613.8
377.7
0.607
0.410
44.3
39.9
4.0 9 10-5 molꢀL-1 with increasing the hydrophobic chain
from 12 to 14, which is not consistent with the tendency of
the common homologous. Generally, hydrocarbon chain of
the surfactant demonstrates weak affinity with water mol-
ecules due to its hydrophobicity, thus making the interfa-
cial free energy between the hydrophobic hydrocarbon
chain and water much higher. In order to reduce this high
interfacial free energy, the hydrophobic hydrocarbon chain
often remains in a coiled state. When the length of the
hydrophobic hydrocarbon chain exceeds a certain value,
the coiled and disorder state of the hydrophobic chain will
be further strengthened, so that it hinders the self-assembly
It is of vital importance whether vesicles could be
formed or not for QAS Cn-2-S as a gene delivery vehicle in
future. Tanford [17] and Israelachvili [18] raised that the
critical packing parameter P predicts the formation of
various surfactant. Different P values are compatible with
different geometric shapes of the aggregates. When P is
less than 1/3, spherical micelles are the preferred form of
aggregation, as the ratio of surfactant head group area is
large in comparison to the hydrophobic part. Cylindrical
micelles form when P is between 1/3 and 1/2. When P is
greater than 1/2, first formed are highly curved bilayer
vesicles and then flat bilayers as P goes to 1. The structure
123

J Surfact Deterg
TEM image of vesicles is formed in Cn-2-S/DOPE aqueous
system, as shown in Fig. 4. The bilayer structure and
existence of inner aqueous phase can be clearly seen,
indicating the QAS Cn-2-S is capable of vesicle formation.
The left presented the different sizes of C12-2-S/DOPE
liposome, mainly large particle-size vesicles. In addition,
the right illustrated the vesicle formation of C14-2-S/DOPE
liposome. Evidently, Lipo C14-2-S displayed ones of
smaller size and better polydispersity compared to Lipo
C12-2-S, which was consistent with the results of size
measurement. According to the critical packing parameter
P, QAS C14-2-S was inclined to form vesicles and QAS
C12-2-S favored flat bilayers, which lead to the size of Lipo
C14-2-S much smaller. In the aqueous system, the average
particle size of liposomes showed comparatively large due
to vesicles aggregation.
Fig. 4 Negatively stained (phosphotungstic acid) TEM image of the
vesicles: Left is Lipo C12-2-S, Right is C14-2-S
of QAS Cn-2-S is single head and two hydrophobic carbon
chains. As for QAS C12-2-S, P is 0.98, which indicates flat
bilayers are formed. In addition, the P value of QAS C14-2-
S is 0.62, which shows curved bilayer vesicles are ener-
getically favored.
Conclusion
Two double chain cationic lipids Cn-2-S derived from thio
galactose were synthesized and confirmed by MS, TOF-
1
MS, H NMR and 13C NMR. QAS C12-2-S appears lower
CMC value and cCMC than QAS C14-2-S counterpart.
Vesicles are formed in aqueous solution and it is consistent
with the result of the critical packing parameter. Moreover,
negatively stained TEM images confirmed the formation of
vesicles.
Particle Size and Zeta-Potential
Particle-size refers to the equivalent diameter of the par-
ticles of the cationic liposomes. Zeta-potential is the
potential of the shear plane, which is an important indicator
to characterize the stability of colloidal dispersions. Posi-
tively charged cationic liposomes can be combined with
the negatively charged DNA to achieve the purpose of gene
transfection. As a result, the particle size and zeta potential
of cationic liposomes directly affect their transfection
efficiency.
Acknowledgments The authors are grateful for the financial sup-
port from the National Natural Science Foundation of China
(20876023).
References
The results on particle sizes and zeta potentials of the
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ded data it is seen that the cationic liposome with a shorter
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liposome over 0.5 could put its gene delivery at a disad-
vantage. As a result, large size and high PDI of Lipo C12-2-
S might have a poor influence on gene transfection effi-
ciency. However, Lipo Cn-2-S shows a positive charge,
which is expected to compound with negatively charged
plasmid DNA and to obtain a satisfactory gene transfection
efficiency. Whether the results of transfection efficiency
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experiments in the future.
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Author Biographies
Weihong Qiao obtained her master’s degree from the Dalian
University of Technology in 1994 and a Ph.D. from the same
university in 2000. Since 1994, she has been teaching and working in
the State Key Laboratory of Fine Chemicals at the Dalian University
of Technology, Liaoning, PR China. Her main field of research is the
design, synthesis, and physical chemistry of surfactants.
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Min Zhou obtained her bachelor’s degree from the China University
of Mining and Technology in 2010. She is working on a master’s
degree in the synthesis and physical chemistry of surfactants at Dalian
University of Technology.
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16. Xiao J, Zhao Z (2003) Principles of surfactant application, 1st
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Limei Luo received a bachelor’s degree and works at Dalian
University of Technology. She is an engineer in the analysis of
molecular spectroscopy.
123