Synthesis and evaluation of mono- and multi-hydroxyl low toxicity pH-sensitive cationic lipids for drug delivery

Article abstract of DOI:10.1016/j.ejps.2019.03.018

Cationic lipids can easily assemble into spherical liposomes in aqueous phase which showed unique superiority in drug and gene delivery. However, the toxicity of cationic lipids is still an obstacle to application. To develop low toxicity cationic lipids, we designed two cationic lipids contained different number of hydroxyl groups. Biocompatible mono-hydroxyl and multi-hydroxyl galactose head group was respectively modified to a biodegradable quaternary amine lipid, and two novel hydroxyl cationic lipids were synthesized and characterized by MS, ¹H NMR and ¹³C NMR. Two lipids showed good surface activity and both of them can assemble to about 80 nm stable small unilamellar vesicles (SUVs) with cholesterol in aqueous phase. Both of lipids showed relatively lower toxicity than the well-known cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In vitro 24 h IC₅₀ of two assemblies were more than 50 μg/mL, which were about 10 μg/mL higher than the IC₅₀ of DOTAP. Multi-hydroxyl galactose lipids group showed much lower toxicity than mono-hydroxyl lipids group. Moreover, Both of the assemblies with lower hemolysis were nearly non-hemolytic risk under the concentration of 30 μg/mL. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) showed that the average sizes of both doxorubicin (DOX) loaded liposomes were about 110 nm. The DOX entrapment efficiencies of galactose liposome and mono-hydroxyl liposome were 58% and 91%, respectively. Both of the DOX loaded liposomes were stable after one month placed at room temperature. Two DOX loaded liposomes showed better anti-cancer effect than free DOX above 5 μg/mL, and they can be internalized into cells and produce more release of DOX inside MCF-7 cells and HepG2 cells at pH 5.0. These results suggested that synthesized lipids are suitable as potential low toxicity cationic drug delivery systems.

Full text of DOI:10.1016/j.ejps.2019.03.018

European Journal of Pharmaceutical Sciences 133 (2019) 69–78
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Synthesis and evaluation of mono- and multi-hydroxyl low toxicity pH-
sensitive cationic lipids for drug delivery
⁎
Zheng Zhao, Weihe Yao, Ning Wang, Chenyu Liu, Hengjun Zhou, Hailiang Chen, Weihong Qiao
State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, Dalian 116024, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cationic lipids can easily assemble into spherical liposomes in aqueous phase which showed unique superiority
in drug and gene delivery. However, the toxicity of cationic lipids is still an obstacle to application. To develop
low toxicity cationic lipids, we designed two cationic lipids contained different number of hydroxyl groups.
Biocompatible mono-hydroxyl and multi-hydroxyl galactose head group was respectively modified to a biode-
gradable quaternary amine lipid, and two novel hydroxyl cationic lipids were synthesized and characterized by
Hydroxyl
Cationic lipid
Low toxicity
pH-sensitive
Drug delivery
1
13
MS, H NMR and C NMR. Two lipids showed good surface activity and both of them can assemble to about
8
0 nm stable small unilamellar vesicles (SUVs) with cholesterol in aqueous phase. Both of lipids showed rela-
tively lower toxicity than the well-known cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
In vitro 24 h IC50 of two assemblies were more than 50 μg/mL, which were about 10 μg/mL higher than the IC50
of DOTAP. Multi-hydroxyl galactose lipids group showed much lower toxicity than mono-hydroxyl lipids group.
Moreover, Both of the assemblies with lower hemolysis were nearly non-hemolytic risk under the concentration
of 30 μg/mL. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) showed that the
average sizes of both doxorubicin (DOX) loaded liposomes were about 110 nm. The DOX entrapment efficiencies
of galactose liposome and mono-hydroxyl liposome were 58% and 91%, respectively. Both of the DOX loaded
liposomes were stable after one month placed at room temperature. Two DOX loaded liposomes showed better
anti-cancer effect than free DOX above 5 μg/mL, and they can be internalized into cells and produce more release
of DOX inside MCF-7 cells and HepG2 cells at pH 5.0. These results suggested that synthesized lipids are suitable
as potential low toxicity cationic drug delivery systems.
1
. Introduction
surface of carriers would damage cell membranes and produce high
toxicity (Lv et al., 2006). The high toxicity limited the dose of lipids
Lipid molecules consisting of hydrophilic head and hydrophobic
used for drug or gene delivery. Many relatively low toxicity cationic
lipids were synthesized such as 1,2-di-O-octadecenyl-3-trimethy-
lammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammo-
nium-propane (DOTAP), which have been widely studied as drug and
gene delivery in the past 40 years (Falsini and Ristori, 2016; Felgner
et al., 1987; Kodama et al., 2018; Stamatatos et al., 1988). The toxicity
of those lipids was still dissatisfactory. Therefore, it is quite necessary to
synthesis and study new cationic lipids with much lower toxicity.
The toxicity of cationic lipids is mainly determined by their head
groups with positive charges. Lots of studies spread about how to re-
duce the toxicity caused by cationic head, and one of the strategies was
head group modified by introducing other groups to cationic head (Lv
et al., 2006). Hydroxyl as a neutral group has shown excellent bio-
compatibility. Banerjee et al. (1999) modified hexadecylamine and
dihexadecylamine with hydroxyl head group, modified lipids showed
part can disperse in aqueous phase and self-assemble into spherical li-
posomes, which is similar to cytomembrane in nature. Since the dis-
covery of this self-assemble phenomenon in 1960s (Standish et al.,
1
965), lipid based self-assembled nanostructures have been widely
studied and applied in drug delivery systems (Chinaeke et al., 2018;
Porter et al., 2007; Texier et al., 2016), and they represented a various
of advantages such as protecting drug from reaction, solubilizing lipo-
philic drugs, prolonging drug release, and enhanced permeability and
retention effect (EPR effect) (Alexis et al., 2008; Chang et al., 2008;
Golombek et al., 2018; Xu et al., 2015).
Cationic lipids as a type of lipids showed unique superiority in drug
and gene delivery (Bennett et al., 1992; Khoury et al., 2014; Saengkrit
et al., 2014; Zhdanov et al., 2002). However, the toxicity of cationic
lipids is an obstacle to application, a huge of positive charge on the
⁎
Corresponding author.
E-mail address: qiaoweihong@dlut.edu.cn (W. Qiao).
https://doi.org/10.1016/j.ejps.2019.03.018
Received 24 January 2019; Received in revised form 13 March 2019; Accepted 22 March 2019
Available online 23 March 2019
0928-0987/ © 2019 Elsevier B.V. All rights reserved.

Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
flying chemical co. LTD, China. Cholesterol (purity 99.5%) was pur-
chased from Beijing oberstar biotechnology liability co. LTD, China.
Milli-Q water was used for the preparation of water based solutions.
2.2. Synthesis and characterization
2
.2.1. Synthesis of 3-dimethylamine-propyl-1,2-ditetradecylcarbamate (a)
3
-Dimethylamine-propyl-1,2-ditetradecylcarbamate was prepared
as described previously (Yang et al., 2009). Briefly, 16.90 g (57.5 mmol)
Bis-(trichloromethyl)‑carbonate in 40 mL toluene was added into three-
necked flask, and 16.16 g (75.7 mmol) tetradecylamine in 50 mL to-
luene was drop-wise added into the flask under ice bath condition. The
mixture was stirred at 120 °C overnight. Then, toluene was removed by
rotary evaporator, and the mixture was distilled with reducing pressure.
Tetradecyl isocyanate was obtained by collecting the fraction at 200 °C
under the pressure of 9 mm Hg.
Fig. 1. Designed lipid molecule structures.
lower toxicity than hexadecylamine and dihexadecylamine. Study also
showed cationic lipids with glycerol head group produced relatively
low toxicity (Banerjee et al., 2001). Those studies have primary proved
that hydroxyl modified can reduce the toxicity of cationic lipids. On the
other side, hydroxyl as head group in amphipathic molecule can benefit
their self-assemble structures, which can form intermolecular hydrogen
bond with near amphipathic molecules, and promote the self-assembles
and optimize the assemble structures (Ramsammy et al., 1983). Besides,
the intermolecular hydrogen bond can interact with the aqueous phase,
increasing the integrity and stability of bilayer structure (Pei et al.,
9
.56 g (45.3 mmol) prepared tetradecyl isocyanate, 2.57 g
(
21.6 mmol) 3-dimethylamino-1,2 propylene glycol and 6 mL triethy-
lamine were dissolved with 50 mL toluene in a 100 mL flask. After the
mixture was refluxed overnight, the solvent was removed by rotary
evaporator. The crude product was purified by column chromatography
(
Chloroform/methanol = 4:1 v/v) (Yield 31%).
2
.2.2. Synthesis of 1-(2-bromethyl)-2,3,4,6-tetra-O-acetyl-D–galactoside
(
b)
2
011).
The objective of this study was to synthesize novel cationic lipids
5
.00 g (14.6 mmol) 2,3,4,6-Tetra-O-acetyl-D-galactopyranosyl bro-
mide and 28.00 g (224 mmol) bromoethanol were mixed into 100 mL
flask, and the mixture was stirred to completely dissolved. Then, 7.00 g
with different number of hydroxyl group, evaluate and compare their
toxicity and performance as cationic drug delivery systems. Two qua-
ternary amine cationic lipids with mono-hydroxyl head group and
multi-hydroxyl galactose head group (shown in Fig. 1) were synthesized
and characterized. Their self-assemble ability was explored including
surface tension, dynamic light scattering (DLS), zeta potential and
transmission electron microscopy (TEM). To evaluate their performance
as drug carriers, doxorubicin (DOX) as an anticancer drug was loaded
into two liposomes. A range of in vitro test was employed to evaluate
the entrapment efficiency (EE), drug release and storage stability of the
DOX loaded liposomes. Besides, human MCF-7 cells and HepG2 cells
were used to test the vitro toxicity of two blank liposomes and the
anticancer performance of DOX loaded liposomes.
Ag
2
CO was added into the flask. After stirred for 30 h in a dark place,
3
the mixture was filtered by 0.22 μm membrane. The filtrate was
dropped into a large amount of water, then filtered. The residue was
recrystallized from ethanol (Yield 45%). mp.106 °C~109 °C. MS: m/z
M + Na]+ 474.1.
[
2
.2.3. Synthesis of N-(2-O-(2,3,4,6-tetra-O-acetyl-D –galactoside)-ethyl)-
N-(2,3-ditetradecylcarbamate-propyl)-N,N-dimethyl ammonium bromide
c)
(
1
.50 g (2.51 mmol) a and 1.25 g (2.75 mmol) b were dissolved with
1
5 mL ethanol in 50 mL flask, and the mixture was refluxed under ni-
trogen protection for 48 h. The solvent was removed by rotary eva-
porator. After purified by column chromatography (Chloroform/am-
monium hydroxide/methanol = 5:1:0.6 v/v/v), viscous yellow liquid
was obtained (Yield 52%). MS: m/z [M-Br]+ 972.7.
2
. Materials and methods
2.1. Materials
2
.2.4. Synthesis of N-(2-O-D–galactoside-ethyl)-N-(2,3-ditetradecylcarbamate
Methanol (purity 99.8%), ethanol (purity 99.8%), dichloromethane
-propyl)-N,N-dimethyl ammonium bromide (Gal-lipid)
(
(
purity 99.8%), chloroform, toluene (purity 99.8%), petroleumether
purity NA) and diethylether (purity 99.8%) were purchased from
1.57 g (1.49 mmol) c and 0.56 g (10.4 mmol) sodium methoxide and
15 mL methanol were added into 50 mL flask. After 1 h stirred at room
temperature, the mixture was got through D113 cation exchange resin
column (chloroform as solvent). Chloroform was removed by rotary
evaporator. Light yellow solid was obtained (Yield 100%). HRMS: m/z
Tianjin fuyu fine chemical co. LTD, China. Acetic anhydride (purity
9
9.8%), anhydrous sodium acetate (purity 99.8%), hydrobromic acid
(
purity 99.8%), chloropropanol (purity 99.8%) and 3-bromo-1-pro-
+
panol (purity 99.8%) were purchased from Shenyang chemical reagent
factory, China. Sodium carbonate (purity 99.8%), sodium chloride
[M-Br] 804.8893.
(
purity 99.8%), sodium sulfate (purity 99.8%), triethylamine (purity
2.2.5. Synthesis of N-(3-hydroxypropyl)-N-(2,3-ditetradecylcarbamate-
propyl) eN,N-dimethyl ammonium bromide (HO-lipid)
9
9.8%) and phosphate buffer saline (PBS buffer, pH 7.4) were pur-
chased from Tianjin guangfu fine chemical research institute, China.
,3,4,6-Tetra-O-acetyl-D-galactopyranosyl bromide (purity 99.5%) was
Similar to the synthesis of Gal-lipid, 1.50 g (2.51 mmol) compound a
and 0.25 g (2.64 mmol) chloropropanol were dissolved with 15 mL
ethanol in a 50 mL flask. The mixture was refluxed under nitrogen
protection for 48 h, and then the solvent was removed. Product was
purified by column chromatography (Chloroform/methanol =4:1 v/v),
2
purchased from Shanghai Aladdin biochemical technology co. LTD,
China. Silver carbonate (purity 99.8%) was purchased from Tianjin
bodi chemical co. LTD, China. Sodium methoxide (purity 99.8%) was
purchased from Tianjin fuchen chemical reagent plant, China. D113
cation exchange resin and silica gel (200–300 mesh) were purchased
from Qingdao meigao group co. LTD, China. Bis-(tri-
chloromethyl)‑carbonate (purity 99.8%) and dimethyl-1, 2-propylene
glycol (purity 99.8%) were purchased from Tokyo chemical industries,
China. Tetradecylamine (purity 99.5%) was purchased from Jiangsu
+
white solid was obtained (Yield 66%). HRMS: m/z [M-Cl] 656.5932.
2.3. Surface tension measurements
Surface tension measurements are an important evaluation of am-
phiphilic properties of amphipathic molecule. Wilhelmy method was
70

Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
used during surface tension measurements by a tensiometer (Krüss Co.,
Germany). The critical micelle concentration (cmc) and the surface
tension of the solution at the critical micelle concentration (γcmc) of
each sample was found by plotting the surface tension against the
logarithm of concentration.
2.8. Size distribution and stability
The particle size distribution, average particle size, polydispersity
index (PDI) and zeta potential of Gal-L, HO-L, Gal-DL and HO-DL so-
lutions were determined by a dynamic light scattering (Nano ZS-90,
Malvern, UK) after 0 week, 1 week and 1 month placed at room tem-
perature.
2.4. Preparation of blank liposomes
2
.9. Morphology observation
Two liposomes were prepared by the thin film hydration and ul-
trasound method. 6.0 mg Gal-lipid (or 6.0 mg HO-lipid) and 3.1 mg
cholesterol were completely dissolved in 6 mL chloroform/methanol
The morphology of blank liposomes and DOX loaded liposomes was
characterized by TEM (HT7700 Exaens, Hitachi, Japan). Gal-L, HO-L,
Gal-DL and HO-DL solutions were respectively dropped on carbon-
coated copper grid (400 mesh, Beijing zhongjingkeji technology co.
Ltd., China) and dried. Then, the samples were negatively stained with
phosphotungstic acid solution (2%). After fully dried at room tem-
perature, the morphology of samples was observed by TEM.
(
(
2:1 v/v). The mixed solution was dried at 37 °C by a rotary evaporator
RE-2000A, Henan gongyi yuhua instrument factory, China) to form a
thin lipid film. One night vacuum dried were needed to remove residual
solvent. Subsequently, 6 mL Milli-Q water was used to hydrate the lipid
film. Then, the hydrated solution was treated under ultrasound at 40 °C
(
ultrasonic operating parameters: work 2 s, pause 5 s, 120 W, total time
2
0 min) using Ultrasonic signal generator (SCIENTZ-IID, Ningbo xinzhi
2.10. Cytotoxicity test in vitro
biotechnology co. LTD, China). After cooling to room temperature, all
the samples were filtered through 0.8 μm filters. Galactose head group
blank liposome (Gal-L) and hydroxyl head group blank liposome (HO-L)
were obtained.
HepG2 cells and MCF-7 cells were respectively cultured in 96-well
4
plate with a density of 10 cells per well, and incubated in a 37 °C, 5%
CO incubator (BB15, Thermo, USA) for 24 h. They were incubated
2
with 100 μL new serum medium containing 0, 0.1, 0.2, 0.5, 1, 3, 5, 8,
2
.5. Preparation of DOX loaded liposomes
10, 20 μg Gal-L or HO-L per well in 6 copies, then incubated for another
2
4 h. The serum medium were pipetted out, and the cells were washed
Similar as the preparation in the Section 2.4. DOX solution
with PBS buffer (pH 7.4) for 3 times, then 100 μL serum-free medium
and 50 uL MTT were added into each well. The cells were incubated for
another 4 h, then the media were replaced with 150 μL DMSO.
Absorbance was measured at 490 nm using a microplate reader
(Multiskan FC, Thermo, USA).
(
DOX·HCl concentration: 1 mg/mL) was used to hydrate the prepared
lipid films. Finally, galactose head group DOX loaded liposome (Gal-
DL) and hydroxyl head group DOX loaded liposome (HO-DL) were
obtained.
2.11. Homolytic test of liposomes
2.6. Drug entrapment efficiency
Fresh red blood cells (RBCs) from healthy volunteers were diluted
0 times with 0.9% NaCl solution. Diluted blood was pipetted into 24-
The entrapment efficiency is defined as the ratio of the amount of
5
DOX encapsulated in the liposomes to that of the total DOX added to the
liposomes dispersion. To get the entrapment efficiency parameter, non-
encapsulated DOX of the solution was removed by dialysis with
well plate with a volume of 1.5 mL per well. 500 μL 0.9% NaCl solution
containing 0, 20, 60, 200, 400 μg Gal-L, HO-L or DOTAP liposomes
were respectively added into 24-well plate. 500 uL 2% Triton X-100
3
500 Da dialysis bag. The dialysis bag contained 5 mL Gal-DL or HO-DL
(
TX-100) solution as positive control was added into 24-well plate as
solution was immersed in 500 mL of PBS buffer (pH = 7.4, renewed per
well. All the samples were tested in 6 copies (Liu et al., 2018). After 4 h
placed in 37 °C, 1.5 mL of blood was pipetted into tubes and centrifuged
to remove intact RBCs. 200 μL supernatant was pipetted into 96-well
plate and the absorbance was measured at 540 nm using a microplate
reader (Multiskan FC, Thermo, USA).
3
0 min) and dialyzed at 25 °C with 500 rpm stirring for 6 h. The final
obtained solution of DOX loaded liposomes without free DOX was used
to determine entrapment efficiency by UV measurement at 485 nm
(
Lambda 750 s, PerkinElmer, USA). 30 μL Above solution was pipetted
and dried under vacuum at 37 °C, then the solid was dissolved in 5 mL
DMF and their UV absorbance were measured. UV absorbance standard
2
.12. Anti-cancer properties of DOX loaded liposomes in vitro
curve
of
DOX
in
DMF
was
measured
manually
2
(
y = 0.01661x − 0.00649 R = 0.999). DOX entrapment efficiency was
HepG2 cells and MCF-7 cells were respectively cultured and in-
then calculated according to the following equation: EE = ED/TL (1)
cubated in 96-well plate, then Gal-DL, HO-DL and free DOX·HCl solu-
tions were respectively added into 96-well plate with 100 μL new serum
medium (All the groups have the same DOX concentration: 0, 0.1, 0.5,
(
EE is the entrapment efficiency, TL is the total amount of DOX added in
the preparation of liposome and ED is the amount of encapsulated DOX
after dialysis).
1
, 5, 10 μg per well). Then 24 h cells viability experiment were con-
tinued as same as the cytotoxicity test in 2.10. All the measurements
2
.7. Drug release in vitro
were tested in 6 copies as well.
1
mL Gal-DL or HO-DL solution removed free DOX were added in
2.13. Cellular uptake assay
dialysis bag, then the dialysis bags were placed in 100 mL pH 7.4 or 5.0
PBS buffer with stirring at the speed of 300 rpm. At appropriate inter-
vals, the 3 mL solutions outside the dialysis bags were pipetted into
tubes for next fluorescence intensity measurement respectively, and the
Cholesterol-PEG-FITC (MW = 3400, 1% mole fraction) was mixed
into cationic lipids. Two cationic liposomes mixed Cholesterol-PEG-
FITC (FITC-liposomes) were prepared (as the Section 2.4) for cellular
uptake assay. HepG2 cells and MCF-7 cells were respectively cultured in
3
mL new PBS solvent was replenished. The fluorescence intensity were
4
determined at 569 nm (excitation wavelength: 478 nm) using a fluor-
escence spectrophotometer (F7000, HITACHI, Japan), and the DOX
concentration of pipetted solution were obtained compared with stan-
dard curves.
12-well plate with a cell density of 8 × 10 cells per well respectively,
and incubated in a 37 °C, 5% CO incubator for 24 h. Then the cells
2
were incubated with 500 μL new serum medium containing FITC-lipo-
somes (100 μg per well) for 4 h in CO incubator, and analyzed by
2
71

Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
Fig. 2. The synthesis route of Gal-lipid.
Fluorescence Inversion Microscope System (XD-202, Nanjing jiangnan
novel optics co. LTD, China).
been characterized by MS and NMR, shown in supplementary material.
3.2. Self-assembly ability of lipids
3
. Results and discussion
Generally, it's necessary to study the self-assembly properties and
3
.1. Synthesis and characterization
the aggregation morphology of lipids in solution before liposomes
loading drugs, which can help instruct drug loading. The surface ten-
sion of synthesized lipids was measured firstly to study the surface
activity and the aggregation ability of lipids itself. Fig. 3 showed the
surface tension of Gal-lipid and HO-lipid aqueous solution at different
concentration. As the concentration of Gal-lipid or HO-lipid increased, a
Designed lipids show similar synthesis routes. Here we focus on
describing the synthesis route of Gal-lipid, as shown in Fig. 2. Designed
molecular structure can be divided into two parts including hydro-
phobic tail and modified head group. Compound a as pre-modified
hydrophobic tail has been synthesized in our research group before
(
Yang et al., 2009). The reason why we choose compound a as pre-
modified structure was relatively low toxicity and good degradability of
carbamate bond under low pH. The head group is another important
part of the lipid structure. As modified head group, galactose with four
hydroxyls has advantages in hydrophilic ability and biocompatibility.
To obtain target cationic lipids, we designed a quaternisation to connect
hydrophobic chain (compound a) with galactose group (compound b).
In the preparation of compound b, 2,3,4,6-Tetra-O-acetyl-D-galacto-
pyranosylbromide as initial reactant was reacted to 2-bromoethanol.
The bromine ethyl linker could provide enough reaction space and
guarantee successful quaternisation reaction. For the quaternisations,
chloroalkane with low reaction activity needed high temperature and
caused the galactose head decomposed in our initial trials. Theoreti-
cally, iodoalkane has the highest reaction activity. But iodoalkane al-
ways showed huge toxicity to cells which was against to our intention.
So we finally choose bromoalkane for the quaternisation, and prepared
compound c successfully. At the last step of synthesis, the compound c
was deprotected by sodium methoxide. D113 cation exchange resin
column was used as a convenient way to purify Gal-lipid, and the yield
of 100% in this synthesis step was satisfactory. All the compounds have
Fig. 3. Surface tension versus concentration of Gal-lipid and HO-lipid.
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Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
Table 1
DLS and zeta potential data of Gal-L and HO-L.
Liposomes
Average size/nm
PDI
Zeta potential/mV
0
week
1 week
4 weeks
0 week
1 week
4 weeks
0 week
1 week
4 weeks
Gal-L
86.14 ± 0.76
75.93 ± 0.64
111.7 ± 0.81
102.4 ± 1.12
89.78 ± 0.68
67.41 ± 0.71
112.4 ± 0.92
98.00 ± 0.84
–
0.245
0.260
0.197
0.156
0.187
0.175
0.210
0.161
–
+52.1
+43.0
+61.8
+49.3
+32.2
+38.9
+41.8
+45.2
–
HO-L
–
–
–
Gal-DL
HO-DL
110.3 ± 0.96
105.3 ± 1.01
0.183
0.231
+27.0
+35.5
Fig. 4. TEMs of Gal-L and HO-L; (a) and (b) Gal-L under different magnification, (c) and (d) HO-L under different magnification.
(
cmc) and surface tension at cmc (γcmc). The cmc of Gal-lipid and HO-
−5
−1
−6
−1
lipid were 3.73 × 10 mol·L and 5.90 × 10 mol·L , respec-
tively. Correspondently, the γcmc of Gal-lipid and HO-lipid solution
−1
−1
were 32.70 mN·m
and 44.52 mN·m . The surface tensions were si-
milar to glycolipid from bacteria (Arutchelvi and Doble, 2010; White
et al., 2013) and indicated that synthesized amphipathic molecules
have good surface activity and potential self-assembly ability.
Above the cmc, amphiphilic molecules would aggregate into self-
assembly in aqueous phase. DLS and TEM were used to characterize the
morphology and behavior of self-assembly of Gal-L and HO-L. Fig. 5
showed the size distribution of Gal-L and HO-L. Both of the cationic
assembly were distributed from 10 to 200 nm, and the average size of
Gal-L was slightly larger than HO-L, which were 86.1 nm and 75.9 nm
respectively (Table.1). TEM (Fig. 4) showed that two lipids have been
aggregated into relatively regular sphere shell, and bilayer membrane
structure can be seen apparently. Most of the self-assembly are about
8
0 nm SUVs which corresponded to DLS data.
The stability of Gal-L and HO-L were evaluated by a range of test
Fig. 5. The size distribution of Gal-L and HO-L.
including the changes of zeta potential, size and PDI (Table.1). The zeta
potential is a key indicator of the stability of liposome systems (Vogel
et al., 2017), and Gal-L and HO-L were respectively +52.1 mV
and + 43.0 mV initially. Both of them were larger than +30 mV, which
sharp decline can be observed, that was the typical characteristic of
amphipathic molecule. The amphiphilic properties of amphipathic
molecule were evaluated by the data of critical micelle concentration
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Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
Fig. 6. The changes of Gal-L hydrogen bonds; blue dotted line represents the intermolecular hydrogen bond and red dotted line represents the intramolecular
hydrogen bond. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. TEMs of Gal-DL and HO-DL; (a) and (b) Gal-DL under different magnification, (c) and (d) HO-DL under different magnification.
represented good stability. Higher zeta potential of Gal-L suggested that
Gal-L possessed higher stability at the initial time. Besides, the changes
of particle size, PDI and zeta potential were compared after the solu-
tions placed 1 week at room temperature. From Table.1, there was no
significant change in particle size and PDI. However, the zeta potential
of Gal-L and HO-L were respectively +32.2 mV and + 38.9 mV after
one week, showing a much larger 19.9 mV decrease of Gal-L compared
with 4.1 mV decrease of HO-L. They respectively decreased 38.2% and
hydroxyl of 4-C were easily formed intramolecular hydrogens because
of the galactose molecular geometries (Çarçabal et al., 2005). The in-
termolecular hydrogen bonding network was partly destroyed, and the
decrease of Gal-lipid polarity further influenced the stability of lipo-
some systems, which resulted in the huge decrease in zeta potential.
While HO-L just have one hydroxyl group has little chance to form
intramolecular hydrogen bond, showed less change in zeta potential.
Nevertheless, both of the liposomes zeta potential were above +30 mV.
Size and PDI showed no significant changes after 1 week placed in room
temperature, which proved the solutions were still stable.
9
.5% to the original zeta potential. We speculated that the huge de-
crease was the changes of hydrogen bonding (shown in Fig. 6). In the
initial state of 0 week, most of hydroxyl groups on the galactose head
group formed intermolecular hydrogen bonding network with H O or
2
3.3. Preparation and characterization of DOX loaded liposomes
other galactose head groups. The Gal-L with much more hydroxyl
formed larger intermolecular hydrogen bonding network stabilized li-
posomes and showed higher zeta potential than OH-L. Gradually, some
weak intermolecular hydrogens was replaced by stronger in-
tramolecular hydrogen bond. For example, the hydroxyl of 3-C and the
DOX·HCl as broad anticancer drugs was used as transported drug. In
order to study the interaction between DOX and liposomes, the DOX
loaded liposomes (Gal-DL and HO-DL) were prepared using the same
conditions as blank liposomes including the lipid concentration and
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European Journal of Pharmaceutical Sciences 133 (2019) 69–78
3.4. Drug release in vitro
Designed lipids have two degradable carbamate bonds in hydro-
phobic tails, and these bonds can be hydrolyzed under acidic or alkaline
conditions. When the drug loaded liposomes are injected into human
body, the drug loaded liposomes will go through different pH micro-
environment. In the beginning, the drug enters the blood circulation
system at the environment of pH 7.4. During the liposomes enter the
cells by endocytosis, the pH of microenvironment will gradually de-
crease to 5.0, which would hydrolyze carbamate bond and destroy the
structure of liposomes. By studying the drug release at different pH, we
can directly obtain the drug release information inside and outside the
cells. As shown in Fig. 8, DOX release of Gal-DL and HO-DL at pH = 7.4
and 5.0 have been studied. Within 5 h, burst releases was observed in
both liposomes, and nearly 40–50% of DOX was released from the li-
posomes. Then the release rate slowed down. After 17 h, the drug re-
lease reached maximum. Ultimately 54% and 60% DOX were released
respectively at pH = 7.4. While the release rate reached 67% and 79%
at pH =5.0 respectively, which was 13% and 19% higher than those at
pH = 7.4. It indicated that the carbamate bonds of the lipids were
broken at the low pH of 5.0, which destroyed the structure of liposomes
and result in the higher release. It means that the low pH of endosome
would crumble the liposomes and induce more release of DOX, when
Gal-DL and HO-DL are internalized into the cells. It further proved that
prepared liposomes were pH sensitive for therapeutic drug carrier.
Fig. 8. DOX release of Gal-DL and HO-DL at pH = 7.4 and 5.0.
3.5. Toxicity test in vitro
Cationic lipids usually showed much higher toxicity than nonionic
lipids at the same concentration. So there was few report directly study
the relationship between toxicity and different gradient concentration.
For a better understanding the relationship of the toxicity and different
gradient concentration, we have studied the toxicity at different con-
centration (from 0 to 200 μg/mL) with MCF-7 cells and HepG2 cells,
and compared the two synthesized lipids with several typical reported
cationic lipids. 24 h Toxicity have been tested after the addition of Gal-L
or HO-L, shown in Fig. 9. As the concentration of lipids increased, the
cell viability decreased. For MCF-7 cells, two lipids showed relatively
low toxicity, more than 80% cells survived at the concentration of
Fig. 9. The cells toxicity of Gal-L and HO-L in vitro.
ultrasound parameter. Fig. 7 was the TEM morphology of Gal-DL and
HO-DL. From Fig. 7 and Fig. 4, we found regular round sphere of blank
liposomes turned to be polygon sphere after loading DOX. It supposed
that DOX was loaded into the liposomes, and changed the structure of
the bilayer membrane. Both of Gal-DL and HO-DL were ~110 nm dis-
tributed vesicles, and the size of vesicles can accumulate in the vascu-
lature of tumor by EPR effect (Golombek et al., 2018; Maeda et al.,
3
0 μg/mL, which showed much lower toxicity than reported cationic
lipids dodecyl trimethyl ammonium bromide (DTAB), tetradecyl tri-
methyl ammonium bromide (TTAB) and cetyl trimethyl ammonium
bromide (CTAB) (the in vitro viability of DTAB, TTAB and CTAB which
were respectively about 20%, 30% and 55% after just 3 h at the con-
centration of 8 μg/mL liposomes) (Pinnaduwage et al., 1989). When
continuously increasing the concentration to 200 μg/mL, 66% and 33%
cells were still alive for Gal-L group and HO-L group respectively. The
toxicity of two blank liposomes to HepG2 cells were slightly higher than
that to MCF-7 cell. At the concentration of 30 μg/mL, the viability of
HepG2 were 73% and 72%. When the concentration reach to 200 μg/
mL, 58% and 38% HepG2 cells were alive for Gal-L group and HO-L
group respectively. The IC50 of synthesized lipids were above 50 μg/mL,
while the IC50 of well-known low toxicity cationic lipid DOTAP is
2
009; Maeda et al., 2013), which were potential passive target lipo-
somes. Fig. 5 showed the liposomes size distribution, the average sizes
of Gal-DL and HO-DL were 111.7 nm and 102.4 nm respectively, which
were approximately 25 nm larger than the blank liposomes. The zeta
potential of loaded drug were +61.8 mV and +49.3 mV respectively
(
Table 1), and indicated the good stability of two DOX loaded lipo-
somes. The stability of solution after one week placed at room tem-
perature has been study as well. There were no significant changes in
particle size and PDI, shown in Table 1. The zeta potential of two li-
posomes was +41.8 mV and +45.2 mV respectively, which was similar
as the trend of blank liposomes. After one month, no significant size and
PDI changes of Gal-DL and HO-DL were observed, also the solutions
were still clear with no precipitation. It proved that the stability of two
DOX loaded liposomes is at least a month at room temperature. Fur-
thermore, the DOX entrapment efficiencies were evaluated by dialysis.
Gal-DL showed a 58% entrapment efficiency of DOX less than 91%
entrapment efficiency of HO-DL, though the two liposomes have similar
particle sizes. It indicated that not only the size of vesicles but the
hydrophilic head structure could influence the entrapment efficiency of
water-soluble drug.
~
40 μg/mL (Wan et al., 2016). Both of the lipids were over 10 μg/mL
higher than DOTAP. In addition, Fig. 9 also showed that Gal-L have
lower toxicity than HO-L. At the concentration of 100 μg/mL, above
8
0% MCF-7 cells still alive in Gal-DL group, while there is less than 45%
MCF-7 cells alive in HO-DL. At the maximum test concentration of
2
00 μg/mL, the viability of HepG2 cells and MCF-7 cells with Gal-L
were respectively 33% higher and 20% higher than that with HO-L. The
results suggested hydroxyl contained head groups can reduce the ca-
tionic lipids toxicity. The more hydroxyl groups the cationic lipid
contained, the lower toxicity it would be.
75

Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
Fig. 10. The hemolytic test of Gal-L and HO-L; (a) The hemolytic capacity of Gal-L, HO-L and DOTAP liposomes compared with TX-100; (b) Contrast images of
centrifuged blood, 1–5: 0.9% NaCl, TX-100, Gal-L 200 μg/mL, HO-L 200 μg/mL and DOTAP 200 μg/mL; (c) Microscope images of erythrocyte, c1-c5: 0.9% NaCl, TX-
1
00, Gal-L 30 μg/mL, HO-L 30 μg/mL and DOTAP 30 μg/mL.
3
.6. Homolytic test of liposomes
group and HO-L group. In short, the hemocompatibility of Gal-L and
HO-L to erythrocytes was excellent and they have a very large appli-
cation advantage in drug delivery.
To further evaluate the hemocompatibility of Gal-L and HO-L, we
did the homolytic test of Gal-L and HO-L compared with DOTAP lipo-
somes. Fig. 10 represented the hemolytic potential of Gal-L, HO-L and
DOTAP liposomes at different concentration. As shown in Fig. 10a, the
hemolysis ratio of DOTAP raised as the concentration increased. At the
maximum test concentration of 200 μg/mL, the hemolysis ratio of
DOTAP reached to 9.5%. While as 0.9% NaCl group, Gal-L and HO-L
had no significant hemolysis under all the test concentration. We can
also intuitively see from Fig. 10b that the hemolysis of DOTAP is ob-
viously higher than Gal-L and HO-L. Moreover, as shown in Fig. 10c, a
set of micrographs of test erythrocytes were displayed. Gal-L group and
HO-L group exhibited typical healthy erythrocytes as 0.9% NaCl group,
which indicated that Gal-L and HO-L were nearly non-hemolytic risk at
the concentration of 30 μg/mL. DOTAP showed much more damage to
erythrocytes. Some erythrocytes of DOTAP group aggregated and
broken, while no aggregation of erythrocytes was observed in Gal-L
3
.7. Anti-cancer properties in vitro and cellular uptake
Free DOX·HCl was employed as control group to compare the anti-
cancer ability of Gal-DL and HO-DL. As shown in Fig. 11, at the DOX
concentration of 1.0 μg/mL, there was no significant differences be-
tween two DOX loaded liposomes and free DOX. At 5.0 μg/mL, the
MCF-7 cells and HepG2 cells viability of DOX loaded liposomes groups
were about 10% lower than free DOX groups. Both MCF-7 cells and
HepG2 cells were almost killed at the concentration of 100 μg/mL in
both Gal-DL groups and HO-DL groups, while the cells were still more
than 20% alive in free DOX group. Overall, two DOX loaded liposomes
have same level anti-cancer ability and were obviously better than free
DOX. To further prove that liposomes can be internalized into cells, we
marked the liposomes with Cholesterol-PEG-FITC and studied the cells
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Z. Zhao, et al.
European Journal of Pharmaceutical Sciences 133 (2019) 69–78
that the prepared liposome carriers can bring more DOX into cells and
promoted the anticancer effect of DOX.
4
. Conclusion
We have successfully synthesized two novel mono-hydroxyl and
multi-hydroxyl galactose head group cationic lipids and characterized
1
13
their structures by MS, H NMR and C NMR. The two lipids showed
good surface activity and can assemble to ~80 nm stable SUVs with
cholesterol. Both of the blank liposomes showed relatively low toxicity,
which showed much lower toxicity than DTAB, TTAB and CTAB. In
vitro 24 h IC50 of two liposomes were more than 50 μg/mL. It was about
1
0 μg/mL higher than well-known low toxicity DOTAP. Multi-hydroxyl
galactose lipids showed much lower toxicity than mono-hydroxyl lipids
in vitro. Moreover, Gal-L and HO-L with lower hemolysis were nearly
non-hemolytic risk under the concentration of 30 μg/mL. Two DOX
loaded liposomes were prepared and characterized. TEM and DLS
showed the average sizes of both drug loaded liposomes were ~
Fig. 11. The lethality of Gal-DL, HO-DL and DOX to MCF-7 cells and HepG2
cells.
1
5
10 nm. The DOX entrapment efficiencies of Gal-DL and HO-DL were
8% and 91%, respectively. Both of the DOX loaded liposomes were
uptake of liposomes by fluorescence inversion microscope system.
Cholesterol-PEG-FITC is a type of fluorochrome derivatives, which can
glow strong yellow-green light under blue ray exciting. It was used as
the marker of liposomes to track cellular uptake of the cationic lipo-
somes. As shown in Fig. 12, bright green can be seen at the region of
MCF-7 cells and HepG2 cells in all merged figures, which means both of
the liposomes have been uptake by both cells. We can further concluded
stable after a month placed at room temperature. Two DOX loaded li-
posomes showed a better anti-cancer effect compared with free DOX
above 5 μg/mL, which can be internalized into cells and produce a more
release of DOX at low pH inside the cancer cells. The above results
indicated that the studied liposomes can be potential low toxicity ca-
tionic drug delivery systems.
Fig. 12. Cellular uptake images of Gal-DL and HO-DL.
77

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European Journal of Pharmaceutical Sciences 133 (2019) 69–78
Acknowledgements
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This work was supported by the International Science & Technology
Cooperation Program of China [Grant Number: 2015DFA41670].
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