Liposomes actively recognizing the glucose transporter GLUT1 and integrin αvβ3 for dual-targeting of glioma

Article abstract of DOI:10.1002/ardp.201800219

The treatment of glioma is a great challenge because of the existence of the blood–brain barrier (BBB). In order to develop an efficient glioma-targeting drug delivery system to greatly improve the brain permeability of anti-cancer drugs and target glioma, a novel glioma-targeted glucose-RGD (Glu-RGD) derivative was designed and synthesized as ligand for preparing liposomes to effectively deliver paclitaxel (PTX) to cross the BBB and target glioma. The liposomes were prepared and characterized for particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis, and cell cytotoxicity. Also, the Glu-RGD modified liposomes showed superior targeting ability in in vitro and in vivo evaluation as compared to naked PTX, non-coated, singly modified liposomes and liposomes co-modified by physical blending. The relative uptake efficiency and concentration efficiency were enhanced by 4.41- and 4.72-fold compared to that of naked PTX, respectively. What is more, the Glu-RGD modified liposomes also displayed the maximum accumulation of DiD-loaded liposomes at tumor sites compared to the other groups in in vivo imaging. All the results in vitro and in vivo suggested that Glu-RGD-Lip would be a potential delivery system for PTX to treat integrin α_vβ₃-overexpressing tumor-bearing mice.

Full text of DOI:10.1002/ardp.201800219

Received: 22 July 2018
Revised: 5 November 2018
Accepted: 11 November 2018
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DOI: 10.1002/ardp.201800219
FULL PAPER
Liposomes actively recognizing the glucose transporter GLUT1
and integrin αvβ3 for dual-targeting of glioma
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Qiuyi Fu
Yi Zhao
Zhongzhen Yang
Qiming Yue
Wenjiao Xiao
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Yang Chen
Yang Yang
Li Guo
Yong Wu
Key Laboratory of Drug Targeting and Drug
Delivery System of Education Ministry,
Department of Medicinal Chemistry, West
China School of Pharmacy, Sichuan University,
Chengdu, P. R. China
Abstract
The treatment of glioma is a great challenge because of the existence of the blood–
brain barrier (BBB). In order to develop an efficient glioma-targeting drug delivery
system to greatly improve the brain permeability of anti-cancer drugs and target
glioma, a novel glioma-targeted glucose-RGD (Glu-RGD) derivative was designed and
synthesized as ligand for preparing liposomes to effectively deliver paclitaxel (PTX) to
cross the BBB and target glioma. The liposomes were prepared and characterized for
particle size, zeta potential, encapsulation efficiency, release profile, stability,
hemolysis, and cell cytotoxicity. Also, the Glu-RGD modified liposomes showed
superior targeting ability in in vitro and in vivo evaluation as compared to naked PTX,
non-coated, singly modified liposomes and liposomes co-modified by physical
blending. The relative uptake efficiency and concentration efficiency were enhanced
by 4.41- and 4.72-fold compared to that of naked PTX, respectively. What is more, the
Glu-RGD modified liposomes also displayed the maximum accumulation of DiD-loaded
liposomes at tumor sites compared to the other groups in in vivo imaging. All the results
in vitro and in vivo suggested that Glu-RGD-Lip would be a potential delivery system for
PTX to treat integrin αvβ3-overexpressing tumor-bearing mice.
Correspondence
Prof. Li Guo and Dr. Yong Wu, Key
Laboratory of Drug Targeting and Drug
Delivery System of Education Ministry,
Department of Medicinal Chemistry, West
China School of Pharmacy, Sichuan University,
Chengdu 610041, P. R. China.
Email: guoli@scu.edu.cn (L.G.),
wyong@scu.edu.cn (Y.W.)
Funding information
Sichuan Science and Technology Program,
Grant number: 2018JY0537; National Natural
Science Foundation of China, Grant numbers:
21472130, 81573286, 81773577; the
Fundamental Research Funds for the Central
Universities, Grant number: 2012017yjsy211
K E Y W O R D S
drug delivery, dual-targeting, glucose, liposomes, RGD peptide
of brain capillaries with the surrounding astrocytes and pericytes,[4]
resulting in a poor effect in the brain. Although increasing the dose of
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1
INTRODUCTION
Over the past few decades, with the rapid development of medical
science, many diseases are gradually being overcome. But the central
nervous system (CNS) diseases, such as brain tumor, have become one
of the most dangerous threats to human health, due to dramatic
increase of brain diseases and their lower recovery.[1–3] So, the
treatment for brain tumor has become a pressing problem that needs
to be solved. However, the poor brain permeability of anti-cancer
drugs (including paclitaxel) limits their penetration across blood–brain
barrier (BBB), which is formed by tightly connected endothelial cells
drugs in treatments may increase the vascular-corrected brain
concentration, it could cause stronger side effects and severer toxicity.
Therefore, there is a huge amount of demand of, not only for paclitaxel
(PTX) but in general, strategies that can effectively deliver drugs,
including PTX, into the brain for the treatment of brain tumor.[5,6]
The dual-targeting drug delivery system was raised by several
groups in 2009. With the development of targeting delivery system,
the strategies for dual-targeting brain tumor were divided into three
types.[7] The first type was to target two types of cells in brain tumor
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Arch Pharm Chem Life Sci. 2019;e1800219.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft
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with different ligands. Gao et al.[8] used IL-13p and RGD peptide to
target glioma and neovasculature, respectively. The second type was
one ligand with two different targeting warheads. Liu's group utilized
R8-RGD to target BBB and glioma.[9] The third type was to target BBB
and glioma with different ligands. Gao and his co-workers used
transferrin to transport across the BBB and glucose analog to
target glioma, which showed superior treatment effect for glioma.[10]
Thus, an ideal dual-targeting drug delivery system should effectively
transport across the BBB and target brain tumor.[11] In this paper,
Over the past years, our group has done a lot of researches on
brain targeting and cancer targeting moieties specially based on
glucose or peptides by active process.[17–19] In addition to targeting the
Warburg effect to deliver drugs to glioma, RGD sequence, an arginine-
glycine-aspartic (Arg-Gly-Asp) tripeptide, has been shown to be
another most promising ligand for targeting a range of malignant
tumors, including glioma, osteocarcinoma, breast cancer and other
human cancers, that are known to overexpress αvβ3 receptors.[20,21]
Meanwhile, the expression of αvβ3 in normal tissue is limited, all of
which make it to be an attractive ligand for glioma-targeting.[22] A lot of
interesting researches had been done on the nanocarriers modified by
RGD.[23–25]
we will explore the possibility to develop
a glioma-targeting
liposomes drug delivery system which uses glucose and RGD
peptide as the mediator to improve the delivery of drugs into brain
tumor tissue.
Herein, we aim to construct a dual-targeting moiety Glu-RGD-
Chol (Figure 1) as liposomes ligand to delivery PTX not only into brain
parenchyma but also into glioma tissue. What's more, the Glu-RGD
modified, non-coated, singly modified and co-modified by physical
blending liposomes were prepared by the lipid film hydration-
ultrasound method, and the characteristics and targeting abilities
were conducted in vitro and in vivo.
Because of the high transport affinity between the transporter and
substance, carrier-mediated transporter (CMT) system seems to be one
of the most promising methods to facilitate the delivery of drugs into
brain, among which GLUT1 expressed on the surface of brain capillary
endothelial cells is considered as one of the most efficient transport
systems that can transport glucose across the BBB into brain
effectively.[12] It is well known that the large and uninterrupted energy
demand of the brain is provided almost exclusively by glucose, which is
transported through the BBB by GLUT1. It is estimated that the
transport value of GLUT1 is 15–3000 fold more than other transporters.
Liposomes modified by glucose have been proposed as a strategy to
improve their brain uptake. Moreover, it has been widely reported that
C-6 position glycosylation is an effective way to heighten the
accumulation of drugs in brain.[12,13] Our previous studies have also
demonstrated that the glucose-modified liposomes could be delivered
into brain specifically and subsequently release the chemotherapeutic
agent, hence improving the concentration in the brain.[1,2,5,6] These
evidences all suggested that glucose could be used as a good carrier for
brain targeting drugs. What is more, it is well known that one unique
characteristic of cancer cells is that they need more glucose than normal
cells, which was known as the Warburg effect. Hence, many malignant
cells including the glioma have an overexpression of glucose trans-
porters (GLUTs), especially GLUT1, to maintain their growth and
survival. Targeting the Warburg effect as an anticancer strategy has
gained a great deal of interest in recent years.[14–16]
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
The synthetic route of ligand Glu-RGD-Chol is illustrated in
Scheme 1. Firstly, cholesterol 1, the starting material, underwent
four steps to generate compound 5,[2] which was then conjugated
with diethanol amine in the presence of isobutyl chloroformate
(IBCF) and N-methyl morpholine (NMM) to give compound 6.
Subsequently, condensation of compound 6 and glucose deriva-
tive 14 in the presence of dicyclohexyl carbodiimide (DCC)
and 4-dimethylamiopyridine (DMAP) gave 7, which was then
coupled with RGD peptide intermediate 20 to generate 8. Then,
after deprotecting the trimethylsilyl (TMS) groups and the
protecting groups of RGD from 8, the desired conjugate Glu-RGD-
Chol was obtained. All the title compounds and important
intermedium were characterized by their respective 1H NMR
and MS.
FIGURE 1 The structure of the ligand Glu-RGD-Chol

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SCHEME 1 Synthesis of ligand Glu-RGD-Chol. Reagents and conditions: (a) TsCl, pyridine, 50°C, 5 h; (b) triethylene glycol, dioxane, reflux,
6 h; (c) t-butyl bromoacetate, n-Bu4N+HSO4, 50% NaOH, toluene, r.t., 16 h; (d) TsOH, tolunene, reflux, 8 h; (e) diethanol amine, IBCF, NMM,
CH2Cl2, −10°C – r.t., 5 h; (f) 14, DCC, DMAP, CH2Cl2, −5°C – r.t., 5 h; (g) 20, DCC, DMAP, CH2Cl2, −5°C – r.t., 10 h; (h) TFA, CH2Cl2, r.t., 3 h;
(i) H2, Pd/C, CH3OH, 50°C, 24 h
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2.2 Preparation and characterization of liposomes
other hand, PTX-loaded liposomes achieved sustained release
behaviors so that the cumulative PTX release of drug-loaded liposomes
was less than 60% after 48 h incubation in PBS. No significant
difference on release properties was observed among PTX-Lip,
PTX-Glu-Lip, PTX-RGD-Lip, PTX-Glu-RGD-Lip and PTX-Glu +
RGD-Lip, and none of these PTX-loaded liposomes displayed burst
initial release patterns.
Proper size and uniform distribution of nanoparticles were required for
both BBB permeation and brain tumor targeting. The particle sizes and
zeta potentials of different liposomes in this study are listed in Table 1.
For the five types of liposomes, encapsulation efficiencies of paclitaxel
were all greater than 85%, respectively. The average particle sizes of all
liposomes were less than 110 nm, and the values of PDI were less than
0.2, fully complied with the conditions. What's more, the transmission
electron microscopy (TEM) of PTX-Glu-RGD-Lip showed that the
liposomes exhibited uniform spherical shape (Figure 2). To our
knowledge, the particle size and zeta potential of liposomes were
crucial to in vivo bio-distribution.[26]
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2.4 In vitro stability of liposomes in serum
Stability of liposomes under biological conditions is an important
parameter governing the activity of the associated therapeutic agent.
Transmittance of different liposomes were monitored in the presence
of 50% FBS. As shown in Figure 4, the transmittance of the liposomes
were above 90% and hardly changed after 48 h incubation with 50%
FBS. This stability study of liposomes indicated that the liposomes
were enough to prevent the interaction between liposomes and serum
protein, which was important to achieve a long blood half-life in vivo.
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2.3 In vitro drug release study
PTX release properties were evaluated in PBS containing 0.1% Tween
80. As shown in Figure 3, free PTX exhibited a rapid release, with over
80% of the drug released into the media within 12 h incubation. On the
TABLE 1 The composition and characterization of different PTX-loaded liposomes (n = 3)
Liposomes
Size (nm)
PDI
Zeta potential (mV)
−2.08 0.12
−3.45 0.29
−3.84 0.34
−4.56 0.19
−5.73 0.65
EE (%)
PTX-Lip
105.8 2.5
106.3 1.4
105.1 2.5
103.9 3.8
107.8 1.9
0.188 0.023
0.193 0.009
0.189 0.010
0.192 0.034
0.196 0.028
90.57 3.84
88.54 5.01
88.97 3.84
85.46 5.69
86.94 7.81
PTX-Glu-Lip
PTX-RGD-Lip
PTX-Glu-RGD-Lip
PTX-Glu + RGD-Lip

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FIGURE 4 The variations of transmittance of different modified
liposomes in 50% FBS (n = 3, mean SD)
showed higher inhibition rate than PTX-loaded liposomes, because
free drugs could be transported into the cells directly without a drug
release process. On the other hand, the cytotoxicity of blank liposomes
was also measured, and all these five kinds of blank liposomes
exhibited no significant cytotoxicity (Figure 6B and D). Therefore, our
liposomal drug delivery systems were safe and non-toxic to be further
used in vivo.
FIGURE 2 The TEM image of PTX-Glu-RGD-Lip
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2.5 Hemolysis assays
Hemocompatibility is a key point for in vivo applications of liposomes.
As shown in Figure 5, hemolysis assay of ligand-modified liposomes
demonstrated that all the liposomes did not show any significant
increase in the hemoglobin release up to 400 nmoles of phospholipids.
PTX-Glu-RGD-Lip did not display concentration-dependent increase
in hemolysis as well and less than 10% hemolysis was always regarded
as non-toxic.
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2.7 Cellular uptake study
The bEnd.3 cells having endothelial properties were widely used as a
model for simulating brain capillary endothelial cells and evaluating the
BBB penetrating capability in vitro. As the transporter GLUT1 and
integrin αvβ3 receptor were verified to be expressed on both bEnd.3
cells and C6 cells, the cellular uptake of different modified liposomes
was studied. Conventional liposomes were used as negative control.
Figure 7A depicted that the uptake of Glu-RGD-Lip was 2.42, 1.99,
2.06, and 1.23 times higher than that of Lip, Glu-Lip, RGD-Lip,
and Glu + RGD-Lip on bEnd.3 cells, respectively. What's more,
Glu-RGD-Lip also showed an excellent uptake on C6 cells, nearly
10-fold higher compared with Lip (Figure 7B). All data showed that
Glu-RGD-Lip had better targeting property to GLUT1 and integrin
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2.6 Cytotoxicity
The cytotoxicity of different liposomes on bEnd.3 cells and C6 cells
was evaluated using MTT assay. As shown in Figure 6A and C, free PTX
FIGURE 3 The PTX release profiles of free PTX, PTX-Lip, PTX-
Glu-Lip, PTX-RGD-Lip, PTX-Glu-RGD-Lip, and PTX-Glu + RGD-Lip in
PBS (pH 7.4) containing 0.1% Tween 80 over 48 h (n = 3,
mean SD)
FIGURE 5 Hemolysis percentage of different liposomes. Values
are represented as mean SD (n = 3)

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FIGURE 6 (A) The cytotoxicity study of PTX-loaded liposomes and free PTX on C6 cells. (B) The cytotoxicity study of blank liposomes on
C6 cells. (C) The cytotoxicity study of PTX-loaded liposomes and free PTX on bEnd.3 cells. (D) The cytotoxicity study of blank liposomes on
bEnd.3 cells (mean SD, n = 3)
αvβ3-expressed cells due to the synergistic effect of the dual-mediated
endocytosis.
C6 cells, and the inhibition rate was calculated to analyze the uptake
mechanism. As shown in Figure 7C and D, the preincubation of free
glucose or free RGD peptide could competitively inhibit the uptake
of Glu-RGD-Lip both on the two cell lines. This further proved that
In order to study the uptake mechanism of Glu-RGD-Lip, a series
of endocytosis inhibitors were pre-incubated with bEnd.3 cells and
FIGURE 7 Cellular uptake of CFPE-labeled liposomes on bEnd.3 cells (A) and C6 cells (B). Uptake mechanism study of Glu-RGD-Lip on
bEnd.3 cells (C) and C6 cells (D) (mean SD, n = 3), *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001 versus control group, N. S.
indicates no significant difference

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the glucose domain and RGD domain of liposomes increased the
cellular internalization by specifically binding to GLUT1 and αvβ3.
Furthermore, the effects of clathrin-mediated endocytosis on the
internalization of the Glu-RGD-Lip were evaluated using chlorprom-
azine and sucrose, a kind of clathrin-coated pits formation blocking
agent. β-Cyclodextrin, agent disrupting caveolae, was used to
evaluate the effect of caveolae-mediated endocytosis on the
internalization of the Glu-RGD-Lip. Amiloride was chosen to block
micropinocytosis. As the results showed, every inhibitor showed
different levels of inhibition effects. 4°C also had a strong effect on
cellular uptake (down to 27.74 and 11.84%, respectively), suggesting
energy-dependent properties of endocytosis. The images by CLSM
(Figure 8) were well consistent with the above results by a flow
cytometer. All the results indicated that Glu-RGD-Lip could be
recognized by glucose transporter GLUT1 and integrin αvβ3 receptor
on the outer membrane of cells via the residues on the surface,
10 mg/kg body weight of paclitaxel. At 5, 15, 30, 60, 120, 240, 480,
and 1440 min after injection, blood sample was collected from the eye
socket of mice, and placed in tubes containing heparin. Then blood and
brain were collected to analyze the concentration of paclitaxel at
different intervals by HPLC method.
The plasma PTX concentration-time profiles are shown in Figure 9
and the pharmacokinetic parameters of PTX from different formula-
tions are summarized in Table 2. The results showed that the area
under the concentration-time profile (AUC0–t) of paclitaxel in the five
types of liposomes was much higher than that of naked paclitaxel
within 24 h. The (AUC0–t) was increased by about 1.83-time for
PTX-Glu-RGD-Lip compared to free PTX. In addition, it was also shown
that PTX-Glu-RGD-Lip could extend the elimination half-life (t1/2) of
free PTX from 235 to 561 min. These data indicated that the liposomes
showed certain stability which would increase the chance to be
transported across BBB.
and then energy-dependently internalized through
a
synthetic
To further evaluate the possibility of the Glu-RGD-mediated
liposomes being transported across BBB, the distribution in brain of
PTX-loaded liposomes and paclitaxel was determined. The concen-
trations of paclitaxel in brain versus time curves are displayed in
Figure 10 and the pharmacokinetic parameters are listed in Table 3.
In brain, it is obvious that all of the liposomes could be delivered to
the brain following i.v. administration. At different time interval, the
concentration of paclitaxel released from liposomes was much higher
than that from paclitaxel original drug during 24 h. The AUC0–t and
Cmax of paclitaxel in brain after i.v. administration of liposomes were
fairly higher than that after the injection of free paclitaxel. The relative
uptake efficiencies (REs) were enhanced to 1.26, 3.80, 1.73, 4.41,
and 4.06 times than that of naked paclitaxel for liposomes PTX-Lip,
PTX-Glu-Lip, PTX-RGD-Lip, PTX-Glu-RGD-Lip, and PTX-Glu +
RGD-Lip, respectively. The concentration efficiencies (CEs) were
also enhanced to 1.03, 3.85, 2.44, 4.72, and 4.61 times than that of
paclitaxel. So these data further proved our conjecture that liposome
with ligand Glu-RGD can deliver and keep paclitaxel in the CNS.
endocytic pathway including clathrin-mediated, caveolae-mediated
and micropinocytosis-mediated endocytosis.
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2.8 Distribution in brain and pharmacokinetic
studies in mice
For in vivo study, PTX-Lip, PTX-Glu-Lip, PTX-RGD-Lip, PTX-Glu-RGD-
Lip, PTX-Glu + RGD-Lip and paclitaxel original drug were injected
through caudal vein of the mice with a single dose equivalent to
FIGURE 9 The concentration curve of paclitaxel versus time in
plasma after i.v. injection of paclitaxel and PTX-loaded liposomes in
mice (n = 5)
FIGURE 8 Qualitative cellular uptake of CFPE-labeled liposomes
on C6 cells

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TABLE 2 Pharmacokinetic parameters of paclitaxel in blood after administration of paclitaxel and liposomes (n = 5)
PTX-Glu +
RGD-Lip
Paclitaxel
PTX-Lip
PTX-Glu-Lip
PTX-RGD-Lip
PTX-Glu-RGD-Lip
AUC(0–t)
(µg/mL · min)
9617.65
15495.21
20914.38
2843.02
13756.15
2008.70
17648.27
1098.55
15338.97
1735.69
2062.56
2124.35
MRT (min)
Tmax (min)
Cmax (µg/mL)
t1/2 (min)
359.99 37.98
5
427.04 73.06
6
413.40 51.05
6
376.30 89.21
6
423.13 15.74
5
425.41 60.51
5
57.60 6.87
235.24 44.79
65.45 4.44
441.10 197.25
56.85 4.54
498.99 96.79
64.81 10.28
370.70 207.57
57.98 3.39
561.23 100.38
59.77 7.20
486.76 130.69
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2.9 In vivo imaging
glucose-RGD derivative was designed and synthesized as liposome
ligand in this work. Glu-RGD-Lip could be recognized by glucose
transporter GLUT1 and integrin αvβ3 receptor on the outer membrane
of cells via the residues on the surface, and then energy-dependently
internalized through a synthetic endocytic pathway including clathrin-
mediated, caveolae-mediated, and micropinocytosis-mediated endo-
cytosis. What is more, Glu-RGD-Lip was proved to increase the
transport of the liposomes across the BBB through GLUT1-mediated
transcytosis and afterwards target the brain glioma through GLUT1
and αvβ3 in vivo. Therefore, the Glu-RGD modification represents a
promising strategy for the development of future brain-specific drug
delivery systems.
Kunming mice bearing C6 glioma were used to estimate the glioma
targeting efficiency of different liposomes. Figure 11 shows the
in vivo images of intracranial C6 glioma bearing mice at different time
points after systemic administration of DiD-loaded liposomes. For
RGD-Lip group, the trace fluorescence in brain region indicated that
modification of RGD peptide had limited increase for liposomes to
penetrate across the BBB. It was perspicuously observed that the
fluorescence signal of Glu-RGD-Lip in the brain was stronger than
other groups from 1 to 24 h after systemic administration, displaying
the strongest glioma targeting capability of Glu-RGD-Lip. All of the
results suggested that Glu-RGD on the surface of liposomes
endowed the liposomes with brain glioma dual-targeted delivery
capability: Firstly this delivery system traversed the BBB via the
recognition of the facilitative GLUT1 on the BBB, and then was
internalized by the glioma cells via GLUT1 and αvβ3 mediated
endocytosis. These findings were well consistent with the results of
in vitro cellular uptake study.
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4
EXPERIMENTAL
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4.1 General
All liquid reagents were distilled before use. All unspecified
reagents were from commercial resources. TLC was performed
using precoated silica gel GF254 (0.2 mm), while column chroma-
tography was performed using silica gel (100–200 mesh). The
melting point was measured on a YRT-3 melting point apparatus
(Shantou Keyi instrument & Equipment Co. Ltd, Shantou, China).
Elemental analyses were performed by Atlantic Microlab
(Atlanta, GA, USA). 1H NMR spectra were taken on a Varian
INOVA 400/600 (Varian, Palo Alto, CA, USA) using CDCl3,
d6-DMSO and CD3OD as solvent. Chemical shifts are expressed
in δ (ppm), with tetramethylsilane (TMS) functioning as the internal
reference, and coupling constants (J) were expressed in Hz. Mass
spectra were recorded on an Agilent 1946B ESI-MS instrument
(Agilent, Palo Alto, CA, USA). Paclitaxel and docetaxel were
obtained from National Institute for Food and Drug Control.
Soybean phospholipids (SPC) were purchased from Kelong
Chemical. Cholesterol (Chol) was purchased from Bio Life Science
& Technology Co., Ltd. (Shanghai, People's Republic of China). A
Diamonsil column (200 × 4.6 mm, 5 mm) was used. A LC-10A liquid
chromatographic system (Shimadzu) and a reverse-phase HPLC
column (ODS-C18 column, 4.6 × 200 mm, 5 µm, SinoChrom, Dalian,
P.R. China) were used.
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3
CONCLUSION
In order to develop an efficient glioma targeting drug delivery system
to greatly improve the ability to target glioma, a novel dual-targeting
FIGURE 10 The concentration curve of paclitaxel versus time in
brain after i.v. injection of paclitaxel and PTX-loaded liposomes in
mice (n = 5)
The InChI codes of the inverstigated compounds are provided as
Supporting Information.

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TABLE 3 Pharmacokinetic parameters of paclitaxel in brain after administration of paclitaxel and liposomes (n = 5)
PTX-Glu +
RGD-Lip
Paclitaxel
PTX-Lip
PTX-Glu-Lip
3062.68 943.86
527.49 86.72
120
PTX-RGD-Lip
1393.83 87.38
396.63 19.90
132
PTX-Glu-RGD-Lip
3555.90 686.12
489.08 29.79
120
AUC(0–t) (µg/g · min)
MRT (min)
Tmax (min)
Cmax (µg/g)
t1/2 (min)
Re
805.85 181.60
1018.84 149.47
566.21 71.85
144
3272.55 259.40
432.68 29.24
120
548.37 42.52
60
1.49 0.27
1.54 0.46
598.10 315.68
1.26
5.74 0.26
718.03 204.11
3.80
3.64 0.49
543.76 251.15
1.73
7.04 1.17
847.43 230.10
4.41
6.87 0.74
526.11 198.60
4.06
310.26 138.69
–
–
Ce
1.03
3.85
2.44
4.72
4.61
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4.2 Chemistry
DMAP, then the mixture was stirred at −5°C for 30 min. Compound 6
(0.17 g, 0.26 mmol) in CH2Cl2 (5 mL) was added to the above reaction
mixture promptly. After stirring for 5 h at room temperature, the
reaction was terminated and then filtered. The filtrate was concen-
trated, and the residue was purified by flash chromatography to yield 7
(0.15 g, 59%) as a yellow oil. 1H NMR (400 MHz, CDCl3, ppm) δ: 0.18 (s,
36H), 0.73 (s, 3H), 0.88 (d, 6H, J = 6.4 Hz), 0.96 (d, 3H, J = 6.4 Hz), 1.03 (s,
3H), 0.73–2.38 (remaining cholesterol protons), 2.62–2.67 (m, 4H),
2.86 (s, 2H), 3.14–3.19 (m, 1H), 3.41–3.89 (m, 21H), 3.99–4.03 (m, 1H),
4.20–4.31 (m, 4H), 4.38 (d, 1H, J = 11.6 Hz), 5.03 (s, 1H), 5.35 (s, 1H).
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4.2.1 Synthesis of compounds 2–5
The synthesis of compounds 2–5 was reported in our previous work.[2]
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4.2.2 Synthesis of compound 6
To a solution of compound 5 (4.00 g, 6.93 mmol) in CH2Cl2 (25 mL) was
added NMM (0.84 mL, 7.63 mmol) and IBCF (0.96 mL, 7.63 mmol), and
the reaction was stirred at −10°C for 15 min. Then diethanol amine
(1.09 g, 10.40 mmol) in CH2Cl2 (5 mL) was added slowly. After stirring
for another 5 h at r.t., the mixture was washed with 1 mol/L HCl,
saturated NaHCO3 and saturated NaCl. The organic layer was dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography to afford 6 (4.09 g, 89%) as a
yellow oil. 1H NMR (400 MHz, CDCl3, ppm) δ: 0.67 (s, 3H), 0.86 (d, 6H,
J = 6.4 Hz), 0.91 (d, 3H, J = 6.4 Hz), 0.99 (s, 3H), 1.01–2.38 (remaining
cholesterol protons), 3.14–3.21 (m, 1H), 3.50–3.85 (m, 20H), 4.28
(s, 2H), 5.34 (d, 1H, J = 4.8 Hz).
|
4.2.4 Synthesis of compound 8
To a solution of compound 20 (44 mg, 0.066 mmol) in 5 mL dichloro-
methane was added DCC (16 mg, 0.079 mmol) and cat. DMAP, then the
mixturewasstirredat−5°C for 30 min. Compound 7(80 mg, 0.066 mmol)
in CH2Cl2 (3 mL) was added to the above reaction mixture slowly. After
stirring for 10 h at room temperature, the reaction was terminated and
then filtered. The filtrate was concentrated, and the residue was purified
by flash chromatography to give 8 (40 mg, 33%) as a yellow oil. 1H NMR
(400 MHz, CDCl3, ppm) δ: 0.13 (s, 36H), 0.66 (s, 3H), 0.86 (d, 6H,
J = 6.4 Hz), 0.91 (d, 3H, J = 6.4 Hz), 0.97 (s, 3H), 1.00–2.35 (remaining
cholesterol protons), 2.48–2.68 (m, 8H), 2.90–2.98 (s, 2H), 3.18–3.19
(m, 1H), 3.34–4.43 (m, 31H), 4.89 (s, 1H), 5.00 (d, 1H, J = 2.4 Hz), 5.05
(s, 2H), 5.10 (s, 2H), 5.33 (s, 1H), 7.28–7.32 (m, 10H), 7.46–7.78 (m, 4H).
|
4.2.3 Synthesis of compound 7
To
a solution of compound 14 (0.15 g, 0.26 mmol) in 15 mL
dichloromethane was added DCC (63 mg, 0.31 mmol) and cat.
|
4.2.5 Synthesis of compound 9
To a solution of compound 8 (50 mg, 0.027 mmol) in CH2Cl2 (8 mL) was
added trifluoracetic acid (2.0 mL) at 0°C and then the reaction was
stirred for 3 h at room temperature. The mixture was concentrated and
the residue was purified by chromatography to give compound 9
(25 mg, 59%) as a colorless oil. 1H NMR (600 MHz, CD3OD, ppm) δ:
0.70 (s, 3H), 1.00 (s, 3H), 0.70–2.37 (remaining cholesterol protons),
2.51–2.62 (m, 10H), 3.62–3.67 (m, 20H), 3.83–3.93 (m, 3H), 4.19–4.33
(m, 9H), 5.08–5.12 (m, 4H), 5.32–5.34 (m, 2H), 7.32 (s, 10H).
|
4.2.6 Synthesis of ligand Glu-RGD-Chol
FIGURE 11 In vivo images of intracranial C6 glioma bearing mice
at different time points after systemic administration of DiD-loaded
liposomes
To a solution of compound 9 (50 mg, 0.019 mmol) in CH3OH (6 mL),
Pd/C (10 mg, 10%) was added. Then, the mixture was stirred in

FU ET AL.
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hydrogen atmosphere at 50°C for 24 h. Pd/C was filtered and the
filtrate was concentrated to give ligand Glu-RGD-Chol (20 mg, 78%) as
a white solid. 1H NMR (400 MHz, CD3OD, ppm) δ: 0.65 (s, 3H), 0.83–
2.50 (remaining cholesterol protons), 2.57–2.64 (m, 10H), 2.81 (s, 3H),
2.87–3.22 (m, 5H), 3.33–3.36 (m, 2H), 3.54–3.66 (m, 27H), 3.76–3.97
(m, 4H), 4.16–4.36 (m, 14H), 4.47–4.50 (m, 1H), 4.62 (br, 1H), 5.08
(d, 2H, J = 3.6 Hz). ESI-MS calculated for C65H108N7O23 [M−H]−
1354.7, found: 1354.4.
release medium. Then the samples were diluted with acetonitrile and
the concentrations of paclitaxel were determined at the wavelength of
225 nm by HPLC.
|
4.5 In vitro stability of liposomes in serum
Turbidity variations were measured to demonstrate the serum stability
of liposomes in the presence of fetal bovine serum (FBS). Briefly,
liposomes were mixed with equal volume of FBS under 37°C with
moderate shaking at 45 rpm. The transmittance of the mixture was
measured at predetermined time points (0, 1, 2, 4, 8, 12, 24, and 48 h)
at 750 nm by a microplate reader (Thermo Scientific Varioskan Flash,
USA).
|
4.2.7 Synthesis of glucose derivatives, RGD peptide
intermediates, ligand Glu-Chol and RGD-Chol
The detailed synthetic procedures of glucose derivatives, RGD peptide
intermediates, ligand Glu-Chol and RGD-Chol are given in the
Supporting Information.
|
4.6 Hemolysis assays
To evaluate the safety of ligands-modified liposomes during body
circulation, hemolysis assay was performed. Fresh mouse blood was
collected in tubes containing heparin sodium. The red blood cells
(RBCs) were separated and collected by centrifugation at
5 × 103 rpm for 5 min and washed several times with PBS until the
supernatant became colorless. After the last wash, the RBCs were
diluted with PBS to a concentration of 2% (v/v). Various concen-
trations of liposomes were incubated with equal volume of 2% RBCs
solutions for 1 h at 37°C with gentle shaking, followed by
centrifugation at 1 × 104 rpm for 10 min. Absorbance of hemoglobin
|
4.3 Preparation and characterization of liposomes
Liposomes were prepared by thin film hydration method. Lipid
compositions of the prepared liposomes were as follows: (1)
conventional liposomes (Lip), SPC/cholesterol (molar ratio = 62:33);
(2) ligand Glu-RGD-Chol modified liposomes (Glu-RGD-Lip), SPC/
cholesterol/ligand Glu-RGD-Chol (molar ratio = 62:33:3); (3) ligand
Glu-Chol modified liposomes (Glu-Lip), SPC/cholesterol/ligand
Glu-Chol (molar ratio = 62:33:3); (4) ligand RGD-Chol modified
liposomes (RGD-Lip), SPC/cholesterol/ligand RGD-Chol (molar
ratio = 62:33:3); (5) ligand Glu-Chol and RGD-Chol co-modified
liposomes (Glu + RGD-Lip), SPC/cholesterol/ligand Glu-Chol/RGD-
Chol (molar ratio = 62:33:3:3). All lipid materials were dissolved in
the mixture solvent chloroform/methanol (v/v = 2:1), and then the
organic solvent was removed by rotary evaporation to form a lipid
film. After kept in vacuum overnight, the obtained film was
hydrated in PBS (pH 7.4) for 0.5 h at 20°C. Then it was further
intermittently sonicated by a probe sonicator at 80 W for 80 s to
form liposomes. PTX-loaded liposomes were prepared with
paclitaxel added to the lipid organic solution prior to the solvent
evaporation. The entrapment efficiency of paclitaxel was deter-
mined by HPLC. Likewise, CFPE-labeled liposomes were prepared
by adding appropriate amount of CFPE to the solution before
the solvent evaporation. The mean size and zeta potential of
Lip, Glu-RGD-Lip, Glu-Lip, RGD-Lip and Glu + RGD-Lip were
detected by Malvern Zeta sizer Nano ZS90 (Malvern Instruments
Ltd., UK).
was measured using
a microplate reader (Thermo Scientific
Varioskan Flash) at 540 nm. The values for 0% and 100% hemolysis
were determined by incubating erythrocytes with PBS or 1% (v/v)
Triton X-100. The hemolysis percentage was calculated using the
following equation:
À
Á À
Á
The percent hemolysis ¼ ASample ꢀ ANegative = APositive ꢀ ANegative ꢁ 100%
where A is the ultraviolet absorbance of hemoglobin.
|
4.7 Cytotoxicity
Murine brain endothelial cells (bEnd.3) and glioma cells (C6) were
cultured in Dulbecco's modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 100 U/mL streptomycin and
100 U/mL penicillin at 37°C in a 5% CO2 humidified environment
incubator (Thermo Scientific, USA).
The cytotoxicity of PTX-loaded liposomes was measured with
MTT assay. Generally, the cells were seeded in a 96-well plate at a
density of 5 × 103 cells/well and cultured for 24 h at 37°C. PTX-
loaded liposomes and free paclitaxel were diluted to predeter-
mined concentrations with PBS, and added into each well for 24 h
incubation. The final concentrations of paclitaxel were in the range
of 0.016–50 µg/mL. Blank liposomes were added at the same
concentration of PTX-loaded liposomes. Then, 20 µL MTT sol-
utions (5.0 mg/mL) was added to the medium and incubated for
another 4 h at 37°C. After removal of the culture medium, the
|
4.4 In vitro drug release study
In vitro paclitaxel release study was performed using dialysis method.
Each PTX-loaded liposomes (0.4 mL) or free paclitaxel were placed into
dialysis tubes (MWCO = 8000–14000 Da) and tightly sealed. Then the
dialysis tubes were placed into 40 mL PBS containing 0.1% (v/v) Tween
80 and incubated under 37°C for 48 h with gentle oscillating at 45 rpm.
At predetermined time points (0, 1, 2, 4, 8, 12, 24, and 48 h), 0.1 mL
release medium was taken out and replaced with equal volume of fresh

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reduced MTT dye was solubilized by DMSO (150 µL) and the
absorbance was read at 490 nm wavelength on an automatic microplate
spectrophotometer. Cell viability (%) was calculated as the following
equation: Atest/Acontrol × 100%, where Atest and Acontrol represented the
absorbance of treated cells and control cells, respectively.
1.0 mL/min. The sample volume injected was 20 μL and the detection
wavelength was 227 nm.
|
4.9.2 Test animals
Adult Kunming mice weighing 20–22 g were obtained from the animal
center of Sichuan University. The animals were left for 2 days to
acclimatize to animal room conditions and were maintained on
standard pellet diet and water ad libitum. Food was withdrawn on
the day before the experiment, but free access to water was allowed.
Since the experiment could be completed within 24 h, there was no
significant change in the mice's body weight during the experiment. All
animals received human care, and the study protocols complied with
the guidelines of Sichuan University animal ethical experimentation
committee. Throughout the experiments, the animals were handled
according to the requirements of the National Act on the use of
experimental animals (People's Republic of China).
|
4.8 Cellular uptake study
|
4.8.1 Cellular uptake
bEnd.3 cells and C6 cells were seeded in 6-well plates at a density of
5 × 105 cells/well and cultured for 24 h at 37°C. Different liposomes
were added into each well with a final concentration of CFPE at
2 μg/mL. After cultured for 4 h at 37°C, trypsin was used to harvest the
cells, which were washed three times with PBS afterward, and finally
resuspended in 0.5 mL PBS. Then, the fluorescent intensity of cells
treated with different liposomes was measured by a flow cytometer
(Cytomics FC 500, Beckman Coulter, USA).
|
4.9.3 Sample preparation
For confocal microscope studies, C6 cells were plated onto a
6-well plate containing cover glass at a density of 5 × 105 cells/well and
cultured for 24 h at 37°C. CFPE-labeled liposomes were added into
each well and allowed for further co-incubation for 4 h. Following that,
the cells were rinsed with cold PBS three times and fixed with 4%
paraformaldehyde for 30 min at room temperature, and then cell nuclei
were stained with DAPI (1 μg/mL) for 5 min. Finally, the samples were
imaged using laser scanning confocal microscope (CLSM) (TCS SP5
AOBS confocal microscopy system, Leica, Germany).
Blood was collected fromthe eye socketofmouseinto a tubecontaining
heparin sodium, andcentrifugedat 5000 rpm for5 min. The supernatant
was collected as plasma sample. The animals were killed by cervical
dislocation, and the brains were removed and flushed with saline for
three times to remove the remaining blood and then the brains were
rolled over onthe filter paper carefully toremovethe main vessel. Allthe
tissues were homogenized with triple amount of saline. An aliquot of
10 µLofinternalstandard (docetaxel, 30 µL, 100 µg/mL) wasaddedinto
100 µL plasma or 100 µL brain homogenate, and extracted with 200 µL
ether. The mixture was vortexed for 5 min, and centrifuged at
10000 rpm for 10 min. The supernatant was transferred to another
centrifuge tube, and dried under air stream at room temperature. The
dry residue was reconstituted with 100 µL of methanol. The solution
was centrifuged at 10000 rpm for 10 min, and then 20 µL of the
supernatant was injected into the HPLC system for analysis.
|
4.8.2 Uptake mechanism study
In order to study the uptake mechanism of Glu-RGD-Lip, bEnd.3
cells and C6 cells were preincubated with various endocytosis
inhibitors, such as chlorpromazine (20 μg/mL), sucrose (137 mg/mL),
β-cyclodextrin (5.68 mg/mL), and amiloride (1.48 mg/mL).[27–29]
What is more, the inhibition of free glucose (5 mg/mL) and free
RGD peptide (0.2 mg/mL), and the effect of temperature were also
performed. Briefly, the cells were preincubated with different
inhibitors for 30 min at 37°C, then the inhibitors were withdrawn
from the wells and CFPE-labeled Glu-RGD-Lip was added. After
cultured for 4 h at 37°C, the cells were treated as described in 4.8.1
and the fluorescent intensity was measured by a flow cytometer.
|
4.9.4 Body distribution study
Kunming mice were randomly divided into groups for each liposome or
paclitaxel. PTX-Lip, PTX-Glu-Lip, PTX-RGD-Lip, PTX-Glu-RGD-Lip,
PTX-Glu + RGD-Lip and paclitaxel were given to the mice via the tail
vein and each was equivalent to the administration dose of paclitaxel
of 10 mg/kg. At 5, 15, 30, 60, 120, 240, 480, and 1440 min after
injection, blood samples were collected from the eye socket of mice
and placed in tubes containing heparin sodium. After exsanguinations,
the mice were killed by cervical dislocation and the brain was removed
and washed twice with saline. The brain was weighed and prepared as
described earlier. The concentration of paclitaxel was analyzed by
HPLC.
|
4.9 Distribution in brain and pharmacokinetic
studies in mice
|
4.9.1 HPLC analysis of liposomes
The naked paclitaxel and paclitaxel encapsulated liposomes (PTX-Lip,
PTX-Glu-Lip, PTX-RGD-Lip, PTX-Glu-RGD-Lip, PTX-Glu + RGD-Lip)
were measured by LC-10A liquid chromatographic system (Shimadzu).
The analytical column was a reverse-phase HPLC column (ODS-C18
column, 4.6 × 200 mm, 5 μm, SinoChrom) maintaining at 30°C. The
mobile phase consisted of water/methanol (67:33) with a low flow rate
|
4.9.5 Statistical analysis
The area under the concentration-time profile (AUC0–t), maximal
concentration (Cmax), and mean residence times (MRT) were calculated

FU ET AL.
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by Data and max Statistics (DAS 2.0, Shanghai, China). Statistical
evaluation was performed using analysis of variance followed by t-test.
A value of p < 0.05 was considered significant. The relative uptake
efficiency (RE) and concentration efficiency (CE) were calculated to
evaluate the brain targeting property of liposome. The value of RE and
CE were defined as follows:
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RE ¼ ðAUC0ꢀtÞL=ðAUC0ꢀtÞP
CE ¼ ðCmaxÞL=ðCmaxÞP,
[12] Y. Zhao, L. Zhang, Y. Peng, Q. M. Yue, L. Hai, L. Guo, Q. T. Wang, Y.
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where L and P represented the liposomes loading PTX and paclitaxel,
respectively.
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Pomper, J. O. Liu, Angew. Chem. 2016, 55, 12035.
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4.10 In vivo imaging
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Kunming mice were anesthetized with 5% chloral hydrate and placed
on a stereotaxic apparatus. C6 cells (2 × 108 cells/mL) were injected
into the striatum (1.8 mm lateral, 0.6 mm anterior to the bregma and
3 mm of depth) of each mouse. After 10 days, the mice were randomly
divided into four groups (three in each group) and were intravenously
injected via the caudal vein of the tumor-bearing mice with DiD-Glu-
Lip, DiD-RGD-Lip, DiD-Glu-RGD-Lip, and DiD-Glu + RGD-Lip. Then,
the mice were imaged with IVI Spectrum system (Caliper, Hopkington,
MA, USA) at 1, 2, 6, and 24 h after injection.
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ACKNOWLEDGMENTS
[23] W. Xiao, J. Y. Xiong, S. Zhang, Y. Xiong, H. J. Zhang, H. L. Gao, Int. J.
Pharmaceut. 2018, 538, 105.
This work was supported by the National Natural Science Foundation
of China (Nos. 81573286, 81773577, and 21472130) and the Sichuan
Science and Technology Program (2018JY0537), the Fundamental
Research Funds for the Central Universities (2012017yjsy211).
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NANO 2018, 13, 1850008.
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J. C. Wang, X. Zhang, Q. Zhang, Biomaterials 2013, 34, 794.
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CONFLICT OF INTEREST
The authors declare no competing financial interest
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Y. K. Xie, X. Y. Sha, X. L. Fang, Biomaterials 2014, 35, 518.
ORCID
SUPPORTING INFORMATION
Yong Wu
http://orcid.org/0000-0003-0719-4963
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
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