Dual-targeting for brain-specific liposomes drug delivery system: Synthesis and preliminary evaluation

Article abstract of DOI:10.1016/j.bmc.2018.08.006

The treatment of glioma has become a great challenge because of the existence of brain barrier (BB). In order to develop an efficient brain targeting drug delivery system to greatly improve the brain permeability of anti-cancer drugs, a novel brain-targeted glucose-vitamin C (Glu-Vc) derivative was designed and synthesized as liposome ligand for preparing liposome to effectively deliver paclitaxel (PTX). The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity were also characterized. What's more, the cellular uptake of CFPE-labeled Glu-Vc-Lip on GLUT₁- and SVCT₂-overexpressed C6 cells was 4.79-, 1.95-, 4.00- and 1.53-fold higher than that of Lip, Glu-Lip, Vc-Lip and Glu + Vc-Lip. Also, the Glu-Vc modified liposomes showed superior targeting ability in vivo evaluation compared with naked paclitaxel, non-coated, singly-modified and co-modified by physical blending liposomes. The relative uptake efficiency was enhanced by 7.53 fold to that of naked paclitaxel, while the concentration efficiency was up to 7.89 times. What's more, the Glu-Vc modified liposomes also displayed the maximum accumulation of DiD-loaded liposomes at tumor sites with the strongest fluorescence in the brain in vivo imaging. Our results suggest that chemical modification of liposomes with warheads of glucose and vitamin C represents a promising and efficient strategy for the development of brain-specific liposomes drug delivery system by utilizing the endogenous transportation mechanism of the warheads.

Full text of DOI:10.1016/j.bmc.2018.08.006

Accepted Manuscript
Dual-targeting for brain-specific liposomes drug delivery system: synthesis and
preliminary evaluation
Yao Peng, Yi Zhao, Yang Chen, Zhongzhen Yang, Li Zhang, Wenjiao Xiao,
Jincheng Yang, Li Guo, Yong Wu
PII:
S0968-0896(18)30915-5
https://doi.org/10.1016/j.bmc.2018.08.006
BMC 14493
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 May 2018
25 July 2018
4 August 2018
Please cite this article as: Peng, Y., Zhao, Y., Chen, Y., Yang, Z., Zhang, L., Xiao, W., Yang, J., Guo, L., Wu, Y.,
Dual-targeting for brain-specific liposomes drug delivery system: synthesis and preliminary evaluation, Bioorganic
& Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.006
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Dual-targeting for brain-specific liposomes drug delivery
system: synthesis and preliminary evaluation
Yao Penga, Yi Zhaoa, Yang Chena, Zhongzhen Yanga, Li Zhanga, Wenjiao Xiaoa,
Jincheng Yangb, Li Guoa*, Yong Wua
aKey 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. of China.
bDepartment of Pharmacy, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing 100021, P. R. of China.
Abstract: The treatment of glioma has become a great challenge because of the existence of brain barrier
(BB). In order to develop an efficient brain targeting drug delivery system to greatly improve the brain
permeability of anti-cancer drugs, a novel brain-targeted glucose-vitamin C (Glu-Vc) derivative was
designed and synthesized as liposome ligand for preparing liposome to effectively deliver paclitaxel (PTX).
The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile,
stability, hemolysis and cytotoxicity were also characterized. What’s more, the cellular uptake of
CFPE-labeled Glu-Vc-Lip on GLUT1- and SVCT2-overexpressed C6 cells was 4.79-, 1.95-, 4.00- and
1.53-fold higher than that of Lip, Glu-Lip, Vc-Lip and Glu+Vc-Lip. Also, the Glu-Vc modified liposomes
showed superior targeting ability in vivo evaluation compared with naked paclitaxel, non-coated,
singly-modified and co-modified by physical blending liposomes. The relative uptake efficiency was
* Correspondence authors. Tel +86 28 85503777 (L. Guo), +86 28 85503235 (Y. Wu).
E-mail addresses: guoli@scu.edu.cn (L. Guo), wyong@scu.edu.cn (Y. Wu).
1

enhanced by 7.53 fold to that of naked paclitaxel, while the concentration efficiency was up to 7.89 times.
What’s more, the Glu-Vc modified liposomes also displayed the maximum accumulation of DiD-loaded
liposomes at tumor sites with the strongest fluorescence in the brain in vivo imaging. Our results suggest
that chemical modification of liposomes with warheads of glucose and vitamin C represents a promising
and efficient strategy for the development of brain-specific liposomes drug delivery system by utilizing the
endogenous transportation mechanism of the warheads.
Keywords: glucose, ascorbic acid, drug delivery, dual-targeting, liposomes, brain barrier
1. Introduction
With the rapid development of medical science, many diseases have been conquered over the past few
decades. While, the central nervous system (CNS) diseases, such as brain tumor, have become one of the
most dangerous threats to human health,1,2 due to dramatic increase of brain diseases and their lower
recovery. So, the treatment of brain tumor needs to be solved urgently. However, the two physiological
barriers that separate the brain from its blood supply, would significantly decrease the accumulation of
anti-cancer drug (including paclitaxel) in the brain and lead to an extremely limited distribution in CNS.3
One is the blood-brain barrier (BBB) and the other is the blood-cerebrospinal fluid barrier (BCSFB).4-10
The barriers control the entry and exit of endogenous and exogenous compounds. Generally, the capability
of molecules to cross the barriers by passive diffusion is related to their molecular weight, lipid solubility,
charge, hydrogen bonding, ionization profile and physicochemical characteristics.11,12 As a result, over
98% of small molecule drugs and all the macromolecular drugs could not reach the therapeutic
concentration in the brain. Although the vascular-corrected brain concentration could be enhanced by
increasing the dose of drug in treatments, it could cause serious side effects and severer toxicity meanwhile.
2

Therefore, there is a huge amount of demand of, not only for paclitaxel (PTX) but in general, strategies
that can effectively deliver drugs into the brain for the treatment of brain tumor. 13-15
Because of the high transport affinity between the transporter and substance, carrier-mediated
transporter (CMT) system has become one of the most promising methods to facilitate the delivery of
drugs into brain.16,17 There are plenty of physiological transport systems for nutrients and endogenous
compounds, for example, glucose transporter 1 (GLUT1), sodium-dependent vitamin C transporter 2
(SVCT2), large neutral amino acid transporter 1 (LAT1) and monocarboxylic acid transporter 1
(MCT1).18-20
GLUT1 and SVCT2 over-expressed on the surface of brain capillary endothelial cells and choroid plexus
epithelium cells respectively, are considered as the most efficient transportation systems.7,12
It is well known that the enormous and sustained energy demand of the brain is provided almost
exclusively by GLUT1-transported glucose (Glu). It is estimated that 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.22,23 Our previous studies have also shown
that the liposomes modified with glucose could be transported into brain specifically and subsequently
release the drugs, hence increasing the concentration in the brain.16,19 These evidences all suggested that
glucose could be used as a good carrier for brain targeting drugs.
In addition to glucose, it was also widely reported that the vitamin C (Vc) derivatives have a superior
brain targeting efficiency. Studies have indicated that there is a highest concentration of Vc in the brain,
which is 10 fold more than that in other organs.24 In general, Vc is transported into the brain mainly
through two different ways. One is the sugar transporter GLUT1, which can transport dehydro-vitamin C
3

(DHVC, the oxidized form of Vc and can be reduced to Vc in brain). The other way is the transporter
SVCT2 which transports Vc into cerebrospinal fluid (CSF) at the choroid plexus, from which Vc can be
further diffused to brain extracellular fluid (ECF), and then taken up into the brain cells from ECF. What’s
more, it was reported that the hydroxyl groups of enediol lactone in C2 and C3 are vital reaction sites for
Vc in the redox process, while the C5- and C6-hydroxyl groups of Vc are not critical for its
transportation.25 Therefore, the modification of Vc is always conducted at C5 and C6 sites. Given all the
evidences, it is also a promising method to utilize Vc and its transporters to improve the brain targeting
ability.
Recently, some studies have explored the possibility of using Glu or Vc as carriers to promote the
permeation across the BB into brain. But single-modification with Glu or Vc has limited targeting
efficiency with 2-4 fold increase.16,18,19 In our previous work, we designed and synthesized dual-brain
targeting ibuprofen prodrugs, which had much better efficiency in vitro and in vivo than the parent drug
and the single-modified prodrugs.11 In this study, we will explore the possibility to develop a
brain-targeting liposomes drug delivery system which uses glucose and vitamin C as the mediators to
improve the brain permeability. We aim to construct a dual-targeting moiety Glu-Vc-Chol as liposomes
ligand to delivery PTX into brain effectively. What’s more, the Glu-Vc modified, non-coated,
singly-modified and co-modified by physical blending liposomes (Glu+Vc-Lip) were prepared by the lipid
film hydration-ultrasound method, and the characteristics and brain target abilities were conducted in vitro
and in vivo.
2. Results and discussion
2.1. Synthesis of liposomes ligands
4

The synthetic route of ligand Glu-Vc-Chol was illustrated in Scheme 1. Firstly, we synthesized the
glycosylated derivative 5 and the ascorbic acid intermediate 9. Briefly, glucose 1 was totally etherified
with chlorotrimethylsilane (TMSCl) and hexamethyl disilazane in pyridine to give
penta-O-trimethylsilyl-glucopyranose 2, which was treated by the mixture of acetone, acetic acid and
methanol to deprotect the C-6 TMS group selectively to obtain compound 3. Then, the linker
4-(benzyloxy)-4-oxobutanoic acid was connected with intermediate 3 to generate compound 4. After
deprotecting the benzyl group from 4, glucose-TMS derivative 5 was obtained.12 The synthesis of
intermediate 9 started from the available material Vc. Treatment of 6 with acetyl chloride in acetone
following our earlier method afforded 5,6-O,O-isopropylidene protected Vc ketol 7. Benzylation of the C-2
and C-3 hydroxy groups of the ketol 7 was accomplished using K2CO3 and benzyl bromide in acetone to
provide 8. Deblocking of the 5,6-O,O-protected derivative of 8 with HCl in CH3CN solution gave
2,3-O,O-dibenzyl Vc intermediate 9.7 What’s more, cholesterol 10 underwent five steps to generate
compound 15,13 which was then conjugated with glycosylated derivative 5 in the presence of DCC and
DMAP to give compound 16. Finally, compound 16 was readily underwent a deprotection reaction to get
compound 17 when subjected the condition of trifluoroacetic acid in dichloromethane, then the
2,3-O-di-benzyl groups of 17 was removed under hydrogen catalyzed by Pd/C to provide the target ligand
Glu-Vc-Chol.
5

Scheme 1. Synthesis of ligand Glu-Vc-Chol. Reagents and conditions: (a) HMDS, TMSCl, pyridine, 0 °C - r.t.; (b) AcOH,
CH3OH, acetone, 0 °C; (c) 4-(benzyloxy)-4-oxobutanoic acid, DCC, DMAP, CH2Cl2, r.t.; (d) Pd/C, H2, methanol, r.t.; (e)
acetone, AcCl, r.t., 24 h; (f) K2CO3, BnBr, acetone, reflux, 5 h; (g) HCl, CH3CN, r.t., 3 h; (h) TsCl, pyridine, 50°C, 5 h; (i)
Triethylene glycol, dioxane, reflux, 6 h; (j) t-butyl bromoacetate, n-Bu4N+HSO4, 50% NaOH, toluene, r.t., 16 h; (k) TsOH,
tolunene, reflux 8 h; (l) 9, DCC, DMAP, CH2Cl2, r.t., 4 h; (m) 5, DCC, DMAP, CH2Cl2, r.t., 5 h; (n) TFA, CH2Cl2, r.t., 1 h; (o)
Pd/C (10%), H2, CH3OH, 1 h.
The synthetic route of ligand Vc-Chol was illustrated in Scheme 2. Briefly, the 2,3-O-di-benzyl groups
of 15 was removed under hydrogen catalyzed by Pd/C to provide the target ligand Vc-Chol. All the title
compounds and important intermediates were characterized by their respective 1H NMR and MS.
6

Scheme 2. Synthesis of ligand Vc-Chol. Reagents and conditions: (a) Pd/C (10%), H2, CH3OH, 1 h.
2.2. Preparation and characterization of liposomes
One of the requirement for liposomes to penetrate BBB is that they must have proper sizes and uniform
distribution. The particle sizes and zeta potentials of different liposomes in this study were listed in Table 1.
The encapsulation efficiencies (EE%) of paclitaxel in the each type of liposomes were all greater than 82%.
The average particle sizes of all liposomes were less than 110 nm, and the values of polymer dispersity
index (PDI) were close to 0.19. What’s more, the transmission electron microscopy (TEM) of
PTX-Glu-Vc-Lip showed that the liposomes exhibited uniform spherical in shape (Fig. 1). To our
knowledge, the particle size and zeta potential of liposomes were crucial to in vivo study.20
Table 1 Particle sizes and zeta potentials of different liposomes and the PTX entrapment efficiency of PTX-loaded
liposomes (n =3, mean ± SD).
Size (nm)
105.8 ± 2.5
106.3 ± 1.4
102.9 ± 1.7
109.2 ± 3.3
108.5 ± 2.6
PDI
Zeta potential (mV)
-2.08 ± 0.12
EE (%)
PTX-Lip
0.188 ± 0.023
0.193 ± 0.009
0.198 ± 0.017
0.185 ± 0.025
0.186 ± 0.014
90.57 ± 3.84
88.54 ± 5.01
89.62 ± 2.97
86.37 ± 4.68
87.51 ± 6.17
PTX-Glu-Lip
-3.45 ± 0.29
PTX-Vc-Lip
-2.88 ± 0.33
PTX-Glu-Vc-Lip
PTX-Glu+Vc-Lip
-4.13 ± 0.20
-4.66 ± 0.45
7

Fig. 1. The TEM image of PTX-Glu-Vc-Lip
2.3. In vitro drug release study
PTX release properties were evaluated in PBS containing 0.1% Tween 80. As shown in Fig. 2, for free
PTX, the release exhibited a rapid property, with over 80% of the drug released into the media within 12 h
incubation. On the other hand, the PTX-loaded liposomes exhibited the sustained release behaviors, with
the cumulative PTX released from liposomes was less than 60% after 48 h incubation. No significant
difference on release properties was observed among PTX-Lip, PTX-Glu-Lip, PTX-Vc-Lip,
PTX-Glu-Vc-Lip and PTX-Glu+Vc-Lip, and none of these PTX-loaded liposomes displayed burst initial
release patterns.
8

Fig.2. The PTX release profiles of free PTX, PTX-Lip, PTX-Glu-Lip, PTX-Vc-Lip, PTX-Glu-Vc-Lip and PTX-Glu+Vc-Lip
in PBS (pH 7.4) containing 0.1% Tween 80 over 48 h (n = 3, mean ± SD).
2.4. In vitro stability of liposomes in serum
It is important for liposomes to have superior stability in biological conditions, which is closely related
to governing the activity of the associated therapeutic agent. Transmittance of different liposomes were
monitored in the presence of 50% FBS. As shown in Fig. 3, 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 able to prevent the interaction between liposomes and serum protein, which was
important for the long blood half-life in vivo.
9

Fig. 3. Serum stability (represented by transmittance) of Lip, Glu-Lip, Vc-Lip, Glu-Vc-Lip and Glu+Vc-Lip in 50% FBS
over 48 h (n = 3, mean ± SD).
2.5.Hemolysis assays
Hemocompatibility is a key point for in vivo applications of liposomes. As shown in Fig. 4, hemolysis
assay of ligand-modified liposomes demonstrated that all the liposomes did not significantly increase the
hemocompatibility (less than 10%) even the concentration of phospholipids up to 400 nmoles, which
indicated that the lipid material was almost no-toxic..
10

Fig. 4. Hemolysis percentage of various concentrations of Lip, Glu-Lip, Vc-Lip, Glu-Vc-Lip and Glu+Vc-Lip after
incubation for 1 h at 37 °C (n = 3, mean ± SD).
2.6.Cytotoxicity
The cytotoxicity of different liposomes on C6 cells was evaluated using MTT assay. As shown in Fig.
5A, free PTX showed higher inhibition rate than PTX-loaded liposomes, which may be because that free
drugs could be transported into the cells directly by passive diffusion, without a drug release process.
What’s more, among the five groups of liposomes, PTX-Glu-Vc-Lip had the lowest cell viability, which
may due to the transport by the two transporters. On the other hand, the cytotoxicity of blank liposomes
was also measured, and all these five kinds of blank liposomes exhibited no significant cytotoxicity (Fig.
5B). Therefore our liposomal drug delivery systems were safe and nontoxic to be further used in vivo.
11

Fig. 5. (A) The cytotoxicity study on C6 cells after treated with various concentrations of PTX-Lip, PTX-Glu-Lip,
PTX-Vc-Lip, PTX-Glu-Vc-Lip and free PTX for 24 h (n = 3, mean ± SD). (B) The cytotoxicity study on C6 cells after
treated with various concentrations of Lip, Glu-Lip, Vc-Lip, Glu-Vc-Lip and Glu+Vc-Lip for 24 h (n = 3, mean ± SD).
2.7.Cellular uptake
To access whether Glu-Vc-Lip could deliver more drugs into cells than other groups of liposomes, the
GLUT1 and SVCT2-positive C6 cells were chosen as the cell model. It could be seen from Fig. 6A that the
uptake of ligand-modified liposomes was significantly higher compared with the conventional liposomes
(Lip). What’s more, the uptake of Glu-Vc-Lip showed 4.79, 1.95, 4.00 and 1.53 time higher than that of
Lip, Glu-Lip, Vc-Lip and Glu+Vc-Lip, respectively, which indicated that the Glu-Vc moiety exhibited
better penetrability in C6 glioma cells.
The images by confocal laser scanning microscope (CLSM) (Fig. 6B) were well consistent with the
above results by a flow cytometer. The green fluorescence can be clearly observed in Glu-Vc-Lip, which
was much stronger than that of other groups. All the results showed that Glu-Vc-Lip had better targeting
property to GLUT1- and SVCT2-expressed C6 cells.
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Fig. 6. Cellular uptake of CFPE-labeled Lip, Glu-Lip, Vc-Lip, Glu-Vc-Lip and Glu+Vc-Lip on C6 cells. (A) Quantitative 4
h cellular uptake of CFPE-labeled liposomes determined by flow cytometer (mean ± SD, n = 3). (B) Qualitative 4 h cellular
uptake of CFPE-labeled liposomes determined by CLSM, green (CFPE-labeled liposomes), blue (DAPI stained nucleus),
light blue (colocalized CFPE and DAPI). Scale bars represent 10 μm. (C) The relative uptake of CFPE-labeled Glu-Vc-Lip
on C6 cells for 4 h after preincubation of different endocytosis inhibitors determined by flow cytometer (mean ± SD, n = 3).
In order to further study the penetrability of Glu-Vc-Lip, the uptake mechanism was conducted by using
a series of endocytosis inhibitors to pre-incubate with C6 cells, and then calculating the inhibition rate. As
shown in Fig. 6C, the preincubation of free glucose or free Vc could competitively inhibited the uptake of
Glu-Vc-Lip on C6 cells. This further proved that the glucose domain and Vc domain of liposomes
increased the cellular internalization by specifically binding to GLUT1 and SVCT2. Furthermore, the
effects of clathrin-mediated endocytosis on the internalization of the Glu-Vc-Lip were evaluated using
chlorpromazine and sucrose, a kind of clathrin-coated pits formation blocking agent. β-cyclodextrin, agent
13

disrupting caveolae, was used to evaluate the effect of caveolae-mediated endocytosis on the
internalization of the Glu-Vc-Lip. Amiloride was chosen to block micropinocytosis. As the results showed,
every inhibitor showed different levels of inhibition effects. 4 oC also had a strong effect on cellular uptake
(down to 8.70%), suggesting energy-dependent properties of endocytosis. All the results indicated that
Glu-Vc-Lip could be recognized by glucose transporter GLUT1 and SVCT2 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 endocytosis.
Fig. 7. Intracellular localization of Lip, Glu-Lip, Vc-Lip, Glu-Vc-Lip and Glu+Vc-Lip on C6 cells determined by CLSM,
green (CFPE-labeled liposomes), blue (DAPI stained nucleus), red (LysoTracker stained lysosome), light blue (colocalized
CFPE and DAPI), yellow (colocalized CFPE and Lyso Tracker). Scale bars represent 10 μm.
To elucidate the intracellular localization of liposomes after uptake, the lysosomes were labeled with
Lyso-Tracker RED and the nucleus were dyed with DAPI, which were imaged by CLSM. Lysosomes were
visualized as the RED fluorescence, while CFPE-labeled liposomes were shown as the green fluorescence.
14

Co-localization of liposomes with the lysosomes was indicated by yellow. As the Fig. 7 showed, after 1h
incubation, it was similar in each group that most of the liposomes taken up by the cells were found to
localize in the lysosome of C6 cells. With the incubation time increasing, the green fluorescence intensity
was much higher than that of 1 h, suggesting that the cellular uptake of the liposomes exhibited a
time-dependent pattern. What’s more, the increased co-localization of Glu-Vc-Lip was poorer after 4h
incubation than other groups, which might be due to a large proportion of the Glu-Vc-lip entered into the
nucleus as the nucleus dyed with DAPI showing a co-localization with the green fluorescence of CFPE. All
the results indicated that the internalization of Glu-Vc-Lip was mediated by lysosomes and a part of it
could escape from lysosomes, which may be because endosomes are the main organelles that internalize
foreign materials and particles and the abundance of Glu-Vc-Lip taken up into cells increased its chances
of entering the nucleus.
2.8 Distribution Study in Plasma and Brain
For in vivo study, PTX-Lip, PTX-Glu-Lip, PTX-Vc-Lip, PTX-Glu-Vc-Lip, PTX-Glu+Vc-Lip and
paclitaxel original drug were injected through the tail vein with the dose of 10 mg/kg body weight of
paclitaxel. At 5, 15, 30, 60, 120, 240, 480 and 1440 min after injection, blood and brain were collected to
analyze the concentration of paclitaxel by HPLC method.
The plasma PTX concentration-time profiles were shown in Fig. 8 and the pharmacokinetic parameters
of PTX from different formulations were summarized in Table 2. The results showed that the liposomes
could significantly enhance the concentration-time profile (AUC0–t) of paclitaxel compared with the naked
paclitaxel within 24 h. The AUC0–t of PTX-Glu-Vc-Lip was increased by about 1.58-time than that of free
PTX. It was also shown that PTX-Glu-Vc-Lip could extend the elimination half-life (t1/2) of free PTX from
218 min to 501 min. These data indicated that the liposomes possessed certain stability in plasma, with
15

more chance to be transported into brain.
Fig. 8. The concentration curve of paclitaxel versus time in plasma after i.v. injection of PTX-Lip, PTX-Glu-Lip,
PTX-Vc-Lip, PTX-Glu-Vc-Lip, PTX-Glu+Vc-Lip and PTX in mice with the dose of 10 mg/kg (mean ± SD, n = 3).
Table 2. Pharmacokinetic parameters of paclitaxel in blood after administration of paclitaxel and
liposomes (n=3)
Paclitaxel
PTX-Lip
PTX-Glu-Lip
PTX-Vc-Lip
PTX-Glu-Vc-Lip
PTX-Glu+Vc-Lip
AUC(0-t) (μg/g·min) 8630.66 ± 1591.81 15557.77 ± 2386.18 19509.51 ± 1590.99 13856.47 ± 1266.75
13647.47 ± 336.26
389.11 ± 2.10
5
15872.73 ± 867.93
375.31 ± 29.02
6.66
MRT (min)
Tmax (min)
Cmax (μg/g)
t1/2 (min)
351.77 ± 41.81
5
390.41 ± 64.74
6.66
386.35 ± 34.68
5
360.81 ± 64.93
8.33
53.33 ± 4.16
218.31 ± 50.88
65.42 ± 5.76
391.75 ± 243.82
56.44 ± 5.91
478.86 ± 113.42
58.92 ± 13.19
403.77 ± 65.02
47.21 ± 9.34
501.41 ± 197.66
58.08 ± 4.02
364.42 ± 98.34
AUC(0-t): the area under the curve from 0 h to t h. MRT: the mean residence time. Tmax: the time to maximum concentration.
Cmax: the maximum concentration. t1/2: the elimination half-life.
To further evaluate the possibility of the Glu-Vc-mediated liposomes being transported across BBB, the
distribution in brain of PTX-loaded liposomes and paclitaxel was determined. The concentrations of
16

paclitaxel in brain versus time curves were displayed in Figure 9 and the pharmacokinetic parameters were
listed in Table 3.
Fig. 9. The concentration curve of paclitaxel versus time in brain after i.v. injection of PTX-Lip, PTX-Glu-Lip, PTX-Vc-Lip,
PTX-Glu-Vc-Lip, PTX-Glu+Vc-Lip and PTX in mice with the dose of 10 mg/kg. (mean ± SD, n = 3).
Table 3. Pharmacokinetic parameters of paclitaxel in brain after administration of paclitaxel and liposomes
(n=3)
Paclitaxel
PTX-Lip
PTX-Glu-Lip
PTX-Vc-Lip
PTX-Glu-Vc-Lip
PTX-Glu+Vc-Lip
AUC(0-t) (μg/g·min) 807.22 ± 235.63 991.39 ± 239.77 3150.24 ± 1317.91 2341.21 ± 264.80
6075.76 ± 918.10
381.14 ± 10.70
100
5164.32 ± 1071.04
441.68 ± 55.97
120
MRT (min)
Tmax (min)
Cmax (μg/g)
t1/2 (min)
Re
547.68 ± 57.65
60
583.91 ± 113.41
160
542.39 ± 107.44
120
523.79 ± 41.51
120
1.48 ± 0.37
1.60 ± 0.50
5.74 ± 0.35
670.81 ± 141.43
3.90
4.00 ± 0.34
942.33 ± 513.48
2.90
11.68 ± 1.96
351.36 ± 56.27
7.53
8.45 ± 1.12
373.26 ± 69.77
6.40
339.52 ± 187.65 576.21 ± 191.59
-
-
1.23
1.08
Ce
3.90
2.70
7.89
5.71
Re: the relative uptake efficiency. Ce: the concentration efficiency.
In brain, it is obvious that all of the liposomes could be delivered to the brain through i.v. administration.
17

At all the predetermined time points, 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 from
liposomes were fairly higher than that of free paclitaxel. The relative uptake efficiencies (Res) of PTX-Lip,
PTX-Glu-Lip, PTX-Vc-Lip, PTX-Glu-Vc-Lip and PTX-Glu+Vc-Lip were enhanced to 1.23, 3.90, 2.90,
7.53 and 6.40 times than that of naked paclitaxel, respectively. The concentration efficiencies (Ces) were
also enhanced to 1.08, 3.90, 2.70, 7.89 and 5.71 times than that of paclitaxel. So these data further proved
our conjecture that liposome with dual-targeting ligand Glu-Vc-Chol had superior brain-targeting
efficiency.
2.9 In vivo imaging
Kunming mice bearing C6 glioma were used to estimate the glioma targeting efficiency of different
liposomes. Fig. 10A showed the in vivo images of intracranial C6 glioma bearing mice at 1, 2, 6, 24 h after
systemic administration of DiD-loaded liposomes. And then the mice were sacrificed at pre-determined
time points, and the brains, hearts, livers, spleens, lungs and kidneys were harvested and imaged. Ex vivo
images of the brains were exhibited in Fig. 10C and other organs were shown in Fig. 10B. The strongest
fluorescence signal of the all the liposomes in the brain was obviously observed 2 h after injection, which
was consistent with the result of the distribution study. The co-modified liposomes Glu-Vc-Lip exhibited
the best ability to accumulate at the tumors with much stronger fluorescence than singly-modified and
non-modified liposomes and even a little stronger than Glu+Vc-Lip in the brains. However, at 2 h and 6 h
after injection, the strong fluorescence signal of different liposomes was also found in the livers and in part
of the spleen, but no obvious signal was observed in heart, lung, and kidney, which may be due to the
metabolism. These in vivo imaging results suggested that Glu-Vc on the surface of liposomes endowed the
liposomes with brain glioma dual-targeted delivery capability. These findings were well consistent with
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the results of in vitro cellular uptake study.
Fig. 10. (A) In vivo images of intracranial C6 glioma bearing mice at 1, 2, 6, 24h after systemic administration of Did-loaded
liposomes. (B) Ex vivo images of the brains. (C) Ex vivo images of isolated organs and they were heart, liver, spleen, lung
and kidney from left to right. A: DiD-Lip, B: DiD-Glu-Lip, C: DiD-Vc-Lip, D: DiD-Glu-Vc-Lip, E: DiD-Glu+Vc-Lip. (n=3)
3. Conclusion
In order to develop an efficient brain targeting drug delivery system to greatly improve the brain
permeability of drugs, a novel dual-targeting glucose-Vc derivative was designed and synthesized as
liposome ligand in this work. Glu-Vc-Lip could be recognized by glucose transporter GLUT1 and SVCT2
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 endocytosis. What’s more, Glu-Vc-Lip significantly increased the brain uptake
of PTX in vivo with the increased Re and Ce, and the in vivo imaging of DiD-Glu-Vc -Lip showed
significantly more fluorescent than other groups, suggesting that both glucose and vitamin C transporters
19

(GLUT1 and SVCT2) might be involved to help the liposomes to penetrate the BB. Therefore, the Glu-Vc
modification represents a promising strategy for the development of future brain-specific drug delivery
systems.
4. Experimental section
4.1. Materials
The melting point was measured on an YRT-3 melting point apparatus (Shantou Keyi instrument &
Equipment Co. Ltd, Shantou, China). NMR spectra were taken on a Varian INOVA 400 MHz (Varian,
Palo Alto, CA, USA) using CDCl3 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). Reversed-phase chromatography performed on C18 chromatographic analysis was carried out using
the HPLC system (Alltech, IL, USA) consisted of a RF-530 fluorescence detector (Shimadzu, Japan) and
Allchorom plus data operator, respectively. A Diamonsil column (200 × 4.6 mm, 5 μm) was used.
Kunming mice rats (20-25 g) were provided by the Laboratory Animal Center of Sichuan University
(Chengdu, China). All animal study procedures were approved by the Sichuan University Animal Ethical
Experimentation Committee according to the requirements of the National Act on the use of experimental
animals (China).
4.2. Chemistry
4.2.1 Synthesis of compound 2-14
The synthesis of compound 2-14 was reported in our previous work.7,12,16
20

4.2.2 Synthesis of compound 15
To a solution of compound 14 (1.62 g, 2.81 mmol) in CH2Cl2 (40 ml) was added DCC (0.87 g, 4.22
mmol) and DMAP (68 mg, 0.56 mmol), and the reaction was stirred at -5 oC for 30 min. Then compound 9
(0.83 g, 2.34 mmol) in CH2Cl2 (10 ml) was added promptly. After stirring for another 4 h at r.t., the
mixture was filtered and the filtrate was concentrated. Then the residue was purified by chromatography to
give 15 (1.78 g, 69.2%) 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 = 6.6 Hz), 0.98 (s, 3H), 0.67-2.37 (remaining cholesterol protons), 3.14-3.19 (m,
1H), 3.61-3.74 (m, 12H), 4.07-4.10 (m, 1H), 4.19 (s, 2H), 4.26-4.30 (m, 1H), 4.37-4.41 (m, 1H), 4.66 (d,
1H, J = 2.0 Hz), 5.07-5.22 (m, 4H), 5.30-5.33 (m, 1H), 7.20-7.22 (m, 2H), 7.34-7.38 (m, 8H).
4.2.3 Synthesis of compound 16
To a solution of compound 5 (0.64 g, 1.12 mmol) in CH2Cl2 (20 ml) was added DCC (0.31 g, 1.50
mmol) and DMAP (18 mg, 0.15 mmol), and the reaction was stirred at -5 oC for 30 min. Then compound
15 (0.69 g, 0.75 mmol) in CH2Cl2 (10 ml) was added promptly. After stirring for another 5 h at r.t., the
mixture was filtered and the filtrate was concentrated. Then the residue was purified by chromatography to
give 16 (0.64 g, 57.9%) as a yellow oil. 1H NMR (600 MHz, CDCl3, ppm) δ: 0.12-0.14 (m, 36H), 0.67 (s,
3H), 0.87 (d, 6H, J = 11.4 Hz), 0.91 (d, 3H, J = 6.0 Hz), 0.98 (s, 3H), 0.67-2.37 (remaining cholesterol
protons), 2.48-2.51 (m, 2H), 2.60-2.63 (m, 2H), 3.15-3.18 (m, 1H), 3.34-3.41 (m, 2H), 3.62-3.71 (m, 13H),
3.77 (t, 1H, J = 8.4 Hz), 3.87-3.90 (m, 1H), 4.13 (d, 2H, J = 4.8 Hz), 4.31-4.41 (m, 3H), 4.80 (d, 1H, J =
1.8 Hz), 4.99 (d, 1H, J = 3.0 Hz), 5.12-5.24 (m, 4H), 5.33-5.42 (m, 2H), 7.23-7.24 (m, 2H), 7.33-7.41 (m,
8H).
4.2.4 Synthesis of compound 17
To a solution of compound 16 (0.21 g, 0.14 mmol) in CH2Cl2 (10 ml) was added trifluoracetic acid (0.21
21

ml, 2.86 mmol) at 0 °C and then the reaction was stirred for 1 h at room temperature. The mixture was
concentrated and the residue was purified by chromatography to give compound 17 (0.14 g, 83.3%) as a
1
colorless oil. H NMR (400 MHz, CDCl3, ppm) δ: 0.67 (s, 3H), 0.86 (d, 6H, J = 5.2 Hz), 0.91 (d, 3H, J =
6.4 Hz), 0.98 (s, 3H), 0.85-2.44 (remaining cholesterol protons), 2.50-2.55 (m, 4H), 3.09 (br, 3H),
3.15-3.20 (m, 2H), 3.33-3.45 (m, 2H), 3.52-3.57 (m, 2H), 3.63-3.70 (m, 14 H), 3.79 (t, 1H, J = 9.2 Hz),
3.99 (br, 1H), 4.14 (s, 2H), 4.28-4.45 (m, 4H), 4.78 (d, 1H, J = 2.4 Hz), 5.07-5.18 (m, 4H), 5.26 (d, 1H, J =
3.2 Hz), 5.33 (d, 1H, J = 4.4 Hz), 5.40 (br, 1H), 7.21-7.23 (m, 2H), 7.33-7.38 (m, 8H).
4.2.5 Synthesis of ligand Glu-Vc-Chol
To a solution of compound 17 (0.10 g, 0.085 mmol) in methanol (10 ml), Pd/C (10 mg, 10%) was added.
Then, the mixture was stirred in hydrogen atmosphere at room temperature for 1 h. Pd/C was filtered, and
the filtrate was concentrated to give ligand Glu-Vc-Chol (81 mg, 95.6%) as a colorless 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 = 6.0 Hz), 0.98 (s, 3H),
0.78-2.44 (remaining cholesterol protons), 2.63 (br, 3H), 3.18 (s, 1H), 3.48-3.66 (s, 16H), 3.49-4.36 (m,
7H), 4.86-5.45 (m, 9H), 5.33 (s, 1H). ESI-MS calculated for C51H80O19K [M+K]+ 1035.5, found 1034.9.
4.2.6 Synthesis of ligand Vc-Chol
To a solution of compound 15 (0.25 g, 0.27 mmol) in methanol (30 ml), Pd/C (30 mg, 10%) was added.
Then, the mixture was stirred in hydrogen atmosphere at room temperature for 1 h. Pd/C was filtered, and
the filtrate was concentrated to give ligand Vc-Chol (0.19 g, 94.4%) as a colorless oil. 1H NMR (400 MHz,
CDCl3, ppm) δ: 0.63 (s, 3H), 0.78 (d, 6H, J = 6.3 Hz), 0.85 (d, 3H, J = 6.6 Hz), 0.94 (s, 3H), 0.63-2.63
(remaining cholesterol protons), 3.22 (m, 1H), 3.66-3.79 (m, 12H), 4.15 (m, 1H), 4.24 (s, 2H), 4.37 (m,
2H), 4.80 ( m, 1H), 5.34 (m, 1H). ESI-MS calculated for C41H66O11Na [M+Na]+ 757.5, found 757.6.
4.2.7 Synthesis of ligand Glu-Chol
22

The synthesis of ligand Glu-Chol was reported in our previous report.15
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-Vc-Chol modified liposomes (Glu-Vc-Lip), SPC/cholesterol/ligand Glu-Vc-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 Vc-Chol modified liposomes (Vc-Lip), SPC/cholesterol/Ligand Vc-Chol (molar
ratio = 62: 33: 3); (5) Ligand Glu-Chol and Vc-Chol co-modified liposomes (Glu+Vc-Lip),
SPC/cholesterol/Ligand Glu-Chol/Vc-Chol (molar ratio = 62: 33: 3: 3). All lipid materials were dissolved
in the chloroform/methanol (v/v = 2:1), and then the organic solvent was removed by rotary evaporation to
get a lipid film, which was further dried in vacuum overnight. Next, the film was hydrated in PBS (pH 7.4)
o
for 0.5 h at 20 C. Finally, it was further sonicated intermittently 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 determined by HPLC.
Likewise, CFPE-labelled liposomes were prepared by adding appropriate amount of CFPE to the solution
before removing the solvent. The mean size and zeta potential of Lip, Glu-Vc-Lip, Glu-Lip, Vc-Lip and
Glu+Vc-Lip were detected by Malvern Zeta sizer Nano ZS90 (Malvern Instruments Ltd., UK).
4.4. In vitro drug release study
In vitro paclitaxel release study was performed using dialysis method. PTX-loaded liposomes (0.4 mL)
or naked paclitaxel were added into dialysis tubes (MWCO = 8000 - 14000 Da) respectively and sealed
tightly. 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 h, 1 h, 2 h, 4 h, 8 h,
23

12 h, 24 h and 48 h), 0.1 ml release medium was taken out and replaced with equal volume of fresh 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
The serum stability of liposomes was evaluate by measuring the turbidity variations in the presence of
fetal bovine serum (FBS). Briefly, each liposome was mixed with FBS (v/v=1:1) under 37 °C with
moderate shaking at 45 rpm. The transmittance of the mixture was measured at predetermined time points
(0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h) by a microplate reader at 750 nm (Thermo Scientific Varioskan
Flash, USA).
4.6. Hemolysis assays
The safety of ligands-modified liposomes during body circulation was demonstrated by hemolysis assay.
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 the
concentration of 2% (v/v). The RBCs solutions were incubated with various concentrations of liposomes
(v/v=1:1) for 1 h at 37 °C with gentle shaking, followed by centrifugation at 1 × 104 rpm for 10 min.
Absorbance of hemoglobin was measured using a microplate reader 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:
A
Sample
A
A
Negative
The percent hemolysis =
× 100%
APostive
Negative
where A is the ultraviolet absorbance of hemoglobin.
4.7. Cytotoxicity
24

The cytotoxicity of PTX-loaded liposomes and blank liposomes against C6 cells (GLUT1 and
SVCT2-positive) 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 oC. PTX-loaded liposomes and free paclitaxel were
diluted to 0.016, 0.08, 0.4, 2, 10, 50 μg/ml of PTX with medium, followed by being added into each well
and incubated for 24 h. What’s more, the blank liposomes were added at the same concentration of
PTX-loaded liposomes. Then, remove the medium and add 200 μl medium containing MTT solution (0.5
mg/ml) to each well. After incubation for another 4 h at 37 °C, the medium was removed and the reduced
MTT dye was solubilized by DMSO (150 μl). The optical density (OD) value was read at 490 nm on an
automatic microplate spectrophotometer. Cell viability (%) was calculated as the following equation:
ODtest / ODcontrol × 100%.
4.8. Cellular uptake study
C6 cells were seeded in 12-well plates at a density of 5 × 105 cells/well and cultured for 24 h at 37 oC to
reach 80% confluence. Different CFPE-labeled liposomes were added into each well with the final
o
concentration of CFPE at 2 µg/ml. After cultured for 4 h at 37 C, the cells were washed with cold PBS.
and trypsin was used to harvest the cells afterward. Then, the cells were washed with cold PBS and finally
resuspended in 0.5 ml PBS to measure the fluorescent intensity by a flow cytometer at Ex 495 nm and Em
515 nm (BD FACS celestra).
For confocal microscope studies, C6 cells were plated onto a 6-well plate containing cover glass at a
o
density of 5 × 105 cells/well and cultured for 24 h at 37 C. CFPE-labeled liposomes were added into each
well and further co-incubation for 4 h. Following that, the cells were fixed with 4% paraformaldehyde for
30 min at room temperature after rinsed with cold PBS for three times, and then cell nuclei were stained
with DAPI (5 µg/ml) for 5 min. Finally, the samples were imaged using CLSM (LSM 800, ZEISS,
25

German). What’s more, the intracellular localization of liposomes was also studied. After co-incubation
with CFPE-labeled liposomes for 1 h or 4 h, the cells were washed with cold PBS and treated with
o
Lyso-Tracker RED (50 nM, Solarbio) for 1 h at 37 C. And then, the cells were fixed and the cell nuclei
were stained. Finally, the samples were imaged using CLSM.
In order to research the uptake mechanism of Glu-Vc-Lip, 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). What’s more, the inhibition of free glucose (5 mg/ml) and free Vc (88
mg/ml), and the effect of temperature were also performed. Briefly, the cells were preincubated with
different inhibitors for 30 min, then the inhibitors were withdrawn from the wells and CFPE-labeled
o
Glu-Vc-Lip was added. After cultured for 4 h at 37 C, the cells were treated as described before and the
fluorescent intensity was measured by a flow cytometer.
4.9. Distribution in brain and pharmacokinetic studies in mice
4.9.1 Sample preparation
Blood was collected from the eye socket of mouse, and centrifuged at 5000 rpm for 5 min to get the
supernatant as plasma sample. Then the animals were sacrificed to get the brains and flushed with saline to
remove the remained blood and the main vessel. All the brain tissues were homogenized with saline
(w/w=1:3). An aliquot of 10 μl of internal standard (docetaxel, 30 μl, 100 μg/ml) was added into 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 dried and the residue was reconstituted with
100 μl 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.
4.9.2 Body distribution study
26

The mice were divided into five groups for each liposome or paclitaxel randomly. PTX-Lip,
PTX-Glu-Lip, PTX-Vc-Lip, PTX-Glu-Vc-Lip, PTX-Glu+Vc-Lip and paclitaxel were given to the mice via
the tail vein at the dose of paclitaxel of 10 mg/kg. At 5 min, 15 min, 30 min, 60 min, 120 min, 240 min,
480 min and 1440 min after injection, the blood the brain samples were prepared described earlier. The
concentration of paclitaxel was analyzed by HPLC.
4.9.3 Statistical analysis
The area under the concentration-time profile (AUC0-t), maximal concentration (Cmax), and mean
residence times (MRT) were calculated 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:
Re = (AUC0- t)L / (AUC0-t)P
Ce = (Cmax)L / (Cmax)P,
where L and P represented the liposomes loading PTX and paclitaxel, respectively.
4.10. In vivo imaging
Kunming mice (male) were anesthetized with 4% chloral hydrate and placed on a stereotaxic apparatus.
5 µl of 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 14 days, the mice were randomly divided into 5 groups
and were intravenously injected via the caudal vein of the tumor-bearing mice with DiD loaded different
liposomes DiD-Lip, DiD-Glu-Lip, DiD-Vc-Lip, DiD-Glu-Vc-Lip and DiD-Glu+Vc-Lip at a dose of 200
µg/kg. Then, the mice were anesthetized with 4% chloral hydrate and imaged with IVIS Lumina Series III
imaging system (LIVIS Lumina III, Perkin Elmer, USA) at 1 h, 2 h, 6 h and 24 h after injection. The mice
27

were sacrificed after heart perfusion with saline at these pre-determined time points. Brains, hearts, livers,
spleens, lungs and kidneys were collected. All the organs were also imaged with IVIS Lumina Series III
imaging system.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No.81573286,
No.81773577, No.21472130 & No.81603218).
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