Design, synthesis and biological evaluation for docetaxel-loaded brain targeting liposome with "lock-in" function

Article abstract of DOI:10.3109/1061186X.2013.865032

Background: Glucose-modified liposome showed a good brain-targeting ability. However, bidirectional transport of glucose transporter-1 (GLUT1) might reversely pump drugs out of the brain before releasing from the liposomes. Purpose: To overcome the bidirectional delivery of GLUT1, the thiamine disulfide system (TDS), with ability of "lock-in", was introduced and a new ligand, L-TDS-G, was designed and synthesized. Methods: The liposome was prepared and characterized for particle size, zeta potential, surface morphological property, encapsulation efficiency and release profile. C6 glioma cells were used as an in vitro model to access the cellular uptake abilities and cytotoxicity of the liposomes. Competition assay was performed to validate the GLUT1-mediated transport mechanism. Furthermore, the brain targeting abilities of the liposomes were evaluated through in vivo. Results: The preliminary evaluation in vivo demonstrated that L-TDS-G-coated liposome has an improved targeting ability and significantly increased the area under the concentration-time of docetaxel in brain as compared to naked docetaxel, non-coated and L-G coated liposomes. The relative uptake efficiency and concentration efficiency were enhanced by 3.82- and 4.99-fold compared to that of naked docetaxel, respectively. Conclusion: The results of this study indicated that L-TDS-G-coated liposome is a promising drug delivery system to enhance the brain concentrations of chemotherapeutic agents.

Full text of DOI:10.3109/1061186X.2013.865032

http://informahealthcare.com/drt
ISSN: 1061-186X (print), 1029-2330 (electronic)
J Drug Target, 2014; 22(3): 251–261
2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.865032
!
ORIGINAL ARTICLE
Design, synthesis and biological evaluation for docetaxel-loaded brain
targeting liposome with ‘‘lock-in’’ function
Xiaocen Li, Boyi Qu, Xiuxiu Jin, Li Hai, and Yong Wu
Key Laboratory of Drug Targeting of Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, P.R. China
Abstract
Keywords
Background: Glucose-modified liposome showed a good brain-targeting ability. However,
bidirectional transport of glucose transporter-1 (GLUT1) might reversely pump drugs out of the
brain before releasing from the liposomes.
Purpose: To overcome the bidirectional delivery of GLUT1, the thiamine disulfide system (TDS),
with ability of ‘‘lock-in’’, was introduced and a new ligand, L-TDS-G, was designed and
synthesized.
Blood-brain barrier, brain targeting,
competition assay, cytotoxicity, glucose,
glucose transporter-1, thiamine disulfide
system
History
Methods: The liposome was prepared and characterized for particle size, zeta potential, surface
morphological property, encapsulation efficiency and release profile. C6 glioma cells were used
as an in vitro model to access the cellular uptake abilities and cytotoxicity of the liposomes.
Competition assay was performed to validate the GLUT1-mediated transport mechanism.
Furthermore, the brain targeting abilities of the liposomes were evaluated through in vivo.
Results: The preliminary evaluation in vivo demonstrated that L-TDS-G-coated liposome has
an improved targeting ability and significantly increased the area under the concentration-
time of docetaxel in brain as compared to naked docetaxel, non-coated and L-G coated
liposomes. The relative uptake efficiency and concentration efficiency were enhanced by 3.82-
and 4.99-fold compared to that of naked docetaxel, respectively.
Received 29 August 2013
Revised 8 October 2013
Accepted 8 November 2013
Published online 5 December 2013
Conclusion: The results of this study indicated that L-TDS-G-coated liposome is a promising
drug delivery system to enhance the brain concentrations of chemotherapeutic agents.
Introduction
clinical use of docetaxel causes several side effects including
neutropenia [9], musculoskeletal toxicity, peripheral neur-
opathy [10] and hypersensitivity reactions [11]. The blood–
brain barrier (BBB), another factor, restricts the penetration
of most drugs, which directly leads to the ineffective
concentration of chemotherapeutic agents in brain and
reduce the efficacy of the therapeutics [12,13]. Therefore,
targeted delivery strategies for improving the accumulation of
effective therapeutic agents in brain and decreasing adverse
effects were attracted considerable attention in recent years,
especially for the application of liposome.
For most of the patients with malignant brain cancers,
conventional treatment consisted of combinations of surgery,
radiation and chemotherapy remains the bulwark of brain
cancer therapy throughout the world. Unfortunately, this
treatment yields only modest increases in the median survival
time from about several months to over a year [1–3]. Many
reasons response for the decreasing efficacy of this treatment,
among which the systemic toxicity of chemotherapeutic
agents comes first [4]. To cure brain cancer, a high-dose
of chemotherapeutic agent is always given, which causes
substantial adverse effects to the central nervous system
(CNS) and the vital normal tissues [5]. Docetaxel, under the
trade name of TaxotereÔ, is one of the most important anti-
cancer drugs. It belongs to the second generation of the taxoid
family and has shown clinical efficacy against non-small lung
[6], ovarian cancer [7] and breast cancer [8]. However, the
As a targeting carrier, liposome is easily modified and
exhibits many advantages, such as, non-toxicity, biocompati-
bility and biodegradability [14]. In the past decades, many
types of modified liposomes have been developed, for
instance: polyethylene glycol (PEG)-modified liposome [15]
and immune liposome [16]. Liposomes modified by PEGs can
prevent themselves from getting trapped by the reticuloendo-
thelial system such as liver and spleen and thus exhibit good
long-circulating character but bare brain targeting [17].
Kreuter et al. reported that immune liposome with OX-26 (a
transferrin receptor monoclonal antibody) covalently attached
to the PEG layer can be highly transferred across the BBB
[18]. However, immune responses might be accompanied
[19]. The drawbacks limited the application and the develop-
ment of above-mentioned liposomes, but these studies still
Address for correspondence: Li Hai, Key Laboratory of Drug Targeting
of Education Ministry, West China School of Pharmacy, Sichuan
University, Chengdu 610041, P.R. China. Tel: þ86 2885503949. Fax:
þ862885503666. E-mail: smile@scu.edu.cn
Yong Wu, Key Laboratory of Drug Targeting of Education Ministry,
West China School of Pharmacy, Sichuan University, Chengdu 610041,
P.R. China. Tel: þ86 2885503235. Fax: þ862885503666. E-mail:
wyong@scu.edu.cn

252 X. Li et al.
J Drug Target, 2014; 22(3): 251–261
offered us hope to develop more effective delivery
system to overcome the low permeability of BBB as well as
the ineffective concentration of chemotherapeutic agents in
CNS.
same as glucose, would be pumped out to the bloodstream
after entering CNS, thus preventing the concentration of
chemotherapeutic agent in brain. Therefore, to overcome the
bidirectional delivery of GLUT1, the thiamine disulfide
system (TDS) [27], with ability of ‘‘lock-in’’, was introduced
and attached to the glucose moiety at the C-6 position by ester
bond. Once entered the BBB, TDS can be reduced by
disulfide reductase and then ring-closed to be a thiazolium,
which is lipophobic and cannot across the BBB via diffusion.
Furthermore, the liposome with this system would be
‘‘locked’’ in the brain, thus it can serve a sustained release
of the chemotherapeutic agent [28].
A small molecule, glucose, then, caught our eyes.
Glucose, providing the uninterrupted energy supply to the
brain, is mainly transported through the BBB by glucose
transporters, especially glucose transporter-1 (GLUT1)
[20,21]. It was estimated that glucose consumption in the
brain represents about 30% of the whole body glucose
consumption [22]. Moreover, it is estimated that there is a
15–3000-fold higher transport value for the GLUT1 than that
for other transporters [23]. Our group reported glucose–
cholesterol (CHOLE) derivative, L-G, as a ligand for brain
targeting liposome. The L-G modified liposome, Lip-G
(Figure 1), showed a good brain-targeting ability [24,25].
We also confirmed that glucose-modified liposome was a
good strategy to across the BBB and GLUT1 was an efficient
transporter.
In account for all of the above, a new ligand, L-TDS-G,
with ‘‘lock-in’’ function, is designed and synthesized.
Docetaxel-loaded liposomes, non-coated (Figure 2), L-G
coated and L-TDS-G coated (Figure 3), were prepared by the
lipid film hydration–ultrasound method, whose characteristics
and brain target abilities were evaluated in vitro and in vivo.
In vitro cytotoxicity and the drug concentration required to
induce the death of 50% C6 glioma cells (IC50) values of the
C6 glioma cells were obtained to evaluate the therapeutic
effects of these liposomes. Moreover, fluorescent coumarin-
6-loaded liposomes, non-coated, L-G coated and L-TDS-G
coated, were prepared for cellular uptake investigation and
competition assay
However, the GLUT1 was found to be a bidirectional
transporter that mediates the blood-to-brain and brain-to-
blood transport of the glucose in either direction across the
BBB, owing to its expression on both the luminal and
abluminal membranes of the endothelial cells of BBB [26].
This indicated that the glucose-modified liposome, Lip-G, the
Figure 1. Structure of ligand L-G schematic
diagram of L-G coated liposome.
Figure 2. Structure of Docetaxel and sche-
matic diagram of non-coated liposome.
Figure 3. Structure of ligand L-TDS-G and schematic diagram of L-TDS-G coated liposome.

DOI: 10.3109/1061186X.2013.865032
Docetaxel-loaded brain targeting liposome 253
Materials and methods
Allchorom plus data operator, respectively. 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 mm ꢀ 200 mm, 5 mm,
SinoChrom, Dalian, P.R. China) were used.
Materials and animals
Docetaxel and diazepam were obtained from National
Institute for Food and Drug Control. Coumarin-6 was
obtained from Sigma-Aldrich (St. Louis, MO). Soybean
phospholipids (SPC) were purchased from Kelong Chemical
Reagent Factory (Chengdu, P.R. China). CHOLE was
purchased from Bio Life Science & Technology Co., Ltd
(Shanghai, P.R. China). All liquid reagents were distilled
before use. The other chemicals and reagents were obtained
from commercial sources.
Kunming mice were purchased from Experiment Animal
Center of Sichuan University (P.R. China). All animal
procedures for this study were approved by the Experiment
Animal Administrative Committee of Sichuan University
(P.R. China).
Cell lines and cell culture
The melting point was measured on a YRT-3 melting point
apparatus (Shantou Keyi instrument & Equipment Co. Ltd.,
Shantou, China). Infrared (IR) spectra were obtained on a
Perkin Elmer983 (Perkin Elmer, Norwalk, CT). 1HNMR
spectra were taken on a Varian INOVA 400 (Varian, Palo
Alto, CA) using CDCl3, dimethylsulfoxide-d6 (DMSO-d6) and
D2O as solvent. Chemical shifts were expressed in ꢀ (ppm),
with tetramethylsilane (TMS) functioning as the internal
reference, coupling constants (J) were expressed in Hz. Mass
spectra were recorded on an Agilent 1946B ESI-MS instru-
ment (Agilent, Palo Alto, CA). Reversed-phase chromatog-
raphy performed on C18 chromatographic analysis was
carried out using the high performance liquid chromatography
(HPLC) system (Alltech, Woodridge, IL) consisted of a
RF-530 fluorescence detector (Shimadzu, Kyoto, Japan) and
C6 glioma cells (own reserved) were maintained in 0.125%
Gibco trypsin solution cultured in 1640 Medium (RPMI
1640) with 10% Hyclone fetal bovine serum (FBS, Thermo
Scientific, Logan, UT) and 1% Gibco penicillin–streptomycin
(Invitrogen, Carlsbad, CA) at 37 ꢁC under an atmosphere of
5% CO2.
Synthesis of glucose–CHOLE derivatives
General procedures for synthesis of glucose–CHOLE
derivative L-TDS-G
The synthetic route of glucose-modified ligand, L-TDS-G,
with ‘‘lock-in’’ function has been illustrated in Scheme 1.
The starting material is commercially available CHOLE.
O
a
b
c
2
O
O
O
O
O
2
HO
O
TsO
HO
1
Cholesterol
2
3
S
O
8
CHO S
N
S
O
O
CHO S
N
O
HO
O
OH
d
2
O
O
O
O
O
O
2
O
O
e
4
5
S
O
O
f
CHO S
N
g
S
O
O
CHO S
N
2
O
HO
O
O
2
O
O
O
O
O
O
TMSO
TMSO
TMSO
O
6
7
OTMS
S
O
h
O
CHO S
N
O
2
O
O
O
O
O
HO
HO
L-TDS-G
HO
OH
Scheme 1. General synthetic route of L-TDS-G. Reagents and conditions: (a) TsCl, pyridine, 50 ꢁC, 10 h, 83%; (b) HOCH2 (CH2OCH2)2 CH2OH,
dioxane, reflux, 8 h, 58.4%; (c) t-butyl bromoacetate, n-Bu4NþHSO4, 50% NaOH, Toluene, overnight, 80.4%; (d) TsOH, Tolunene, reflux, 91.9%;
(e) DCC, DMAP, CH2Cl2, r.t., 77.4%; (f) HClO4, CH2Cl2, 50.44%; (g) DCC, DMAP, CH2Cl2, overnight, 73.2%; (h) CF3COOH, CH3NO2,
CH2Cl2, 66%.

254 X. Li et al.
J Drug Target, 2014; 22(3): 251–261
Na S O
5H O
2 3
2
2
O
S
Br
S
ONa
b
O
O
a
10
Br
O
N
S
S
O
S
O
CHO
N
-
N
Br
OH
O
c
O
O
OH
H
S
11
8
12
OH
OTMS
OH
O
OH
d
e
HO
HO
O
TMSO
TMSO
TMSO
O
TMSO
TMSO
HO
D-Glucose
OTMS
TMSO
OTMS
13
9
Scheme 2. General synthetic route of TDS system and glucose-TMS derivative. Reagents and conditions: (a) CH3CN, 85 ꢁC, 91.4%; (b) EtOH/H2O,
80 ꢁC, 14 h, 87.7%; (c) NaOH (aq.), EA/H2O, overnight, 86.0%; (d) HMDS, TMSCl, pyridine, 0 ꢁC, overnight, 93.1%; (e) AcOH, MeOH, Acetone, r.t.,
4 h, 75.2%.
After stirring with toluenesulfonyl chloride in pyridine
at 50 ꢁC for 10 h, tosyl protecting group was introduced.
Cholesteryl tosylate and triethylene glycol, then, were
refluxed were dioxane for 8 h to afford compound 2, which
reacted with t-butyl bromoacetate in 50% NaOH/toluene
solution overnight to give 3. After deprotected the t-butyl
group, 4 was generated. Thus, condensation of compounds 4
and 8, the TDS part, in the presence of dicyclohexyl
carbodiimide (DCC) and 4-dimethylamiopyridine (DMAP)
gave ester 5, which then deprotected the t-butyl group to
expose the carboxyl group and obtained 6. Intermediate 6
condensed with glucose-TMS derivative 9 and gave 7, which
then deprotected the trimethylsilyl group, resulting in the
desired product L-TDS-G.
magnesium sulfate and concentrated on a rotary evaporator.
The residue was purified on a silica-gel chromatography
column to get product 2 as a light brown oil (30.3 g, 58.4%).
1H NMR (400 MHz, CDCl3): ꢀ 0.66 (s, 3H, CHOLE CH3-18),
0.85 (d, 6H, J ¼ 6.4 Hz, CHOLE CH3-26 and -27), 0.91
(d, 3H, J ¼ 6.4 Hz, CHOLE CH3-21), 0.99 (s, 3H, CHOLE
CH3-19), 0.67–2.38 (remaining CHOLE protons), 3.20
(m, 1H, –OCH–), 3.59–3.75 (m, 12H, OCH2CH2O ꢀ 3),
5.34 (m, 1H, CHOLE H-6). IR (KBr, cm–1): ꢁ: 3394, 2935,
1458, 1378, 1107. MS (m/z): 541.43 [M þ Na]þ.
Synthesis of the compound 3. To a solution of 2 (10.3 g,
19.85 mmol) in toluene was added 50%NaOH (aq., 50 ml)
at 0 ꢁC. Then t-butyl-bromoacetate (4.8 ml, 29.78 mmol) and
n-Bu4NþHSO4 (636.4 mg, 1.98 mmol) was added to the
–
The synthetic route of key intermediate 9, the TDS part,
was described in Scheme 2. The starting material 11, 5-(2-
Hydroxyethyl)-4-methylthiazole, is commercially available.
Compound 12 was carried out with t-butyl bromoacetate in
acetonitrile to generate quaternary thiazolium salt 13 in good
yield. Hydrolysis of compound 13 in the solution of sodium
hydroxide was treated with thiosulfate salt 10 prepared from
isopropyl bromide by sodium thiosulfate, resulting in inter-
mediates 8. Glucose-TMS derivative 9 was synthesized in
the synthetic route shown in Scheme 2. D-Glucose was totally
etherified with chlorotrimethylsilane and selectively depro-
tected the trimethylsilyl group by the mixture of acetone,
acetic acid and methanol to obtain the partially silylated
intermediate 9.
Synthesis of the compound 1. To a solution of CHOLE
(40 g, 0.103 mol) in 3300 ml pyridine (distilled) was added
paratoluensulfonyl (29.6 g, 0.155 mol). The reaction mixture
was stirred at 50 ꢁC for 10 h, and then concentrated in vacuo
to residue, which was neutralized with HCl solution to
pH ¼ 7. The suspension was stirred overnight and
filtrated to give a white solid afford (55.1 g, 83%; m.p.:
132–133 ꢁC).
above solution. The reaction mixture was stirred overnight at
room temperature. The toluene layer was separated, and the
water layer was extracted with ethyl ether, the organic extract
was dried by anhydrous magnesium sulfate and concentrated
in vacuo, to afford a crude product, and was purified on
a silica-gel chromatography column to give a yellow oil
(10.1 g, 80.4%).1H NMR (400 MHz, CDCl3): ꢀ 0.66 (s, 3H,
CHOLE CH3-18), 0.85 (d, 6H, J ¼ 6.4 Hz, CHOLE CH3-26
and -27), 0.91 (d, 3H, J ¼ 6.6 Hz, CHOLE CH3-21), 0.99
(s, 3H, CHOLE CH3-19), 0.64–2.39 (remaining CHOLE
protons), 1.47 (s, 9H, –C(CH3)3), 3.20 (m, 1H, CHOLE
H-3), 3.58–3.79 (m, 12H, OCH2CH2O ꢀ 3), 4.02 (s, 2H,
–CH2COO–) and 5.34 (m, 1H, CHOLE H-6). IR (KBr, cm–1):
ꢁ: 2939, 1754, 1155 and 1112. MS (m/z): 655.52 [MþNa]þ.
Synthesis of the compound 4. To a solution of compound 3
(4.5 g, 7.11 mmol) in 50 mL toluene, p-toluenesulfoic acid
(0.24 g, 1.4 mmol) was added, the reaction mixture was
refluxed for 4 h. Toluene was removed in vacuo, and the crude
product was purified by chromatography to afford a yellow oil
1
(3.77 g, 91.9%). H NMR (400 MHz, CDCl3): ꢀ 0.66 (s, 3H,
CHOLE CH3-18), 0.86 (d, 6H, J ¼ 6.4 Hz, CHOLE CH3-26
and -27), 0.92 (d, 3H, J ¼ 6.6 Hz, CHOLE CH3-21), 0.99
(s, 3H, CHOLE CH3-19), 0.66–2.39 (remaining CHOLE
protons), 3.20 (m, 1H, CHOLE H-3), 3.61–3.81 (m, 12H,
OCH2CH2O ꢀ 3), 4.17 (s, 2H, –CH2COO–) and 5.35 (m, 1H,
CHOLE H-6). IR (KBr, cm–1): 3482, 2938, 1754, 1151 and
1112. MS (m/z): 577.83 [M þ H]þ.
Synthesis of the compound 2. Cholesteryl tosylate 1
(55.1 g, 0.102 mol) and triethyleneglycol (69 ml, 0.551 mol)
were refluxed in dioxane for 8 h. Then reaction mixture
was concentrated in vacuo, and the residue was redissolved
in dichoromethane, washed with brine, dried over anhydrous

DOI: 10.3109/1061186X.2013.865032
Docetaxel-loaded brain targeting liposome 255
Synthesis of the compound 5. To a solution of compound
4 (3.70 g, 6.41 mmol l) in dichloromethane (DCM) was added
DCC (1.5 g, 7.69 mmol) and DMAP (93.9 mg, 0.769 mmol),
then stirred at room temperature for 30 min. A solution of
compound 8 (2.23 g, 6.41 mmol) in DCM was added to the
above reaction mixture. After stirring at room temperature for
3 h, the reaction was terminated and then filtered. The filtrate
was concentrated, and the residue was purified by flash
(s, 1H, –CHO). IR (KBr, cm–1): ꢁ 2927, 2869, 1736, 1725,
1715, 1242, 1125 and 1072. MS (m/z): 1302.40 [M þ H]þ.
Synthesis of the ligand L-TDS-G. A solution of compound
7 (1.5 g, 0.60 mmol) in DCM:MeCN (1:1) was added
trifluoroacetic acid (1.8 ml, 24.3 mmol) in drop at 0 ꢁC and
then stirred at room temperature for 2 h. The reaction mixture
was concentrated, and the residue was purified by chroma-
tography to give L-TDS-G as a yellow solid (770 mg, 66%,
m.p.: 108–110 ꢁC). 1H NMR (400 MHz, CDCl3): ꢀ 0.67
(s, 3H, CHOLE CH3-18), 0.86 (d, 6H, J ¼ 6.4 Hz, CHOLE
CH3-26 and -27), 0.91 (d, 3H, J ¼ 6.4 Hz, CHOLE CH3-21),
0.99 (s, 3H, CHOLE CH3-19), 0.67–2.37 (remaining CHOLE
protons), 1.25 (d, 6H, J ¼ 6.4Hz, –CH(CH3)2), 2.06 (s, 3H,
¼CCH3), 2.97 (s, 3H, J ¼ 7.2 Hz, ¼CCH2–), 3.18 (m, 1H,
–CH(CH3)2), 3.46 (m, 2H, glucose H-3 and -4), 3.63–3.69
(m, 12H, OCH2CH2O ꢀ 3), 3.72 (s, 2H, –C(O)CH2), 3.87
(m, 1H, glucose H-2), 4.03 (m, 2H, glucose H-6), 4.15 (s, 2H,
–NCH2), 4.29 (t, 2H, J ¼ 6.8 Hz, –CH2COO–), 4.43 (s, 1H,
glucose H-5), 4.63 (s, 1H, glucose H-1), 5.33 (s, 1H, CHOLE
H-6) and 8.01 (s, 1H, –CHO). IR (KBr, cm–1): ꢁ 3290, 2925,
2868, 1742, 1739, 1721, 1239, 1121 and 1065. MS (m/z):
1036.25 [M þ Na]þ.
1
chromatography to yield 5 as a yellow oil (4.5 g, 77.4%). H
NMR (400 MHz, CDCl3): ꢀ 0.67 (s, 3H, CHOLE CH3-18),
0.87 (d, 6H, J ¼ 6.4 Hz, CHOLE CH3-26 and -27), 0.91 (d,
3H, J ¼ 6.4 Hz, CHOLE CH3-21), 0.99 (s, 3H, CHOLE CH3-
19), 0.67–2.38 (remaining CHOLE protons), 1.26 (d, 6H,
J ¼ 6.4 Hz, –CH(CH3)2), 1.47 (s, 9H, –C(CH3)3), 2.06 (s, 3H,
¼CCH3), 2.97 (t, 2H, J ¼ 6.8 Hz, ¼CCH2), 3.17 (m, 1H,
–CH(CH3)2), 3.63–3.83 (m, 12H, OCH2CH2O ꢀ 3), 4.01 (s,
2H, –NCH2), 4.15 (s, 2H, –C(O)CH2), 4.28 (t, 2H, J ¼ 6.8Hz,
–CH2COO–), 5.36 (m, 1H, CHOLE H-6), 8.02 (s, 1H,
–CHO). IR (KBr, cm–1): ꢁ 2934, 2867, 1740, 1725, 1715,
1620, 1450, 1251, 1105 and 1072. MS (m/z): 886.54
[MþNa]þ.
Synthesis of the compound 6. A solution of HClO4 (0.5 ml
in 1 m DCM) was added to the DCM solution of compound 5
(3.8 g, 4.18 mmol) at 0 ꢁC. After stirring at room temperature
for 7 h, the reaction was terminated and was concentrated.
The residue was purified by flash chromatography to yield 6 as
Synthesis of the TDS intermediate 8 and the glucose-TMS
derivative 9. The synthesis of TDS intermediate 8 and
the glucose-TMS derivative 9 were reported in our previous
work [28].
1
a yellow oil (1.8 g, 50.44%). H NMR (400 MHz, CDCl3): ꢀ
Synthesis of glucose–CHOLE derivative (L-G)
0.67 (s, 3H, CHOLE CH3-18), 0.85 (d, 6H, J ¼ 8 Hz, CHOLE
CH3-26 and -27), 0.91 (d, 3H, J ¼ 6.4 Hz, CHOLE CH3-21),
0.99 (s, 3H, CHOLE CH3-19), 0.67–2.38 (remaining CHOLE
protons), 1.25 (d, 6H, J ¼ 6.4 Hz, –CH(CH3)2), 2.10 (s, 3H,
¼CCH3), 2.98 (t, 2H, J ¼ 6 Hz, ¼CCH2-), 3.19 (m, 1H,
–CH(CH3)2), 3.64–3.79 (m, 12H, OCH2CH2O ꢀ 3), 4.11
(s, 2H, –NCH2), 4.20 (s, 2H, –C(O)CH2), 4.35 (t, 2H,
J ¼ 6 Hz, –CH2COO–), 5.36 (m, 1H, CHOLE H-6) and 8.10
(s, 1H, –CHO). IR (KBr, cm–1): ꢁ 3305, 2940, 2933, 2869,
1734, 1728, 1712, 1642, 1108 and 1069. MS (m/z): 874.25
[M þ Na]þ.
The synthetic route of the glucose–CHOLE derivative L-G,
which was also started from CHOLE, was reported
previously [24].
Preparation of docetaxel or coumarin-6-loaded
lipsomes
Conventional (non-coated), L-G coated and L-TDS-G coated
liposomes loaded with docetaxel/coumarin-6 were prepared
according to the lipid film hydration-ultrasound method and
the formulaes are listed in Table 1. Briefly, lipid materials
(SPC and CHOLE) and ligand, docetaxel/coumarin-6 were
dissolved in chloroform, which then was removed by rotary
evaporation at 37 ꢁC, and then kept under vacuum to remove
the solvent completely. After incubated in phosphate buffered
saline (PBS, PH ¼ 6.5, 0.02 M) for 1 h at 37 ꢁC with constant
mixing, the liposome solution was ultrasonicated with an
ultrasonic cell grinder (JY92-II, Scientz Biotechnology Co.,
Ltd., Ningbo, P.R. China) at 300W for 75 s. The final
concentration of docetaxel/coumarin-6 was 0.375 mg/ml or
0.5 mg /ml.
Synthesis of the compound 7. A stirred solution of
compound 6 (1.4 g, 1.64 mmol) in DCM was treated with
DCC (407 mg, 1.97 mmol) and DMAP (24.1 mg, 0.197 mmol)
and kept at room temperature for 30 min. Then glucose-TMS
derivative 9 (770 mg, 1.64 mmol) was added. After stirring
overnight at room temperature, the reaction mixture was
filtered, and the filtrate was concentrated and the residue
was purified by chromatography to offer a yellow oil (1.56 g,
1
73.2%). H NMR (400 MHz, CDCl3): ꢀ 0.13–0.19 (m, 36H,
–Si(CH3)3 ꢀ 4), 0.67 (s, 3H, CHOLE CH3-18), 0.86 (d, 6H,
J ¼ 6.4 Hz, CHOLE CH3-26 and -27), 0.91 (d, 3H, J ¼ 6.4 Hz,
CHOLE CH3-21), 0.99 (s, 3H, CHOLE CH3-19), 0.67–2.38
(remaining CHOLE protons), 1.26 (d, 6H, J ¼ 6.4 Hz,
–CH(CH3)2), 2.06 (s, 3H, ¼CCH3), 2.96 (t, 2H, J ¼ 7.2 Hz,
¼CCH2-), 3.17 (m, 1H, –CH(CH3)2), 3.36 (dd, 1H, J1 ¼2.8 Hz,
J2 ¼ 9.2 Hz, glucose H-4), 3.41 (t, 1H, J ¼ 8.8 Hz, glucose H-
3), 3.63–3.74 (m, 12H, OCH2CH2O ꢀ 3), 3.72 (s, 2H,
–C(O)CH2), 3.78 (t, 1H, J ¼ 8.8Hz, glucose H-2), 3.92 (m,
1H, glucose H-6), 4.09 (m, 1H, glucose H-6’), 4.17 (s, 2H,
–NCH2), 4.27 (t, 2H, J ¼ 6.8Hz, –CH2COO–), 4.40 (dd, 1H,
J1 ¼ 2 Hz, J2 ¼ 11.6 Hz, glucose H-5), 4.99 (d, 1H, J ¼ 2.8 Hz,
glucose H-1), 5.33 (d, 1H, J ¼ 4.8 Hz, CHOLE H-6) and 8.04
Table 1. Formulas of liposome.
Molar Docetaxel Coumarin-6
(mg)
Batches
Lipid compositions
ratio
(mg)
DTX-Lip
DTX-Lip-G
SPC:CHOLE
SPC:CHOLE:L-G
8:4
8:4:1
DTX-Lip-TDS-G SPC:CHOLE:L-TDS-G 8:4:1
3.75
3.75
3.75
–
–
–
CM6-Lip
CM6-Lip-G
CM6-Lip-TDS-G SPC:CHOLE:L-TDS-G 8:4:1
SPC:CHOLE
SPC:CHOLE:L-G
8:4
8:4:1
0.5
0.5
0.5
Abbreviations: DTX, docetaxel; CM6, couarim-6; Lip, liposome; SPC,
soybean phospholipid; CHOLE, cholesterol.

256 X. Li et al.
J Drug Target, 2014; 22(3): 251–261
Characterization of liposome
Cellular uptake and competition assay
Particle size and zeta potential
The coumarin-6-loaded liposomes were subjected to conflu-
ent C6 glioma cell line, which were seeded into six-well assay
plates at 5 ꢀ 103 viable cells/well. After the cells reached
confluence, the cells were incubated with 100 ml of 10 mg/ml
each liposomes (CM6-Lip, CM6-Lip-G and CM6-Lip-
TDS-G) in the 1640 supplemented with 10% FBS and 1%
Gibco penicillin–streptomycin at 37 ꢁC for 4 h. Then the
suspension was discarded and the wells were washed twice
with PBS. After trypsin was added and incubated for 5 min at
37 ꢁC, the wells were washed with PBS. Next, cells were
transferred to Eppendorf tubes and centrifuged (3000 rpm,
3 min). The fluorescence intensity of each sample was
measured through a flow cytometry.
The average diameters, polydispersity indices (PDI) and
zeta-potentials of docetaxel/courmain-6-loaded liposomes
(conventional, L-G coated and L-TDS-G coated) were
determined using a Malvern Zetasizer Nano ZS90 instru-
ment (Malvern Instruments Ltd., Worcestershire, UK).
Prior to measurement, the liposome samples were
diluted 10 times with to nearly 1 mg/ml of docetaxel/
courmain-6.
Morphological study
The surface morphological property of liposome DTX-Lip-
TDS-G was observed by a field-emission transmission
electron microscopy system (Tecnai G2 F20 S-TWIN,
FEI Company, Hillsboro, OR). The sample of DTX-Lip-
TDS-G was prepared by placing one drop on a copper
grid and dried naturally, then observed directly without any
staining.
For the competition assay, 1 mg D-glucose was added to the
wells with confluent C6 glioma cells. After 30 min incubation
at 37 ꢁC, 100 ml of each coumarin-6-loaded liposome (at a
concentration of 10 mg/ml) was added. After incubating for 4 h,
the solution was removed and the above-mentioned steps were
followed. The fluorescence intensity of the stock solution
diluted by FBS-free 1640 supplemented of each sample was
taken as control. Transport ratio (%) was defined as the
fluorescence intensity of sample to that of control.
Determination of docetaxel/coumarin-6 encapsulation
efficiency
The docetaxel encapsulated liposomes (DTX-Lip, DTX-Lip-
G and DTX-Lip-TDS-G) were measured; acetonitrile: 0.1%
phosphoric acid solution (55:45) was used as the mobile
phase with a flow rate of 1.0 ml/min and a column tempera-
ture of 25 ꢁC; and the UV detector was set to monitor the
signal at 230 nm corresponding to the maximum absorbance
for the docetaxel.
Cytotoxicity of docetaxel-loaded liposomes
Cytotoxicity of docetaxel-loaded liposomes was investigated
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. C6 glioma cells were seeded into 96-
well plates at 5 ꢀ 103 viable cells/well with 1640 medium
(RPMI 1640) and incubated overnight to allow cell attach-
ment. After that the cells were incubated with these three
docetaxel-loaded liposomes and docetaxel at the concentra-
tions of 0, 0.025, 0.25, 2.5 and 25 mg/ml equivalent to
docetaxel for 24 h. The medium was removed, and the wells
were washed twice with PBS. Following that, 20 ml of MTT
solution (5 mg/ml) prepared in the RPMI 1640 medium was
added to each well of the plate, and the plates were further
incubated for 4 h. Finally, MTT in medium was removed and
150 ml of DMSO was added into each well of transformed
MTT crystals and the absorbance of the transformed MTT
solution in the wells was measured at 570 nm wavelength
using a microplate reader [32].
The entrapment efficiency of docetaxel was determined
by measuring the concentration of free drug (unentrapped) in
aqueous medium [29]. Briefly, to docetaxel-loaded liposomes
(1 ml) were add 340 ml of 1 M HCl and 1.5 ml of PBS
(pH ¼ 6.5), centrifuged at 20 000 rpm for 2 h at 4 ꢁC and
900 ml of the supernatant fluid was transferred and diluted to
2 ml to determine the free drug. Another 1 ml of the
docetaxel-loaded lipsomes were added to 1.84 ml of PBS,
900 ml of which was added to 1 ml of isopropanol and
diluted to 2 ml to determine the total drug content through the
HPLC method mentioned above.
To the coumarin-6-loaded liposomes, free coumarin-6
was removed by dialyzing method in Tris buffer, and
the encapsulation efficiency (EE%) was determined by
fluorospectrophotometry (Ex ¼ 465 nm, Em ¼ 502 nm).
In vivo study
Methodology evaluation
In vitro drug release
The chromatograms of blank mouse plasma and brain tissues
showed no peaks at the retention time of docetaxel (12 min
approximately) and the internal standard diazepam (8 min
approximately). A good baseline separation of docetaxel,
diazepam and other major components from mouse plasma
and brain tissue samples was achieved under this chromato-
graphic condition.
The in vitro drug release from the docetaxel-loaded liposomes
(DTX-Lip, DTX-Lip-G and DTX-Lip-TDS-G) was studied
by dialysis bag diffusion method. In brief, 1 ml of liposomes
samples (equivalent to 103 mg of docetaxel) were placed
in the dialysis bag (molecular weight cut off 1000 Da) sealed,
and immersed into 20 ml of PBS (pH 7.4) containing
0.1% (w/v) Tween 80 [30,31]. The entire system was kept
at 37 ꢂ 0.5 ꢁC with continuous shaking at 100 rpm/min.
Release medium samples were taken at various intervals
and replaced by fresh medium. The drug contents in
the samples were determined by HPLC method described
earlier.
Pharmacokinetics in plasma and brain of docetaxel-loaded
liposomes
Kunming mice weighing 20–22 g were randomly divided into
groups, and the mice were injected the docetaxel-loaded
liposome samples and naked docetaxel through the tail vein

DOI: 10.3109/1061186X.2013.865032
Docetaxel-loaded brain targeting liposome 257
Determination of docetaxel/coumarin-6 EE
at an equivalent dose of 5 mg/kg body weight of docetaxel,
respectively. Then, mice from each group were sequentially
sacrificed at 30 min, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h after
injection, and their brain and blood were collected and
analyzed by HPLC method described above.
The encapsulation efficiencies of the docetaxel-loaded lipo-
somes were 93.54 ꢂ 2.41%, 84.79 ꢂ 3.46% and 82.83 ꢂ 5.37%
for liposome DTX-Lip, DTX-Lip-G and DTX-Lip-TDS-G,
respectively. Similarly, the encapsulation efficiencies of the
coumarin-6-loaded liposome CM6-Lip, CM6-Lip-G and
CM6-Lip-TDS-G were 92.65 ꢂ 5.37, 82.87 ꢂ 4.24% and
85.63 ꢂ 6.72%, respectively. There was a slight decrease in
the docetaxel/coumarin-6 EE in the ligand-coated liposomes.
The encapsulation efficiencies of all the liposomes met the
requirements of this experiment.
Statistical analysis
The area under the concentration-time (AUC0-t), Cmax and
mean residence time (MRT) were calculated by Data and Max
Statistics (DAS 2.0, Shanghai, China). The relative uptake
efficiency (RE) and concentration efficiency (CE) were
calculated to evaluate the brain targeting properties of the
liposomes. The values of RE and CE were defined as follows:
In vitro drug release
Figure 5 exhibited the accumulated percentage release of
docetaxel from the drug-loaded liposomes and naked
docetaxel in 80 h. The naked docetaxel showed initial burst
RE ¼ ðAUC0ꢃt
ÞLip=ðAUC0ꢃtÞDTX
CE ¼ ðCmax Lip=ðCmaxÞDTX
Þ
where Lip and DTC represented liposome samples and naked
Docetaxel, respectively.
Data are expressed as mean ꢂ standard deviation.
Statistical analysis was performed with SPSS 16.0 (SPSS
Inc., Chicago, IL). Differences were considered significant at
a level of p50.05. Mean values of EE, in vitro data and the
pharmacokinetic parameters were compared using one-way
analysis of variance and Tukey’s post hoc test with the
assumption of equal variances. If the data were not normally
distributed, a Games–Howell method was used.
Results
Characterization of liposomes
Particle size, polydispersity and zeta potential analysis
The mean particle size and polydispersity of docetaxel/
coumarin-6-loaded liposomes were shown in Table 2. PCS
measurements were undertaken in multimodal analysis to get
a true reflection of particle size distribution. The particle size
distribution curves for all the samples were unimodal. The
polydispersity of all the liposomes showed quite narrow size
distribution, which is nearer to 0.2. The particle size results
indicate that the ligand Lip-G coating leads to decrease in the
liposome size. Instead, an increase in the sizes of liposomes
was observed with the Lip-TDS-G coating, and a negative
zeta potential was observed in all the liposomes.
Figure 4. The transmission electron microscope (TEM) image of
liposome DTX-Lip-TDS-G.
60.00%
Docetaxel
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
DTX-Lip
DTX-Lip-G
DTX-Lip-TDS-G
Morphological study
The transmission electron microscope image showed that the
liposome DTX-Lip-TDS-G (L-TDS-G coated) exhibited
uniform spherical in shape and was around 100 nm scale in
diameter, which demonstrating the successful formation of
the L-TDS-G coated liposome (Figure 4).
ꢀ
ꢁꢀ
ꢂꢀ
ꢃꢀ
ꢄꢀ
Time (h)
Figure 5. In vitro drug release curve of docetaxel loaded liposomes in
PBS (PH 7.4) (n ¼ 3).
Table 2. The characterization of different docetaxel/coumarin-loaded liposomes (n ¼ 3).
Batches
Average size (nm)
Polydispersity index (PDI)
Encapsulating efficiency (EE, %)
Zeta-potential (mV)
DTX-Lip
121.6 ꢂ 5.6
118.3 ꢂ 8.4
133.4 ꢂ 6.1
110.6 ꢂ 3.2
103.6 ꢂ 5.9
123.4 ꢂ 7.5
0.219 ꢂ 0.024
0.264 ꢂ 0.019
0.245 ꢂ 0.022
0.189 ꢂ 0.029
0.232 ꢂ 0.014
0.275 ꢂ 0.024
93.54 ꢂ 2.41
84.79 ꢂ 3.46
82.83 ꢂ 5.37
92.65 ꢂ 6.94
82.87 ꢂ 4.24
85.63 ꢂ 6.72
–64.1 ꢂ 9.2
–44.8 ꢂ 1.6
–37.6 ꢂ 6.5
–60.1 ꢂ 10.5
–47.6 ꢂ 4.8
–33.6 ꢂ 3.5
DTX-Lip-G
DTX-Lip-TDS-G
CM6-Lip
CM6-Lip-G
CM6-Lip-TDS-G

258 X. Li et al.
J Drug Target, 2014; 22(3): 251–261
release, while the three liposomes showed controlled release
for more than three days without any burst release. After 80 h
of dialysis in PBS (PH 7.4), the percentage of docetaxel
released from the liposomes was 56.09%, 31.59%, 20.9% and
41.56% for naked docetaxel, liposome DTX-Lip, DTX-Lip-G
and DTX-Lip-TDS-G, respectively.
Liposome DTX-Lip-TDS-G resulted in higher cytotoxicity to
C6 glioma cells compared with naked docetaxel, liposome
DTX-Lip and DTX-Lip-G in all equivalent docetaxel con-
centration levels. Furthermore, the cell viability was signifi-
cantly decreased to 54.37%, 49.90%, 52.36% and 44.11% of
the control value for naked docetaxel, DTX-Lip, DTX-Lip-G
and DTX-Lip-TDS-G, respectively, after C6 glioma cells
were treated with the concentration of 50 mg/ml samples
for 24 h.
Cellular uptake and competition assay
The cellular uptake of C6 glioma cells after incubation
with three coumarin-6-loaded liposomes were shown in
Figure 6. The average fluorescence intensity of each samples
were 221, 358, 436 and 646, respectively. It can be noticed
that the cellular uptake of the L-TDS-G coated liposome
(CM-Lip-TDS-G) was significantly higher compared with
the naked coumarin-6, conventional liposome (CM-Lip) and
L-G coated liposome (CM-Lip-G), which indicated that
more CM-Lip-TDS-G was transferred into C6 glioma
cells during the test time. Moreover, it can be observed
that glucose-modified liposomes (CM-Lip-G and CM-Lip-
TDS-G) exhibited better penetrability across C6 glioma
cells. To validate the mechanism of glucose transport,
competition assay was performed. When the model was pre-
incubated with free glucose to saturate GLUT1 on the
cell surface, the transport ratios of CM-Lip-G and
CM-Lip-TDS-G were significantly reduced to the level of
CM-Lip (Figure 7).
In order to demonstrate the advantages of the docetaxel-
loaded liposomes, a quantitative index of inhibitory concen-
tration, IC50, was calculated and listed in Table 3. After 24 h
incubation, IC50 for each type of the liposomes versus
naked docetaxel determined from the cytotoxicity data
were 55.82 ꢂ 1.01, 49.65 ꢂ 0.67 and 25.65 ꢂ 0.89 versus
59.09 ꢂ 1.43 mg/ml, respectively. The IC50 values also implied
that the three docetaxel-loaded liposomes were 5.5%, 16.0%
and 56.6% more efficient than naked docetaxel after
incubated for 24 h, respectively.
In vivo test of liposome
The curves in Figure 9 reflected the docetaxel concentrations
of docetaxel and the three docetaxel-loaded liposomes in
plasma. The pharmacokinetic parameters of docetaxel in
plasma were shown in Table 4. Docetaxel of liposome DTX-
Lip, DTX-Lip-G and DTX-Lip-TDS-G presented a higher
AUC0–t compared to naked docetaxel with a ratio of 1.10,
1.85 and 1.83, respectively. The Cmax of docetaxel in plasma
after intravenous (i.v.) administration liposome DTX-Lip,
DTX-Lip-G and DTX-Lip-TDS-G were 1.08, 2.25 and 2.98
times than that of naked docetaxel.
Cytotoxicity
Cell viability was measured by MTT method. In vitro
cytotoxicities of the docetaxel in the non-coated liposome,
the L-G coated liposome and the L-TDS-G coated liposome
were investigated in comparison with docetaxel at the same
equivalent drug concentration on C6 glioma cells after 24 h
incubation at 37 ꢁC and the results are shown in Figure 8.
The curves in Figure 10 reflected the docetaxel concen-
trations of naked docetaxel and the three docetaxel-loaded
liposomes in brain. The pharmacokinetic parameters of
docetaxel in brain were shown in Table 5. The REs were
700
600
500
400
300
200
100
0
Docetaxel
100
90
80
70
60
50
40
30
20
10
0
DTX-Lip
DTX-Lip-G
DTX-Lip-TDS-G
Coumarin-6
CM-Lip
CM-Lip-G
CM-Lip-TDS-G
Figure 6. Cellular uptake efficiency of coumarin-6 loaded liposomes.
30
25
20
15
10
5
Concentration of docetaxel (µg/ml)
Figure 8. Cytotoxicity results of naked docetaxel and docetaxel loaded
liposomes.
Table 3. The IC50 values of docetaxel and docetaxel loaded liposomes
for C6 glioma cells (n ¼ 6).
0
Coumarin-6
CM-Lip
CM-Lip-G CM-Lip-TDS-G
Batches
Dcetaxel
DTX-Lip DTX-Lip-G DTX-Lip-TDS-G
Figure 7. The transport ratios (%) of coumarin-6 across the C6 glioma
cells.
IC50 (mg/ml) 59.09 ꢂ 1.43 55.82 ꢂ 1.01 49.65 ꢂ 0.67 25.65 ꢂ 0.89

DOI: 10.3109/1061186X.2013.865032
Docetaxel-loaded brain targeting liposome 259
800
Docetaxel
1000
800
600
400
200
0
Docetaxel
DTX-Lip
DTX-Lip
DTX-Lip-G
DTX-Lip-TDS-G
600
400
200
0
DTX-Lip-G
DTX-Lip-TDS-G
ꢀ
ꢅ
ꢆꢀ
ꢆꢅ
ꢁꢀ
ꢁꢅ
0
5
10
15
20
25
Time(h)
Time(h)
Figure 9. The concentration of docetaxel in plasma versus time after i.v.
injection in mice. Error bars show the value of SD (n ¼ 3).
Figure 10. The concentration of docetaxel in brain versus time after i.v.
injection in mice. Error bars show the value of SD (n ¼ 3).
Table 4. Pharmacokinetic parameters of docetaxel in plasma after i.v. injection of docetaxel and liposomes (n ¼ 3).
Pharmacokinetic parameters
AUC(0–t) (ng/mlꢄh)
MRT (h)
T1/2 (h)
Tmax (h)
Cmax (ng/ml)
Docetaxel
DTX-Lip
DTX-Lip-G
DTX-Lip-TDS-G
3800.5 ꢂ 173.4
4320.5 ꢂ 93.2
7044.9 ꢂ 68.8*
6966.3 ꢂ 326.1z
9.66 ꢂ 0.17
10.86 ꢂ 0.44
10.04 ꢂ 0.69
8.51 ꢂ 0.25y
16.24 ꢂ 2.50
20.13 ꢂ 1.53
19.89 ꢂ 2.65
8.97 ꢂ 1.99
4
3.3
2
233.6 ꢂ 13.3
253.1 ꢂ 22.1
526.7 ꢂ 57.5y
696.9 ꢂ 87.7y
2
*p50.001 versus Docetaxel.
yp50.05 versus Docetaxel.
zp50.01 versus Docetaxel.
Table 5. Pharmacokinetic parameters of docetaxel in brain after i.v. injection of docetaxel and liposomes (n ¼ 3).
Pharmacokinetic
parameters
AUC(0-t) (ng/gꢄh)
MRT (h)
T1/2 (h)
Tmax (h)
Cmax (ng/g)
Re
CE
Docetaxel
DTX-Lip
DTX-Lip-G
DTX-Lip-TDS-G
3365.9 ꢂ 125.1
6045.4 ꢂ 358.5y
8839.0 ꢂ 322.1z
12864.3 ꢂ 647.9y
11.43 ꢂ 0.15
11.03 ꢂ 0.40
10.32 ꢂ 0.05y
9.01 ꢂ 0.26y
98.11 ꢂ 61.70
35.67 ꢂ 5.90
29.22 ꢂ 18.08
11.15 ꢂ 1.11
4
1.67
1
171.9 ꢂ 10.3*
334.4 ꢂ 29.5
707.7 ꢂ 62.1
858.2 ꢂ 111.9y
–
–
1.79
2.62
3.82
1.94
4.12
4.99
1
*p50.05 versus Docetaxel.
yp50.01 versus Docetaxel.
zp50.001 versus Docetaxel.
enhanced to 1.79, 2.62 and 3.82 times than that of naked
docetaxel for liposome DTX-Lip, DTX-Lip-G and
DTX-Lip-TDS-G, respectively. Furthermore, the CEs were
increased to 1.94-, 4.12- and 4.99-fold compared to naked
docetaxel. In addition, a higher Cmax of docetaxel compared
to docetaxel or any other liposome group can be observed
in both Figure 10 and Table 5.
of the drugs in CNS. Thus, in this study, we introduced TDS
system, with ‘‘lock-in’’ function, to the ligand and developed
a novel brain-targeting liposome system.
Particle size of liposomes plays a critical role in their
clearance by the sinusoidal splenic filtration of human and
rats at the interendothelial cell slits in the walls of venous
sinuses. Therefore, liposomes must be small enough and a
generally limit in diameter for brain-targeted liposomes is
200 nm. The particle sizes of all the liposomes prepared in
this study were under 150 nm and the size distributions were
quite narrow with PDIs under 0.3, which provided a favorable
size condition for the vitro and vivo studies of the liposomes
[36]. Furthermore, the particle size results indicated that L-G
coating leads to slight decrease in the liposome size; while
an increase in the sizes of liposomes was observed with
L-TDS-G coating (Table 2).
The zeta potentials of all the liposomes were found to be
negatively charged. Typically, the non-coated liposome
(DTX-Lip or CM6-Lip) showed more negative charge than
the L-G and L-TDS-G coated liposomes on the surface
(Table 2). This may be attributed to the hydroxyl groups or
aldehyde group of the coated layers of the liposomes, which
Discussion
Docetaxel, a well-known anticancer drug, has limited brain
uptake due to the existence of BBB [33]. Many strategies have
been studied to overcome the low penetration of BBB, among
which liposome, one of the particulate drug-delivery systems,
has been investigated extensively to increase the targeting
efficiency of drugs to the brain [34]. Previous work of our
group demonstrated that liposome with ligand L-G, glucose
linked to CHOLE with ester bond, modified on the surface
was a useful tool through the specific endogenous transporters
GLUT1 localized on the BBB [35]. However, the bidirectional
transport characteristic of GLUT1 has been neglected, which
may be one of the reasons caused the low concentrations

260 X. Li et al.
J Drug Target, 2014; 22(3): 251–261
can combine with the hydrogen ions in aqueous solution
thus reducing the negatively charge of the liposome surface.
Moreover, the zeta potentials confirmed the presence of
ligand (L-G or L-TDS-G) coated layer on the liposome
surface.
and the non-coated liposomes when killing the same amount
cancer cells.
In vivo studies were performed to investigate the distribu-
tion of the drug-loaded liposomes, especially for DTX-Lip-
TDS-G. The result showed that AUC0–t of docetaxel of three
liposomes in plasma was higher than that of naked docetaxel
with a ratio of 1.10, 1.85 and 1.83, respectively. The Cmax of
docetaxel in plasma after i.v. administration of liposome
DTX-Lip, DTX-Lip-G and DTX-Lip-TDS-G were 1.08,
2.25 and 2.98 times than that of naked docetaxel. These data
indicate that the liposomes showed certain stability, which
would increase the chance to be transported across BBB.
The MRTs of docetaxel in plasma after i.v. administration
of DTX-Lip, DTX-Lip-G and DTX-Lip-TDS-G were 1.12,
1.04, 1.07 times than that of docetaxel, respectively, and the
MRT of DTX-Lip-G and DTX-Lip-TDS-G was the same as
DTX-Lip or a slight increase. The half lives (T1/2) and MRT
of docetaxel by these liposomes indicated that the liposomes
can prolong the circulation time of docetaxel, by delaying its
metabolic rate in vivo. So it is obvious that all the liposomes
can keep the certain concentration of docetaxel in plasma,
which might increase the chance for the drug to be delivered
across BBB.
The influence of surface coatings on the drug release was
also studied. Naked docetaxel showed a burst release, while
the docetaxel-loaded liposomes exhibited a controlled release
(Figure 5). The docetaxel encapsulated in the liposomes
has a total release in PBS (pH 7.4) at 31.59%, 20.9% and
41.56% for liposome DTX-Lip, DTX-Lip-G and DTX-Lip-
TDS-G, respectively. The relative low total drug release
of the liposomes may be explained by the low solubility
of the docetaxel in PBS; furthermore, the unsatisfactory
results also confirmed the controlled release of the liposomes.
The release curves illustrated there was significant increase
in the surface coated liposomes (DTX-Lip-G and DTX-
Lip-TDS-G) compared with the non-coated liposome
(DTX-Lip), which may be explained by the influence of
hydrophilic and solubilization property of L-G or L-TDS-G
on docetaxel [37].
Cellular uptake studies were performed to access the
intracellular uptake of the liposomes with C6 glioma cells.
It was shown in Figure 6 that the fluorescence intensity of
L-TDS-G coated liposome (CM6-Lip-TDS-G) was 2.92,
1.80 and 1.48 times higher than that of naked docetaxel,
CM6-Lip (non-coated) and CM6-Lip-G (L-G coated),
respectively, which suggested that liposome might be much
easier up taken by C6 glioma cells when transporting to the
brain tissue. The higher cellular uptake efficiency of CM6-
Lip-TDS-G may be due to the TDS system, which improved
the liposolubility of the liposome; after entering the BBB,
the TDS part can be reduced to a thiazolium, which would
be ‘‘locked’’ in the C6 glioma cells and then there might be
a sustained accumulation of liposome CM6-Lip-TDS-G.
To confirm the increased uptake is indeed contribution of
the ligand, excess free D-glucose was pre-incubated in the
competition assay to saturate the glucose transporter GLUT1
on the C6 glioma cells [38]. The results showed that adding D-
glucose significantly reduced the transport ratios of DTX-
Lip-G and DTX-Lip-TDS-G to the level of CM6-Lip.
Therefore, it can be concluded that the transport mechanism
of glucose-modified liposomes might be mediated by GLUT1.
In vitro cytotoxicities of the docetaxel-loaded liposomes
were investigated in comparison with docetaxel on C6 glioma
cells, and the results are shown in Figure 8. It is obviously that
the docetaxel-loaded liposomes achieved the higher cytotox-
icity compared with naked docetaxel in all concentrations,
which may be due to the high permeabilities of the liposomes
to enter the C6 glioma cells and the effect of controlled
docetaxel release from the liposomes. Especially, L-TDS-G
coated liposome DTX-Lip-TDS-G resulted in highest cyto-
toxicity (cell viability was decreased to 44.11% of the control
value), which was constant with its highest cellular uptake
result among all three. Moreover, the IC50 values implied
that liposome DTX-Lip-TDS-G was 56.6% more efficient
than naked docetaxel after incubated for 24 h, while that
of DTX-Lip and DTX-Lip-G was 5.5% and 16.0%.
The results indicated that DTX-Lip-TDS-G require much
lower drug concentration in comparison with the L-G coated
It can be observed that the concentrations of docetaxel
of liposomes with coated surface layer were much higher
than that from uncoupled liposomes and naked docetaxel at
different time interval. The AUC0–t and Cmax of docetaxel in
brain after i.v. administration of liposomes were fairly higher
than that after the injection of naked docetaxel, and it is worth
noting that the L-TDS-G coated liposome DTX-Lip-TDS-G
showed an increase in many pharmacokinetic parameters.
Typically, the RE and CE were enhanced to 3.82 and 4.99
times that of naked docetaxel, respectively. So these data
further proved our conjecture that liposome with ligand
having the ‘‘lock-in’’ function can deliver and keep docetaxel
in the CNS. Furthermore, the pharmacokinetic parameters
suggested that the brain targeted ability of this designed
liposome is better compared to DTX-Lip, DTX-Lip-G.
Moreover, the higher REs and CEs of modified liposomes
demonstrated that exposing glucose residues outside the
membrane of coupled liposomes could recognize GLUT1;
furthermore, the TDS system could be reduced and ring-
closed to be a thiazolium in the BBB; thus, the liposome
with this system was ‘‘locked’’ in the brain and then it can
serve a sustained release of the chemotherapeutic agent.
Therefore, liposome DTX-Lip-TDS-G showed a better
affinity. Moreover, the higher docetaxel concentration of
DTX-Lip-TDS-G shown in brain also confirmed the bidir-
ectional transport characteristic of GLUT1 as well as the
effectiveness of TDS system.
Conclusion
Generally, to overcome the bidirectional delivery of GLUT1,
the TDS, with the ability of ‘‘lock-in’’, was introduced, and
a new ligand L-TDS-G coated liposome was designed
and prepared in this work. The experimental results confirmed
the hypothesis that liposome with L-TDS-G coated was
a promising brain targeting drug delivery system since
it behaved higher transendothelial ability, higher brain

DOI: 10.3109/1061186X.2013.865032
Docetaxel-loaded brain targeting liposome 261
peptide dalargin after its delivery across the blood-brain barrier
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concentration, an increased AUC0–t and a better RE value
compared to naked docetaxel, non-coated and L-G coated
liposomes in brain. Furthermore, the results indicated that the
glucosyl thiamine disulfide moiety could act as a vector,
transporting the liposome across the BBB and then constantly
releasing drug in CNS.
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Declaration of interest
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22. Pardridge WM. Drug and gene targeting to the brain via blood-
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2005;1277:49–62.
The authors report no declarations of interest.
This work was supported by the National Natural Science
Foundation of China (No.81072532 and No.81102324) and
the Ph.D. Programs Foundation of Ministry of Education of
China (No. 20110181130011).
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