Design, preparation and evaluation of different branched biotin modified liposomes for targeting breast cancer

Article abstract of DOI:10.1016/j.ejmech.2020.112204

A series of liposome ligands (Bio-Chol, Bio-Bio-Chol, tri-Bio-Chol and tetra-Bio-Chol) modified by different branched biotins that can recognize the SMVT receptors over-expressed in breast cancer cells were synthesized. And four liposomes (Bio-Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip) modified by above mentioned ligands as well as the unmodified liposome (Lip) were prepared to study the targeting ability for breast cancer. The cytotoxicity study and apoptosis assay of paclitaxel-loaded liposomes showed that tri-Bio-Lip had the strongest anti-proliferative effect on breast cancer cells. The cellular uptake studies on mice breast cancer cells (4T1) and human breast cancer cells (MCF-7) indicated tri-Bio-Lip possessed the strongest internalization ability, which was 5.21 times of Lip, 2.60 times of Bio-Lip, 1.67 times of Bio-Bio-Lip and 1.17 times of tetra-Bio-Lip, respectively. Moreover, the 4T1 tumor-bearing BALB/c mice were used to evaluate the in vivo targeting ability. The data showed the enrichment of liposomes at tumor sites were tri-Bio-Lip > tetra-Bio-Lip > Bio-Bio-Lip > Bio-Lip > Lip, which were consistent with the results of in vitro targeting studies. In conclusion, increasing the density of targeting molecules on the surface of liposomes can effectively enhance the breast cancer targeting ability, and the branching structure and spatial distance of biotin residues may also have an important influence on the affinity to SMVT receptors. Therefore, tri-Bio-Lip could be a promising drug delivery system for targeting breast cancer.

Full text of DOI:10.1016/j.ejmech.2020.112204

European Journal of Medicinal Chemistry 193 (2020) 112204
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, preparation and evaluation of different branched biotin
modified liposomes for targeting breast cancer
,
**
Baolan Tang a, Yao Peng a, Qiming Yue a, Yanchi Pu a, Ru Li a, Yi Zhao b, Li Hai a, Li Guo a
,
,
*
Yong Wu a
a Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-
Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
b Department of Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of liposome ligands (Bio-Chol, Bio-Bio-Chol, tri-Bio-Chol and tetra-Bio-Chol) modified by
different branched biotins that can recognize the SMVT receptors over-expressed in breast cancer cells
were synthesized. And four liposomes (Bio-Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip) modified by
above mentioned ligands as well as the unmodified liposome (Lip) were prepared to study the targeting
ability for breast cancer. The cytotoxicity study and apoptosis assay of paclitaxel-loaded liposomes
showed that tri-Bio-Lip had the strongest anti-proliferative effect on breast cancer cells. The cellular
uptake studies on mice breast cancer cells (4T1) and human breast cancer cells (MCF-7) indicated tri-Bio-
Lip possessed the strongest internalization ability, which was 5.21 times of Lip, 2.60 times of Bio-Lip, 1.67
times of Bio-Bio-Lip and 1.17 times of tetra-Bio-Lip, respectively. Moreover, the 4T1 tumor-bearing BALB/
c mice were used to evaluate the in vivo targeting ability. The data showed the enrichment of liposomes
at tumor sites were tri-Bio-Lip > tetra-Bio-Lip > Bio-Bio-Lip > Bio-Lip > Lip, which were consistent with
the results of in vitro targeting studies. In conclusion, increasing the density of targeting molecules on the
surface of liposomes can effectively enhance the breast cancer targeting ability, and the branching
structure and spatial distance of biotin residues may also have an important influence on the affinity to
SMVT receptors. Therefore, tri-Bio-Lip could be a promising drug delivery system for targeting breast
cancer.
Received 27 December 2019
Received in revised form
2 March 2020
Accepted 2 March 2020
Available online 5 March 2020
Keywords:
Biotin
Active targeting ligand
Breast cancer
Drug delivery
Liposome
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
treatment in contrast to basic therapeutic approaches. Chemo-
therapeutic agents have been reformulated into liposomes or
According to the latest WHO statistics, breast cancer as a highly
heterogeneous systemic disease is the leading threaten of cancers
in women worldwide, with high incidence and high mortality [1].
Despite significant progress has been made in the breast cancer
treatment during the past decades, the current therapeutic effect is
still far from optimal outcomes. Surgery, radiotherapy, chemo-
therapy and hormone therapy are the regular clinical approaches,
yet they have the disadvantages of low specificity and inevitably
severe side effects [2,3].
nanoparticle which improve accumulation within the tumor site.
For example, the commercial availability of Doxil® and Abraxane®
have been extensively used for breast cancer adjuvant therapy [4].
However, these nanomedicines can only be passively enriched in
tumor sites through the enhanced permeability and retention effect
(EPR effect), which were originally designed to be broad-spectrum
anticancer agents rather than specific for breast cancer treatment.
Thus, their therapeutic effect for breast cancer are barely satisfac-
tory. For these reasons, finding novel targeting drug delivery sys-
tems (TDDS) are the focus of current researches on breast cancer
treatment.
Nanomedicine is a promising alternative for breast cancer
As drug carriers, nanomaterials have many advantages in cancer
treatment, such as easy surface modification, adjustable particle
size and surface charge, high porosity and large specific surface area
etc [5e10]. At present, many kinds of TDDS have been applied to the
* Corresponding author.
** Corresponding author.
E-mail addresses: guoli@scu.edu.cn (L. Guo), wyong@scu.edu.cn (Y. Wu).
https://doi.org/10.1016/j.ejmech.2020.112204
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
treatment for breast cancer, like micelles, albumin, and gold
nanoparticles [11e17]. Among these nanocarriers, liposome is
considered to be the most mature TDDS, which has similar bio-
logical structure to cell membrane, good biocompatibility and
safety [18e21].
precoated silica gel GF254 (0.2 mm), while column chromatography
wasperformedusingsilicagel(100e200mesh).1HNMRspectrawere
taken on a Varian INOVA 400 or 600 (Varian, Palo Alto, CA, USA) using
CDCl3 or DMSO‑d6 as solvent. Chemical shifts are expressed in
d
(ppm), with tetramethylsilane (TMS) functioning as the internal
The surface ligand functionalization of nanocarriers directly
affects the targeting ability of TDDS, which has long been consid-
ered as a crucial factor for the targeting efficiency. So far, many
types of ligands (e.g., cyclic RGD, folic acid, biotin, hyaluronic acid,
human epidermal receptor 2, galactose, glycyrrhizin and
bisphosphonates) have been employed for active tumor-targeting
drug delivery [22e24]. For example, biotin, as a water-soluble
small molecule (244 Da) vitamin that can not be synthesized by
human or any mammalian cells [25e27], has great advantages such
as simple structure, single functional group, smaller steric hin-
drance, and easy to be modified on the surface of nanocarriers.
Moreover, the sodium-dependent multivitamin transporter (SMVT)
has been proved to be the main transporter of biotin [28,29]. It has
been reported that SMVT was overexpressed in several aggressive
cancer lines such as breast cancer (MCF-7, 4T1, JC, MMT06056),
while at low levels in normal cells [30e33]. These evidences sug-
gest that biotin would be a promising ligand for targeting breast
cancer.
Along with the functionalization of surface ligands on nano-
carriers, researchers have focused on the modification of ligands.
And few studies have been carried out on the density of ligand
residues on the surface of nanocarriers, which is also essential for
ligands to enhance their targeting ability, for example, increasing
the percentage of single-branched modified nanomaterials, using
multi-branched ligand modified nanomaterial and so on. Moreover,
multi-branched ligand seems more promising compared with the
single-branched ligand. For instance, Wu’s group explored multi-
valent glucosides with high affinity as ligands for brain targeting
liposomes [34]; and Zhang’s group studied the targeting efficiency
of RGD-modified nanocarriers with different ligand intervals in
response to integrin avb3 clustering [35]; in addition, Punit P. Seth
used triantennary N-acetylgalactosamine conjugated antisense ol-
igonucleotides for targeted delivery to hepatocytes [36]. All of the
three examples used the branched ligands to modify the nano-
carriers and made a great breakthrough.
reference, and coupling constants (J) were expressed in Hz. Paclitaxel
were obtained from National Institute for Food and Drug Control.
Soybean phospholipids (SPC) were purchased from Kelong Chemical
(Chengdu, China). Cholesterol (Chol) was purchased from Bio Life
Science & Technology Co., Ltd (Shanghai, China). D-(þ)-Biotin was
purchased from Shanghai Darui Finechemical Co., Ltd (Shanghai,
China). 1, 2-dioleoyl-snglycero-3-phosphoethanolamine-N-(carbox-
yfluorescein) (CFPE) were purchased from Avanti Polar Lipids (USA).
40-6-Diamidino-2-phenylindole (DAPI) and 3-(4, 5-Dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from
Beyotime
Institute
Biotechnology
(Haimen,
China).
4-
chlorobenzenesulfonate salt (DiD) were purchased from Biotium
(USA). Annexin V-FITC/PI apoptosis detection kit was obtained from
KeyGEN Biotech (China).
2.2. Synthesis of ligands
2.2.1. Synthesis of ligand Bio-Chol and ligand Bio-Bio-Chol
The synthesis of ligand Bio-Chol and ligand Bio-Bio-Chol was
reported in our previous work [37].
2.2.2. Synthesis of compound 2e5
The synthesis of compound 2e5 was reported in our previous
work [37,38].
2.2.3. Synthesis of compound 7
Diethanolamine 6 (1.00 g, 9.51 mmol) was dissolved in 30 mL
acetonitrile, and tert-butoxycarbonyl anhydride (2.49 g,
11.41 mmol) was added under room temperature agitation. After
stirring for 4 h at room temperature, the mixture was concentrated
in vacuo. The residue was purified by flash column chromatography
to afford compound 7 (1.82 g, 93%) as a colorless oil. 1H NMR
(400 MHz, CDCl3, ppm)
(s, 2H), 1.47 (s, 9H).
d: 3.80 (s, 4H), 3.44 (d, 4H, J ¼ 11.6 Hz), 2.83
In our previous study, we have synthesized ligands Bio-Chol,
Bio-Bio-Chol modified by biotin (Fig. 1), and preliminarily dis-
cussed the density of targeting molecular and different ligand
modification methods on the targeting ability of liposomes for
breast cancer [37]. The results showed that increasing the density
of targeting molecules on the surface of liposomes for SMVT
recognition can effectively enhance the targeting ability of lipo-
somes for breast cancer; furthermore, at the same density of tar-
geting molecules, liposomes modified by branched ligand had
stronger breast cancer targeting ability. But there is still a lot of
room to explore and improve the breast targeting ability of
branched biotin modified liposomes.
Based on our previous study, tri-Bio-Chol and tetra-Bio-Chol
modified by biotin with different branches were designed and
synthesized in this paper (Fig. 1). And different types of biotin-
modified liposomes were prepared by lipid film hydration-
ultrasound method using paclitaxel (PTX) as a model drug for
breast cancer targeting study in vitro and in vivo.
2.2.4. Synthesis of compound 8
To a solution of D-(þ)-Biotin (2.38 g, 9.76 mmol) in mixed sol-
vent of 45 mL dichloromethane and 15 mL N, N-dimethylforma-
mide was added 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI, 2.80 g, 14.61 mmol), DMAP (1.78 g,
14.61 mmol) and N, N-Diisopropylethylamine (DIPEA, 4.84 mL,
29.28 mmol) at room temperature, then compound 7 (500 mg,
2.44 mmol) in 10 mL dichloromethane was added slowly. After
stirring for 10 h, the mixture was washed with 1 mol/L HCl and
saturated NaCl. The organic layer was dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography to afford compound 8 (1.24 g, 77%) as
a yellowish solid; mp: 108e110 ꢀC. 1H NMR (400 MHz, CDCl3, ppm)
d: 4.54e4.52 (m, 2H), 4.35 (s, 2H), 4.19 (s, 4H), 3.54e3.43 (m, 4H),
3.18e3.12 (m, 2H), 2.95e2.90 (m, 2H), 2.75 (d, 2H, J ¼ 7.2 Hz), 2.38
(s, 4H), 1.76e1.65 (m, 8H), 1.47 (s, 9H), 1.29e1.26 (m, 4H).
2.2.5. Synthesis of compound 9
Compound 8 (3.22 g, 5.77 mmol) was dissolved in 20 mL
dichloromethane, and then trifluoroacetic acid (10 mL) was added
under room temperature agitation. After stirring for 4 h at room
temperature, the mixture was concentrated in vacuo to afford
3.47 g dark green oil, then 30 mL of ice ether was added to separate
out the yellowish solid. After centrifugation and drying, 2.94 g
compound 9 as a yellowish solid was obtained.
2. Materials and methods
2.1. Materials
All liquid reagents were distilled before use. All unspecified re-
agents were from commercial resources. TLC was performed using

B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
3
Fig. 1. The structure of ligands Bio-Chol, Bio-Bio-Chol, tri-Bio-Chol and tetra-Bio-Chol.
2.2.6. Synthesis of compound 10
2.2.8. Synthesis of compound 12
To a mixture of succinic anhydride (1.12 g, 11.2 mmol) and
dichloromethane (25 mL), triethylamine (3.12 mL, 22.4 mmol) and
the solution of compound 9 (3.76 g, 5.59 mmol) in 45 mL
dichloromethane were added at room temperature subsequently.
After stirring for 6 h, the mixture was washed with 1 mol/L HCl and
saturated NaCl. The organic layer was dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography to afford compound 10 (2.14 g, 58%)
Compound 11 (600 mg, 0.43 mmol) was dissolved in 6 mL
dichloromethane, and 2 mL trifluoroacetic acid was added under
room temperature agitation. After stirring for 30 min at room
temperature, the mixture was washed with saturated NaHCO3 and
NaCl. The organic layer was dried over anhydrous Na2SO4, filtered
and concentrated in vacuo to afford compound 12 (525 mg, 94%) as
pale yellow jelly. 1H NMR (600 MHz, CDCl3, ppm)
d: 5.35 (s, 1H),
4.54e4.06 (m, 11H), 3.72e3.42 (m, 14H), 3.18e3.12 (m, 3H),
2.92e2.66 (m, 10H), 2.37e2.32 (m, 4H), 2.29e0.86 (remaining
cholesterol & lys & biotin protons), 1.44 (s, 9H), 0.99 (s, 3H), 0.91 (d,
3H, J ¼ 10.2 Hz), 0.86 (d, 6H, J ¼ 10.8 Hz), 0.67 (s, 3H).
as a colorless oil. 1H NMR (400 MHz, CDCl3, ppm)
d: 12.09 (br, 1H),
4.33e4.29 (m, 2H), 4.19e4.12 (m, 4H), 4.09e4.06 (m, 2H), 3.61 (t,
2H, J ¼ 5.6 Hz), 3.50 (t, 2H, J ¼ 5.6 Hz), 3.18e3.12 (m, 2H), 3.10 (s,
2H), 2.85e2.80 (m, 2H), 2.59e2.56 (m, 4H), 2.50 (s, 4H), 2.44e2.41
(m, 2H), 1.64e1.42 (m, 8H), 1.37e1.26 (m, 4H).
2.2.9. Synthesis of ligand tri-Bio-Chol
To a solution of D-(þ)-Biotin (150 mg, 0.61 mmol) in mixed
solvent of 24 mL dichloromethane and 8 mL N, N-dimethylforma-
mide was added HATU (278 mg, 0.73 mmol) and DIPEA (202 mL,
2.2.7. Synthesis of compound 11
To a solution of compound 10 (1.16 g, 1.76 mmol) in mixed sol-
vent of 40 mL dichloromethane and 10 mL N, N-dimethylforma-
1.22 mmol) at ꢁ5 ꢀC. After the reaction was stirred at the temper-
ature for 30 min, compound 12 (525 mg, 0.41 mmol) in 13 mL
dichloromethane was added slowly. After stirring for another 10 h,
the mixture was washed with 1 mol/L HCl and saturated NaCl. The
organic layer was dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography to afford ligand tri-Bio-Chol (521 mg, 84%) as a
pale yellow solid; mp: 121e123 ꢀC. 1H NMR (600 MHz, CDCl3, ppm)
mide
was
added
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU, 802 mg,
2.11 mmol) and DIPEA (873
m
L,5.28 mmol) at ꢁ5 ꢀC, and the reac-
tion was stirred at the temperature for 30 min. Then compound 5
(876 mg, 1.17 mmol) in 12 mL dichloromethane was added slowly.
After stirring for another 6 h, the mixture was washed with 1 mol/L
HCl and saturated NaCl. The organic layer was dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was puri-
fied by flash column chromatography to afford compound 11
(1.32 g, 81%) as a pale yellow jelly. 1H NMR (600 MHz, CDCl3, ppm)
d: 5.34 (s, 1H), 4.54e4.05 (m, 13H), 3.92e3.52 (m, 14H), 3.20e3.16
(m, 6H), 2.91e2.72 (m, 10H), 2.36 (d, 4H, J ¼ 12.0 Hz), 2.24e0.86
(remaining cholesterol & lys & biotin protons), 0.99 (s, 3H), 0.91 (d,
3H, J ¼ 6.0 Hz), 0.87 (d, 3H, J ¼ 6.6 Hz), 0.68 (s, 3H). HR-MS
calculated for
1534.8431.
C
77H125N9O15S3Na [MþNa]þ 1534.8426, found
d: 5.34 (s, 1H), 4.57e4.09 (m, 11H), 3.75e3.39 (m, 14H), 3.20e3.15
(m, 3H), 3.07 (t, 2H, J ¼ 6.0 Hz), 2.92e2.66 (m, 8H), 2.36 (d, 4H,
J ¼ 10.2 Hz), 2.23e0.86 (remaining cholesterol & lys & biotin pro-
tons), 1.44 (s, 9H), 0.99 (s, 3H), 0.92 (d, 3H, J ¼ 6.6 Hz), 0.87 (d, 6H,
J ¼ 6.0 Hz), 0.68 (s, 3H).
2.2.10. Synthesis of ligand tetra-Bio-Chol
To a solution of compound 10 (408 mg, 0.62 mmol) in 10 mL

4
B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
dichloromethane was added HATU (354 mg, 0.93 mmol) and DIPEA
collected in tubes containing heparin sodium. The red blood cells
(RBCs) were separated and collected by centrifugation at
5 ꢂ 103 rpm for 10 min and washed with PBS until the supernatant
became colorless. Subsequently, the RBCs were diluted with PBS to
a concentration of 2% (v/v). Various concentrations (10, 25, 50, 100,
(308
m
L, 1.86 mmol) at ꢁ5 ꢀC. The reaction was stirred at the tem-
perature for 30 min, and then compound 12 (397 mg, 0.31 mmol) in
10 mL dichloromethane was added slowly. After stirring for another
10 h, the mixture was washed with 1 mol/L HCl and saturated NaCl.
The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography to afford ligand tetra-Bio-Chol (415 mg, 70%) as a
pale yellow solid; mp: 154e157 ꢀC. 1H NMR (600 MHz, CDCl3, ppm)
200 300, 400 mmol/L) of liposomes were co-incubated with equal
volume of 2% RBCs solutions for 1 h at 37 ꢀC with moderate shaking.
After centrifugation at 10000 rpm for 10 min, the absorbance of
hemoglobin was measured using a microplate reader at 540 nm.
The values for 0% and 100% hemolysis were determined by incu-
bating erythrocytes with PBS or 1% (v/v) Triton X-100. The hemo-
lysis percentage was calculated using the following equation:
d: 5.34 (s, 1H), 4.55e4.04 (m, 19H), 3.90e3.40 (m, 18H), 3.19e3.14
(m, 7H), 2.90e2.61 (m, 14H), 2.37e2.33 (m, 10H), 2.27e0.86
(remaining cholesterol & lys & biotin protons), 0.99 (s, 3H), 0.91 (d,
3H, J ¼ 6.6 Hz), 0.87 (d, 3H, J ¼ 6.6 Hz), 0.68 (s, 3H). HR-MS
95H152N12O21S4Na [MþNa]þ 1949.0037, found
ASample ꢁ ANegative
calculated for
1949.0052.
C
The percent hemolysis ¼
ꢂ 100%
APostive ꢁ ANegative
where A is the absorbance of hemoglobin.
2.3. Preparation and characterization of liposomes
2.7. Cytotoxicity
Liposomes were prepared by thin film hydration method. Lipid
composition of the liposomes was as follows: soybean phospho-
lipid (SPC)/cholesterol/ligand ¼ 64/33/3 (molar ratio). All lipid
materials were dissolved in a mixture solvent chloroform/methanol
(v/v ¼ 2/1), and then the organic solvent was removed by rotary
evaporation to form a lipid film. After overnight in vacuum, the
obtained film was hydrated in PBS (pH 7.4) at 20 ꢀC for 0.5 h. Li-
posomes were then formed by further intermittent sonication at
80 W for 3 min by a probe sonicator.
PTX-loaded liposomes were prepared by adding paclitaxel to the
lipid organic solution prior to the evaporation of solvent. Likewise,
CFPE-labeled liposomes and DiD-loaded liposomes were prepared
by adding appropriate amount of CFPE or DiD to the solution
respectively. The entrapment efficiency of paclitaxel was deter-
mined by HPLC (Shimadzu, Kyoto, Japan). The average particle size
and zeta potential of Lip, Bio-Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-
Bio-Lip were measured using Malvern Zeta sizer Nano ZS90 (Mal-
vern Instruments Ltd., U.K).
The cytotoxicity of PTX-loaded liposomes and free PTX against
4T1 cells and MCF-7 cells (the biotin receptor positive cell lines) as
well as the normal cell line L929 cells were measured with MTT
assay. Generally, the cells were seeded in a 96-well plate at a
density of 5 ꢂ 103 cells/well and cultured for 24 h at 37 ꢀC. PTX-
loaded liposomes and free paclitaxel were diluted to concentra-
tions of 20
0.01 g/mL with medium, and added into each well. After incuba-
tion for 48 h, 20
mg/mL、5 mg/mL、1 mg/mL、0.5 mg/mL、0.1 mg/mL and
m
mL MTT solutions (5.0 mg/mL) was added to the
wells and incubated for another 4 h at 37 ꢀC. After removal of the
culture medium, the reduced MTT dye was solubilized by DMSO
(150 mL) and the absorbance was read at 490 nm wavelength by an
automatic microplate spectrophotometer. Cells with drug-free
medium were used as control. Cell viability (%) was calculated as
the following equation: Atest/Acontrol ꢂ 100%, where Atest and Acontrol
represented the absorbance of treated cells and control cells,
respectively.
2.4. In vitro drug release study
2.8. Apoptosis assay
In vitro paclitaxel release study was performed using dialysis
method. Free PTX was prepared as follows: PTX was dissolved in a
mixture of ethanol-Cremophor ELP35 with a volume ratio of 1:1.
Each PTX-loaded liposome (0.4 mL) or free paclitaxel was placed in
an 8000e14000 Da dialysis bag and sealed. Then the dialysis bags
were incubated in 40 mL PBS containing 0.1% (v/v) Tween 80 at
37 ꢀC for 48 h with oscillating at 45 rpm. At 0 h, 1 h, 2 h, 4 h, 8 h,
12 h, 24 h and 48 h, 0.1 mL release medium was sampled and
replaced with equal volume of fresh release medium. Then the
samples were diluted with acetonitrile and the concentrations of
paclitaxel were measured at the wavelength of 227 nm by HPLC.
To evaluate the cell apoptosis induced by PTX-loaded liposomes
and free PTX, 4T1 cells were treated with PTX-loaded liposomes
and free PTX (PTX concentration of 0.01
37 ꢀC. And then, cells were harvested, washed three times with cold
PBS and resuspended in 500 L binding buffer. Then 5 L Annexin
V-FITC and 5 L PI were added and incubated with the cells for
mg/mL) for 24 h under
m
m
m
15 min in the dark. Finally, the stained cells were analyzed by a flow
cytometer (BD FACSCelesta, BD, USA). PTX-Lip group was set as
control.
2.9. Cellular uptake
2.5. In vitro stability of liposomes in serum
4T1 cells and MCF-7 cells were seeded in 12-well plates at a
density of 3 ꢂ 105 cells/well and cultured for 24 h at 37 ꢀC. CFPE-
labeled different liposomes were added into each well with a
The serum stability of the liposomes was conducted by the
turbidimetric method. Briefly, liposomes were mixed with an equal
volume of fetal bovine serum (FBS) under 37 ꢀC with moderate
agitation at 45 rpm. Samples were taken at predetermined time
points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h), and the trans-
mittance was measured at 750 nm by a microplate reader (Thermo
Scientific Varioskan Flash, USA).
final concentration of CFPE at 2 mg/mL. After cultured for 4 h at
37 ꢀC, trypsin was used to harvest the cells, which were washed
three times with PBS afterward, and finally resuspended in 0.3 mL
PBS. Then, the fluorescent intensity of cells was measured by a flow
cytometer.
For qualitative experiments, 4T1 cells and MCF-7 cells were
plated onto a 6-well plate containing cover glass at a density of
3 ꢂ 105 cells/well and cultured for 24 h at 37 ꢀC. CFPE-labeled li-
posomes were added into each well with a final concentration of
2.6. Hemolysis assays
Hemolysis assays were performed to assess the biosecurity of
liposomes during the blood circulation. Fresh mouse blood was
CFPE at 2
mg/mL and allowed for further co-incubation for 4 h.

B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
5
Scheme 1. The synthetic route of cholesteric component. Reagents and conditions: (a) TsCl, pyridine, 50 ꢀC, 5 h; (b) Triethylene glycol, dioxane, reflux, 6 h; (c) N-Boc-N0-Fmoc-L-
Lysine, DCC, DMAP, CH2Cl2, -5 ꢀC- r.t., 8 h; (d) DBU, CH2Cl2, r.t., 20 min.
Following that, the cells were rinsed with cold PBS three times and
fixed with 4% paraformaldehyde for 30 min at room temperature,
and then cell nuclei were stained with DAPI (0.1 mg/mL) for 5 min.
Finally, the samples were imaged using laser scanning confocal
microscope (CLSM) (LSM800, Carl Zeiss AG, Germany).
subcutaneous 4T1 breast tumor model [39]. Briefly, the female
BALB/c mice were subcutaneously injected with 0.1 mL of cell
suspension containing 2 ꢂ 107 4T1 cells/mL in the right flank at day
0. After 14 days, the mice were randomly divided into five groups
and were intravenously injected via the caudal vein with DiD-
loaded liposomes at a dose of 200
mg/kg DiD. 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, 4 h, 8 h, 12 h and 24 h after injection. The mice were
sacrificed after heart perfusion with saline. Tumors, hearts, livers,
2.10. In vivo imaging
Tumor targeting capability of DiD-loaded liposomes Lip, Bio-Lip,
Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip was investigated in
Scheme 2. The synthetic route of targeted component. Reagents and conditions: (e) (Boc)2O, CH3CN, r.t., 4 h; (f) Biotin, EDCI, DMAP, CH2Cl2,r.t., 10 h; (g) CF3COOH, CH2Cl2, r.t., 4 h;
(h) Et3N, succinic anhydride, CH2Cl2, r.t., 6 h.

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B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
Scheme 3. The synthetic route of ligand tri-Bio-Chol. Reagents and conditions: (i) Compound 10, HATU, DIPEA, CH2Cl2, r.t., 6 h; (j) CF3COOH, CH2Cl2, r.t., 30 min; (k) Biotin, HATU,
DIPEA, CH2Cl2, DMF, ꢁ5 ꢀC- r.t., 10 h.
spleens, lungs and kidneys were collected. All the tissues and or-
gans were also imaged with IVIS Lumina Series III imaging system.
(Boc)2O, then was conjugated with biotin in the presence of EDCI
and DMAP to obtain compound 8. The protective group of com-
pound 8 was subsequently removed and reacted with succinic
anhydride to obtain compound 10.
2.11. Statistical analysis
As shown in Scheme 3, after the cholesteric component and
targeted component were synthesized respectively, compound 11
was obtained from compound 5 and compound 10 under the action
of the condensation agent HATU. Subsequently, the Boc protection
group of compound 11 was removed by CF3COOH to afford com-
pound 12, and finally the ligand tri-Bio-Chol was synthesized by
condensation of compound 12 with biotin. All the title compounds
and important intermedium were characterized by their respective
1H NMR and MS.
Data were analyzed using an F-test with subsequent T-tests
(equal variance) for the comparison between two different groups.
For 3 or more groups, one-way ANOVA with Tukey’s multiple
comparison test was used. A value of p < 0.05 was considered
significant.
3. Results and discussion
3.1. Synthesis of ligand tri-Bio-Chol
The synthetic route of ligand tri-Bio-Chol can be divided into
three parts: (1) synthesis of cholesteric component (Scheme 1), (2)
synthesis of targeted component (Scheme 2), (3) coupling of
cholesteric component and targeted component (Scheme 3).
In the first part, compound 3 was synthesized by 2 steps reac-
tion from cholesterol 1, which was then conjugated with N-Boc-N0-
3.2. Synthesis of ligand tetra-Bio-Chol
Scheme 4 showed the synthetic route of ligand tetra-Bio-Chol.
Firstly, using cholesterol as raw material, compound 11 was ob-
tained by multi-step reaction in accordance with the synthetic
method of ligand tri-Bio-Chol as described in section 3.1. Then, the
protective group of compound 11 was removed by CF3COOH to
afford compound 12. Finally, compound 10 was conjugated with
compound 12 in the presence of HATU and DIPEA to obtain ligand
tetra-Bio-Chol.
Fmoc-L-Lysine in the presence of DCC and DMAP to obtain com-
pound 4, the Fmoc protective group of compound 4 was later
removed by DBU to afford compound 5. In the second part, com-
pound 7 was synthesized by the reaction of diethanolamine with

B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
7
Scheme 4. The synthetic route of ligand tetra-Bio-Chol. Reagents and conditions: (a) CF3COOH, CH2Cl2, r.t., 30 min; (b) Compound 10, HATU, DIPEA, CH2Cl2, DMF, ꢁ5 ꢀC- r.t., 10 h.
Table 1
The composition and characterization of different paclitaxel-loaded liposomes (mean SD, n ¼ 3).
Liposomes
Size(nm)
PDI
EE (%)
Zeta potential (mV)
PTX-Lip
PTX-Bio-Lip
PTX-Bio-Bio-Lip
PTX-tri-Bio-Lip
PTX-tetra-Bio-Lip
106.8 3.5
105.3 2.1
110.9 3.4
102.2 2.7
113.2 1.9
0.146 0.025
0.133 0.019
0.128 0.036
0.119 0.022
0.154 0.014
90.26 2.13
88.54 3.43
87.62 1.77
89.13 4.37
86.78 2.08
ꢁ3.27 0.23
ꢁ3.59 0.31
ꢁ2.63 0.14
ꢁ2.72 0.41
ꢁ2.33 0.26
Fig. 2. The TEM image of PTX-tri-Bio-Lip (A) and PTX-tetra-Bio-Lip (B).
Fig. 3. The PTX release profiles of free PTX and different PTX-loaded liposomes
(mean SD, n ¼ 3).
3.3. Characterization of liposomes
Proper size, uniform distribution, zeta potential, and high
encapsulation efficiencies (EE%) of nanoparticles are crucial to
reach the targeted site and play their therapeutic effect [40]. As can
be seen from Table 1, for all types of liposomes, the PTX were well
encapsulated in the liposomes, whose encapsulation efficiencies
were all greater than 86%. In addition, the average particle sizes of
all liposomes ranged from 102 to 114 nm, and the values of PDI
were less than 0.2. Besides, the liposomes were weak negative
charged, which is helpful for escaping the absorption of the retic-
uloendothelial system and immune response. What’s more, the

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B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
obvious aggregation of liposomes were found. The results indicated
that these liposomes were stable enough for obtaining a longer
half-life of blood in vivo [41].
3.6. Hemolysis assays
Hemocompatibility is a key factor to the application of lipo-
somes in vivo. As shown in Fig. 5, with the increase of lipid con-
centration, the hemolysis rates of five groups of PTX-loaded
liposomes were not significantly increased, and the hemolysis rate
was still less than 10% even the lipid concentration was up to
400 mM, which indicated that PTX-loaded liposomes modified by
biotin had good biosecurity. m.
3.7. Cytotoxicity
In vitro cytotoxicity of different PTX-loaded liposomes against
breast cancer cells and normal cells was performed by MTTassays. As
shown in Fig. 6A and B, with the increase of paclitaxel concentration,
the inhibition of PTX-Lip, PTX-Bio-Lip, PTX-Bio-Bio-Lip, PTX-tri-Bio-
Lip and PTX-tetra-Bio-Lip on4T1 and MCF-7 cells graduallyincreased.
Compared with other types of PTX-loaded liposomes, PTX-tri-Bio-Lip
modified by tri-Bio-Chol showed stronger cytotoxicity, especially for
4T1 cells. In addition, free paclitaxel showed better inhibitoryactivity,
because the free drugs could enter the cell through passive diffusion
directly and take effect without a drug release process, which indi-
rectly indicated the sustained release ofliposomes. Asfor normalcells
(Fig. 6C), the cytotoxicity of different PTX-loaded liposomes against
L929 cells was much weaker than breast cancer cells. And the biotin
modified liposomes showed lower inhibition comparing with free
PTX and non-modified liposomes, which may contribute to the
selectivity between the biotin and SMVT receptors over-expressed on
breast cancer cells.
Fig. 4. The variations of transmittance of different PTX-loaded liposomes in 50% FBS
(mean SD, n ¼ 3).
3.8. Apoptosis assay
Annexin V-FITC/PI apoptosis detection kit was used to study the
cell apoptosis induced by PTX-loaded liposomes. As shown in Fig. 7,
the percentage of apoptosis and necrotic cells was 40.01 1.98% for
free PTX, 23.75 2.85% for PTX-Lip, 25.55 1.59% for PTX-Bio-Lip,
35.26 1.68% for PTX-Bio-Bio-Lip, 43.29 0.84% for PTX-tri-Bio-Lip
Fig. 5. Hemolysis percentage of different PTX-loaded liposomes (mean SD, n ¼ 3).
PTX-tri-Bio-Lip and PTX-tetra-Bio-Lip exhibited uniform spherical
in shape and suitable size (Fig. 2) under transmission electron
microscopy (TEM). The Characterization of liposomes showed the
liposomes can accumulate in tumor through EPR effect.
and 36.84
1.69% for PTX-tetra-Bio-Lip, respectively. Compared
with other groups, PTX-tri-Bio-Lip modified by tri-Bio-Chol had a
stronger effect on inducing apoptosis of 4T1 cells, which was
consistent with the results of in vitro cytotoxicity.
3.4. In vitro drug release study
3.9. Cellular uptake study
To mimic the drug release behavior in vivo, the PTX-loaded li-
posomes were co-incubated with 1% Tween 80 in PBS. As shown in
Fig. 3, the release of free paclitaxel was relatively rapid, after 12 h of
incubation, more than 85% of drugs were released into the medium.
The PTX loaded in liposomes was released slowly with no signifi-
cant sudden release, whose cumulative release amount was less
than 72% after 48 h of co-incubation with PBS. The results showed
that these PTX-loaded liposomes could improve the drug release
behavior and have sustained release effects. With the increase of
targeted molecular density, there was no significant difference in
the drug release behavior of PTX-loaded liposomes in each group.
In order to primarily evaluate the selectivity, affinity and
endocytosis of breast cancer cells for different branched biotin-
modified liposomes, we measured the cellular uptake of CFPE-
labeled liposomes on biotin receptor-positive cell line 4T1 cells
and MCF-7 cells. As shown in Fig. 8A and B, both 4T1 cells and MCF-
7 cells showed the strongest uptake capacity of tri-Bio-Lip modified
by tri-Bio-Chol. On 4T1 cells, the fluorescence intensity of tri-Bio-
Lip was 5.21 times of Lip, 2.60 times of Bio-Lip, 1.67 times of Bio-
Bio-Lip and 1.17 times of tetra-Bio-Lip, respectively. The uptake of
tri-Bio-Lip on MCF-7 cells was 2.90, 2.27, 1.70 and 1.33 times higher
than that of Lip, Bio-Lip, Bio-Bio-Lip and tetra-Bio-Lip, respectively.
As shown in Fig. 8C and D, the qualitative uptake experiment of
laser confocal imaging showed that tri-Bio-Lip exhibited the
highest uptake levels on 4T1 cells and MCF-7 cells, which was
consistent with the quantitative results of flow cytometry.
3.5. In vitro stability of liposomes in serum
The liposomes must possess appropriate stability in the circu-
latory system to reach the targeted site. As shown in Fig. 4, within
48 h of co-incubation with 50% FBS, the transmittances of five
groups of PTX-loaded liposomes were all higher than 91%, and no
By comparing the uptake of Lip, Bio-Lip, Bio-Bio-Lip, tri-Bio-Lip
and tetra-Bio-Lip on 4T1 cells and MCF-7 cells, the results showed

B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
9
Fig. 6. (A) represents the cytotoxicity study of PTX-loaded liposomes and free PTX on 4T1 cells, (B) represents the cytotoxicity study of PTX-loaded liposomes and free PTX on MCF-
7 cells; (C) represents the cytotoxicity study of PTX-loaded liposomes and free PTX on L929 cells; *, **, *** and **** represent p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 versus
PTX-Lip group, (mean SD, n ¼ 3).
Fig. 7. (A), (B), (C), (D), (E) and (F) represent the apoptosis study of free paclitaxel, PTX-Lip, PTX-Bio-Lip, PTX-Bio-Bio-Lip, PTX-tri-Lip and PTX-tetra-Bio-Lip on 4T1 cells, respectively.
(G) represents the percentage of apoptosis and necrotic cells after free PTX and PTX-loaded liposomes on 4T1 cells. **, *** represent p < 0.01 and p < 0.001 versus PTX-Lip group,
respectively; N. S. indicates none significant difference, (mean SD, n ¼ 3).

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B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
Fig. 8. (A) and (B) represent cellular uptake of CFPE-labeled liposomes determined by flow cytometer on 4T1 cells and MCF-7 cells, respectively; *, ** and *** represent p < 0.05,
p < 0.01 and p < 0.001 versus Lip group, N. S. indicates none significant difference, (mean SD, n ¼ 3). (C) and (D) represent cellular uptake of CFPE-labeled liposomes determined
by CLSM on 4T1 cells and MCF-7 cells, respectively; green (CFPE-labeled liposomes), blue (DAPI stained nucleus), light blue (colocalized CFPE and DAPI), scale bars represent 20 mm,
(mean SD, n ¼ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
that the in vitro targeting ability of five groups of liposomes from
strong to weak was tri-Bio-Lip > tetra-Bio-Lip > Bio-Bio-Lip > Bio-
Lip > Lip. It can be seen that from single-branched biotin to tri-
branched biotin-modified liposomes, increasing the density of
targeting molecules can significantly enhance the targeting ability
for breast cancer, which is consistent with our previous study [37].
But it was also found that tetra-Bio-Lip < tri-Bio-Lip, which was
similar to other reports about the affinity between different
branched ligands and receptors [36,42-44]. Thus, we concluded
that in addition to targeting molecular density, the branching
structure and spatial distance of biotin residues of ligands may also
have an important influence on the affinity between liposomes
modified by different branched biotin and SMVT receptor.
parameters of PTX in plasma showed the area under the
concentration-time curve (AUC0et) of PTX in biotin modified lipo-
somes were much higher than that of naked PTX within 24 h,
meanwhile, the mean residence time (MRT) and the elimination
half-life (t1/2) of biotin modified liposomes was much longer
comparing with the naked PTX and unmodified liposome. The re-
sults showed PTX-Bio-Bio-Lip and PTX-Bio-Lip possessed better
blood circulation characteristics, which facilitated the liposomes to
reach and deliver its cargo to the target site. Moreover, the distri-
bution in tissues and tumors also showed the PTX-Bio-Bio-Lip
accumulated more than other groups in the breast tumor. Based
on these studies and the in vitro targeting results mentioned above
in this study, we speculated that tri-Bio-lip and tetra-Bio-lip also
have good blood circulation characteristics and stronger tumor
targeting ability in vivo.
3.10. In vivo distribution and targeting
4T1 tumor-bearing BALB/c female mice were used to estimate
the breast cancer targeting efficiency of liposomes modified by
different branched biotin. As shown in Fig. 9A, in vivo image results
showed that DiD-loaded liposomes modified by biotin accumulated
In our previous study [37], the distribution in plasma and tissues
of PTX-Bio-Bio-Lip and PTX-Bio-Lip as well as the PTX-Lip were
evaluated in 4T1 tumor-bearing BALB/c mice. The pharmacokinetic

B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
11
Fig. 9. (A) represents the imaging of different types of DiD-loaded liposomes in tumor-bearing mice, (B) represents the semi-quantitative fluorescence intensity analysis of different
types of DiD-loaded liposomes in isolated tumor tissues 8 h after administration, (C) represents the imaging of different types of DiD-loaded liposomes in isolated tumor tissues, (D)
represents the imaging of different types of DiD-loaded liposomes in isolated organs (from left to right are heart, liver, spleen, lung and kidney). **, *** represent p < 0.01 and
p < 0.001 versus Lip group, respectively, N. S. indicates none significant difference, (mean SD, n ¼ 3).
in tumor tissues 4 h after administration, with weak fluorescence
intensity, and 8 h after administration reached the peak. As shown
in Fig. 9C, in vitro image results of isolated tumor tissues showed
that the fluorescence intensity of five groups of DiD-loaded lipo-
somes reached its peak in tumor tissues 8 h after administration
which was consistent with the results in Fig. 9A. As shown in
Fig. 9B, the semi-quantitative results of fluorescence intensity of
tumor tissues showed that the fluorescence intensity of tri-Bio-Lip
was 3.59, 2.97, 1.40 and 1.29 times higher than that of Lip, Bio-Lip,
Bio-Bio-Lip and tetra-Bio-Lip at 8 h, respectively. As shown in
Fig. 9D, in vitro image results of isolated organs showed that five
groups of DiD-loaded liposomes were mainly distributed in liver
and spleen after administration, especially in liver.
The results of in vivo studies indicated that the in vivo targeting
ability of five groups of liposomes from strong to weak was tri-Bio-
Lip > tetra-Bio-Lip > Bio-Bio-Lip > Bio-Lip > Lip, which was
consistent with the results of in vitro targeting studies. The in vivo
studies further proved that increasing the density of targeting
molecules of ligands can effectively enhance the breast cancer
targeting ability of liposomes, and the branching structure and
spatial distance of biotin residues of ligands may also have an
important influence on the affinity between liposomes modified by
different branched biotin and SMVT receptor.
Lee and other researchers have studied the affinity between
different branched galactose (Gal)/N-acetylgalactosamine (GalNAc)
residues and asialoglycoprotein receptor (ASGPR) [36,42-44]. The
results showed that the affinity of different branched galactose
residues with receptor were: tetra-branched galactoside > tri-
branched galactoside [ bi-branched galactoside [ mono-galac-
toside, and the affinity of tri-branched galactoside with receptor
was 50e100 times higher than that of mono-galactoside, but the
affinity of tetra-branched galactoside with ASGPR was not signifi-
cantly higher than that of tri-branched galactoside. Further
research indicated that for the same bi-branched polysaccharides,
the affinity of galactoside with ASGPR with the branching distance
at 15 Å was higher than that of branching distance at 23 Å. The
results of Biessen et al. [45] also showed that the affinity of galac-
toside with ASGPR was the strongest when the distance between

12
B. Tang et al. / European Journal of Medicinal Chemistry 193 (2020) 112204
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Both the targeting evaluation in vitro and in vivo indicated tri-
Bio-Lip can significantly improve the targeting ability of liposome
for breast cancer, we speculated that PTX-tri-Bio-Lip could be a
promising drug delivery system for the treatment of breast cancer.
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4. Conclusion
In summary, a series of liposome ligands Bio-Chol, Bio-Bio-Chol,
tri-Bio-Chol and tetra-Bio-Chol modified by different branched bi-
otins were designed and synthesized in this study. And different
types of liposomes Bio-Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip
with different targeting molecular density were prepared for tar-
geting breast cancer. The cytotoxicity study and apoptosis assay of
paclitaxel-loaded liposomes showed that PTX-tri-Bio-Lip had the
strongest anti-proliferative effect on breast cancer cells. The cellular
uptake studies on breast cancer cells indicated tri-Bio-Lip
possessed the strongest internalization ability. Moreover, the
in vivo image on 4T1 tumor-bearing BALB/c mice showed the
enrichment of liposomes at tumor sites were tri-Bio-Lip > tetra-
Bio-Lip > Bio-Bio-Lip > Bio-Lip > Lip. Both the targeting evaluation
in vitro and in vivo indicated that increasing the density of targeting
molecules of ligands can effectively enhance the breast cancer
targeting ability of liposomes. Besides, the branching structure and
spatial distance of biotin residues may also have an important in-
fluence on the affinity between liposomes modified by different
branched biotin and SMVT receptors. Among these liposome li-
gands, tri-Bio-Chol can significantly improve the targeting ability of
liposome for breast cancer, making it a potential breast cancer
targeting ligand. This study not only enhances the targeting ability
of biotin for breast cancer, but also makes a great difference to
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Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgement
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This work was supported by the National Natural Science
Foundation of China (No. 81573286, No. 81773577
& No.
81903448), the Sichuan Science and Technology Program
(2018JY0537), the Fundamental Research Funds for the Central
Universities (2012017yjsy211) and the Open Research Subject of
Healthy, Xihua University (szjj2017-036).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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