Liposomes modified with double-branched biotin: A novel and effective way to promote breast cancer targeting

Article abstract of DOI:10.1016/j.bmc.2019.05.039

Although active targeting liposomes with cancer-specific ligands can bind and internalize into cancer cells, only a few high-efficiency liposomes have been developed so far because traditional single branched ligand modified liposomes generally failed to deliver adequate therapeutic payload. In this paper, we broke the traditional design concept and synthesized the double branched biotin modified cholesterol (Bio₂-Chol) for the first time. On this basis, different biotin density modified liposomes ((Bio-Chol)Lip, (Bio-Chol)₂Lip and (Bio₂-Chol)Lip) were successfully prepared and used as active targeting drug delivery systems for the treatment of breast cancer. The in vitro and in vivo breast cancer-targeting ability of these liposomes were systemically studied using paclitaxel (PTX) as the model drug. And the uptake mechanism of (Bio₂-Chol)Lip was investigated. The results showed that (Bio₂-Chol)Lip had the best breast cancer-targeting ability compared with naked paclitaxel, unmodified Lip, (Bio-Chol)Lip and (Bio-Chol)₂Lip. In particular, the relative uptake efficiency (RE) and concentration efficiency (CE) of (Bio₂-Chol)Lip were respectively enhanced by 5.61- and 5.06-fold compared to that of naked paclitaxel. Both distribution data and pharmacokinetic parameters suggested that the double branched biotin modified liposome ((Bio₂-Chol)Lip) is a very promising drug delivery carrier for breast cancer.

Full text of DOI:10.1016/j.bmc.2019.05.039

Accepted Manuscript
Liposomes modified with double-branched biotin: A novel and effective way to
promote breast cancer targeting
Runxin Lu, Lin Zhou, Qiming Yue, Qijun Liu, Xiaojing Cai, Wenjiao Xiao, Li
Hai, Li Guo, Yong Wu
PII:
S0968-0896(19)30490-0
https://doi.org/10.1016/j.bmc.2019.05.039
BMC 14926
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 March 2019
23 May 2019
27 May 2019
Please cite this article as: Lu, R., Zhou, L., Yue, Q., Liu, Q., Cai, X., Xiao, W., Hai, L., Guo, L., Wu, Y., Liposomes
modified with double-branched biotin: A novel and effective way to promote breast cancer targeting, Bioorganic
&
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.039
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Liposomes modified with double-branched biotin: A novel and
effective way to promote breast cancer targeting
Runxin Lu, Lin Zhou, Qiming Yue, Qijun Liu, Xiaojing Cai, Wenjiao Xiao, Li Hai, Li Guo*, Yong Wu*
Key Laboratory of Drug Targeting and Drug Delivery System of Education Ministry, Department of Medicinal
Chemistry, West China School of Pharmacy, Sichuan University, Chengdu 610041, P. R. of China
*Corresponding authors. guoli@scu.edu.cn (L. Guo), wyong@scu.edu.cn (Y. Wu).
Abstract Although active targeting liposomes with cancer-specific ligands can bind and internalize into cancer
cells, only a few high-efficiency liposomes have been developed so far because traditional single branched ligand
modified liposomes generally failed to deliver adequate therapeutic payload. In this paper, we broke the traditional
design concept and synthesized the double branched biotin modified cholesterol (Bio -Chol) for the first time. On
2
this basis, different biotin density modified liposomes ((Bio-Chol)Lip, (Bio-Chol) Lip and (Bio -Chol)Lip) were
2
2
successfully prepared and used as active targeting drug delivery systems for the treatment of breast cancer. The in
vitro and in vivo breast cancer-targeting ability of these liposomes were systemically studied using paclitaxel (PTX)
as the model drug. And the uptake mechanism of (Bio -Chol)Lip was investigated. The results showed that
2
(
(
(
Bio -Chol)Lip had the best breast cancer-targeting ability compared with naked paclitaxel, unmodified Lip,
2
Bio-Chol)Lip and (Bio-Chol) Lip. In particular, the relative uptake efficiency (RE) and concentration efficiency
2
CE) of (Bio -Chol)Lip were respectively enhanced by 5.61- and 5.06-fold compared to that of naked paclitaxel.
2
Both distribution data and pharmacokinetic parameters suggested that the double branched biotin modified
liposome ((Bio -Chol)Lip) is a very promising drug delivery carrier for breast cancer.
2
Keywords: Biotin; Active targeting; Breast tumor; Drug delivery; Liposome
1
.
Introduction
Breast cancer is the most common type of cancer in women and is the second leading cause of cancer-related
deaths worldwide [1]. According to report of the World Health Organization (WHO), 17.5 million breast
cancer-related deaths can be expected per year, by 2050 [2]. Currently, comprehensive treatments for breast cancer
include surgery, radiotherapy, immunotherapy and chemotherapy [3]. Among these treatments, chemotherapy plays
an crucial role in improving the survival rate of breast cancer patients. However, its clinical applications are
restricted largely by the high toxicity and non-selectivity [4]. Therefore, considerable attention has been focused on
1

the modification of classic chemotherapy drugs and the exploitation of new drug delivery systems [5-8].
Tumor-specific active targeting drug delivery system has been widely noted due to its great potential which
permits encapsulated chemotherapy drugs to be distributed less in the normal tissue and more in the tumor site.
Liposome-based drug delivery systems have attracted considerable attention because of its outstanding merits of
their biodegradable, non-toxic and biocompatible features [9-11]. Therefore, the use of targeted liposomes to
encapsulate chemotherapy drugs has become an international research hotspot and main development direction in
the treatment of breast cancer.
The intense metabolic activity of cancer cell arises from their rapid growth and is accompanied by the intense
consumption of essential vitamins [12]. The receptors involved in vitamin internalization are over-expressed on the
surface of cancer cells to maintain extensive vitamin uptake and may serve as useful tumor-specific targets for
liposomes [12]. Therefore, using vitamin modified liposomes as carriers of chemotherapy drugs recently represent
an attractive and valuable strategy for the treatment of cancer. Interestingly, among vitamins, biotin seems to be the
most promising targeting agent [13-14]. Biotin (vitamin B7, vitamin H), a water-soluble vitamin, plays an essential
role in cell proliferation, signal transduction and many other cellular functions [13]. So far, the sodium-dependent
multivitamin transporter (SMVT) on the cell surface has been proved as the key transporter for biotin [15].
Importantly, this transporter has been found to be over-expressed in breast cancer cells (such as 4T1 and MCF7)
compared to normal cells [16]. Thus, biotinylation is a promising strategy to enhance the binding/affinity of
liposomes to breast tumor cells, leading to increasingly curative anti-tumor therapy [17].
Although active targeting liposomes with cancer-specific ligands can bind and internalize into cancer cells,
only a few high-efficiency liposomes have been developed so far because traditional single branched ligand
modified liposomes generally failed to deliver adequate therapeutic payload [18]. Simultaneously, treatment with
these liposomes is likely to lead to the development of drug-resistant cancers. The same problem exists in single
branched biotin modified active targeting drug delivery systems currently used to cure breast cancer. Therefore, we
want to develop a novel active targeting liposome which can deliver more drugs into the breast cancer cells than
traditional single branched biotin modified liposomes.
We assumed that increasing the density of biotin on the surface of liposomes would increase SMVT-mediated
recognition and transshipment. There are two methods to achieve this goal. The one is to simply increase the
dosage of single branched biotin modified cholesterol in the preparation of liposomes, and the other is to alter the
biotin amount attached to a single cholesterol, keeping the dosage of biotin modified cholesterol unchanged.
In the past decades, several radioactively labeled bis-biotin complexes had been developed to increase the
2

effects of radiotherapy [19]. Besides, Lo and his co-workers successfully prepared luminescent bis-biotin
complexes as the signal amplifiers for heterogeneous recognition assay [20]. Inspired by these literatures, we
designed and synthesized the single branched biotin modified cholesterol (Bio-Chol) and double branched biotin
modified cholesterol (Bio -Chol) for the first time. Liposomes ((Bio-Chol)Lip, (Bio-Chol) Lip and (Bio -Chol)Lip)
2
2
2
modified with different biotin density were prepared to investigate the effect of biotin density on uptake of breast
cancer cells (Fig. 1). Thereinto, (Bio-Chol) Lip had twice single branched biotin modified cholesterol compared
2
with (Bio-Chol)Lip, and (Bio -Chol)Lip had the double branched biotin modified cholesterol keeping the dosage of
2
biotin modified cholesterol unchanged.
We want to know whether the drug delivery capacity of (Bio-Chol) Lip and (Bio -Chol)Lip is twice that of
2
2
(Bio-Chol)Lip when the density of biotin on the surface of liposomes is doubled. In addition, when the dosage of
biotin is kept constant, whether the double branched biotin modified lipsome (Bio -Chol)Lip has a stronger drug
2
delivery ability than (Bio-Chol) Lip. In order to improve the reliability of results, unmodified liposome was also
2
prepared for contrastive study. The cellular uptake efficiency, in vivo breast cancer targeting properties and
therapeutic efficacy of these paclitaxel (PTX) loaded liposomes were studied systemically. In addition, the uptake
mechanism of (Bio -Chol)Lip was investigated.
2
Figure 1. Schematic diagrams of (Bio-Chol)Lip, (Bio-Chol) Lip, (Bio -Chol)Lip, Bio-Chol and Bio -Chol.
2
2
2
2
. Materials and methods
3

2.1. Materials
All liquid reagents were distilled before use. All unspecified reagents were from commercial resources. TLC
was performed using precoated silica gel GF254 (0.2 mm), while column chromatography was performed using
1
silica gel (100-200 mesh). H NMR spectra was taken on a Varian INOVA 400 (Varian, Palo Alto, CA, USA) using
CDCl , d -DMSO and D O as solvents. Chemical shifts are expressed in δ (ppm), with tetramethylsilane (TMS)
3
6
2
functioning as the internal reference, and coupling constants (J) were expressed in Hz. Mass spectra were recorded
on an Agilent 1946B ESI-MS instrument (Agilent, Palo Alto, CA, USA). Paclitaxel and docetaxel were obtained
from National Institute for Food and Drug Control. Soybean phospholipids (SPC) was purchased from Kelong
Chemical (Chengdu, China). Cholesterol (Chol) was purchased from Bio Life Science & Technology Co., Ltd
(Shanghai, China). D-(+)-Biotin was purchased from shanghai darui fine chemical Co., Ltd (Shanghai, China).
1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(carboxyfluorescein) (CFPE) was purchased from Avanti Polar
Lipids (USA). 4’-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). Sodium azide, amiloride,
chlorpromazine, filipin, biotin-NHS, valeric acid, ascorbic acid, pantothenic acid and thiamine were obtained from
Sigma-Aldrich (St. Louis, MO, USA). 4-chlorobenzenesulfonate salt (DiD) was purchased from Biotium (USA).
Annexin V-FITC/PI apoptosis detection kit was obtained from KeyGEN Biotech (China). A Diamonsil column
(200 × 4.6 mm, 5 mm) was used. A LC-10A liquid chromatographic system (Shimadzu) and a reverse-phase HPLC
column (ODS-C18 column, 4.6mm × 200 mm, 5 mm, SinoChrom, Dalian, China) were used.
2
2
.2. Synthesis of compounds 2-5
The synthesis of compounds 2-5 were reported in our previous work [21].
.3. Synthesis of compound 6
To a solution of compound 5 (4.00 g, 6.93 mmol) in CH Cl (30 mL) was added NMM (0.84 mL, 7.63 mmol)
2
2
o
and IBCF (0.96 mL, 7.63 mmol), and the reaction was stirred at -10 C for 30 min. Then, diethanol amine (1.09 g,
0.40 mmol) in CH Cl (15 mL) was added slowly. After stirring for another 6h at room temperature, the mixture
1
2
2
was washed with 1 mol/L HCl and saturated NaCl solution. The organic layer was dried over anhydrous Na SO ,
2
4
and concentrated in vacuo. The residue was purified by flash column chromatography to afford compound 6 (4.09 g,
1
8
9%) as a pale yellow oil. H NMR (400 MHz, CDCl , ppm) δ: 0.67 (s, 3H), 0.86 (d, 6H, J = 6.4 Hz), 0.91 (d, 3H,
3
J = 6.4 Hz), 0.99 (s, 3H), 1.01-2.38 (remaining cholesterol protons), 3.14-3.21 (m, 1H), 3.50-3.85 (m, 20H), 4.28 (s,
4

+
2
H), 5.34 (d, 1H, J = 4.8 Hz). HRMS (ESI) Calcd. for C H NO Na [M + Na] 686.4966, found 686.4968.
39 69 7
2
.4. Synthesis of ligand Bio -Chol
2
To a solution of D-(+)-Biotin (147 mg, 0.604 mmol) in CH Cl (10 mL) and N,N-Dimethylformamide (5 mL)
2
2
was added EDCI (145 mg, 0.755 mmol), DMAP (92 mg, 0.755 mmol) and DIPEA (156 L, 1.21 mmol), and the
reaction was stirred at room temperature for 30 min. Then compound 6 (100 mg, 0.151 mmol) in CH Cl (5 mL)
2
2
was added slowly. After stirring for another 14h at room temperature, the mixture was washed with 1 mol/L HCl
and saturated NaCl solution. The organic layer was dried over anhydrous Na SO , and concentrated in vacuo. The
2
4
residue was purified by flash column chromatography to afford ligand Bio -Chol (110 mg, 65%) as a white
2
1
vesicular solid. H NMR (400 MHz, CDCl , ppm) δ: 0.67(s, 3H), 0.86 (d, 6H, J = 6.4 Hz), 0.91 (d, 3H, J = 6.4 Hz),
3
0
3
.99 (s, 3H), 1.02-2.23 (remaining cholesterol & biotin protons), 2.34 (s, 4H), 2.75-2.78 (m, 2H), 2.90-2.93 (m, 2H),
.15 (s, 3H), 3.63-3.70 (m, 16H), 4.25-4.34 (m, 8H), 4.53 (s, 2H), 5.33 (s, 1H). HRMS (ESI) Calcd. for
+
C H N O S Na [M + Na] 1138.6518, found 1138.6520.
5
9
97
5
11 2
2.5. Synthesis of ligand Bio-Chol
To a solution of D-(+)-Biotin (57 mg, 0.23 mmol) in CH Cl (5 mL) and N,N-Dimethylformamide (2 mL) was
2
2
added EDCI (56 mg, 0.29 mmol), DMAP (35 mg, 0.29 mmol) and N,N-Diisopropylethylamine (DIPEA) (75 mg,
.58 mmol), and the reaction was stirred at room temperature for 30 min. Then compound 3 (100 mg, 0.19 mmol)
in CH Cl (5 mL) was added slowly. After stirring for another 14h at room temperature, the mixture was washed
0
2
2
with 1 mol/L HCl and saturated NaCl solution. The organic layer was dried over anhydrous Na SO , and
2
4
concentrated in vacuo. The residue was purified by flash column chromatography to afford ligand Bio-Chol (110
1
mg, 76%) as a white vesicular solid. H NMR (400 MHz, CDCl , ppm) δ: 0.69 (s, 3H), 0.88 (d, 6H, J = 6.4 Hz),
3
0
2
1
.93 (d, 3H, J = 7.2 Hz), 1.01 (s, 3H), 0.88-2.04 (remaining cholesterol & biotin protons), 2.19-2.25 (m, 1H),
.36-2.41 (m, 3H), 2.82 (m, 2H), 3.16-3.24 (m, 2H), 3.65-3.73 (m, 10H), 4.22-4.30 (m, 2H), 4.41 (s, 1H), 4.61 (s,
+
H), 5.36 (s, 1H). HRMS (ESI) Calcd. for C H N O SNa [M+ Na] 767.5003, found 767.5005.
43
72
2
6
2.6. Preparation and characterization of liposomes
Liposomes were prepared by the thin film hydration method described previously [22]. Lipid composition of
the prepared liposomes were as follows: (1) unmodified liposomes (Lip): SPC/cholesterol (molar ratio = 62: 33); (2)
ligand Bio -Chol modified liposomes ((Bio -Chol)Lip): SPC/cholesterol/Bio -Chol (molar ratio = 62: 33: 3); (3)
2
2
2
ligand Bio-Chol modified liposomes ((Bio-Chol)Lip): SPC/cholesterol/Bio-Chol (molar ratio = 62: 33: 3); (4)
5

ligand Bio-Chol modified liposomes ((Bio-Chol) Lip): SPC/cholesterol/Bio-Chol (molar ratio = 62: 33: 6). All
2
lipid materials were dissolved in the mixture solvent chloroform/methanol (v/v = 2:1), and then the organic solvent
was removed by rotary evaporation to form a lipid film. After kept in vacuum overnight, the obtained film was
o
hydrated in PBS (pH = 7.4) for 0.5h at 20 C. Then it was further intermittently sonicated by a probe sonicator at 80
W for 90 s to form liposomes.
Free PTX was prepared as follows: PTX was dissolved in a mixture of ethanol-Cremophor ELP35 with a
volume ratio of 1:1. PTX-loaded liposomes were prepared with PTX added to the lipid organic solution prior to the
solvent evaporation. The entrapment efficiency (EE) of PTX was determined by high performance liquid
chromatography (HPLC, Agilent 1200 series, Palo Alto, CA). Likewise, CFPE-labelled or DiD-loaded liposomes
were prepared by adding appropriate amount of CFPE or DiD to the solution before the solvent evaporation.
The mean size and zeta potential of Lip, (Bio-Chol)Lip, (Bio-Chol) Lip and (Bio -Chol)Lip were detected by
2
2
Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., U.K).
2
.7. In vitro drug release study
In order to investigate the PTX release behavior of all liposomes, an in vitro drug release assay was conducted
using dialysis method [23]. Each PTX-loaded liposome (0.8 mL) or free PTX were placed into dialysis tubes
MWCO = 8000-14000 Da) and tightly sealed. Then the dialysis tubes were placed into 40 mL PBS containing
.1% (v/v) Tween 80 and incubated under 37 °C for 48h with gentle oscillating at 45 rpm. At predetermined time
(
0
points (0h, 1h, 2h, 4h, 8h, 12h, 24h and 48h), 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 PTX were
determined at the wavelength of 227 nm by HPLC.
2.8. In vitro stability of liposomes in serum
Turbidity variations were measured to demonstrate the serum stability of liposomes in the presence of fetal
bovine serum (FBS). Briefly, liposomes were mixed with equal volume of FBS under 37 °C with moderate shaking
at 45 rpm. At predetermined time points (0h, 1h, 2h, 4h, 8h, 12h, 24h and 48h), 200 μL of the sample was pipetted
out and onto a 96-well plate to measure the transmittance of the mixture at 750 nm by a microplate reader (Thermo
Scientific Varioskan Flash, USA).
2.9. Hemolysis assays
To evaluate the safety of ligand-modified liposomes during body circulation, hemolysis assay was performed.
6

Fresh mice blood was collected in tubes containing heparin sodium. The red blood cells (RBCs) were separated and
3
collected by centrifugation at 5 × 10 rpm for 10 min, and washed several times with PBS until the supernatant
became colorless. After the last wash, the RBCs were diluted with PBS to a concentration of 2% (v/v). Various
o
concentrations of liposomes were incubated with equal volume of 2% RBCs solutions for 1h at 37 C with
4
moderate shaking at 45 rpm, followed by centrifugation at 1 × 10 rpm for 10 min. Absorbance of hemoglobin was
measured using a microplate reader (Thermo Scientific Varioskan Flash) at 540 nm.
RBC hemolysates in PBS solutions and in 1% Triton X-100 were used as negative and positive controls,
respectively.
A
Sample
A
A
Negative
The percent hemolysis =
× 100%, where A is the absorbance of hemoglobin.
APostive
Negative
2
.10.
SMVT over-expressed cells (4T1 and MCF7 cells) and SMVT negative expressed B16 cell [16] were used in
this study. MCF-7 cells (Human breast cancer cell line), 4T1 cells (Mouse breast cancer cell line) and B16 cells
Mouse skin melanoma cell line) were cultured in Dulbecco's Modified Eagles Medium (DMEM) and RPMI 1640
Cell line and Cell culture
(
supplemented with 10% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin at 37 °C in a 5% CO humidified
2
environment incubator (Thermo Scientific, USA).
2.11.
In vitro cellular uptake study
5
4T1 cells, MCF-7 cells and negative cell line B16 cells were seeded in 12-well plates at a density of 3 × 10
o
cells/well and cultured for 24h at 37 C. Different CFPE-labeled liposomes were added into each well with a final
o
concentration of CFPE at 2 µg/mL. After cultured for 4h at 37 C, the cells were washed three times with cold PBS,
trypsinized, and finally resuspended in 0.3 mL PBS. Then, the fluorescent intensity of cells treated with different
liposomes was measured by a flow cytometer (BD FACSCelesta, BD, USA).
For qualitative experiments, 4T1 cells, MCF-7 cells and B16 cells were plated onto a 6-well plate containing
5
o
cover glass at a density of 3 × 10 cells/well and cultured for 24h at 37 C. CFPE-labeled liposomes were added
into each well with a final concentration of CFPE at 2 µg/mL and allowed for further co-incubation for 4h.
Following that, the cells were rinsed with cold PBS for three times and fixed with 4% paraformaldehyde for 30 min
at room temperature, and then cell nuclei were stained with DAPI (5 µg/mL) for 5 min. Finally, the samples were
imaged using Laser Scanning Confocal Microscope (CLSM) (LSM800, Carl Zeiss AG, Germany).
2.12.
Uptake mechanism study
7

In order to study the uptake mechanism of how (Bio -Chol)Lip was delivered into cells. Endocytosis
2
inhibition assay and SMVT transport verification experiment were carried out. For Endocytosis inhibition assay,
4T1 cells and MCF-7 cells were preincubated with various endocytosis inhibitors, such as sodium azide (50 mM),
amiloride (5 mM), chlorpromazine (5 μg/ml) and filipin (3.5 μg/ml), meanwhile the effect of temperature (both
37 °C and 4 °C）were also studied.
+
SMVT is a Na -dependent multivitamin transporter that mediates cellular uptake of biotin, pantothenic acid,
and lipoic acid [15]. Biotin transported by SMVT appears to be time-, concentration-, sodium ion-dependent but
independent of chloride ions. It was also significantly inhibited in the presence of biotin and other SMVT substrates,
such as pantothenic acid. The carboxyl group was considered to be a key feature for interaction with SMVT,
structurally unrelated vitamins had no influence in SMVT transporting [15].
Therefore, to confirm the involvement of SMVT, time-, concentration-, ion dependence assays and substrate
specificity test were executed. Uptake of (Bio -Chol)Lip on 4T1, MCF7 cells was assessed at different time and
2
various concentrations. To investigate the effect of sodium ions on the uptake of (Bio -Chol)Lip, equimolar
2
quantities of choline chloride and dipotassium phosphate were added to replace sodium chloride and sodium
phosphate in the buffer, respectively. In a similar way to delineate the effect of chloride ions, sodium, potassium
and calcium chlorides in the buffer were replaced with equimolar quantities of sodium phosphate, potassium
phosphate and calcium acetate respectively. Structural requirements of the carrier mediated system were delineated
by performing uptake experiments in the presence of competitive inhibitors such as unlabeled biotin (10 mM),
pantothenic acid (10 mM). Further we examined the influence of free carboxylic group (COOH) on biotin uptake
by incubating cells with structural analogs such as valeric acid (10mM) which possess a free carboxylic acid group,
and biotin-NHS (10 mM) which devoid free carboxylic group. Similar study was performed in the presence of
other vitamins such as ascorbic acid (10 mM) and thiamine (10 mM) to examine the effect of structurally unrelated
vitamins on uptake of biotin in 4T1 cells and MCF-7 cells.
Briefly, the cells were preincubated with different inhibitors for 30 min at 37 °C, then the inhibitors were
o
withdrawn from the wells and CFPE-labeled (Bio -Chol)Lip was added. After cultured for 4h at 37 C, the cells
2
were treated as described in 2.11. and the fluorescent intensity was measured by a flow cytomete (BD FACSCelesta,
BD, USA).
2
2
.13.
In vitro cytotoxicity study
.13.1. MTT assay
8

The cytotoxicity of PTX-loaded liposomes and free PTX against 4T1 cells and MCF-7 cells were measured
with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide (MTT) assay. Generally, the cells were
3
o
seeded in a 96-well plate at a density of 5 × 10 cells/well and cultured for 24h at 37 C. PTX-loaded liposomes and
free PTX were diluted to predetermined concentrations with medium, and added into each well for 48h incubation.
The final concentrations of PTX were in the range of 0.01-20 μg/mL.
Blank liposomes and the solvent of free PTX (ethanol-cremophor ELP 35 mixture, v/v =1: 1) were added at
the same concentration of PTX-loaded liposomes as well.
Then, 20 μL MTT solutions (5.0 mg/mL in PBS) was added into each well and incubated for another 4h at
37 °C. After removal of the culture medium, cells were dissolved by 150 μL dimethyl sulfoxide. The absorbance
was measured at 490 nm wavelength on an automatic microplate spectrophotometer (Thermo Scientific Varioskan
Flash, USA). Cell viability (%) was calculated as the following equation: Atest / Acontrol × 100%, where Atest and
Acontrol represented the absorbance of treated cells and control cells, respectively.
2.13.2. Apoptosis assay
The quantitative analysis of cell apoptosis induced by different PTX-loaded liposomes was determined by an
Annexin V-FITC/PI apoptosis detection kit, 4T1 cells were treated with PTX-loaded liposomes (PTX concentration
o
is 0.01 μg/ml) for 24h under 37 C. Free PTX group was used as control. Cells were harvested, washed three times
with cold PBS and resuspended in 500 μL binding buffer. Then 5 μL Annexin V-FITC and 5μL PI were added and
incubated with the cells for 15 min in the dark. Finally, the stained cells were analyzed by a flow cytometer (BD
FACSCelesta, BD, USA).
2
2
.14.
.14.1. Animals
Female BALB/c mice (5–6 weeks, 18–22 g) were purchased from Dashuo Biotechnology Co., Ltd. (Chengdu,
Distribution in vivo and pharmacokinetic studies in mice
China), and all animals experiments were performed according to the experimental guidelines of the Animal
Experimentation Ethics Committee of Sichuan University. Throughout the experiments, the animals were handled
according to the requirements of the National Act on the use of experimental animals (China).
2.14.2. Tumor model
7
Female BALB/c mice were anesthetized, and 4T1 cells ( 1× 10 cells/mL, 100 μL) were slowly injected into
9

3
the right upper thigh subcutaneous region. Tumors were allowed to grow to an average volume of 70-100 mm
2
(
tumor volume = length × width × 0.52) before the in vivo bio-distribution and imaging.
2.14.3. Sample preparation
Blood was collected from the eye socket of mice into tubes containing heparin sodium, and centrifuged at
500 rpm for 10 min. The supernatant was collected as plasma sample.
5
In addition, the organs (heart, liver, spleen, lung and kidney) and tumor tissues were removed after excretion
of blood by cardiac perfusion with saline, and washed twice, then flushed with saline for three times to remove the
blood remained, finally, the organs and tumor tissues were rolled over on the filter paper carefully to remove the
main vessel. All the tissues were homogenized with triple amount of saline.
An aliquot of 30 μL or 10 μL of internal standard (docetaxel, 100 μg/mL) was added into 100 μL plasma or
100 μL tissues homogenate, and extracted with 400 μL ether. The mixture was vortexed for 5 min, and centrifuged
at 10000 rpm for 15 min. The supernatant was transferred to another centrifuge tube, and dried under air stream at
room temperature. The dry residue was reconstituted with 100 μL of methanol. The solution was centrifuged at
10000 rpm for 15 min, and then 20 μL of the supernatant was injected into the HPLC system for analysis.
2.14.4. In vivo bio-distribution
Tumor targeting capability of free PTX, PTX-Lip, PTX-(Bio-Chol)Lip, PTX-(Bio-Chol) Lip and
2
PTX-(Bio -Chol)Lip for intravenous injection was investigated in subcutaneous 4T1 model. The xenograft
2
4T1-breast tumor bearing mice were randomly divided into groups (3 mice each) for the four types of liposomes
and PTX. PTX-Lip, PTX-(Bio-Chol)Lip, PTX-(Bio-Chol) Lip, PTX-(Bio -Chol)Lip and free PTX were given to
2
2
the mice via the tail vein and each was equivalent to the administration dose of PTX of 10 mg/kg. At 5 min, 15 min,
0 min, 60 min, 120 min, 240 min, 480 min, 720 min and 1440 min after injection, blood samples were collected
3
from the eye socket of mice, and placed in tubes containing heparin sodium. Likewise, at the predetermined time
points after injection, the organs and tumor tissues were removed after excretion of blood by cardiac perfusion with
saline. All the tissues were prepared as described in 2.14.3. The concentration of PTX was analyzed by HPLC.
2.14.5. In vivo imaging
The active targeting ability of Lip, (Bio-Chol)Lip, (Bio-Chol) Lip, (Bio -Chol)Lip was studied in 4T1-breast
2
2
tumor bearing BALB/c mice. The 4T1-breast tumor bearing BALB/c mice were randomly divided into four groups
4 each group) and were intravenously injected via the caudal vein with DiD-Lip, DiD-(Bio-Chol)Lip,
(
10

DiD-(Bio-Chol) Lip and DiD-(Bio -Chol)Lip at a dose of 200 μg DiD/kg. The DiD fluorescence signal (Ex 620 nm,
2
2
Em 670 nm) was imaged by the IVIS Lumina Series III imaging system (Perkin-Elmer, USA) at 1h, 2h, 8h and 24h
after injection.
2.15.
Statistical analysis
The area under the concentration-time profile (AUC ), maximal concentration (Cmax), and mean residence
0
-t
times (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 breast tumor targeting capability
of liposome. The value of RE and CE were defined as follows:
RE = (AUC ) / (AUC )
0-t P
0- t L
CE = (C ) / (C ) ,
max L
max P
where L and P represented the liposomes loading PTX and free PTX, respectively.
The results were expressed as means ± standard deviations (s.d.) of the mean. Data comparison between two
different groups were analyzed using an F-test with subsequent T-tests (equal variance). Statistical comparisons
were performed by one-way ANOVA for multiple groups. Significant differences between or among groups were
indicated by *p < 0.05, **p < 0.01 and ***p < 0.001, respectively.
3
.
Results and discussion
3
.1. Synthesis of ligand Bio -Chol
2
11

o
Scheme 1. The synthetic route of ligand Bio -Chol. Reagents and conditions: (a) TsCl, pyridine, 50 C, 5h. (b) Triethylene glycol,
2
+
-
dioxane, reflux, 6h. (c) t-butyl bromoacetate, n-Bu N HSO , 50% NaOH, toluene, r.t., 16h. (d) TsOH, toluene, reflux, 8h. (e)
4
4
o
Diethanol amine, IBCF, NMM, CH Cl , -10 C, r.t., 5h; (f) D-(+)-Biotin, EDCI, DMAP, DIPEA, CH Cl , r.t., 12h.
2
2
2
2
The synthetic route of ligand Bio -Chol was illustrated in Scheme 1. Firstly, compound 5 was synthesized
2
from cholesterol (1) in four steps, which was then conjugated with diethanol amine in the presence of isobutyl
chloroformate (IBCF) and N-methyl morpholine (NMM) to obtain compound 6. Finally, compound 6 reacted with
D-(+)-biotin to give the target compound Bio -Chol under the condensation condition. The target compound and
2
1
important intermediates were characterized by H NMR and mass spectra.
3.2. Synthesis of ligand Bio-Chol
Scheme 2 showed the synthetic route of ligand Bio-Chol. Similarly, compound 3 was synthesized from
cholesterol (1) in two steps, then was conjugated with D-(+)-biotin in the presence of EDCI and DMAP to obtain
ligand Bio-Chol.
o
Scheme 2 .The synthetic route of ligand Bio-Chol. Reagents and conditions: (a) TsCl, pyridine, 50 C, 5h. (b) Triethylene glycol,
dioxane, reflux, 6h. (c) D-(+)-Biotin, EDCI, DMAP, DIPEA, CH Cl , r.t., 12h.
2
2
3.3. Characterization of liposomes
Proper size, uniform distribution and zeta potential of nanoparticles are crucial to cellular uptake efficiency, in
vivo bio-distribution and so on [25]. The mean diameter, encapsulation efficiency and zeta potential of these
liposomes were exhibited in Table 1. Dynamic light scattering (DLS) showed the average particle sizes of these
liposomes were about 110 nm with a polydispersity index (PDI) less than 0.2. Liposomes are all negatively charged,
and their zeta potentials are mainly in the range of -2 to -3 mV.
PTX could be successfully loaded into these liposomes with entrapment efficiency (EE%) above 87%,
suggesting that PTX was well loaded, and were suitable for the follow-up in vitro and in vivo assays (Table 1).
12

In addition, the PTX-(Bio -Chol)Lip showed a regular circular shape and a suitable size under the
2
transmission electron microscope (TEM) (Fig. 2). The above results indicated that liposomes we prepared could be
used for subsequent studies, such as cell uptake, in vivo distribution, etc.
Table 1. The composition and characterization of different paclitaxel-loaded liposomes (n = 3, mean ± SD).
Liposomes
PTX-Lip
Size(nm)
103.3 ± 2.1
108.1 ± 1.5
105.6 ± 2.7
112.4 ± 2.5
PDI
EE (%)
Zeta potential (mV)
-3.06 ± 0.22
0.135 ± 0.016
0.147 ± 0.023
0.121 ± 0.042
0.119 ± 0.008
91.23 ± 2.48
87.45 ± 1.36
87.02 ± 2.06
88.15 ± 1.64
PTX-(Bio-Chol)Lip
-3.53 ± 0.17
PTX-(Bio-Chol) Lip
-2.18 ± 0.32
2
PTX-(Bio -Chol)Lip
-2.67 ± 0.18
2
Figure 2. The TEM image of PTX-(Bio -Chol)Lip.
2
3.4. In vitro PTX release study
PTX release properties were evaluated in PBS containing 0.1% Tween 80. As shown in Fig. 3, free PTX
exhibited a rapid release, with more than 80% of the drug released into the medium after 12h incubation.
PTX-loaded liposomes achieved sustained release behaviors with cumulative PTX release less than 75% within 48h
incubation in PBS. Besides, it was also noted that no significant difference in release properties was observed
among PTX-Lip, PTX-(Bio-Chol)Lip, PTX-(Bio-Chol) Lip and PTX-(Bio -Chol)Lip, and none of these four kinds
2
2
of PTX-loaded liposomes displayed a sudden initial release pattern. All the results showed that biotin modification
didn't change the release profile of PTX in liposomes.
13

Figure 3. The PTX release profiles of free PTX, PTX-Lip, PTX-(Bio-Chol)Lip, PTX-(Bio-Chol) Lip and PTX-(Bio -Chol)Lip in
2
2
PBS (pH 7.4) containing 0.1% Tween 80 over 48h (n = 3, mean ± SD).
3.5. In vitro stability of liposomes in serum
Liposomal particle stability against physiological condition is prerequisite for the further application in vivo,
so 50% fetal bovine serum (FBS) was used to mimic the in vivo condition. Transmittance variations of different
liposomes as important parameters were monitored in our study to explore the serum stability of liposomes. As
shown in Fig. 4, the transmittance of liposomes were above 90% and showed no detectable change within 48h. The
results indicated that liposomes were sufficient to prevent the interaction between liposomes and serum proteins,
which was important for obtaining a longer half-life of blood in vivo [25].
Figure 4. The variations of transmittance of different modified liposomes in 50% FBS (n =3, mean ± SD).
3.6. Hemolysis assays
Hemocompatibility is the key to the application of liposomes in vivo. As shown in Fig. 5, the hemolysis test
14

showed that even when the concentration of phospholipids increased to 400 nM, the hemoglobin release of all
liposomes did not increase significantly.
PTX-(Bio -Chol)Lip did not display concentration-dependent increase in hemolysis as well and less than 10%
2
hemolysis was always regarded as non-toxic [25]. It was reported that cationic delivery vectors could cause cell
lysis and hemoglobin release [28]. The result of hemolysis assays was also consistent with the negative potential of
biotin-modified liposomes shown in Table 1. In conclusion, all types PTX-loaded liposomes could not interact with
negatively charged membrane of the erythrocytes because of electrostatic repulsion of negatively charge,
suggesting that PTX-loaded liposomes of biotin modification showed minimal undesirable effects.
Figure 5. Hemolysis percentage of different liposomes. Values are represented as mean ± SD (n =3).
3.7. Cellular uptake study
To demonstrate the preference of biotin-modified liposomes for the breast cancer cells, the cellular uptake of
different biotin density modified liposomes was studied and unmodified liposome was used as a negative control.
SMVT over-expressed cells (4T1 and MCF7 cells) and SMVT negative expressed cells (B16 cells) were used to
investigate the cellular uptake in vitro.
As flow cytometry results showed (Fig. 6A-B), in SMVT over-expressed cells, liposomes modified with
Bio -Chol have stronger affinity with the transporters than other types of liposomes. (Bio -Chol)Lip displayed more
2
2
uptake than Lip (3.17-time higher), (Bio-Chol)Lip (2.50-time higher), and (Bio-Chol) Lip (2.17-time higher) in
2
4T1 cells. The same trend was observed in MCF7 cells (Fig. 6B). In contrast, no significant difference on
fluorescence intensity was observed among groups in the SMVT negative expressed cells (B16 cells) (Fig. 6C).
Notably, similar results were obtained from confocal images (Fig. 6D-F).
15

Figure 6. Cellular uptake of CFPE-labeled Lip, (Bio-Chol)Lip, (Bio-Chol) Lip and (Bio -Chol)Lip on 4T1 cells, MCF-7 cells and
2
2
B16 cells were measured. (a) (A), (B), (C) represented cellular uptake of CFPE-labeled liposomes measured by a flow cytometer on
T1 cells, MCF-7 cells and B16 cells, respectively (mean ± SD, n =3). (b) (D), (E), (F) represented qualitative 4h cellular uptake of
4
16

CFPE-labeled liposomes determined by CLSM on 4T1 cells, MCF-7 cells and B16 cells, respectively (mean ± SD, n =3), green
CFPE-labeled liposomes), blue (DAPI stained nucleus), light blue (colocalized CFPE and DAPI). Scale bars represent 20 μm. *, **
(
and *** represent p < 0.05, p < 0.01 and p < 0.001 versus Lip group, N. S. indicates none significant difference.
Both flow cytometry and confocal images indicated that (Bio -Chol)Lip having a higher cellular
2
internalization in SMVT over-expressed cells but no effect on the uptake of SMVT negative expressed B16 cells.
These data suggested that increasing the density of biotin on the surface of liposomes can significantly increase its
ability to deliver drugs into breast cancer cells. Furthermore, when the amount of biotin residue on the liposome
surface was the same, the liposomes modified by double branched biotin ((Bio -Chol)Lip) had better targeting
2
ability for breast cancer than those modified by single biotin ((Bio-Chol) Lip), especially in 4T1 cells (3.17-time
2
higher than Lip). Above all, (Bio -Chol)Lip had the greatest targeting efficacy on breast cancer cells.
2
3.8. Cellular Uptake mechanism study
The cellular uptake mechanism of (Bio -Chol)Lip was also investigated, and a series of endocytosis inhibitors
2
were pre-incubated with 4T1 cells and MCF-7 cells. Chlorpromazine, filipin and amiloride were chosen to block
clathrin-mediated endocytosis, caveolin-mediated endocytosis and micropinocytosis respectively [28]. 4°C and
NaN (oxidative phosphorylation inhibitor) were selected to study the effect of energy. As the results showed (Fig.
3
7
A and 7C), these endocytosis inhibitors showed different levels of inhibition effects. Besides, 4 °C and NaN also
3
displayed strong impact on cellular uptake (down to 15% and 75% respectively), which indicated the uptake of
(
Bio -Chol)Lip was energy-dependent.
2
The (Bio -Chol)Lip uptake in 4T1 and MCF7 cells was shown to be time and concentration dependent (Fig.
2
S1). The effect of sodium ions on the rate of (Bio -Chol)Lip uptake was delineated by performing the experiment in
2
sodium-free buffer. As shown in Fig. 7B and 7D, a marked decrease (> 45%) in (Bio -Chol)Lip uptake was evident
2
in the absence of sodium ions. On the other hand, no significant difference was observed in the uptake when
chloride in the incubation buffer was displaced. Hence the uptake process in both the cell lines was found to be
highly sodium dependent and chloride independent.
Furthermore, (Bio -Chol)Lip uptake was significantly inhibited in the presence of biotin and pantothenic acid
2
which were well known as substrates for SMVT (Fig. 7B and 7D). Interestingly, a significant inhibition in
(
Bio -Chol)Lip uptake was observed in presence of NHS- biotin (devoid of free carboxylic group) and valeric acid
2
(which has free carboxylic group) in 4T1 cell, but no marked inhibition of NHS- biotin in MCF7. Our results
correspond to earlier reports suggesting that a free carboxylic group may be essential for specific binding to the
17

transporter [15]. (Bio -Chol)Lip uptake was unaltered in the presence of other structurally unrelated vitamins
2
(
ascorbic acid, thiamine). These results along with previous reports suggest that (Bio -Chol)Lip internalization in
2
accordance with the characteristics of SMVT transport [15].
In conclusion, all the results indicated that (Bio -Chol)Lip could be recognized by sodium-dependent
2
multivitamin transporter (SMVT) on the membrane of breast tumor cells via the residues on the surface, and then
energy-dependently internalized through a synthetic endocytic pathway including clathrin-mediated, caveolae-
mediated and micropinocytosis-mediated endocytosis.
Figure 7. The relative uptake of CFPE-labeled (Bio -Chol)Lip on 4T1 cells (A, B) and MCF-7 cells (C, D) for 4h after preincubation
2
of different inhibitors determined by flow cytometer (mean ± SD, n =3). *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001
versus control group, respectively, N. S. indicates none significant difference.
3.9. Cytotoxicity
It was demonstrated that cellular uptake of (Bio -Chol)Lip was significantly higher than others. To further
2
verify the Bio -Chol endowed (Bio -Chol)Lip with antiproliferation and the potent active targeting ability of SMVT,
2
2
in vitro cytotoxicity of different liposomes against 4T1 cells and MCF-7 cells was performed by MTT assays.
As shown in Fig. 8A and 8C, free PTX showed higher inhibition rate than PTX-loaded liposomes, because
free drugs could be transported into the cells directly and without a drug release process. In addition, according to
18

the trend of cell viability, PTX-(Bio -Chol)Lip displayed significant higher inhibition rate than PTX-(Bio-Chol)Lip
2
and PTX-(Bio-Chol) Lip against 4T1 cells and MCF-7 cells simultaneously. Accordingly, the results suggested that
2
Bio -Chol could remarkably enhance the cytotoxicity of drug loaded liposomes.
2
On the other hand, the cytotoxicity of liposomes without drug loaded was also measured. Blank Lip,
(
Bio-Chol)Lip, (Bio-Chol) Lip, and (Bio -Chol)Lip at different concentrations all exhibited little toxicity as the cell
2 2
viability was more than 89% even at a relatively high concentration, while free PTX solvent (ethanol-cremophor
ELP 35 mixture, v/v = 1: 1 ) displayed an anti-proliferation effect as the concentration increased (Fig. 8B and 8D).
Therefore, our liposomal drug delivery systems were safe and nontoxic to be further used in vivo.
Figure 8. (a) The cytotoxicity study on 4T1 cells (A), MCF-7 cells (C) after treated with various concentrations of PTX-Lip,
PTX-(Bio-Chol)Lip, PTX-(Bio-Chol) Lip, PTX-(Bio -Chol)Lip and free PTX for 48h (n =3, mean ± SD). (b) The cytotoxicity study
2
2
on 4T1 cells (B), MCF-7 cells (D) after treated with various concentrations of free PTX solvent, Lip, (Bio-Chol)Lip, (Bio-Chol) Lip
2
and (Bio -Chol)Lip for 48h (n =3, mean ± SD). *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001 versus blank Lip group,
2
respectively
Annexin V-FITC/PI apoptosis detection kit was used to study the cell apoptosis induced by PTX-loaded
liposomes. As shown in Fig. 9, the percentage of apoptosis and necrotic cells was 34.59 ± 3.46% for free PTX,
2
3
2.57 ± 1.90% for PTX-Lip, 22.42 ± 3.62% for PTX-(Bio-Chol)Lip, 29.39 ± 2.82% for PTX-(Bio-Chol) Lip and
2
5.83 ± 3.00% for PTX-(Bio -Chol)Lip, respectively. Bio -Chol modified PTX loaded liposomes and free PTX
2
2
induced significant stronger cell apoptosis effects, and the results were consistent with the cytotoxicity experiments
19

in vitro.
What’s more, the cytotoxicity and apoptosis study of PTX formations confirmed that (Bio -Chol)Lip could
2
remarkably improve the cellular internalization of drug delivery systems, release more therapeutic agents into the
cytoplasm and achieve stronger inhibition effect in vitro.
Figure 9. (a) The apoptosis study of 4T1 cells after incubation with free PTX (A), PTX-Lip (B), PTX-(Bio-Chol)Lip (C),
PTX-(Bio-Chol) Lip (D), PTX-(Bio -Chol)Lip (E). (b) The percentage of apoptosis and necrotic cells after free PTX and PTX-loaded
2
2
liposomes treatment (F). *, ** and represent p < 0.05, p < 0.01 and versus PTX-Lip group, N. S. indicates none significant difference
n =3, mean ± SD).
(
3.10.
Bio-distribution study in plasma and tissues
A perfect delivery system for tail vein administration should have enough circulation time in the bloodstream
to reach and diffuse through all tissues and deliver its cargo to the target site. 4T1 tumor-bearing BALB/c mice
were used to estimate the plasma distribution of different formulations. We firstly assessed the distribution of the
PTX-Lip, PTX-(Bio-Chol)Lip, PTX-(Bio-Chol) Lip, PTX-(Bio -Chol)Lip and free PTX in plasma. The plasma
2
2
PTX concentration-time profiles were illustrated in Fig. 10 and the corresponding pharmacokinetic parameters of
PTX from different formulations were summarized in Table 2.
It was shown that the area under the concentration-time curve (AUC ) of PTX in four types of liposomes
0
–t
were much higher than that of naked PTX within 24h after the same dosage (10 mg/kg) was administrated (Table 2).
Meanwhile, PTX-(Bio -Chol)Lip could extend the elimination half-life (t ) of free PTX from 359 min to 601 min
2
1/2
(p < 0.001) while the smaller-sized particles had longer blood retention [28]. These results suggested that the
clearance of PTX in plasma was delayed owing to the sustained release of liposomes. Meanwhile, these data also
indicated that all the four types of liposomes showed certain stability which would increase the chance transport to
20

tumor tissues.
Figure 10. The concentration curve of paclitaxel versus time in plasma after i.v. injection of paclitaxel and PTX-loaded liposomes in
mice (n = 3, mean ± SD).
Table 2. Pharmacokinetic parameters of paclitaxel in blood after administration of paclitaxel and liposomes (n =3).
Parameters
Paclitaxel
AUC(0-t) ( g/mL·min)
16360.94 ± 1569.49
25169.06 ± 1208.74
35319.11 ± 8067.41
31447.98 ± 6352.58
27872.99 ± 1585.19
MRT (min)
340.70 ± 15.46
399.96 ± 22.99
500.99 ± 62.67
470.13 ± 21.04
458.93 ± 3.88
Tmax (min)
Cmax (μg/mL)
74.17 ± 1.59
82.07 ± 7.02
81.66 ± 4.73
77.14 ± 13.69
80.29 ± 5.08
t1/2 (min)
5
5
5
5
5
358.89 ± 48.86
340.87 ± 46.28
729.22 ± 529.13
536.38 ± 154.64
600.68 ± 217.75
PTX-Lip
PTX-(Bio-Chol)Lip
PTX-(Bio-Chol) Lip
2
PTX-(Bio -Chol)Lip
2
To further evaluate the breast tumor targeting capability of formulations, the distribution of liposomes in
various organs and tumor tissues were also studied in 4T1 tumor-bearing BALB/c mice.
As shown in Fig. 11 and Table 3, it was clearly observed that the PTX of PTX-(Bio -Chol)Lip accumulated
2
more than other groups in the breast tumor from 15 min to 1440 min after administration (21985.38 ± 993.94
μg/g·h, Fig. 11), displaying the strongest breast tumor targeting ability of PTX-(Bio -Chol)Lip.
2
PTX-loaded liposomes and naked PTX all reached the maximum distribution in the breast tumor at 480 min
after injection. Especially, the relative absorption efficiency (REs) of PTX-(Bio -Chol)Lip reached 5.61 times (p <
2
0.001) that of PTX, the concentration efficiencies (CEs) were also enhanced to 5.06 times (p < 0.001). These
findings showed that PTX-(Bio -Chol)Lip had a higher affinity for SMVT on breast tumor, enabling more PTX to
2
be transported to targeted tissues with minimal side effect. All these results suggested that PTX-(Bio -Chol)Lip had
2
the potential as breast tumor targeting carriers for hydrophobic anticancer agents.
21

Figure 11. In vivo distribution of 4T1-tumor bearing mice at different time points after i.v. injection of paclitaxel and PTX-loaded
liposomes in mice, including heart (A), liver (B), spleen (C), lung (D), kidney (E) and tumor (F). Data represent mean±SD. (n = 3,
mean ± SD).
Table 3. Pharmacokinetic parameters of paclitaxel in tumor tissues after administration of paclitaxel and liposomes (n =3).
Parameters
AUC(0-t) (μg/g·min)
MRT (min) Tmax (min)
Cmax (μg/g)
RE
CE
Paclitaxel
PTX-Lip
3920.90 ± 140.91
6779.30 ± 720.60
533.07 ± 55.59
556.63 ± 4.79
480
480
480
480
480
7.54 ± 0.77
11.30 ± 2.56
17.36 ± 1.11
22.57 ± 1.81
38.16 ± 0.82
-
-
1.73
3.03
4.05
5.61
1.50
2.30
3.00
5.06
PTX-(Bio-Chol)Lip
11888.57 ± 150.29 479.12 ± 10.83
15867.44 ± 189.01 504.30 ± 5.86
21985.38 ± 993.94 507.30 ± 31.38
PTX-(Bio-Chol) Lip
2
PTX-(Bio -Chol)Lip
2
3.11.
In vivo imaging
To investigate the in vivo targeting efficiency and monitoring the accumulation behavior in breast tumor site,
DiD-labeled liposomes were employed. As shown in Fig. 12, images in vivo indicated that PTX-(Bio -Chol)Lip
2
presented the highest fluorescence intensity and the best accumulation in tumor region from 1h to 24h in contrast to
other liposomes after systemic administration. The four types of liposomes showed weak fluorescence signal at
tumor site 2h later, and the fluorescence intensity reached its maximum at 8h which was in accordance with the
results of bio-distribution. These results displayed the strongest breast tumor targeting capability of (Bio -Chol)Lip.
2
22

Figure 12. In vivo images of 4T1-tumor bearing mice at different time points after systemic administration of DiD-loaded liposomes.
A: DiD-Lip, B: DiD-(Bio-Chol)Lip, C: DiD-(Bio-Chol) Lip, D: DiD-(Bio -Chol)Lip (n =4).
2
2
These bio-distribution and in vivo imaging results further proved that PTX-(Bio -Chol)Lip possessed a high
2
selective breast tumor transporting ability not only in vitro but also in vivo. Hence, PTX-(Bio -Chol)Lip could be
2
superior to other groups in the therapeutic efficacy of breast cancer.
4
.
Conclusion
In conclusion, we have successfully designed and developed a series of biotin modified liposomes as active
targeting drug delivery systems for the therapy of breast cancers. In particular, we broke the traditional design
concept of using single branched ligands to modify liposomes and prepared the double branched biotin modified
liposome for the first time. Excitingly, in the in vitro uptake and in vivo distribution experiments, the double
branched biotin modified liposome ((Bio -Chol)Lip) exhibited excellent targeting ability of breast cancer. In
2
contrast, SMVT negative expressed cells have very little uptake of (Bio -Chol)Lip, thus ensuring very low systemic
2
toxicity. In addition, cytotoxicity and apoptosis assays showed that PTX-(Bio -Chol)Lip exhibited better
2
therapeutic effects for breast cancer than other PTX-loaded liposomes. All these results indicate that increasing the
density of ligand on the surface of liposomes can significantly improve tumor targeting ability of liposomes.
Especially, the modification of cholesterol by double branched ligands can further improve the active targeting of
liposomes while keeping the dosage of cholesterol low. We believe that the strategy of modifying cholesterol with
23

multi-branched ligands will open a novel and exciting chapter in the design of tumor-specific diagnostic reagents
and active targeting drug delivery systems.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No. 81573286, No. 81773577
&
No. 21472130), the Sichuan Science and Technology Program (2018JY0537) and the Fundamental Research
Funds for the Central Universities (2012017yjsy211).
Conflict of Interest
The authors declare no competing financial interest.
Appendix A. Supporting Information
Supplementary data associated with this article can be found in the online version at.
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