Dual-targeting liposomes with active recognition of GLUT5 and αvβ3 for triple-negative breast cancer

Article abstract of DOI:10.1016/j.ejmech.2019.111720

At present, chemo- and radiotherapies remain to be the mainstream methods for treating triple-negative breast cancer (TNBC), which is known for poor prognosis and high rate of mortality. Two types of novel dual-targeting TNBC liposomes (Fru-RGD-Lip and Fru+RGD-Lip) that actively recognize both fructose transporter GLUT₅ and integrin α_vβ₃ were designed and prepared in this work. Firstly, a Y-shaped Fru-RGD-chol ligand, where a fructose and peptide Arg-Gly-Asp (RGD) were covalently attached to cholesterol, was designed and synthesized. Then, the Fru-RGD-Lip was constructed by inserting Fru-RGD-chol into liposomes, while Fru+RGD-Lip was obtained by inserting both Fru-chol and RGD-chol (with the molar ratio of 1:1) into liposomes. The particle size, zeta potential, encapsulation efficiency and serum stability of the paclitaxel-loaded liposomes were characterized. The results indicated that the paclitaxel-loaded Fru-RGD-Lip had the strongest growth inhibition against GLUT₅ and α_vβ₃ overexpressed MDA-MB-231 and 4T1 cells. The cellular uptake of Fru-RGD-Lip on MDA-MB-231 cells and 4T1 cells was 3.19- and 3.23-fold more than that of the uncoated liposomes (Lip). The uptake of Fru+RGD-Lip was slightly lower, giving a 2.81- and 2.90-fold increase than that of Lip in two cell lines, respectively. The mechanism study demonstrated that the cellular uptake of both dual-targeting liposomes was likely to be recognized and mediated by GLUT₅ and α_vβ₃ firstly, then endocytosed through comprehensive pathways in an energy-dependent manner. Moreover, Fru-RGD-Lip displayed the maximum accumulation, which was 2.62-fold higher than that of Lip for instance, at the tumor sites compared to other liposomes using in vivo imaging. Collectively, the liposomes co-modified by fructose and RGD have enormous potential in the development of targeted TNBC treatment, especially the covalently modified Fru-RGD-Lip, making it a promising multifunctional liposome.

Full text of DOI:10.1016/j.ejmech.2019.111720

European Journal of Medicinal Chemistry 183 (2019) 111720
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Dual-targeting liposomes with active recognition of GLUT5 and avb3
for triple-negative breast cancer
,
, **
Yanchi Pu a, Hao Zhang a, Yao Peng a, Qiuyi Fu a, Qiming Yue a, Yi Zhao a b, Li Guo a
,
,
*
Yong Wu a
a Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug, Sichuan
Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu, 610041, PR China
b Department of Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
At present, chemo- and radiotherapies remain to be the mainstream methods for treating triple-negative
breast cancer (TNBC), which is known for poor prognosis and high rate of mortality. Two types of novel
dual-targeting TNBC liposomes (Fru-RGD-Lip and FruþRGD-Lip) that actively recognize both fructose
transporter GLUT5 and integrin avb3 were designed and prepared in this work. Firstly, a Y-shaped Fru-
RGD-chol ligand, where a fructose and peptide Arg-Gly-Asp (RGD) were covalently attached to choles-
terol, was designed and synthesized. Then, the Fru-RGD-Lip was constructed by inserting Fru-RGD-chol
into liposomes, while FruþRGD-Lip was obtained by inserting both Fru-chol and RGD-chol (with the
molar ratio of 1:1) into liposomes. The particle size, zeta potential, encapsulation efficiency and serum
stability of the paclitaxel-loaded liposomes were characterized. The results indicated that the paclitaxel-
loaded Fru-RGD-Lip had the strongest growth inhibition against GLUT5 and avb3 overexpressed MDA-
MB-231 and 4T1 cells. The cellular uptake of Fru-RGD-Lip on MDA-MB-231 cells and 4T1 cells was
3.19- and 3.23-fold more than that of the uncoated liposomes (Lip). The uptake of FruþRGD-Lip was
slightly lower, giving a 2.81- and 2.90-fold increase than that of Lip in two cell lines, respectively. The
mechanism study demonstrated that the cellular uptake of both dual-targeting liposomes was likely to
be recognized and mediated by GLUT5 and avb3 firstly, then endocytosed through comprehensive
pathways in an energy-dependent manner. Moreover, Fru-RGD-Lip displayed the maximum accumula-
tion, which was 2.62-fold higher than that of Lip for instance, at the tumor sites compared to other li-
posomes using in vivo imaging. Collectively, the liposomes co-modified by fructose and RGD have
enormous potential in the development of targeted TNBC treatment, especially the covalently modified
Fru-RGD-Lip, making it a promising multifunctional liposome.
Received 18 June 2019
Received in revised form
24 August 2019
Accepted 18 September 2019
Available online 18 September 2019
Keywords:
Liposome
Dual-targeting
TNBC
Fructose
RGD peptide
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
breast cancer are ineffective against TNBC due to the loss of ther-
apeutic targets. At present, the main treatments of TNBC are still
Globally, breast cancer has the highest incidence among
women. As a malignant tumor, it seriously affects women's health
[1]. Triple-negative breast cancer (TNBC), with deficiencies of es-
trogen receptor (ER), progesterone receptor (PR), and human
epidermal growth factor receptor 2 (Her-2), is one of the most
invasive subtypes of breast cancer, accounting for approximately
20% of all breast cancer [2,3]. The drugs normally used to treat
surgery, chemotherapy and radiotherapy, with poor prognosis and
quite high mortality rate [4]. Therefore, the dilemma faced by TNBC
is urgently to be alleviated.
Commonly used chemotherapeutic drugs, such as paclitaxel
(PTX) and doxorubicin (DOX), are highly toxic to normal cells.
Therefore, one approach to increase their efficacy is to enhance the
selectivity of such drugs, so that higher dose of drugs could be
applied [5]. Current research are mainly focused on designing novel
targeting drug delivery system (TDDS) [6]. What attracts much
attention is to search ligands that can selectively bind with specific
receptors and/or transporters, such as glucose transporters, integ-
rin receptors, folate receptors, and ferritin receptors and etc [7],
* 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.2019.111720
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
overexpressed at the target sites (especially cancer cells).
TNBC was designed and synthesized with fructose and RGD pep-
tide as targeting molecules, lysine as a linker and cholesterol as a
lipid material, in which C1eOH of fructose and primary amino
group of RGD were the attachment sites.
The synthetic route of ligand Fru-RGD-chol was illustrated in
Scheme 1. Firstly, the C2,3,4,5-OH of D-fructose was protected by a
The metabolic behavior of tumor is different from normal cells,
characterized by the sharp increase of glucose uptake and glycolysis
even in oxygen sufficient conditions, which was known as the
Warburg effect [8]. Hence, the rapid proliferation and Warburg
effect make a large amount of glucose consumption, causing a low-
glucose microenvironment in cancer cells [9].
D
-fructose, the sec-
propylidene group to obtain compound 2. The C1eOH was
extended by reacting with succinic anhydride to generate com-
pound 3. Secondly, the cholesterol derivative 6, gained on the basis
of our previous study [34], was conjugated with N-Boc-N0-Fmoc-L-
lysine to obtain the intermediate 7 with two attachment sites. After
that, compound 8 was generated by removing the Fmoc group, and
immediately reacted with RGD derivative 21 (Scheme S1) to give
compound 9 (NMM, IBCF, DCM). Similarly, compound 10 was
generated by removing the Boc group, and it was further coupled
with the fructose derivative 3 to obtain compound 11 (DIPEA, EDCI,
DMAP). Finally, the protective group of fructose and RGD were
removed by trifluoroacetic acid and Pd/C/H2 respectively, to pro-
vide Fru-RGD-chol.
The synthetic route of ligand Fru-chol was illustrated in Scheme
2. Briefly, condensation of the fructose derivative 3 and the
cholesterol derivative 6 gave compound 12 (DCC/DMAP). After
deprotecting the propylidene groups, Fru-chol was gained. The
synthesis of RGD-chol followed our previous work [28]. All the title
compounds and important intermediates were characterized by
their respective 1H NMR and HRMS.
ond most abundant monosaccharide in nature, is largely used by
cancer cells to alleviate the deficiency of energy. Therefore, the
cellular uptake of fructose in breast cancer is much more than that
of the normal breast cells, and even for other tumor cells are 8e10
times higher. Yet the uptake of glucose in breast cancer cells is only
slightly changed comparing to normal breast cells [10e12]. Among
the 14 subtypes of hexose transporters (GLUTs), only GLUT2 and
GLUT5 were able to transport fructose [13]. The substrate of GLUT5
is almost only fructose, and about 90% of fructose is transported by
GLUT5 [14,15]. Therefore, GLUT5 is a specific transporter for fruc-
tose. GLUT5 has been reported to be overexpressed in breast cancer
cells and tumor tissues, but not in their normal counterpart
[16e18]. Thus, the substrate specificity of GLUT5 and its relatively
concentrated expression tissue site give it the advantage to be an
efficient target for the diagnosis and treatment of TNBC [19e21].
In addition to the GLUTs, integrin receptors, which are consid-
ered as a biomarker of tumors, are also overexpressed in many
tumor cells and neovascular endothelial cells. Integrins belong to
the transmembrane glycoprotein family, and mediate cell-cell and
cell-matrix interactions [22], which is related to the adhesion and
migration of tumor cells and the formation of tumor blood vessels
[23,24]. Among the 26 subtypes, avb3 is the most concerned [25].
Various subtypes of integrin receptors can recognize the same
peptide Arg-Gly-Asp (RGD). Therefore, RGD is broadly used as a
specific ligand for integrins. It has been widely reported that avb3 is
overexpressed on the surface of a variety of tumor cells, including
triple-negative breast cancer cells [26]. Therefore, avb3 may act as a
promising target of TNBC.
2.2. Preparation and characterization of liposomes
Liposomes were prepared by the thin film hydration method
and the prescription followed previous work [28,35]. The charac-
terizations of different liposomes in this study were listed in
Table 1. The results showed that the entrapment efficiency (EE%) of
these five PTX-loaded liposomes were greater than 80%, suggesting
PTX was well loaded in liposomes. The particle sizes of the PTX-
loaded liposomes were all around 110 nm, and the dispersity in-
dex (polymer dispersity index, PDI) was around 0.2, implying the
liposomes were evenly distributed. Besides, the Zeta potential was
weak negative electricity (about ꢀ5 mV), which can decrease the
absorption of the reticuloendothelial system and immune response
[36e38]. All of results contributed to achieve the passive targeting
through the enhanced permeability and retention effect (EPR ef-
fect) [39,40].
Fructose [21] and RGD as well as their derivatives [27] have been
reported to be individually conjugated with nanocarriers for tar-
geting TNBC. However, the targeting ability mediated by a single
receptor or transporter can hardly achieve the expectation. So, to
utilize more than one receptor that is specifically overexpressed on
the tumor cells may further enhance the targeting ability. The dual-
targeting strategy has been confirmed by many reports, such as
glucose and RGD co-modified liposomes [28], p-aminophenyl-a-D-
mannopyranoside (MAN) and transferrin (TF) co-modified dauno-
rubicin liposomes [29], and etc. In this work, by targeting both
GLUT5 and avb3 that are overexpressed on TNBC cells, we report
two types of novel liposomes co-modified with fructose and RGD to
improve the targeting ability. One is by physical mixing of two li-
gands (with the molar ratio of 1:1) with the liposome (FruþRGD-
Lip). The other is to covalently link two ligands first to form a Y-
shaped ligand with both fructose and RGD, then mixed with the
liposomes (Fru-RGD-Lip). The characteristics of liposomes and their
targeting ability to TNBC were evaluated in vitro and in vivo.
2.3. In vitro stability of liposomes in serum
In order to reach the target site effectively and regulate the ac-
tivity of loaded chemotherapeutic agents, the liposome must have a
certain stability in the blood circulation. The turbidity method was
used to monitor the stability of PTX-loaded liposomes in 50% fetal
bovine serum (FBS) [41]. As Fig. 1 showed, after being incubated at
37 ꢁC for 48 h, the transmittance of the five liposomes were still
more than 90%. Besides, there was no obvious aggregation phe-
nomenon, indicating that the prepared liposomes were stable in
serum for a long time.
2. Results and discussion
2.1. Synthesis of liposomes ligands
2.4. In vitro drug release study
According to the structure-activity relationship of the specific
affinity between fructose and GLUT5, and the phosphorylation of
fructose when metabolized in TNBC cells, the fructose can only be
modified on C1eOH [30e32]. Moreover, for the RGD tripeptide, it
has little impact on affinity when modifying the primary amino
The drug release properties were evaluated in phosphate buffer
(PBS, pH 7.4) containing 0.1% Tween 8041. The results in Fig. 2
showed that the release of free paclitaxel was fastest, with more
than 85% in 12 h and more than 90% in 48 h. The release of PTX in
liposomes was much slower, about 70% within 48 h. In addition,
there was no obvious burst release and significant difference in the
release characteristics among the five liposomes. The data
group [33], while the other groups are essential for binding to avb3
.
Therefore, the Y-shaped ligand (Fru-RGD-chol) for dual-targeting

Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
3
Scheme 1. Synthesis of ligand Fru-RGD-chol. Reagents and conditions: (a) 5% H2SO4-Acetone, 0 ꢁC- r. t., 2 h, 73%; (b) Succinic anhydride, DMAP, Et3N, toluene, 110 ꢁC, 2.5 h, 97%; (c)
TsCl, Pyridine, 0 ꢁCe55 ꢁC, overnight, 90%; (d) Triethylene glycol, dioxane, reflux, 6 h, 61%; (e) N-Boc-N0-Fmoc-L-lysine, DCC, DMAP, DCM, ꢀ5 ꢁC- r. t., 8 h, 93%; (f) DBU, DCM, r. t.,
20 min, 93%; (g) compound 21, NMM, IBCF, DCM, ꢀ5 ꢁC- r. t., overnight, 81%; (h) TFA, DCM, r. t., 20 min, 99%; (i) compound 3, DIPEA, EDCI, DMAP, DCM, ꢀ5 ꢁC- r. t., overnight, 77%; (j)
TFA, H2O, r. t., 24 h; then, H2, Pd/C (10%), CH3OH, 45 ꢁC, 48 h.
Scheme 2. Synthesis of ligand Fru-chol. Reagents and conditions: (a) DCC, DMAP, DCM, 0 ꢁC- r. t., 3 h, 77%; (b) TFA, H2O, r. t., 24 h, 38%.

4
Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
Table 1
Particle sizes, zeta potentials and the PTX entrapment efficiency of five different PTX-loaded liposomes (n ¼ 3, mean SD).
Liposomes
Size (nm)
PDI
EE (%)
Zeta potential (mV)
PTX-Lip
PTX-Fru-Lip
PTX-RGD-Lip
PTX-Fru-RGD-Lip
PTX-(FruþRGD)-Lip
108.5 4.7
108.8 2.2
110.8 6.2
112.0 2.5
113.6 2.1
0.203 0.024
0.209 0.017
0.198 0.015
0.201 0.019
0.186 0.017
86.65 1.53
81.47 1.11
82.60 2.09
82.16 2.92
80.42 3.02
ꢀ5.07 0.14
ꢀ4.54 0.17
ꢀ4.27 0.24
ꢀ4.74 0.19
ꢀ4.20 0.17
the results were shown in Fig. 3. In the lipid concentration range of
10e800 mol/L, the five liposomes did not cause significant he-
m
moglobin release, and the hemolysis rate was less than 10%, which
was regarded as non-toxic. All the characterizations indicated that
the liposomes were fit for further in vivo and in vitro study.
2.6. Cytotoxicity
Cytotoxicity of different PTX-loaded liposomes on human triple-
negative breast cancer cells (MDA-MB-231) [17,42] and murine
triple-negative breast cancer cells (4T1) [17,43]overexpressing
GLUT5 and avb3 simultaneously, was evaluated by MTT assay. The
results in Fig. 4A and B showed that PTX-Fru-RGD-Lip manifested
the significant cytotoxicity compared to the other liposomes, with
the highest inhibition of proliferation at each concentration. In
addition, the group of free paclitaxel exhibited the lowest cell
viability, probably because the free drugs could enter the cell
through passive diffusion directly and take effect without a drug
release process. The data further demonstrated the sustained
release effect of the liposomes, which is consistent with the in vitro
drug release study.
Fig. 1. Transmittance of PTX-Lip, PTX-Fru-Lip, PTX-RGD-Lip, PTX-Fru-RGD-Lip and
PTX-FruþRGD-Lip after incubation in 50% serum (n ¼ 3, mean SD).
In order to further investigate the toxicity of the lipid material,
the five types of blank liposomes were diluted to the corresponding
concentration of the PTX-loaded liposomes. The result was shown
in Fig. 4C and D. The unloaded lipid material showed no significant
toxicity to cells even up to 800 mmol/L. At each lipid concentration,
Fig. 2. In vitro drug release behavior of PTX-Lip, PTX-Fru-Lip, PTX-RGD-Lip, PTX-Fru-
RGD-Lip, PTX-FruþRGD-Lip and free PTX in PBS containing 0.1% Tween 80 within 48 h
(n ¼ 3, mean SD).
the cell viability was above 80%. Therefore, the unloaded lipid
material was safe and nonvenomous to be further used in vitro and
in vivo.
2.7. Cellular uptake
The CFPE-labeled liposomes were prepared to detect the lipo-
somes entered cells. It can be seen from Fig. 5A and B, for MDA-MB-
231 and 4T1 cells, Fru-RGD-Lip displayed the strongest cellular
uptake, followed by FruþRGD-Lip. The cellular uptake of Fru-RGD-
Lip on MDA-MB-231 cells and 4T1 cells was 3.19- and 3.23-fold
more than that of the uncoated liposomes (Lip). The uptake of
FruþRGD-Lip was slightly lower, giving a 2.81- and 2.90-fold in-
crease than that of Lip in two cell lines, respectively.
The qualitative study by confocal laser scanning microscope
(CLSM) in Fig. 5C and D was well consistent with the results of
quantitative by flow cytometry. The target efficacy of the five
different liposomes on both TNBC cells was Fru-RGD-
Lip > FruþRGD-Lip > RGD-Lip > Fru-Lip > Lip. In addition, the green
fluorescence was mainly located in the cytoplasm, and the light
blue fluorescent signal was rarely to be seen. The result indicated
that the liposome was mainly located in the cytoplasm and rarely
entered the nucleus, which was facilitating to the release of the
drug after the phospholipid bilayer structure being destroyed un-
der the acidic environment of the lysosome.
Fig. 3. Hemolysis rate of various concentrations of PTX-Lip, PTX-Fru-Lip, PTX-RGD-Lip,
PTX-Fru-RGD-Lip and PTX-FruþRGD-Lip (n ¼ 3, mean SD).
indicated that the liposome can significantly improve the release
behavior of the drug with a sustained release effect.
2.5. Hemolytic evaluation
2.8. Uptake mechanism
The biosecurity of liposomes in the blood circulation is limited
by hemocompatibility, so the hemolytic assay was conducted and
In order to investigate the uptake mechanism of Fru-RGD-Lip

Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
5
Fig. 4. The cytotoxicity of PTX-loaded liposomes on MDA-MB-231 cells (A) and 4T1 cells (B) for 24 h. The cytotoxicity of blank liposomes on MDA-MB-231 cells (C) and 4T1 cells (D)
for 24 h (n ¼ 3, mean SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus PTX-Lip).
and FruþRGD-Lip, receptor competition inhibitors and endocytosis
inhibitors were used to explore the uptake mechanism study. It can
be seen in Fig. 6 that both of free fructose and RGD can inhibit the
cellular uptake of Fru-RGD-Lip and FruþRGD-Lip to a certain extent,
especially the fructose showed significant inhibition in the cellular
uptake of FruþRGD-Lip on 4T1 cells, whose relative cellular uptake
was only 50.43%. Receptor competition studies proved that the
cellular uptake of Fru-RGD-Lip and FruþRGD-Lip was mediated by
GLUT5 and the integrin avb3. Chlorpromazine, filipin and amiloride
hydrochloride [44e46] were chosen as the inhibitors of clathrin-
mediated, caveolin-mediated and macropinocytosis endocytosis.
For Fru-RGD-Lip, the cellular uptake was inhibited by the three
inhibitors, while for FruþRGD-Lip, only chlorpromazine and ami-
loride hydrochloride showed inhibition. Sodium azide and tem-
perature can significantly affect the uptake of liposomes, especially
the low temperature, the relative uptake rate dropped below 20%,
indicating they were energy-dependent. In conclusion, the mech-
anism demonstrated that the cellular uptake of both dual-targeting
liposomes was likely to be recognized and mediated by GLUT5 and
avb3, and endocytosed through comprehensive pathways with
energy-dependent.
The single modified liposome DiD-Fru-Lip and DiD-RGD-Lip also
improved a little compared to unmodified liposomes. The five li-
posomes reached the maximum accumulation in tumor at 12 h
after injection, and the semi-quantitative data also showed the
fluorescence intensity of DiD-Fru-RGD-Lip, DiD-FruþRGD-Lip, DiD-
Fru-Lip and DiD-RGD-Lip were 2.62, 1.85, 1.68 and 1.56 times of
DiD-Lip respectively. After 24 h, the dual-targeting liposomes
remaining at the tumor site were more than other groups. More-
over, the liposomes mainly accumulated in liver, the main meta-
bolic organs, and reached the peak concentration at 6 h while most
of them were eliminated at 24 h. And other organs were less
enriched. These results indicated that the tumor targeting ability of
the five liposomes was Fru-RGD-Lip > FruþRGD-Lip
> Fru-
Lip > RGD-Lip > Lip, which was almost consistent with the in vitro
targeting results.
It should be pointed out that the targeting ability of RGD-Lip and
FruþRGD-Lip didn't exhibit as excellent as in vitro, which may be
that RGD-modified liposomes are more easily taken up by the liver
[47e49], resulting in a slightly less enrichment at the tumor site. It
can be proved that liposomes co-modified with fructose and RGD,
especially covalently modified liposomes, can further significantly
enhance the ability of liposomes to target TNBC based on singly
modified liposomes. The improved targeting ability would effi-
ciently enhance the therapeutic effect and reduce unwanted side
effects to normal tissues.
2.9. In vivo imaging
The tumor targeting ability of five liposomes in vivo was further
investigated in a tumor-bearing mouse model established by sub-
cutaneous inoculation of 1 ꢂ 106 cells in the right flank. The in vivo
images of 4T1-bearing Balb/C were evaluated at different time
points after systemic administration of DiD-labeled liposomes. As
displayed in Fig. 7, DiD-Fru-RGD-Lip showed the strongest tumor
targeting ability at each time point, followed by DiD-FruþRGD-Lip.
3. Conclusion
Two types of novel dual-targeting TNBC liposomes (Fru-RGD-Lip
and FruþRGD-Lip) that actively recognize both fructose transporter
GLUT5 and integrin avb3 were designed and prepared in this work.

6
Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
Fig. 5. Quantitative cellular uptake of CFPE-labeled liposomes on MDA-MB-231 cells (A) and 4T1 cells (B) determined by flow cytometer (n ¼ 3, mean SD; *P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001 versus CFPE-Lip). Qualitative cellular uptake of CFPE-labeled liposomes on MDA-MB-231 cells (C) and 4T1 cells (D) determined by CLSM, green (CFPE-
labeled liposomes), blue (DAPI stained nucleus), light blue (colocalized CFPE and DAPI). Scale bars represent 10
the reader is referred to the Web version of this article.)
mm. (For interpretation of the references to color in this figure legend,
The dual-targeting liposomes were not only co-modified by Fru-
chol and RGD-chol in a physical mixing manner (FruþRGD-Lip),
but also innovatively modified by the covalently linked Y-shaped
Fru-RGD-chol ligand (FruþRGD-Lip). Fru-RGD-Lip and FruþRGD-
Lip exhibited a variety of attractive properties such as improved
cellular internalization, identified cellular uptake pathways,
enhanced anti-proliferation effects on tumor cells, and surprisingly
efficient accumulation at the tumor site. Besides, Fru-RGD-Lip
showed much better targeting ability than FruþRGD-Lip, making
it a superior multifunctional liposome. Moreover, Fru-RGD-lip may
40-6-diamidino-2-pheylindole (DAPI) and 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
from Beyotime Institute Biotechnology (Haimen, China). 1,10-Dio-
ctadecyl-3,3,30,30-tetramethyl indodicarbocyanine perchlorate
(DiD) were purchased from Solarbio (Being, China). Sodium azide,
amiloride, chlorpromazine and filipin were obtained from Sigma
Aldrich (St. Louis, MO, USA). Paclitaxel (PTX)was obtained from
National Institute for Food and Drug Control. All liquid reagents
were distilled before use. The other chemicals and reagents were
obtained from commercial sources. Kunming mice and Balb/C mice
were purchased from Chengdu Dashuo Experimental Animal Co.,
Ltd (Chengdu, China).
The melting point was measured on a YRT-3 melting point
apparatus (Shantou Keyi Instrument & Equipment Co. Ltd., Shan-
tou, China). 1H NMR spectra were taken on a Varian INOVA 400 or
600 (Varian, Palo Alto, CA) using CD3Cl and CD3OD as solvent. High-
resolution mass spectroscopy data of the product were collected on
a Waters Micromass GCT or a Bruker Apex IV FTMS instrument.
Optical rotation was obtained from Rudolph Research Analytical
Autopol VI automatic polarimeter. Reversed-phase chromatog-
raphy performed on C18 chromatographic analysis was carried out
also be applied to other tumors that overexpress GULT5 and avb3
.
The idea of dual-targeting liposomes reported in this work could
also provide new insight in the development of other novel tar-
geted drug delivery system.
4. Experimental section
4.1. 1General
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy-
fluorescein) (CFPE) were purchased from Avanti Polar Lipids (USA).

Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
7
Fig. 6. The relative uptake of CFPE-Fru-RGD-Lip on MDA-MB-231 cells (A) and 4T1 cells (B); The relative uptake of CFPE-FruþRGD-Lip on MDA-MB-231 cells (C) and 4T1 cells (D).
(mean SD, n ¼ 3; *P < 0.05, **P < 0.01, ***P < 0 0.001, ****P < 0.0001 versus control).
using the high-performance liquid chromatography (HPLC) system
(Shimadzu, Kyoto, Japan). Particle size and potential were deter-
mined using Sizer Nano ZS90 laser particle size and Zeta potential
analysis (Malvern, UK). Cellular uptake was detected by flow
cytometry (BD FACS Celesta, USA) and laser confocal microscopy
(Carl Zeiss LSM800, Germany); in vivo imaging experiment was
conducted by small animal in vivo imager (PerkinElmer IVIS Lumina
III, USA).
9.23 mmol) and 4-dimethylaminopyridine (DMAP, 94 mg,
0.77 mmol) were dissolved in a mixed solution of toluene (30 mL)
and triethylamine (1 mL) under argon. And the reaction was
refluxed at 110 ꢁC for 2.5 h. Then, toluene was removed, and the
residue was dissolved in a mixture of CH2Cl2 (38 mL) and H2O
(54 mL), and pH was adjusted to neutral with acetic acid. The
mixture was extracted with CH2Cl2 (40 mL ꢂ 3), and the organic
phase was concentrated to give 3 as a colorless oil (2.69 g, 97%). 1H
NMR (600 MHz, CDCl3, ppm) d: 1.34 (s, 3H), 1.41 (s, 3H), 1.48 (s, 3H),
1.54 (s, 3H), 2.70 (s, 4H), 3.75e3.78 (m, 1H), 3.89e3.92 (m, 1H),
4.06e4.09 (m, 1H), 4.23e4.25 (m, 1H), 4.31 (s, 1H), 4.42e4.45 (m,
1H), 4.60e4.61 (m, 1H). [ a ]D20: ꢀ23.0 (c 0.5, CHCl3).
4.2. Chemistry
4.2.1. Synthesis of compound 2
The anhydrous -fructose 1 (2.00 g, 11.11 mmol) was added to a
D
mixed solution of anhydrous acetone (39 mL) and sulfuric acid
(1.95 mL) at 0 ꢁC. And the reaction solution was stirred at room
temperature (r.t.) for 2 h. Then, the mixture was neutralized by
aqueous sodium hydroxide (0.10 g, 152 mmol, 55 mL) and filtered.
The filtrate was concentrated and re-dissolved in CH2Cl2 (100 mL),
followed by washed with H2O (100 mL ꢂ 2) and saturated NaCl
(100 mL ꢂ 3). The organic layer was dried over anhydrous Na2SO4
and concentrated. The residue was purified by recrystallized with
petroleum ether to give 2 as a white solid (2.10 g, 73%). Mp:
94e96 ꢁC (literature Mp [50]: 95e96 ꢁC). [ a ]2D0: ꢀ22.0 (c 0.5,
CHCl3).
4.2.3. Synthesis of compound 5e6
The synthesis of compound 5e6 was reported in our previous
work [34].
4.2.4. Synthesis of compound 7
To a solution of N-Boc-N0-Fmoc-L-lysine (5.00 g, 10.67 mmol) in
CH2Cl2 (25 mL), was added dicyclohexylcarbodiimide (DCC, 2.93 g,
14.23 mmol) and DMAP (174 mg, 1.42 mmol), and the reaction was
stirred at ꢀ5 ꢁC for 30 min. Then a solution of compound 6 (3.69 g,
7.11 mmol) in CH2Cl2 (5 mL) was slowly added dropwise to the re-
action. The reaction was stirred at r. t. for 8 h. After the by-product
was filtrated and the filtrate was concentrated, compound 7 was
obtained by silica gel column chromatography (petroleum/
4.2.2. Synthesis of compound 3
Compound 2 (2.00 g, 7.69 mmol), succinic anhydride (923 mg,
Acetone ¼ 8/1) as
a
pale yellow solid (6.41 g, 93%).1H NMR

8
Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
Fig. 7. (A) In vivo images of the 4T1-bearing Balb/C mice at 2 h, 6 h, 12 h, 16 h and 24 h after systemic administration of DiD-labeled liposomes. (B) Ex vivo images of tumors. (C)
Semi-quantitative fluorescence intensity of isolated tumors; (D) Ex vivo images of isolated organs, and they were heart, liver, spleen, lung and kidney from left to right. (a ¼ DiD-Lip,
b ¼ DiD-Fru-Lip, c ¼ DiD-RGD-Lip, d ¼ DiD-Fru-RGD-Lip, e ¼ DiD-FruþRGD-Lip. n ¼ 3, mean SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.)
(400 MHz, CDCl3, ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91
4.2.5. Synthesis of compound 8
(d, 3H, J ¼ 6.4 Hz), 0.98 (s, 3H), 1.43 (s, 9H), 0.85e2.36 (remaining
cholesterol & Lys protons), 3.10e3.19 (m, 3H), 3.62e3.64 (m, 8H),
3.71 (t, 2H, J ¼ 4.6 Hz), 4.22 (t, 1H, J ¼ 6.8 Hz), 4.30e4.31 (m, 2H),
4.35e4.44 (m, 3H), 5.32e5.33 (m, 1H), 7.32 (t, 2H, J ¼ 7.2 Hz), 7.40 (t,
2H, J ¼ 7.2 Hz), 7.61 (d, 2H, J ¼ 7.2 Hz), 7.77 (d, 2H, J ¼ 7.2 Hz). HRMS
To a solution of compound 7 (2.58 g, 2.66 mmol) in CH2Cl2
(20 mL) was added 1,8-diazabicycloundec-7-ene (DBU, 1.194 mL,
7.98 mmol), and the reaction was stirred at r. t. for 20 min. The
mixture was washed with H2O (50 mL ꢂ 3) and saturated NaCl
(100 mL ꢂ 2) successively, and the organic layer was concentrated
and purified by silica gel column chromatography (CH2Cl2/
CH3OH ¼ 15/1) to give compound 8 as a pale yellow solid (1.85 g,
(ESI) calculated for
C
59H88N2NaOþ9 [MþNa]þ-991.6382, found
991.6388. [ a ]D20: ꢀ12.6 (c 0.5, CHCl3).
93%).1H NMR (400 MHz, CDCl3, ppm)
d: 0.67 (s, 3H), 0.86 (d, 6H,

Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
9
J ¼ 6.4 Hz), 0.91 (d, 3H, J ¼ 6.4 Hz), 0.99 (s, 3H), 1.43 (s, 9H),
0.85e2.38 (remaining cholesterol & Lys protons), 3.12e3.21 (m,
3H), 3.63e3.66 (m, 8H), 3.70e3.75 (m, 4H), 4.14e4.15 (m, 1H),
4.31e4.43 (m, 2H), 5.34 (s, 1H), 8.69 (s, 2H). [ a ]D20: ꢀ7.6 (c 0.5,
CHCl3).
4.2.9. Synthesis of compound Fru-RGD-chol
Compound 11 (100 mg, 0.06 mmol) was added to a mixed sol-
vent of TFA (3 ml) and H2O (3 ml), and the reaction was stirred at r. t
for 24 h. Then TFA was removed, and the residue was diluted with
water and extracted with CH2Cl2 (30 mL ꢂ 3). The organic layer was
washed with saturated NaCl (30 mL ꢂ 2) and concentrated to give a
pale yellow semi-solid (86 mg). The pale yellow semi-solid was
further dissolved in of methanol (10 mL), and 10% Pd/C (10 mg) was
added. The reaction was stirred under a hydrogen pressure (0.4
Mpa) at 45 ꢁC for 48 h. Then, Pd/C was removed by filtration, and
the solvent in filtrate was removed. The residue was washed with
diethyl ether (5 mL ꢂ 5) to remove the small polar impurity
(10 mL ꢂ 5) to give a white solid (21.86 mg). 1H NMR (400 MHz,
4.2.6. Synthesis of compound 9
4-methylmorpholin (NMM, 0.127 mL, 1.15 mmol) and isobutyl
chloroformate (IBCF, 0.151 mL, 1.15 mmol) were added to a solution
of compound 8 (900 mg, 1.34 mmol) in CH2Cl2 (10 mL), and the
reaction was stirred at ꢀ5 ꢁC for 30 min. Then, a solution of com-
pound 21 (715 mg, 0.96 mmol) in CH2Cl2 (10 mL), was slowly added
dropwise. The reaction was stirred at r. t. overnight. The mixture
was washed with hydrochloric acid (1 N, 50 mL ꢂ 2) and saturated
NaCl (50 mL ꢂ 2). The organic layer was concentrated and purified
by silica gel column chromatography (CH2Cl2/CH3OH ¼ 50/1) to
give compound 9 as a pale yellow foamy solid (1.09 g, 81%). 1H NMR
CD3OD, ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.8 Hz), 0.91 (d, 3H,
J ¼ 6.4 Hz), 0.98 (s, 3H), 0.86e2.37 (remaining cholesterol and Lys
protons), 2.50e2.79 (m, 10H), 2.90e3.05 (m, 2H), 3.17e3.20 (m,
2H), 3.62e3.76 (m, 12H), 3.77e3.94 (m, 5H), 4.18e4.35þ(m, 8H), 4.61
(br, 1H). HRMS (ESI) calculated for C66H110N8NaO21 [MþNa]þ-
1361.7678, found 1361.7674.
(400 MHz, CDCl3, ppm)
d: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91
(d, 3H, J ¼ 6.4 Hz), 0.98 (s, 3H), 1.41 (s, 9H), 0.85e2.37 (remaining
cholesterol & Lys protons & Arg protons), 2.54 (s, 4H), 2.78 (br, 2H),
3.05 (s, 2H), 3.13e3.21 (m, 1H), 3.29 (s, 2H), 3.61e3.63 (m, 8H), 3.67
(s, 2H), 3.93 (s, 2H), 4.19e4.28 (m, 2H), 4.44 (s, 2H), 4.91 (br, 1H),
5.04 (s, 2H), 5.09e5.10 (m, 2H), 5.32e5.33 (m, 1H), 7.25e7.33 (m,
10H). [ a ]D20: ꢀ6.2 (c 0.5, CHCl3).
4.2.10. Synthesis of compound 12
To a solution of compound 3 (1.04 g, 2.89 mmol) in CH2Cl2
(30 mL), were added DCC (795 mg, 3.86 mmol) and DMAP (47 mg,
0.39 mmol), and the reaction was stirred at ꢀ5 ꢁC for 30 min. A
solution of compound 6 (1.00 g, 1.93 mmol) in CH2Cl2 (20 mL) was
added dropwise and the reaction was stirred at r. t for 3 h. The
solvent was removed, and the residue was re-dissolved by ethyl
acetate (50 mL), and then filtrated. The filtrate was concentrated
and purified by silica gel column chromatography (petroleum
ether/ethyl acetate ¼ 5/1) to give compound 12 as a pale yellow
4.2.7. Synthesis of compound 10
Compound 9 (1.092 g, 0.78 mmol) was added to a mixture of
trifluoroacetic acid (TFA, 3 mL) and CH2Cl2 (9 mL), and the reaction
was stirred at r. t. for 20 min. The solvent was removed and the
residue was re-dissolved with CH2Cl2 (50 mL), followed by washed
with H2O (50 mL ꢂ 1), saturated NaHCO3 (50 mL ꢂ 1), and saturated
NaCl (50 mL ꢂ 2). The organic layer was concentrated to give
compound 10 as a light yellow solid (0.999 g, 99%). 1H NMR
viscous oil (1.78 g, 77%). 1H NMR (400 MHz, CDCl3, ppm)
d: 0.67 (s,
3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91 (d, 3H, J ¼ 6.4 Hz), 0.99 (s, 3H), 1.34
(s, 3H), 1.41 (s, 3H), 1.48 (s, 3H), 1.54 (s, 3H), 0.86e2.39 (remaining
cholesterol protons), 2.69 (s, 4H), 3.14e3.22 (m, 1H), 3.64e3.66 (m,
8H), 3.69e3.72 (m, 2H), 3.75e3.78 (m, 1H), 3.89e3.93 (m, 1H),
4.05e4.08 (m, 1H), 4.23e4.27 (m, 3H), 4.31 (d, 1H, J ¼ 2.6 Hz),
4.42e4.44 (m, 1H), 4.59e4.62 (m, 1H), 5.34 (s, 1H). HRMS (ESI)
calculated for C49H80O12Naþ [MþNa]þ-883.5542, found 883.5550. [
a ]D20: ꢀ17.6 (c 0.5, CHCl3).
(400 MHz, CDCl3, ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91
(d, 3H, J ¼ 6.4 Hz), 0.98 (s, 3H), 0.86e2.36 (remaining cholesterol &
Lys protons & Arg protons), 2.56 (br, 4H), 2.94e3.20 (m, 5H), 3.27 (s,
2H), 3.62e3.65 (m, 10H), 3.92 (br, 2H), 4.25 (s, 2H), 4.42e4.55 (m,
2H), 4.90 (br, 1H), 5.04e5.09 (m, 4H), 5.33 (s, 1H), 7.28e7.32 (m,
10H). [ a ]D20: ꢀ6.2 (c 0.5, CH3OH).
4.2.11. Synthesis of Fru-chol
Compound 12 (3.49 g, 4.00 mmol) was added to a mixed solvent
of TFA (20 mL) and H2O (20 mL), and the reaction was stirred at r. t.
for 24 h. The TFA was removed, then the mixture was neutralized
with Na2CO3, and extracted by Ethyl acetate (50 mL ꢂ 3). The
organic layer was concentrated and purified by silica gel column
chromatography (petroleum ether/ethyl acetate ¼ 3/1) to give a
pale yellow viscous oil (1.197 g, 38%).1H NMR (400 MHz, CDCl3,
4.2.8. Synthesis of compound 11
To a solution of compound 3 (415 mg, 1.15 mmol) in CH2Cl2
(10 mL), were added N,N-diisopropylethylamine (DIPEA, 0.38 mL,
2.30 mmol), 1-(3-dimethylaminopropyl) 3-ethylcarbodiimide hy-
drochloride (EDCI, 294 mg, 1.54 mmol) and DMAP (188 mg,
1.54 mmol), and the reaction was stirred at ꢀ5 ꢁC for 30 min. A
solution of compound 10 (999 mg, 0.77 mmol) in CH2Cl2 (10 mL)
was added dropwise, and the mixture was stirred at r. t. overnight.
The mixture was washed with HCl solution (1 N, 30 mL ꢂ 2) and
saturated NaCl (30 mL ꢂ 2). The organic layer was concentrated,
and the residue was purified by silica gel column chromatography
CH2Cl2/CH3OH ¼ 8/1) to give compound 11 as a white foamy solid
ppm)
d
: 0.67 (s, 3H), 0.86 (d, 6H, J ¼ 6.4 Hz), 0.91 (d, 3H, J ¼ 6.4 Hz),
1.00 (s, 3H), 0.86e2.38 (remaining cholesterol protons), 2.68 (s, 4H),
3.16e3.21 (m, 1H), 3.42e3.50 (m, 7H), 3.62e3.69 (m, 10H),
4.25e4.36 þ(m, 4H), 5.34 (s, 1H). HRMS (ESI) calculated for
C
43H72NaO12[MþNa]þ- 803.4916, found 803.4912.
(977 mg, 77%). 1H NMR (400 MHz, CDCl [3], ppm)
d
: 0.67 (s, 3H),
4.3. Preparation and characterization of liposomes
0.86 (d, 6H, J ¼ 6.6 Hz), 0.91 (d, 3H, J ¼ 6.2 Hz), 0.98 (s, 3H), 1.33 (s,
3H), 1.39 (s, 3H), 1.46 (s, 3H), 1.52 (s, 3H), 0.86e2.36 (remaining
cholesterol & Lys protons & Arg protons), 2.51e2.75 (m, 8H),
2.90e3.06 (m, 2H), 3.13e3.33 (m, 5H), 3.62e3.64 (m, 8H),
3.66e3.69 (m, 2H), 3.72e3.76 (m, 1H), 3.87e3.93 (m, 3H),
3.99e4.02 (m, 1H), 4.22e4.26 (m, 3H), 4.30e4.31 (m, 1H),
4.42e4.47 (m, 3H), 4.59e4.61 (m, 1H), 4.88e4.91 (m, 1H), 5.02e5.14
(m, 4H), 5.33 (s, 1H), 7.28e7.34 (m, 10H). HRMS (ESI) calculated for
We selected the optimal prescription: cholesterol: soybean
phospholipid (SPC): ligand ¼ 33:64:3 (molar ratio), and lipid:
paclitaxel ¼ 30:1 (quality ratio), hydration solution was PBS
(pH ¼ 7.4). Briefly, the prescribed amount of lipid material and PTX
were accurately weighed to be dissolved with chloroform-
methanol mixed solution (V/V ¼ 2/1). And then the organic sol-
vent was removed by rotary evaporation at 37 ꢁC to give a complete
thin film. The thin film was hydrated in PBS for 0.5 h at 20 ꢁC.
Finally, it was further sonicated intermittently (80 W, 5S, 5S) for
C
85H127N9NaOþ23 [MþNa]þ-1664.8937, found 1664.8939. Mp:
46e48 ꢁC.[ a ]D20: ꢀ6.0 (c 0.5, CHCl3).

10
Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
3 min to give slightly milky liposome solution. Likewise, CFPE-
labeled liposomes or DiD-labeled liposomes were prepared with
appropriate amount of CFPE or DiD added to the solution before
removing the solvent.
The encapsulation efficiency of PTX-loaded liposomes was
detected by HPLC after the unencapsulated free paclitaxel and PTX-
loaded liposomes were separated by cryo-centrifugation [51]. The
encapsulation efficiency (EE%) of the PTX-loaded liposome was
calculated according to the formula: EE% ¼ A after centrifugation/A before
centrifugation ꢂ 100%. After the prepared liposome was diluted to a
suitable concentration with ultrapure water, the particle size and
zeta potential of the liposome were measured by Malvern Zeta sizer
Nano ZS90.
DMSO (150
measured at 570 nm. The cell survival rate was calculated as: cell
and OD
sample represented the value of the cells treated with blank medium
and various drugs, respectively.
ml). The optical density absorption value (OD) was
Viability (%) ¼ OD sample/OD control ꢂ 100%. The OD
control
4.8. Cellular uptake
MDA-MB-231 cells and 4T1 cells were seeded in a 12-well plate
at a density of 1 ꢂ 105 cells/well and 2 ꢂ 105 cells/well respectively,
and cultured to reach 80% confluence. The CFPE-labeled liposomes
were added with a lipid concentration at 0.3
mmol/mL and a CFPE
concentration at 2 g/mL. After being incubated for 2 h, the cells
m
were washed twice with pre-cooled PBS, followed by trypsinized
and collected. Finally, the cells were resuspended in PBS, and the
fluorescence intensity was measured by flow cytometry.
4.4. In vitro drug release study
In vitro drug release of different PTX-loaded liposomes was
investigated by dialysis method. 0.4 mL PTX-loaded liposomes or
free PTX were placed in a dialysis bag of 8000e12000 Da. Then, the
dialysis bag was sealed tightly and placed in 40 mL dialysis medium
(1% Tween 80 in PBS, v/v). After being incubated at 37 ꢁC with
gently waggling, 0.1 ml release medium of each group was taken
out at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h, to be analyzed
by HPLC as the previous work described [28].
For confocal microscope studies, MDA-MB-231 and 4T1 cells
were seeded at a density of 5 ꢂ 105 or 1 ꢂ 106 cells/well in a 6-well
plate pre-coated with cover glasses, and cultured for 24 h. Then,
CFPE-labeled liposomes were added to incubate for 2 h. After that,
cells were fixed with 4% paraformaldehyde for 30 min and stained
with 5 mg/mL DAPI for 5 min. The samples were sealed with glyc-
erin and imaged with a laser confocal microscope.
4.9. Uptake mechanism
4.5. In vitro stability of liposomes in serum
In order to illuminate the uptake mechanism of Fru-RGD-Lip
and FruþRGD-Lip, MDA-MB-231 cells and 4T1 cells were pre-
incubated with receptor competition inhibitors (10 g/L fructose or
Each group of PTX-loaded liposomes were mixed with an equal
volume of FBS and co-incubated at 37 ꢁC. Then, the transmittance of
the sample was measured at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h
by a microplate reader at 750 nm. The control group was deter-
mined by the transmittance measured immediately after the lipo-
somes were mixed with the PBS, whose transmittance was
regarded as 100%.
200
mg/mL RGD), endocytosis inhibitors (10 mg/mL chlorpromazine,
1
m
g/mL filipin or 2 mg/mL amiloride hydrochloride), and energy
inhibitor (1 mg/mL NaN3) at 37 ꢁC for 30 min. Then, the CFPE-
labeled liposomes were added to incubate for another 2 h. The
low temperature group was set up, which was incubated for 2 h at
4 ꢁC. Finally, the cells were treated as described in 4.8 and measured
by flow cytometry.
4.6. Hemolysis assays
The blood of 18e22 g Kunming mice was taken from the eyelids
and collected in a centrifuge tube coated with heparin sodium, and
centrifuged at 4 ꢁC (10000 rpm ꢂ 10 min) to discard the superna-
tant. And then the red blood cells (RBCs) were washed with PBS
until the supernatant was colorless. Subsequently, the RBCs were
resuspended in PBS to the concentration of 2% (w/v). The PTX-
loaded liposomes were gradually diluted with PBS to give lipid
4.10. In vivo imaging
4T1 cells were injected into female Balb/C mice to establish a
tumor model. After 14 days, the mice were given DiD-labeled li-
posomes through tail vein injection at a dose of 500 mg/kg DiD.
After administration of 2 h, 6 h, 12 h, 16 h and 24 h, each group of
mice were anesthetized with 4% chloral hydrate and imaged with
IVIS Lumina Series III imaging system. And then, the heart, liver,
spleen, lung, kidney and tumor were taken out immediately after
the mice were sacrificed by heart perfusion, and also imaged. The
average fluorescence intensity of the ex vivo tumors were calcu-
lated by the Image software.
concentrations of 800, 400, 300, 200, 150, 75, 50, 25, 10 mmol/L.
Finally, various concentrations of liposomes were co-incubated
with an equal volume of 2% RBCs at 37 ꢁC for 1 h. After being
centrifuged at 10,000 rpm for 10 min, the absorbance of the su-
pernatant was detected with a microplate reader at 540 nm. The
positive control and the negative control were determined by co-
incubation of RBCs with 1% Triton X-100 and PBS, whose hemoly-
sis rate was set to 100% and 0% respectively. The hemolysis rate of
each group of liposomes was calculated as: percent hemolysis % ¼
(A sample - A negative)/(A positive - A negative) ꢂ 100%.
4.11. Statistical analysis
Data was analyzed by the Graphpad Prism 6 software. Statistical
comparisons were performed by one-way ANOVA for multiple
groups. The results were analyzed by student's t-test. Significant
differences between or among groups were indicated by *P < 0.05,
**P < 0.01, ***P < 0.001, ****P < 0.0001.
4.7. Cytotoxicity
MDA-MB-231 cells or 4T1 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. The
PTX-loaded liposomes or free PTX were diluted to a PTX concen-
Acknowledgement
tration of 20, 5, 2, 0.5 0.1 and 0.02
After being incubated for 24 h, the medium was discarded and
replaced by 200 L of serum-free medium containing MTT (0.5 mg/
mL), and the plates were further incubated for 4 h. Finally, the
medium was removed, and the reduced MTT dye was solubilized by
m
mol/L, and added to the plates.
This work was supported by the National Natural Science
Foundation of China (No. 81573286,
& No. 81773577 &
m
No.81903448); the Sichuan Science and Technology Program
(2018JY0537); the Sichuan Youth Science and Technology Innova-
tion Research Team Funding (2016TD0001); the Fundamental

Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
11
6-deoxy-6-[F]fluoro-fructose as a radiotracer for PET imaging of GLUT5 in
breast cancer, Nucl. Med. Biol. 38 (4) (2011) 461e475, https://doi.org/
10.1016/j.nucmedbio.2010.11.004.
Research Funds for the Central Universities (2012017yjsy211); and
the Open Research Subject of Healthy, Xihua University (szjj2017-
036).
ꢀ
[20] S. Kannan, V.V. Begoyan, J.R. Fedie, S. Xia, Ł.J. Weselinski, M. Tanasova, S. Rao,
Metabolism-driven high-throughput cancer identification with GLUT5-
specific molecular probes, Biosensors 8 (2) (2018) 39e50, https://doi.org/
10.3390/bios8020039.
Appendix A. Supplementary data
[21] Z. Jiacheng, B. Krzysztof, L. Hongxu, D. Aydan, G. Michael, M.H. Stenzel,
Fructose-coated nanoparticles:
negative breast cancer therapy, Chem. Commun. 50 (100) (2014)
15928e15931, https://doi.org/10.1039/C4CC06651K.
a promising drug nanocarrier for triple-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111720.
[22] R.O. Hynes, Integrins: a family of cell surface receptors, Cell 48 (4) (1987)
549e554, https://doi.org/10.1016/0092-8674(87)90233-9.
[23] M.J. Humphries, Integrin structure, Biochem. Soc. Trans. 28 (4) (2000)
311e339, https://doi.org/10.1042/0300-5127:0280311.
References
[24] A. Duro-Castano, E. Gallon, C. Decker, M.J. Vicent, Modulating angiogenesis
with integrin-targeted nanomedicines, Adv. Drug Deliv. Rev. (2017) 101e119,
https://doi.org/10.1016/j.addr.2017.05.008.
[25] P. Carmeliet, Mechanisms of angiogenesis and arteriogenesis, Nat. Med. 6 (4)
(2000) 389e395, https://doi.org/10.1038/74651.
[26] B. Katja, M. Claudia, B. Jürgen, Expression profiling reveals genes associated
with transendothelial migration of tumor cells: a functional role for alphav-
beta3 integrin, Int. J. Cancer 121 (9) (2010) 1910e1918, https://doi.org/
10.1002/ijc.22879.
[27] K. Wu, C. Ru, Z. Jian, F. Meng, D. Chao, Z. Zhong, Micellar nanoformulation of
lipophilized bortezomib: high drug loading, improved tolerability and tar-
geted treatment of triple negative breast cancer, J. Mater. Chem. B 5 (28)
(2017) 5658e5667, https://doi.org/10.1039/C7TB01297G.
[1] A.M. Brewster, C.M. Mariana, B. Powel, Epidemiology, biology, and treatment
of triple-negative breast cancer in women of African ancestry, Lancet Oncol.
15 (13) (2014) 625e634, https://doi.org/10.1016/S1470-2045(14)70364-X.
[2] J.S. Reis-Filho, A.N.J. Tutt, Triple negative tumours: a critical review, Histo-
pathology 52 (1) (2010) 108e118, https://doi.org/10.1111/j.1365-
2559.2007.02889.x.
[3] E.A. Rakha, I.O. Ellis, Triple-negative/basal-like breast cancer: review, Pathol-
ogy 41 (1) (2009) 40e47, https://doi.org/10.1080/00313020802563510.
[4] O. Catherine, V. Giuseppe, D.L. Angelo, Management of triple negative breast
cancer, Breast 19 (5) (2010) 312e321, https://doi.org/10.1016/
j.breast.2010.03.026.
[5] A.J. Primeau, R. Augusto, H. David, L. Lothar, I.F. Tannock, The distribution of
the anticancer drug Doxorubicin in relation to blood vessels in solid tumors,
Clin. Cancer Res. 11 (1) (2005) 8782e8788, https://doi.org/10.1158/1078-
0432.CCR-05-1664.
[6] E. Bernabeu, L. Gonzalez, M. Cagel, E.P. Gergic, M.A. Moretton, D.A. Chiappetta,
Novel Soluplus(®)-TPGS mixed micelles for encapsulation of paclitaxel with
enhanced in vitro cytotoxicity on breast and ovarian cancer cell lines, Colloids
[28] Q. Fu, Y. Zhao, Z. Yang, Q. Yue, W. Xiao, Y. Chen, Y. Yang, L. Guo, Y. Wu, Li-
posomes actively recognizing the glucose transporter GLUT1 and integrin
alphav beta3 for dual-targeting of glioma, Arch. Pharm. 352 (2) (2019)
1800219e1800229, https://doi.org/10.1002/ardp.201800219.
[29] Y. Xue, W. He, L. Wan-Liang, D. Ju, G. Jia, T. Wei, M. Ying, Z. Yan, L. Ruo-Jing,
Y. Ting-Yuan, Dual-targeting daunorubicin liposomes improve the therapeutic
efficacy of brain glioma in animals, J. Control. Release Off. J. Control. Release
Soc. 141 (2) (2010) 183e192, https://doi.org/10.1016/j.jconrel.2009.09.020.
Surfaces
B Biointerfaces 140 (2016) 403e411, https://doi.org/10.1016/
j.colsurfb.2016.01.003.
[7] A.S. Abu Lila, T. Ishida, Liposomal delivery systems: design optimization and
current applications, Biol. Pharm. Bull. 40 (1) (2017) 1e10, https://doi.org/
10.1248/bpb.b16-00624.
[8] S. Huaming, N. Ben, S. Hongbin, Metabolic targeting of cancers: from molec-
ular mechanisms to therapeutic strategies, Curr. Med. Chem. 16 (13) (2009)
1561e1587, https://doi.org/10.2174/092986709788186255.
[9] X.D. Xu, S.X. Shao, H.P. Jiang, Y.W. Cao, Y.H. Wang, X.C. Yang, Y.L. Wang,
X.S. Wang, H.T. Niu, Warburg effect or reverse Warburg effect? A review of
cancer metabolism, Oncol. Res. Treat. 38 (3) (2015) 117e122, https://doi.org/
10.1159/000375435.
[10] G. Gayatri, Z.K. Sabine, J. Anitha, R. Robert, L. Jelena, S. Ananth, B.J. Kjerstin,
C. John, D. Ludger, S.S. Gambhir, GLUT5 is not over-expressed in breast cancer
cells and patient breast cancer tissues, PLoS One 6 (11) (2011) 26902e26909,
https://doi.org/10.1371/journal.pone.0026902.
€
[30] A. Tatibouet, J. Yang, C. Morin, G.D. Holman, Synthesis and evaluation of
fructose analogues as inhibitors of the d-fructose transporter GLUT5, Bioorg.
Med. Chem. 8 (7) (2000) 1825e1833, https://doi.org/10.1016/S0968-0896(00)
00108-5.
[31] Y. Jing, D. James, T.T. Arnaud, H. Yasumaru, G.D. Holman, Development of
high-affinity ligands and photoaffinity labels for the D-fructose transporter
GLUT5, Biochem. J. 367 (2) (2002) 533e539, https://doi.org/10.1042/
bj20020843.
[32] J. Levi, C.O.G. Zhen, Fluorescent fructose derivatives for imaging breast cancer
cells, Bioconjug. Chem. 18 (3) (2007) 628e634, https://doi.org/10.1021/
bc060184s.
[33] J.P. Xiong, T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S.L. Goodman,
M.A. Arnaout, Crystal structure of the extracellular segment of integrin alpha
Vbeta3 in complex with an Arg-Gly-Asp ligand, Science 296 (5565) (2002)
151e155, https://doi.org/10.1126/science.1069040.
[34] B. Qu, X. Li, M. Guan, X. Li, L. Hai, Y. Wu, Design, synthesis and biological
evaluation of multivalent glucosides with high affinity as ligands for brain
targeting liposomes, Eur. J. Med. Chem. 72 (1) (2014) 110e118, https://
doi.org/10.1016/j.ejmech.2013.10.007.
[11] B.J. Trayner, T.N. Grant, F.G. West, C.I. Cheeseman, Synthesis and character-
ization of 6-deoxy-6-fluoro-d-fructose as a potential compound for imaging
breast cancer with PET, Bioorg. Med. Chem. 17 (15) (2009) 5488e5495,
https://doi.org/10.1016/j.bmc.2009.06.034.
ꢀ
ꢀ
[12] S.P. Zamora-Leon, D.W. Golde, I.I. Concha, C.I. Rivas, F.,. Delgado-Lopez,
J.,. Baselga, F.,. Nualart, J.C. Vera, Expression of the fructose transporter GLUT5
in human breast cancer, Proc. Natl. Acad. Sci. U.S.A. 93 (5) (1996) 1847e1852,
https://doi.org/10.2307/38426.
[35] Y. Peng, Y. Zhao, Y. Chen, Z. Yang, L. Zhang, W. Xiao, J. Yang, L. Guo, Y. Wu,
Dual-targeting for brain-specific liposomes drug delivery system: synthesis
and preliminary evaluation, Bioorg. Med. Chem. 26 (16) (2018) 4677e4686,
https://doi.org/10.1016/j.bmc.2018.08.006.
[13] C.A. Colville, M.J. Seatter, T.J. Jess, G.W. Gould, H.M. Thomas, Kinetic analysis of
the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xen-
opus oocytes: substrate specificities and effects of transport inhibitors, Bio-
chem. J. 290 (Pt3) (1993) 701e706, https://doi.org/10.1111/j.1745-
4603.1989.tb00424.x.
[14] T.,. Kayano, C.F. Burant, H.,. Fukumoto, G.W. Gould, Y.S. Fan, R.L. Eddy,
M.G. Byers, T.B. Shows, S. Seino, G.I. Bell, Human facilitative glucose trans-
porters. Isolation, functional characterization, and gene localization of cDNAs
encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle,
and adipose tissue and an unusual glucose transporter pseudogene-like,
J. Biol. Chem. 265 (22) (1990) 13276e13282, https://doi.org/10.0000/
PMID1695905.
[36] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Drug targeting to tumors:
principles, pitfalls and (pre-) clinical progress, J. Control. Release 161 (2)
(2012) 175e187, https://doi.org/10.1080/10717544.2018.1431978.
[37] E. Wisse, F. Braet, D. Luo, R.D. Zanger, D. Jans, E. Crabbe, A.N. Vermoesen,
Structure and function of sinusoidal lining cells in the liver, Toxicol. Pathol. 24
(1) (1996) 100e111, https://doi.org/10.1177/019262339602400114.
[38] K. Murugan, Y.E. Choonara, P. Kumar, D. Bijukumar, L.C.D. Toit, V. Pillay, Pa-
rameters and characteristics governing cellular internalization and trans-
barrier trafficking of nanostructures, Int. J. Nanomed. 10 (2015) 2191e2206,
https://doi.org/10.2147/IJN.S75615.
[39] Z. Lei, F. Yu, A.J. Cole, B. Chertok, A.E. David, J. Wang, V.C. Yang, Gum Arabic-
coated magnetic nanoparticles for potential application in simultaneous
magnetic targeting and tumor imaging, AAPS J. 11 (4) (2009) 693e699,
https://doi.org/10.1208/s12248-009-9151-y.
[40] R. Tong, J. Cheng, Anticancer polymeric nanomedicines, Polym. Rev. 47 (3)
(2007) 345e381, https://doi.org/10.1080/15583720701455079.
[41] K. Shi, J. Li, Z. Cao, P. Yang, Y. Qiu, B. Yang, Y. Wang, Y. Long, Y. Liu, Q. Zhang,
A pH-responsive cell-penetrating peptide-modified liposomes with active
[15] A.R. Manolescu, K. Witkowska, A. Kinnaird, T. Cessford, C. Cheeseman, Facil-
itated hexose transporters: new perspectives on form and function, Physi-
ology 22 (4) (2007) 234e240, https://doi.org/10.1152/physiol.00011.2007.
[16] I. Stuart W, T. Paul, Glucose transporters (GLUT and SGLT): expanded families
of sugar transport proteins, Br. J. Nutr. 89 (1) (2003) 3e9, https://doi.org/
10.1079/BJN2002763.
[17] X. Fan, H. Liu, M. Liu, Y. Wang, L. Qiu, Y. Cui, Increased utilization of fructose
has a positive effect on the development of breast cancer, Peerj 5 (2) (2017)
3804e3818, https://doi.org/10.7717/peerj.3804.
recognizing of integrin
avb3 for the treatment of melanoma, J. Control.
Release 217 (2015) 138e150, https://doi.org/10.1016/j.jconrel.2015.09.009.
[42] K. Bauer, C. Mierke, J. Behrens, Expression profiling reveals genes associated
with transendothelial migration of tumor cells: a functional role for alphav-
beta3 integrin, Int. J. Cancer 121 (9) (2007) 1910e1918, https://doi.org/
10.1002/ijc.22879.
[18] G. Alejandro, U. Viviana, R. Federico, R. Karin, A.J. Ya?Ez, M.D.L.A. García,
ꢀ
R.A. Medina, C. Monica, B. Sofía, C. Tamara, Differential subcellular distribution
of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural
localization of GLUT1 and GLUT5 in breast tumor tissues, J. Cell. Physiol. 207
(3) (2010) 614e627, https://doi.org/10.1002/jcp.20606.
[43] A. Srivatsan, M. Ethirajan, S.K. Pandey, S. Dubey, X. Zheng, T.H. Liu, M. Shibata,
J. Missert, J. Morgan, R.K. Pandey, Conjugation of cRGD peptide to chlorophyll
[19] M. Wuest, B.J. Trayner, T.N. Grant, H.S. Jans, J.R. Mercer, D. Murray, F.G. West,
A.J.B. Mcewan, F. Wuest, C.I. Cheeseman, Radiopharmacological evaluation of

12
Y. Pu et al. / European Journal of Medicinal Chemistry 183 (2019) 111720
a based photosensitizer (HPPH) alters its pharmacokinetics with enhanced
tumor-imaging and photosensitizing (PDT) efficacy, Mol. Pharm. 8 (4) (2011)
1186e1197, https://doi.org/10.1021/mp200018y.
cyclic RGD peptide for tumor targeting, J. Drug Target. 12 (5) (2004) 257e264,
https://doi.org/10.1080/10611860410001728040.
[48] Z. Hu, L.Y.P. Fang, Arg-Gly-Asp (RGD) peptide conjugated poly (lactic acid)-
poly(ethylene oxide) micelle for targeted drug delivery, J. Biomed. Mater.
Res. A 85A (3) (2010) 797e807, https://doi.org/10.1002/jbm.a.31615.
[49] X. Xiao-Bing, H. Yue, L. Wan-Liang, Z. Xuan, Z. Hua, N. Tsuneji, Z. Qiang,
Enhanced intracellular delivery and improved antitumor efficacy of doxoru-
[44] H.Y. Nam, S.M. Kwon, H. Chung, S.-Y. Lee, S.-H. Kwon, H. Jeon, Y. Kim, J.H. Park,
J. Kim, S. Her, Cellular uptake mechanism and intracellular fate of hydro-
phobically modified glycol chitosan nanoparticles, J. Control. Release 135 (3)
(2009) 259e267, https://doi.org/10.1016/j.jconrel.2009.01.018.
[45] M. Tamaru, H. Akita, T. Fujiwara, K. Kajimoto, H. Harashima, Leptin-derived
peptide, a targeting ligand for mouse brain-derived endothelial cells via
macropinocytosis, Biochem. Biophys. Res. Commun. 394 (3) (2010) 587e592,
https://doi.org/10.1016/j.bbrc.2010.03.024.
[46] X. Kai, L. Yuanpei, L. Juntao, J.S. Lee, X. Wenwu, A.M. Gonik, R.G. Agarwal,
K.S. Lam, The effect of surface charge on in vivo biodistribution of PEG-
oligocholic acid based micellar nanoparticles, Biomaterials 32 (13) (2011)
3435e3446, https://doi.org/10.1016/j.biomaterials.2011.01.021.
bicin by sterically stabilized liposomes modified with a synthetic RGD
mimetic, J. Control. Release 107 (2) (2005) 262e275, https://doi.org/10.1016/
j.jconrel.2005.03.030.
[50] M. NILSSON, Constituents of pollen, Acta Chem. Scand. 10 (3) (1956) 413e415.
[51] F. Han, S. Kairong, H. Guanlian, Y. Yuting, K. Qifang, L. Libao, Z. Li, C. Wenfei,
D. Mingling, C. Yantao, Tumor-targeted paclitaxel delivery and enhanced
penetration using TAT-decorated liposomes comprising redox-responsive
poly(ethylene glycol), J. Pharm. Sci. 104 (3) (2015) 1160e1173, https://
doi.org/10.1002/jps.24291.
[47] P.K. Dubey, V. Mishra, S. Jain, S. Mahor, S.P. Vyas, Liposomes modified with