Targeted Delivery of a Mannose-Conjugated BODIPY Photosensitizer by Nanomicelles for Photodynamic Breast Cancer Therapy

Article abstract of DOI:10.1002/chem.201702935

The targeted delivery of a photosensitizer (PS) with appropriate carriers represents an attractive means of selectively delivering cargo to target tissues or subcellular compartments for photodynamic therapy (PDT). Herein, a three-arm distyryl BODIPY derivative conjugated with mannose units (denoted by BTM) that can co-assemble with Tween 80 to form nanomicelles (BTM-NMs) for targeted PDT is reported. MDA-MB-231 breast cancer cells recognized and specifically internalized BTM-NMs via mannose-receptor-mediated endocytosis with preferential accumulation in the lysosomes. These NMs could disassemble in cell lysosomes and subsequently induce highly efficient singlet oxygen (¹O₂) generation upon light irradiation. ¹O₂ disrupted the lysosomal membrane and promoted the escape of BTM from the lysosome into the cytoplasm, thereby resulting in the efficient and selective killing of cancer cells through PDT. This study may provide a new strategy for designing targeted PDT systems to fight cancer.

Full text of DOI:10.1002/chem.201702935

A Journal of
Accepted Article
Title: Targeted Delivery of Mannose-Conjugated BODIPY
Photosensitizer by Nanomicelles for Photodynamic Breast
Cancer Therapy
Authors: Jian Yin, Quan Zhang, Ying Cai, Qiu-Yan Li, Lin-Na Hao,
Zheng Ma, and Xiao-Jun Wang
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201702935
Link to VoR: http://dx.doi.org/10.1002/chem.201702935
Supported by

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Targeted Delivery of Mannose-Conjugated BODIPY
Photosensitizer by Nanomicelles for Photodynamic Breast
Cancer Therapy
Quan Zhang,[a] Ying Cai,[a] Qiu-Yan Li,[b] Lin-Na Hao,[a] Zheng Ma,[b] Xiao-Jun Wang,*[b] and Jian Yin*[a]
Abstract: The targeted delivery of a photosensitizer (PS) with
appropriate carriers represents an attractive means of selectively
delivering cargo to target tissues or subcellular compartments for
photodynamic therapy (PDT). Herein, we report a three-arm distyryl
BODIPY derivative conjugated with mannose units (denoted by
BTM) that can co-assemble with Tween 80 to form nanomicelles
(BTM-NMs) for targeted PDT. MDA-MB-231 breast cancer cells
recognized and specifically internalized BTM-NMs via mannose
receptor-mediated endocytosis with preferential accumulation in the
lysosomes. These NMs could disassemble in cell lysosomes and
subsequently induce highly efficient singlet oxygen (1O2) generation
upon light irradiation. 1O2 disrupted the lysosomal membrane and
promoted the escape of BTM from the lysosome into the cytoplasm,
thereby resulting in the efficient and selective killing of cancer cells
through PDT. This study may provide a new strategy for designing
targeted PDT systems to fight cancer.
nanoparticles,[11] have been developed to deliver PSs for cancer
PDT. Compared with free PSs, PS-loaded nanocarriers have
obvious advantages, including improving bioavailability,
prolonging the blood circulation time, and facilitating the passive
targeting and accumulation of the PS at the tumour sites via the
enhanced permeability and retention (EPR) effect.[12] Moreover,
grafting targeting units, such as antibodies and folic acid, onto
the nanocarrier surface allows active targeting, thus reducing the
side effects by decreasing the interactions of nanocarriers with
healthy cells.[13]
Conjugating hydrophobic PSs with carbohydrates can make
them amphiphilic, thereby improving their solubility in
physiological fluids and promoting cellular recognition via
specific carbohydrate-protein interactions on cell surfaces.[14] For
example, a significant increase in the in vitro photosensitizing
efficiency of porphyrins conjugated with mannose compared to
that of their unconjugated analogue has been reported.[15] The
mannose receptor (MR) is overexpressed on the surfaces of
some
malignant
cells,[16]
and
mannose-functionalized
Introduction
nanocarriers can be internalized into cancer cells via receptor-
mediated endocytosis.[17] Although the individual binding of
mannose with the MR is weak, this interaction can be improved
by incorporating multiple mannose ligands onto one nanocarrier,
a process that increases the binding intensity and specificity
between the mannose-functionalized nanocarriers and
overexpressed MRs on cancer cells.[18] To the best of our
knowledge, no study has been reported on the use of
carbohydrate-conjugated PS to construct nanocarriers, and thus
to make them applicable in targeted PDT.
Photodynamic therapy (PDT), a minimally invasive treatment
of tumours and other diseases, has attracted an increasing
amount of attention.[1] The PDT process comprises three key
elements: light, oxygen, and a photosensitizer (PS). Cancer
treatment via PDT involves the uptake of a PS by tumour tissue
and irradiation with light of the appropriate wavelength. Upon
irradiation, the excited PS transfers energy to surrounding
molecular oxygen, resulting in generation of highly cytotoxic
reactive oxygen species (ROS), primarily singlet oxygen (1O2).[2]
To date, an extensive series of PSs have been developed,
including porphyrins,[3] chlorins,[4] phthalocyanines,[5] and boron
dipyrromethene (BODIPY).[6] However, most PSs are
hydrophobic organic molecules and cannot target malignant
tissues,[7] which considerably hampers their practical
applications for PDT.
In this work, we reported a new delivery system for
a
mannose-conjugated BODIPY PS (Scheme 1). A three-arm
distyryl BODIPY PS conjugated with mannose units was
synthesized (denoted by BTM) and co-assembled with Tween
80 to form BTM-loaded nanomicelles (denoted by BTM-NMs).
The molecular design of BTM is based on the following factors:
(1) 2,6-Halogenation (I) of the BODIPY core renders it an
efficient PS for ROS generation because of the heavy atom
effect; (2) Incorporating distyryl moieties into the BODIPY core is
expected to shift the absorption to the long wavelength region
To overcome the solubility and target specificity limitations of
PSs, many nanocarriers, including liposomes,[8] polymeric
nanoparticles,[9]
nanomicelles
(NMs),[10]
and
inorganic
with
a maximum of 665 nm, which enables deep-tissue
penetration; (3) Because of its inherent amphiphilicity, BTM can
co-assemble with Tween 80 to generate stable and well-defined
NMs in aqueous media; and (4) NMs can provide a platform for
the multivalent presentation of mannose, which increases their
selectivity for MDA-MB-231 breast cancer cells with
overexpressed MRs. Targeted PDT with the BTM-NMs was
demonstrated through their phototoxicity to MDA-MB-231 cancer
cells and nontoxicity to MCF-10A normal cells.
[a]
[b]
Prof. Q. Zhang, Y. Cai, L.-N. Hao, Prof. J. Yin
Key Laboratory of Carbohydrate Chemistry and Biotechnology
Ministry of Education, School of Biotechnology
Jiangnan University, Wuxi, 214122 (P. R. China)
E-mail: jianyin@jiangnan.edu.cn
Dr. Q.-Y. Li, Z. Ma, Prof. X.-J. Wang
Jiangsu Key Laboratory of Green Synthetic Chemistry for Functional
Materials, School of Chemistry and Materials Science
Jiangsu Normal University, Xuzhou, 221116, (P. R. China)
E-mail: xjwang@jsnu.edu.cn
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201702935
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Results and Discussion
HO
HO
O
HO
HO
N
O
N
O
N
O
O
B
I
I
N
N
Co-assembly
F
F
+
O
O
O
O
HO
O
HO
HO HO
O
HO
HO
HO
O
O
HO
O
O
N
N
N
N
N
N
BTM
Tween 80
BTM-NMs
Light(665 nm)
Receptor
HO HO
O
HO
HO
N
O
N
3O2
O
N
O
O
I
I
N
N
B
F
F
Cancer cells
O
O
O
O
HO
O
HO
HO
O
HO
HO
HO
HO
1O2
HO
O
O
O
O
N
N
N
N
N
N
HO HO
HO
HO
O
N
O
N
N
O
Nucleus
O
O
I
I
N
N
B
F
F
O
O
Lysosome
O
O
HO
HO
HO
O
HO
HO
HO
O
HO
HO
O
O
O
O
N
N
N
N
N
N
Scheme 1. Schematic illustration of the preparation of BTM-NMs and their application in targeted PDT.
The synthesis of BTM is shown in Scheme 2. First, compound
1 was prepared according to reported procedures,[19] and the
experimental details are provided in the Supporting Information
(Scheme S1). Compound 1 was reacted with 2,4-dimethylpyrrole
in the presence of trifluoroacetic acid, and then 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) and BF3⋅Et2O were added to
afford 2, which was further transformed via reaction with 1-iodo-
2,5-pyrrolidinedione (NIS) into 3. Compound 4 was obtained in
59% yield by reacting 3 with 1 in the presence of piperidine.
Mannose was conjugated to 4 via the copper-catalysed click
reaction between 4 and 5, and BTM was obtained in 41% yield
BTM was also characterized by FT-IR spectroscopy and UV-Vis
spectrophotometry. In the FT-IR spectrum of compound 4, the
absorption band at 2100 cm-1 is attributed to the stretching
vibration of the azide groups (Figure S9, Supporting Information).
After the click reaction between compounds 4 and 5, the
spectrum of BTM shows no absorption band at 2100 cm-1. The
strong stretching absorption band appearing at 1660 cm-1
appears due to the formation of the triazole linkage. In the UV-
Vis spectrum, BTM has a maximum absorption at 665 nm that
strictly followed the Lambert-Beer law, indicating that BTM did
not form aggregates in dimethylformamide (DMF) (Figure S10,
Supporting Information).
after
high-performance
liquid
chromatography (HPLC)
purification. The chemical structures of the intermediate
products and BTM were characterized using NMR and MS
(Figure S1−S8, Supporting Information). To verify the molecular
weight and purity of BTM, HPLC analysis was performed, and
the results are shown in Figure 1. The HPLC profile showing one
retention time of BTM at 10.05 min indicates the high purity of
the compound. The MS data show that the molecular weight of
BTM (m/z, M+Na+) is 1949.31, which is consistent with the
calculated value (m/z, M+Na+, 1949.30). These experimental
results confirm that BTM has been synthesized successfully.
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10.1002/chem.201702935
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H
N3
O
N
O
O
Information) with a fluorescence quantum yield (Ф) of 0.17
relative to zinc(II) phthalocyanine (ZnPc) (0.28).[20] When the
water content in DMF was increased, an obvious decrease in
the fluorescence intensity of BTM was observed (Figure 2a).
When the fw in DMF was 70%, BTM clearly formed well-
dispersed nanoaggregates and exhibited a fluorescent decrease
at 678 nm (Figure 2b). After DMF was removed by dialysis, a
stable NM solution with a concentration of 0.4 mg mL-1 was
obtained, and the BTM content in the BTM-NMs was estimated
as 39 wt%.
a)
TFA, DCM
O
O
N3
O
H
b) DDQ c) Et3N, BF3 • Et2
O
O
N
N
B
F
F
1
2
N3
O
O
O
B
N3
O
O
O
NIS
I
I
1
N
N
DCM
Piperidine
F
F
I
I
N
N
(a)
(b)
300
B
300
250
200
150
100
F
F
O
O
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
95%
O
O
250
200
150
100
50
O
O
N3
N3
3
4
HO
HO
HO
O
HO
N
50
0
O
N
N
O
O
O
0
HO HO
O
HO
HO
650
700
750
800
850
900
0
20
40
60
80
100
O
%of Water
Wavelength (nm)
5
I
I
N
N
B
(c)
(d)
CuSO4/NaVc, H2O/DMSO
F
F
O
O
O
O
HO
HO
HO
O
HO
HO
HO
O
HO
HO
O
O
O
O
N
N
N
N
N
N
BTM
Scheme 2. Synthesis of BTM.
1 925-20% ACN #7 RT: 0 .11 A V :
T:
1
NL : 1.5 3E 6
+
c
E S I Q1 MS [900 .00 0-20 00.000]
194 9.3 1
100
Figure 2. (a) Fluorescence spectra (excitation at 610 nm) of BTM recorded in
DMF solutions with increasing water content. The coloured lines indicate the
volume percentage of water during the measurements. (b) Corresponding
plots of emission intensity changes for BTM versus water content. (c) TEM
image of as-prepared BTM-NMs when the fw in DMF was 70%. (d) TEM image
of larger nanoaggregates when the fw in DMF was 80%.
95
Before purification
After purification
90
BTM(M + Na+)
calcd = 1949.30
85
80
75
70
65
60
55
50
45
40
0
5
10
15
20
25
The morphology of the resulting BTM-NMs was determined
using transmission electron microscopy (TEM). According to
TEM, the BTM-NMs were roughly spherical in shape (Figure 2c)
and were approximately 29.0 ± 3.3 nm in size. Notably, larger
nanoaggregates with a wide size distribution (~10 nm to 200 nm)
were obtained when the fw in DMF was 80% during the
nanoaggregate preparation process (Figure 2d). In addition,
irregular nanoaggregates were obtained when a 30% BTM
solution in DMF was mixed with 70% water without 0.1 vol%
Tween 80 (Figure S12, Supporting Information). These results
confirm that BTM could co-assemble with Tween 80 to generate
BTM-NMs when the fw in DMF was 70%. Dynamic light
scattering (DLS) analysis showed that the average
hydrodynamic diameter of the BTM-NMs in water was
35
30
Time (min)
25
20
15
10
5
1 930. 97
1824 .00
985 .79
1441 .95
18 05 .42
97 6.61
1661 .63
1 357.19
1 726.74
1199 .25
1200
14 71.0 6
1912 .70
1900
998.2 5
1 000
15 97.63
1 600
11 16 .50
0
90 0
1100
1300
1400
15 00
170 0
1 800
20 00
m /z
Figure 1. Mass spectrum of BTM. Inset: The HPLC profiles of BTM before and
after purification.
After successfully synthesizing BTM, we proceeded to
prepare the BTM-NMs using a reprecipitation technique, i.e.,
slowly adding a DMF solution of BTM into deionized water
containing 0.1 vol% Tween 80. To investigate the aggregation
process, we measured the fluorescence spectra of BTM
aggregates formed in the DMF/water mixture by varying the
fraction of water (fw). In pure DMF, BTM showed strong
fluorescence emission at 678 nm (Figure S11, Supporting
approximately 56.8
± 4.4 nm (PDI, 0.298) (Figure S13,
Supporting Information). The physiological stability of the BTM-
NMs was evaluated by suspending them in culture medium
containing 10% serum. No significant size change was observed
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after incubating the BTM-NMs in this medium for at least 72 h,
indicating that the BTM-NMs have good stability under
physiological conditions (Figure S14, Supporting Information).
observations were further confirmed using ImageJ software
analysis, which showed a statistically significant decrease in the
red fluorescent intensity in BTM-NM–treated MDA-MB-231 cells
after adding free mannose (Figure S15, Supporting Information).
These results suggest that the BTM-NMs were taken up
significantly more in the absence of mannose than in the
presence of mannose because of MR-mediated endocytosis.
Thus, BTM-NMs not only selectively target cancer cells but also
improve the internalization of PSs within targeted cancer cells.
The 1O2 generation efficiency was studied in DMF by
(a)
(b)
measuring
the
absorbance
decrease
of
1,3-
diphenylisobenzofuran (DPBF) upon 665-nm LED light
irradiation (Figure S16, Supporting Information). The 1O2
quantum yield (ФΔ) of BTM was determined to be 0.58 using
ZnPc as the standard PS (ФΔ = 0.60 in DMF).[21] Moreover, the
1O2 generation capability of BTM-NMs was evaluated using a
chemical
method
with
9,10-
anthracenediylbis(methylene)dimalonic acid (ABDA) as the 1O2
trapping agent.[22] ABDA reacts with 1O2 to produce the
corresponding endoperoxide and then change its absorption. A
typical absorption spectrum of ABDA in water shows two
characteristic bands at 412 and 435 nm, and no significant
change was observed when an aqueous solution of ABDA was
irradiated with a 665-nm LED light (20 mW cm-2, 10 min) (Figure
S17, Supporting Information). However, the absorption of ABDA
decreased continuously when a mixture of ABDA and BTM-NMs
in water was irradiated under the same conditions. Thus, these
results confirmed the efficient generation of 1O2 from BTM-NMs
(c)
Figure 3. CLSM images of (a) MDA-MB-231 cancer cells and (b) MCF-10A
cells treated with BTM-NMs (at a BTM dose of 8 μg mL-1) for 24 h. CLSM
images of (c) MDA-MB-231 cancer cells treated with BTM-NMs (at a BTM
dose of 8 μg mL-1) with mannose (1 mM) competition. For each panel, the
three images from left to right show cell nuclei stained by DAPI (blue), BTM
molecules in cells (red), and overlays of the two sets of images, respectively.
in aqueous conditions.
The intracellular 1O2 production of the BTM-NMs was
investigated using fluorescence microscopy imaging with the
probe dichlorofluorescein diacetate (DCFH-DA), which can be
oxidized by 1O2 into dichlorofluorescein (DCF), a compound that
emits green fluorescence.[23] MDA-MB-231 cancer cells were
incubated with BTM-NMs for 24 h, and then DCFH-DA was
added, followed by irradiation with the 665-nm LED light (20 mW
cm-2) for 10 min. After being washed with PBS, the cells were
monitored in real time via excitation at 488 nm using a confocal
microscope. As shown in Figure 4a, obvious DCF fluorescence
can be detected after 665-nm light irradiation, whereas no
fluorescence was observed without irradiation. In addition, the
morphology of the MDA-MB-231 cancer cells significantly
changed after irradiation, indicating that the BTM-NPs can
induce serious photodynamic cellular damage and eventual
apoptosis. Moreover, the fluorescence intensity clearly
decreased in the presence of sodium azide, a well-known 1O2
scavenger (Figure 4b).[24] These results indicate that BTM can
efficiently produce intracellular 1O2 in the irradiated region after
the internalization of BTM-NMs in MDA-MB-231 cancer cells via
receptor-mediated endocytosis. Based on our cell experiment
results, 1O2 was produced from endocytosed BTM-NMs in the
cells (Figure 3), and the red fluorescence of BTM was observed
inside the cell (Figure 3). Thus, we can conclude that BTM-NMs
disassemble into BTM molecules in the MDA-MB-231 cancer
cells after being internalized via receptor-mediated endocytosis.
The cellular uptake behaviour of BTM-NMs was monitored
using confocal laser scanning microscopy (CLSM), as shown in
Figure 3. MDA-MB-231 breast cancer cells with high MR
expression (MR+) were chosen as a positive control,[14b,16b] and
normal MCF-10A cells with low MR expression (MR-) were used
as a negative control. Both MDA-MB-231 and MCF-10A cells
were treated with BTM-NMs for 24 h. After the cells were
washed with a phosphate buffered saline (PBS, pH 7.4) solution,
the cell nuclei were stained with 4’,6-diamidino-2-phenylindole
(DAPI) before observation. The blue fluorescence from DAPI
signifies the cell nucleus, and the red fluorescence arises from
the BTM molecules. More red fluorescent spots were observed
inside MDA-MB-231 cancer cells than in MCF-10A cells, in
which a very weak fluorescence was observed. These results
indicated a much higher uptake of the BTM-NMs by the MDA-
MB-231 cancer cells (Figure 3a) than by the MCF-10A cells
(Figure 3b), which was due to the low level of MR expression on
the cellular membranes of the latter cell line. To confirm the
association between BTM-NM uptake and MR expression,
competition inhibition studies were performed using free
mannose to compete with the BTM-NMs for binding with MR on
the surface of cancer cells, potentially reducing the uptake of
BTM-NMs into MDA-MB-231 cells. As shown in Figure 3c, when
MDA-MB-231 cells were incubated with both BTM-NMs and free
mannose under the same conditions, a significant inhibition of
BTM-NM uptake due to MR competition was observed. These
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lysosomes were yellow because of the overlap between the
green (LysoTracker) and red (BTM) fluorescent spots. Figure 5
shows that the red BTM fluorescence had a colocalization
percentage of approximately 91% with the lysosomes in the
absence of irradiation, indicating effective endocytosis into the
lysosomes upon cell internalization. However, BTM exhibited a
poor colocalization of 45% with the lysosomes in the presence of
irradiation, suggesting that BTM could escape from the
lysosome after irradiation and was distributed in the cytoplasm.
Interestingly, the lysosomal disruption was significantly inhibited
in the presence of sodium azide under irradiation, as under
these conditions a colocalization percentage between BTM and
lysosomes of 82% was observed. These results reveal that
lysosomal disruption is attributable to the damage caused by the
intracellular 1O2 generated upon irradiation.
Brightfield
Fluorescence
Merge
(a)
Untreated
BTM-NMs
BTM-NMs +NaN3
NaN3
(b)
To observe the effects of targeted PDT, a glucose-conjugated
BODIPY PS (BTG) was synthesized using similar methods to
BTM, except glucose units were used instead of mannose units.
Subsequently, glucose-functionalized nanomicelles (BTG-NMs)
were prepared as a control sample. MDA-MB-231 cancer cells
were incubated with BTM-NMs or BTG-NMs at the same PS-
equivalent dose (8 μg mL-1). After 24 h, the cells were stained
with DAPI to examine changes in the nuclear morphology after
PDT.[25] As shown in Figure 6a, the nuclei in MDA-MB-231 cells
treated with BTG-NMs and irradiated with 665 nm LED light
irradiation (20 mW cm-2) did not change. By contrast, the nuclei
of MDA-MB-231 cancer cells treated with BTM-NMs under the
same irradiation conditions were significantly damaged. In
addition, BTG-NMs and BTM-NMs were individually incubated
with MDA-MB-231 cancer cells under the same conditions for 24
h. After being washed three times with PBS, the cells were
irradiated with 665 nm LED light (20 mW cm-2) for 30 min and
then co-stained with calcein-AM and PI.[26] Green fluorescence
represents live cells stained with calcein-AM, and red
fluorescence denotes dead cells stained with PI. After being
washed another three times with PBS, the cells were monitored
using a confocal microscope. As shown in Figure 6b, only a few
cells incubated with BTG-NMs were photodamaged, whereas
almost no live cells were found after they were treated with
BTM-NMs. These results confirmed that the BTM-NMs have
greater cellular uptake and intracellular ROS production than the
BTG-NMs. Moreover, we can clearly observe that barely any
cells in the irradiation region are alive, which further
demonstrates that BTM-NMs can specifically kill cancer cells
under 665-nm light irradiation (Figure S18, Supporting
Information).
Figure 4. (a) CLSM images of MDA-MB-231 cancer cells treated with BTM-
NMs (at a BTM dose of 8 μg mL-1) for 24 h followed by DCFH-DA staining and
irradiation (665 nm, 20 mW cm-2) for 15 min. (b) Detection of intracellular 1O2
by DCFH-DA staining in MDA-MB-231 cancer cells incubated with BTM-NMs
(at a BTM dose of 8 μg mL-1) in the presence or absence of sodium azide. The
green fluorescence is from the product of DCFA-DA oxidation by 1O2.
LysoTracker
BTM-NMs
Merge
Figure 5. CLSM images of live MDA-MB-231 cells after 24 h of incubation with
BTM-NMs (at a BTM dose of 8 μg mL-1) with or without irradiation. When
colocalized, the green and red spots produce a yellow colour, which is due to
BTM molecules trapped inside the cell lysosomes.
To investigate the intracellular distribution of the BTM-NMs,
MDA-MB-231 breast cancer cells were incubated with the
nanocarriers for 24 h and then treated with LysoTracker, a
marker of late endosomal and lysosomal vesicles. CLSM was
employed to observe the BTM-NM intracellular distribution with
and without irradiation. Green fluorescence denotes the
LysoTracker-labelled lysosomes, and red fluorescence is from
the intracellular BTM molecules. The NMs located inside the
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(a) 120
(b)120
100
80
Untreated
BTG-NMs
BTM-NMs
BTG-NMs
BTM-NMs
BTG-NMs
BTM-NMs
**
**
**
100
80
60
40
20
0
**
**
60
**
(a)
40
20
0
0
2
4
6
8
10
12
0
2
4
6
8
10
12
Concentration (g mL-1
)
Concentration (g mL-1
)
(c)
(d)
BTG-NMs
BTM-NMs
BTG-NMs
BTM-NMs
100
100
80
60
40
20
0
80
60
40
20
0
(b)
*
**
**
**
30
0
5
10
15
20
25
30
0
5
10
15
20
25
Figure 6. (a) CLSM images of cell nuclei stained by DAPI for assessing
nuclear damage after light irradiation (665 nm, 20 mW cm-2) for 30 min. Scale
bar: 20 μm. (b) CLSM images of a live/dead assay (calcein-AM/PI) performed
on co-stained MDA-MB-231 cells after being incubated with BTG-NMs or
BTM-NMs and then being irradiated (665 nm, 20 mW cm ) for 30 min. The
cells were co-stained with calcein-AM (green, live cells) and PI (red, dead
Time (min)
Time (min)
Figure 7. Viability of (a) MCF-10A normal cells and (b) MDA-MB-231 cancer
cells after being incubated for 24 h with different PS concentrations of BTG-
NMs or BTM-NMs and then being irradiated (665 nm, 20 mW cm-2) for 30 min.
Time-dependent PDT effects on (c) MCF-10A normal cells and (d) MDA-MB-
231 cancer cells incubated with BTG-NMs or BTM-NMs at the same PS-
equivalent dose of 12 μg mL-1 and then irradiated (665 nm, 20 mW cm-2) for
different times.
-2
cells). Scale bar: 100 μm.
The targeted PDT efficacy of the BTM-NMs was further
evaluated
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay.[27] First, MDA-MB-231
(MR+) and MCF-10A (MR-) cells were incubated with different
concentrations of either BTG-NMs or BTM-NMs. After being
washed with PBS, the cells were irradiated with the 665-nm LED
light for 30 min. For the MCF-10A cells, the two samples showed
similar cell viability (Figure 7a). Conversely, MDA-MB-231 cells
treated with the BTM-NMs exhibited much lower cell viability
than those treated with the BTG-NMs (Figure 7b), indicating that
the targeting effect of BTM-NMs through MR-mediated
endocytosis increased the internalization of the NMs and thus
led to a high percentage of BTM being taken up by MDA-MB-
231 cells. Next, we assessed the phototoxicity of BTG-NMs and
BTM-NMs with different light irradiation times. Whereas the
phototoxicity of the BTM-NMs was found to be similar to that of
the BTG-NMs for MCF-10A normal cells (Figure 7c), a significant
difference between the two NMs was observed for MDA-MB-231
cancer cells. As the irradiation time was prolonged, the
phototoxicity of the BTM-NMs gradually became higher than that
of the BTG-NMs (Figure 7d). These results confirm the time-
dependent PDT effect and MR-mediated cancer cell targeting
properties of the BTM-NMs. Furthermore, the dark toxicity of the
BTG-NMs and BTM-NMs was also investigated (Figure S19,
Supporting Information). Without light irradiation, a negligible
toxicity to both MDA-MB-231 cells and MCF-10A cells was
observed for the two NMs, even at a high concentration (30 μg
mL-1). No concentration-dependent toxic effect was found,
indicating that the internalization of the present dose of NMs is
minimally cytotoxic.
Conclusions
In summary, a three-arm distyryl BODIPY PS conjugated with
mannose units was synthesized and used to prepare mannose-
functionalized nanomicelles for targeted PDT against MDA-MB-
231 breast cancer cells. After selectively being internalized into
MDA-MB-231 breast cancer cells via receptor-mediated
endocytosis, the BTM-NMs can disassemble into BTM
molecules in the cell lysosomes and efficiently generate 1O2
upon light irradiation. 1O2 disrupted the lysosomal membrane
and promoted the escape of BTM from lysosome into the
cytoplasm, thereby facilitating 1O2 reaching organelles such as
the nucleus and mitochondria for enhanced photodynamic cell
damage. The BTM-NMs exhibit excellent targeted delivery of the
PS that not only leads to selective cancer cell death but also
minimizes damage to normal cells after irradiation. Therefore,
the current research has demonstrated the potential of this
system to deliver PSs for enhanced PDT of breast cancer cells.
Experimental Section
Materials
2-[2-(2-chloroethoxy)ethoxy]ethanol, ABDA, boron trifluoride diethyl
etherate (BF3⋅Et2O), copper sulfate pentahydrate (CuSO4·5H2O), DAPI,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone
dichloromethane (DCM, 99.5%),
(DDQ),
2,4-dimethylpyrrole,
DCFH-DA,
4-
(dimethylamino)pyridine (DMAP), DMF (99.5%), DPBF, MTT, Dulbecco’s
modified Eagle’s medium (DMEM), ethyl acetate (EtOAc, 99.5%), foetal
bovine serum (FBS), 4-hydroxybenzaldehyde, NIS, LysoTracker probes
(Green DND), α-D-mannose, methanol (MeOH, 99.5%), PBS (pH 7.4),
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10.1002/chem.201702935
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piperidine, sodium ascorbate, tetrahydrofuran (THF, 99.0%), p-
toluenesulfonic acid (TsOH), p-toluenesulfonyl chloride (TsCl),
triethylamine (Et3N), trifluoroacetic acid (TFA), and trimethylsilyl
trifluoromethanesulfonate (TMSOTf) were purchased commercially and
used without further purification. All aqueous solutions were prepared
using ultrapure water from a Milli-Q system (Millipore, USA).
70.79, 70.14, 69.80, 67.57, 50.70, 29.70, 22.70, 17.18, 15.99, 14.13.
ESI-MS: m/z calcd for C25H28BF2I2N5O3: 749.03; found: 771.97 [M+Na]+.
Synthesis of compound 4
To a solution of compound 3 (0.3 g, 0.40 mmol) in toluene (55 mL) were
added compound 1 (0.34 g, 1.22 mmol), piperidine (3 mL), and a
catalytic amount of TsOH under a nitrogen atmosphere, and then the
resulting solution was stirred for 5 h at 140 °C. After being cooled to room
temperature, the reaction mixture was diluted with DCM (400 mL) and
washed twice with water (600 mL). The organic layer was dried over
anhydrous Na2SO4, and the solvent was evaporated in vacuo. The crude
product was purified by column chromatography on silica gel
(DCM/EtOAc=25:2) to provide compound 4 (0.30 g, yield: 59%). 1H NMR
(400 MHz, CDCl3) δ=1.50 (s, 6H), 3.41 (q, J=5.9, 5.0, 6H), 3.75 (m, 18H),
3.92 (m, 6H), 4.21 (m, 6 H), 6.97 (d, J=8.8, 4H), 7.06 (d, J=8.6, 2H), 7.16
(d, J=8.6, 2H), 7.59 (m, 6H), 8.12 (d, J=16.9, 2H). 13C NMR (101 MHz,
CDCl3) δ=158.90, 149.39, 144.70, 137.98, 132.23, 128.77, 128.59,
128.20, 126.46, 115.82, 114.47, 113.98, 69.92, 69.75, 69.10, 68.78,
66.56, 49.70, 28.67, 21.67, 16.69. ESI-MS: m/z calcd for
C51H58BF2I2N11O9: 1271.26; found: 1294.40 [M+Na]+.
Characterization
1H NMR spectra were obtained on
a Bruker Avance spectrometer
operating at 400 MHz. HPLC was performed on an Agilent HPLC system
equipped with a UV detector (665 nm). The analytical column was an
Agilent Zorbax XDB-C18 (5 μm particles, 4.6 × 150 mm). The following
two eluents were used: A contained 100% H2O, and B contained 100%
CH3OH. B was increased from 70% to 100% over 20 min at a flow rate of
0.8 mL min-1. TEM images were obtained on a HITACHI H-7650 TEM at
80 kV. UV-Vis absorption spectra and fluorescence spectra were
recorded on a Hitachi U-3900 UV-Vis spectrophotometer and a Hitachi F-
7000 spectrophotometer, respectively. Hydrodynamic diameter and zeta
potential measurements were performed at 25 °C on a Malvern Zetasizer
NanoZS instrument. All the measurements were performed with the NMs
suspended in filtered water or filtered cell culture medium at
a
concentration of 30 μg mL-1. A Nikon confocal microscope was used for
cell imaging. An LED lamp with a light density of 20 mW cm-2, which was
monitored using a light power meter (NEWPORT Model 842-PE), was
used.
Synthesis of BTM
To a solution of compound 4 (100 mg, 78.67 μmol) in DMF and H2O (8
mL, 15:1, v/v) were added compound
5 (86 mg, 394.12 μmol),
CuSO4·5H2O (6.5 mg, 26.09 μmol), and sodium ascorbate (8.7 mg, 43.92
μmol) at room temperature. The reaction mixture was stirred at room
temperature for 48 h. After the insoluble solid was removed by filtration,
the solvent was removed in vacuo. The residue was dissolved in H2O
and purified using C18 reversed-phase HPLC. The product was
lyophilized to afford BTM (62 mg, 41%). 1H NMR (400 MHz, DMSO-d6)
δ=1.47 (s, 6H), 3.36 – 3.63 (m, 30H), 3.66 – 3.79 (m, 10H), 3.84 (m, 5H),
4.15 (m, 6H), 4.46 – 4.59 (m, 14H), 4.67 (dd, J=12.2, 2.9, 3H), 4.73 (m,
7H), 7.07 (d, J=7.3, 4H), 7.16 (d, J=8.3, 2H), 7.34 (d, J=8.3, 2H), 7.44 (d,
J=5.3, 2H), 7.58 (d, J=8.4, 4H), 8.10 (m, 5H). 13C NMR (101 MHz,
DMSO-d6) δ=160.38, 143.86, 139.14, 129.41, 129.09, 124.91, 115.78,
99.46, 70.68, 70.26, 70.03, 69.18, 67.86, 67.48, 61.77, 58.51, 56.96,
49.81, 17.77. ESI-MS: m/z calcd for C78H100BF2I2N11O27: 1926.30; found:
1949.31 [M+Na]+.
Synthesis of compound 1
The synthetic procedures are described in the Supporting Information.
Synthesis of compound 2
To a solution of compound 1 (1.1 g, 3.94 mmol) in anhydrous DCM (120
mL) were added 2,4-dimethylpyrrole (0.9 g, 9.46 mmol) and TFA (0.3 mL)
at room temperature. The mixture was stirred under
a nitrogen
atmosphere for 12 h. To this solution was added DDQ (2.0 g, 8.16 mmol),
and the resulting mixture was stirred for 30 min at room temperature.
After the mixture was cooled in an ice bath, Et3N (10 mL, 135.4 mmol)
was added dropwise to the mixture. After 30 min, BF3⋅Et2O (12 mL, 95.12
mmol) was added dropwise, and the reaction mixture was stirred for 10 h
at 0 °C and allowed to warm to room temperature. After the mixture was
diluted with DCM (500 mL) and washed with water (400 mL), the organic
layer was dried over anhydrous Na2SO4. Following evaporation of the
solvent, the product was purified by column chromatography on silica gel
(DCM/EtOAc=50:1) to provide compound 2 (0.58 g, yield: 30%). 1H NMR
(400 MHz, CDCl3) δ=1.42 (s, 6H), 2.54 (s, 6H), 3.40 (t, J=5.0, 2H), 3.71
(m, 4H), 3.77 (m, 2H), 3.92 (dd, J=5.6, 3.8, 2H), 4.19 (dd, J=5.7, 3.8, 2H),
5.97 (s, 2H), 7.02 (d, J=8.6, 2H), 7.15 (d, J=8.6, 2H). ESI-MS: m/z calcd
for C25H30BF2N5O3: 497.24; found: 519.96 [M+Na]+.
Preparation of BTM-NMs and BTG-NMs
BTM (5 mg) was dissolved in DMF (5 mL) and used as a stock solution.
Then, 0.6 mL of the stock solution was added dropwise into deionized
water (1.4 mL) containing 0.1 vol% Tween 80 under vigorous stirring. An
NM dispersion of the BTM-NMs in aqueous solution (0.4 mg/mL) was
obtained after dialyzing (MW 3500) with water to remove excess Tween
80 and DMF. BTG-NMs were prepared using BTG (5 mg) according to
the same steps as those described for BTM-NM formation. To determine
the concentration of BTM in the NM suspension, water was first removed
by lyophilization, and the solid sample of the NMs was dissolved in DMF.
The amount of BTM in DMF was calibrated based on a standard curve of
concentration vs. absorbance determined by UV-Vis spectroscopy.
Synthesis of compound 3
To a solution of compound 2 (0.3 g, 0.61 mmol) in DCM (30 mL) was
added 1-iodo-2,5-pyrrolidinedione (NIS) (0.33 g, 1.47 mmol) at room
temperature, and the resulting mixture was stirred in the dark for 5 h.
After the solvent was removed under reduced pressure, the residue was
purified by column chromatography on silica gel (DCM/EtOAc=100:1) to
afford compound 3 (0.42 g, yield: 92%). 1H NMR (400 MHz, CDCl3)
δ=1.44 (s, 6H), 2.64 (s, 6H), 3.41 (t, J=5.0, 2H), 3.71 (m, 4H), 3.80 (m,
J=5.3, 2.1, 2H), 3.94 (dd, J=5.6, 3.7, 2H), 4.21 (dd, J=5.7, 3.7, 2H), 7.13
(d, J=8.6, 2H), 7.05 (d, J=8.6, 2H). 13C NMR (101 MHz, CDCl3) δ=159.76,
156.57, 145.35, 141.53, 131.70, 130.17, 129.05, 126.88, 115.50, 70.95,
Detection of singlet oxygen generation in water
ABDA was used to evaluate the 1O2 generation of the BTM-NMs in water.
In this method, ABDA was dissolved in water (3 mL, 0.01 μg mL-1), and
then a solution of BTM-NMs in water (20 μL, 0.5 mg mL-1) was added.
The resulting solution was then subjected to light irradiation from a 665
nm LED lamp with a light density of 20 mW cm-2 that was set near
(approximately 2 cm) to the quartz cuvette. At each predetermined
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10.1002/chem.201702935
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interval, the absorption spectrum of the solution was recorded. ABDA in
Acknowledgements
water (3 mL, 0.01 μg mL-1) was used as a control.
This work was financially supported by National Natural Science
Foundation of China (51403081 and 21302072), Natural
Science Foundation of Jiangsu Province (BK20140137), the
Fundamental Research Funds for the Central Universities
(JUSRP51629B), the Open Foundation of Key Laboratory of
Carbohydrate Chemistry & Biotechnology Ministry of Education
(KLCCB-KF201705), Max Planck Society International Partner
Group Program, High-end Foreign Experts Recruitment Program,
and The Thousand Talents Plan (Young Professionals).
Cell culture
MDA-MB-231 (breast cancer) cells and MCF-10A (breast normal) cells
were cultured in DMEM containing 10% FBS, penicillin (100 U mL-1), and
streptomycin (100 μg mL-1) in a humidified atmosphere with 5% CO2 at
37 °C.
Cell uptake of BTM-NMs
MDA-MB-231 cells were seeded in 35-mm plastic-bottomed Ibidi μ-
dishes and allowed to grow for 24 h. After incubation with BTM-NMs (at a
BTM dose of 8 μg mL-1) in culture medium for 24 h, the cells were
washed three times with PBS (pH 7.4) and then fixed with 4.0%
formaldehyde at room temperature for 15 min. After being washed with
PBS, the cells were stained with DAPI (1 μg mL-1) for 15 min. After being
washed again with PBS, the cells were observed under a confocal
microscope (100 × oil objective, 408/561 nm excitation).
Conflict of interest
The authors declare no conflict of interest.
Keywords: BODIPY• breast cancer • photodynamic therapy •
Localization of BTM-NMs
photosensitizer • targeted delivery
MDA-MB-231 cells or MCF-10A cells were seeded according to the
abovementioned procedure. The cells were washed three times with PBS
(pH 7.4) and then incubated with BTM-NMs (at a BTM dose of 8 μg mL-1)
in culture medium at 37 °C. After 24 h of incubation, the medium was
removed, and the cells were washed three times with PBS and treated
with LysoTracker Green DND-99 solution (50 nM) in DMEM at 37 °C for
30 min. After being washed with fresh medium, the cells were irradiated
with or without an LED light (665 nm, 20 mW cm-2) for 15 min. Finally, the
cells were examined under an inverted Nikon confocal microscope (100 ×
oil objective, 488/561 nm excitation).
[1]
a) D. E. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3,
380-387; b) S. B. Brown, E. A. Brown, I. Walker, Lancet Oncol. 2004, 5,
497-508; c) A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer
2006, 6, 535-545; d) J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans, K. S.
Samkoe, S. Verma, B. W. Pogue, T. Hasan, Chem. Rev. 2010, 110,
2795-2838; e) J. M. Dabrowski, L. G. Arnaut, Photochem. Photobiol.
Sci. 2015, 14, 1765-1780; f) Y. Shen, A. J. Shuhendler, D. Ye, J.-J. Xu,
H.-Y. Chen, Chem. Soc. Rev. 2016, 45, 6725-6741.
[2]
[3]
a) M. Ethirajan, Y. Chen, P. Joshi, R. K. Pandey, Chem. Soc. Rev.
2011, 40, 340-362; b) J. Zhao, W. Wu, J. Sun, S. Guo, Chem. Soc. Rev.
2013, 42, 5323-5351; c) J. Zhao, K. Xu, W. Yang, Z. Wang, F. Zhong,
Chem. Soc. Rev. 2015, 44, 8904-8939; d) J. Zhou, J. Song, L. Nie, X.
Chen, Chem. Soc. Rev. 2016, 45, 6597-6626.
Phototoxicity assay
The cells were seeded into a 96-well plate at density of 1 × 104 cells per
well within DMEM (100 μL) for 24 h. The culture medium was then
replaced with fresh culture medium (100 μL) containing either BTM-NMs
or BTG-NMs at different PS concentrations. After 24 h, the cells were
washed three times with PBS, and the wells were refilled with 100 μL of
fresh culture medium. The plates were irradiated with 665 nm LED light
for 30 min. The distance between the 96-well plate and light source is
approximately 2 cm, and the light power at the 96-well plate position is 20
mW cm-2. The cells were further cultured for 12 h, and the culture
medium from each well was replaced with 100 μL of medium containing
MTT (0.5 mg mL-1). After another 4 h of incubation, culture supernatants
were removed, and DMSO (100 μL) was added into each well. The plate
was gently shaken for 10 min, and the absorbance at 570 nm was
recorded on a BioTek Synergy H4 microplate reader. The relative cell
viability compared to the control wells treated with only medium was
calculated using [A]test/[A]control, where [A]test and [A]control are the average
absorbance of the test and control samples, respectively.
a) E. D. Sternberg, D. Dolphin, C. Bruckner, Tetrahedron 1998, 54,
4151-4202; b) S. K. Pushpan, S. Venkatraman, V. G. Anand, J. Sankar,
D. Parmeswaran, S. Ganesan, T. K. Chandrashekar, Curr. Med.
Chem.: Anti-Cancer Agents 2002, 2, 187-207; c) A. E. O'Connor, W. M.
Gallagher, A. T. Byrne, Photochem.Photobiol.2009, 85, 1053-1074.
a) M. Taniguchi, J. S. Lindsey, Chem. Rev. 2017, 117, 344-535; b) J. D.
Spikes, J. Photochem. Photobiol., B 1990, 6, 259-274.
[4]
[5]
a) R. Bonnett, Chem. Soc. Rev. 1995, 24, 19-33; b) L. M. Moreira, F. V.
dos Santos, J. P. Lyon, M. Maftoum-Costa, C. Pacheco-Soares, N. S.
da Silva, Aust. J. Chem. 2008, 61, 741-754.
[6]
[7]
a) M.-R. Ke, S.-L. Yeung, D. K. P. Ng, W.-P. Fong, P.-C. Lo, J. Med.
Chem. 2013, 56, 8475−8483; b) H. He, P.-C. Lo, S.-L. Yeung, W.-P.
Fong, D. K. P. Ng, Chem. Commun. 2011, 47, 4748-4750; c) A.
Kamkaew, S. H. Lim, H. B. Lee, L. V. Kiew, L. Y. Chung, K. Burgess,
Chem. Soc. Rev. 2013, 42, 77-88.
a) D. K. Chatterjee, L. S. Fong, Y. Zhang, Adv. Drug Delivery Rev.
2008, 60, 1627−1637; b) M. R. Detty, S. L. Gibson, S. J. Wagner, J.
Med. Chem. 2004, 47, 3897-3915.
Statistical analysis
[8]
[9]
a) A. S. Derycke, P. A. de Witte, Adv. Drug Delivery Rev. 2004, 56, 17-
30; b) H.-I. Chang, M.-K. Yeh, Int. J. Nanomed. 2012, 7, 49-60.
a) E. R. Gillies, J. M. J. Frechet, Drug Discovery Today 2005, 10, 35-
43; b) W. Hu, H. Ma, B. Hou, H. Zhao, Y. Ji, R. Jiang, X. Hu, X. Lu, L.
Zhang, Y. Tang, Q. Fan, W. Huang, ACS Appl. Mater. Interfaces 2016,
8, 12039-12047; (c) Y. Zhang, C. Teh, M. Li, C. Y. Ang, S. Y. Tan, Q.
Qu, V. Korzh, Y. Zhao, Chem. Mater. 2016, 28, 7039-7050.
Statistical analysis was performed using Student’s t-test. Data were
collected from three or more replicates for each experiment and are
presented as the mean
± standard deviation. Differences were
considered to be statistically significant (*p<0.05, **p<0.01).
[10] a) C. J. F. Rijcken, O. Soga, W. E. Hennink, C. F. van Nostrum, J.
Controlled Release 2007, 120, 131-148; b) J. Tian, L. Ding, H. Ju, Y.
This article is protected by copyright. All rights reserved.

10.1002/chem.201702935
Chemistry - A European Journal
FULL PAPER
Yang, X. Li, Z. Shen, Z. Zhu, J.-S. Yu, C. J. Yang, Angew. Chem. Int.
Mongin, M. Blanchard-Desce, A. Morere, M. Garcia, J. O. Durand, L.
Raehm, Angew. Chem. Int. Ed. 2011, 50, 11425 – 11429; Angew.
Chem. 2011, 123, 11627–11631.
Ed. 2014, 53, 9544-9549; Angew. Chem. 2014, 126, 9698-9703.
[11] a) L. Pan, J. Liu, J. Shi, Adv. Funct. Mater. 2014, 24, 7318-7327; b) K.
Yang, L. Feng, X. Shi, Z. Liu, Chem. Soc. Rev. 2013, 42, 530-547; c) X.
Ma, S. Sreejith, Y. Zhao, ACS Appl. Mater. Interfaces 2013, 5, 12860-
12868; d) X. Ma, Q. Qu, Y. Zhao, ACS Appl. Mater. Interfaces 2015, 7,
10671-10676.
[18] a) M. Delbianco, P. Bharate, S. Varela-Aramburu, P. H. Seeberger,
Chem. Rev. 2016, 116, 1693–1752; b) Z. Ye, Q. Zhang, S. Wang, P.
Bharate, S. Varela-Aramburu, M. Lu, P. H. Seeberger, J. Yin, Chem.
Eur. J. 2016, 22, 15216-15221; c) Q. Zhang, Y. Cai, X.-J. Wang, J.-L.
Xu, Z. Ye, S. Wang, P. H. Seeberger, J. Yin, ACS Appl. Mater.
Interfaces 2016, 8, 33405-33411.
[12] a) D. K. Chatterjee, L. S. Fong, Y. Zhang, Adv. Drug Delivery Rev.
2008, 60, 1627-1637; b) S. S. Lucky, K. C. Soo, Y. Zhang, Chem. Rev.
2015, 115, 1990-2042.
[19] J. M. Poolman, C. Maity, J. Boekhoven, L. van der Mee, V. A. A. le
Sage, G. J. M. Groenewold, S. I. van Kasteren, F. Versluis, J. H. van
Esch, R. Eelkema, J. Mater. Chem. B 2016, 4, 852-858.
[13] a) H. Koo, M. S. Huh, I.-C. Sun, S. H. Yuk, K. Choi, K. Kim, I. C. Kwon
Acc. Chem. Res. 2011, 44, 1018−1028; b) S. Cui, D. Yin, Y. Chen, Y.
Di, H. Chen, Y. Ma, S. Achilefu, Y. Gu, ACS Nano 2013, 7, 676-688; c)
L. Huang, Z. Li, Y. Zhao, Y. Zhang, S. Wu, J. Zhao, G. Han, J. Am.
Chem. Soc. 2016, 138, 14586-14591.
[20] X.-J. Jiang, S.-L. Yeung, P.-C. Lo, W.-P. Fong, D. K. P. Ng, J. Med.
Chem. 2011, 54, 320-330.
[21] H. Lu, N. Kobayashi, Chem. Rev. 2016, 116, 6184-6261.
[22] N. M. Idris, M. K. Gnanasammandhan, J. Zhang, P. C. Ho, R.
Mahendran, Y. Zhang, Nat. Med. 2012, 18, 1580-1585.
[14] a) X. Chen, L. Hui, D. A. Foster, C. M. Drain, Biochemistry 2004, 43,
10918-10929; b) S. Singh, A. Aggarwal, S. Thompson, J. o. P. C. Tomé,
X. Zhu, D. Samaroo, M. Vinodu, R. Gao, C. M. Drain, Bioconjugate
Chem. 2010, 21, 2136-2146; c) F. Hammerer, G. Garcia, S. Chen, F.
Poyer, S. Achelle, C. Fiorini-Debuisschert, M.-P. Teulade-Fichou, P.
Maillard, J. Org. Chem. 2014, 79, 1406-1417.
[23] X. Wang, H. Fang, Z. Huang, W. Shang, T. Hou, A. Cheng, H. Cheng, J.
Mol. Med. 2013, 91, 917−927.
[24] J. Hu, Y. Tang, A. H. Elmenoufy, H. Xu, Z. Cheng, X. Yang, Small 2015,
11, 5860-5887.
[15] I. Laville, S. Pigaglio, J.-C. Blais, F. Doz, B. Loock, P. Maillard, D. S.
Grierson, J. Blais, J. Med. Chem. 2006, 49, 2558-2567.
[25] Q. Zhang, X. Wang, P.-Z. Li, K. T. Nguyen, X.-J. Wang, Z. Luo, H.
Zhang, N. S. Tan, Y. Zhao, Adv. Funct. Mater. 2014, 24, 2450−2461.
[26] S. H. Kim, J. E. Lee, S. M. Sharker, J. H. Jeong, I. In, S. Y. Park,
Biomacromolecules 2015, 16, 3519−3529.
[16] a) J. H. Ahire, I. Chambrier, A. Mueller, Y. Bao, Y. Chao, ACS Appl.
Mater. Interfaces 2013, 5, 7384–7391; b) L. Yin, Y. Chen, Z. Zhang, Q.
Yin, N. Zheng, J. Cheng, Macromol. Rapid Commun. 2015, 36,
483−489.
[27] (a) Q. Zhang, K. G. Neoh, L. Xu, S. Lu, E. T. Kang, R. Mahendran, E.
Chiong, Langmuir 2014, 30, 6151−6161; (b) H.-J. Xiang, H. P. Tham, M.
D. Nguyen, S. Z. F. Phua, W. Q. Lim, J.-G. Liu, Y. Zhao, Chem.
Commun. 2017, 53, 5220-5223.
[17] a) D. Brevet, M. Gary-Bobo, L. Raehm, S. Richeter, O. Hocine, K. Amro,
B. Loock, P. Couleaud, C. Frochot, A. Morere, P. Maillard, M. Garcia, J.
O. Durand, Chem. Commun. 2009, 1475–1477; b) M. Gary-Bobo, Y.
Mir, C. Rouxel, D. Brevet, I. Basile, M. Maynadier, O. Vaillant, O.
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Quan Zhang, Ying Cai, Qiu-
Breast cancer cells recognize
and internalize nanomicelles
co-assembled from a
Yan Li, Lin-Na Hao, Zheng Ma,
Xiao-Jun Wang,* Jian Yin*
Light(665nm)
Receptor
mannose-conjugated
BODIPY photosensitizer and
Tween 80. These
HO HO
HO
HO
O
3O2
N
O
N
N
O
O
O
B
I
I
N
N
F
F
Targeted Delivery of
Mannose-Conjugated
BODIPY Photosensitizer by
Nanomicelles for
Photodynamic Breast Cancer
Therapy
Cancer cells
nanomicelles exhibit
O
O
O
1O2
O
HO
HO
HO HO
HO
HO
O
O
HO
HO
O
O
excellent targeted delivery of
the photosensitizer that not
only leads to selective cancer
cell death but also minimizes
damage to normal cells after
665-nm LED light irradiation.
O
O
N
H
O
H
O
HO
O
N
N
H
N
O
N
N
N
O
N
N
O
Nucleus
O
O
I
I
N
N
B
F
F
Lysosome
O
O
O
O
H
HO
HO
O
HO
O
H
O
HO
O
HO
O
H
O
O
O
N
O
N
N
N
N
N
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