Comparative study of two near-infrared coumarin-BODIPY dyes for bioimaging and photothermal therapy of cancer

Article abstract of DOI:10.1039/c9tb01165j

Near-infrared (NIR) responsive agents for cancer bioimaging and photothermal therapy are available and significant. Herein, we employed two easily available dyes, boron-dipyrromethane and coumarin, to synthesize a pair of coumarin-borondipyrromethane dyes with different conjugate degrees (BDC and BSC). The difference in conjugate degree made their photophysical properties poles apart. After the self-assembling of BDC and BSC, the newly constructed nanoparticles (BDC NPs and BSC NPs) demonstrated good biocompatibility. Moreover, BDC NPs exhibited a good photothermal effect under irradiation of 808 nm laser, which could effectively inhibit the growth of HeLa cells, and BSC NPs could quickly show a conspicuous fluorescence in the HeLa cells. The exploration demonstrates that the two organic dyes prepared with different conjugation degrees could provide new options for photothermal therapy of cancer and rapid bioimaging.

Full text of DOI:10.1039/c9tb01165j

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PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
particles
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Received 00th January 20xx, Accepted Comparison Study of Two Near‐Infrared Coumarin‐BODIPY Dyes
00th January 20xx
for Bioimaging and Photothermal Therapy of Cancer
DOI: 10.1039/x0xx00000x
Yuandong Zhang,a Nan Song,a Yuanyuan Li,c Zhiyu Yang,a Li Chen,*a Tingting Sun, b Zhigang Xie*b
Near‐infrared (NIR) responsive agents for cancer bioimaging and photothermal therapy are available and significant. Herein,
we employed two easily available dyes, boron‐dipyrromethane and coumarin, to synthesize
a pair of
coumarin‐borondipyrromethane dyes with different conjugate degrees (BDC and BSC). The difference of conjugate degree
made their photophysical properties poles apart. After self‐assembling of BDC and BSC, the newly constructed nanoparticles
(BDC NPs and BSC NPs) demonstrated good biocompatibility. Moreover, BDC NPs exhibited a good photothermal effect
under irradiation of 808 nm laser which could effectively inhibit the growth of HeLa cells, and BSC NPs could quickly show a
conspicuous fluorescence in the HeLa cells. The exploration demonstrates that the two organic dyes prepared with different
conjugation degrees could provide new options for photothermal therapy of cancer and rapid bioimaging.
advantage are widely expanded in the fields of carbon dots45‐47
quantum dots48, fluorescent proteins49, 50, and organic dyes51‐55
,
.
Introduction
Non‐invasive treatment and bioimaging have attracted the
researchers’ interest. Photothermal therapy (PTT) is one of the
emerging non‐invasive treatment methods of cancer with low
toxicity, high selectivity, and perfect biological adaptability1‐8
BODIPY and its derivatives have certain advantages, such as high
molar extinction coefficient and photochemical stability56, 57. The
core of BODIPY is easily modified, in particular, the 3,5positions are
regularly employed to introduce the vinyl group which could broaden
the conjugation degree of the molecule and cause the red shift of
absorption wavelength to the NIR region. Coumarin, a common
heterocyclic compound, owns an easily modified structure, as a
result, it has a wide variety of derivatives and is always applied as
.
Photothermal agents could convert the irradiated photon into heat,
and the increasing temperature would induce the heat susceptive
cancer cells apoptosis9‐12. Near‐infrared (NIR) light could penetrate
deep tissues effectively, as a result, the absorption of photothermal
agents in this interval is of great significance. Photothermal agents
are commonly classified into inorganic nanomaterials such as gold
nanomaterials13‐15 and metal‐organic frameworks (MOFs)16‐19, and
organic
nanomaterials like porphyrin derivatives20‐22, cyanine dyes23‐25, and
boron‐dipyrromethenes (BODIPYs)26‐31. Organic photothermal agents
possess many advantages in terms of biocompatibility, metabolism,
and potential biodegradability. Non‐invasive bioimaging has been
bioluminescent probe58‐60
.
Knoevenagel condensation reaction was typically a series of
reactions which carbonyl compounds and the compounds with active
methyl group under a base catalyzed condition were dehydrated to
afford the α, β‐ unsaturated compounds. In synthesis of BODIPY
derivatives, Knoevenagel condensation reaction was facilely and
versatilely employed to efficiently tune BODIPY dyes for near‐infrared
absorption or emission61‐63
.
validated as a widely
applicable tool for clinical diagnostics and laboratory studies32,
33
Currently, the researchers usually use coumarin‐BODIPY
derivatives as sensitive fluorescent probes64‐67. Nevertheless, the
coumarin‐BODIPY derivatives with near‐infrared absorption and
.
Today, a wide variety of bioimaging techniques such as ultrasound
imaging (US)34, 35, CT imaging36, 37, magnetic resonance imaging
(MRI)38‐40 and positron emission computed tomography (PET)41,
42
employed as
a photothermal agent or a rapid fluorescence
and fluorescence imaging43, 44 are deeply developed. Fluorescence
imaging holds numbers of advantages such as nontoxicity, rapid
feedback and high sensitivity. NIR
bioimaging agent have not been reported yet.
In this work, we synthesized two coumarin‐BODIPY derivatives
(BDC and BSC) by Knoevenagel condensation reaction with
acetonitrile as solvent (Scheme 1). The difference between BDC and
BSC is a 7‐diethylaminocoumarin, which is linked to BODIPY by a π‐π
double bond. This dissimilarity has generated many differences
between BDC and BSC in their photophysical properties, as well as
their nanoparticles in photothermal effects, bioimaging, and
photothermal toxicity. BSC possessed more excellent fluorescence
performance, which laid a foundation for BSC NPs in bioimaging. BDC
retained a longer absorption wavelength and a low fluorescence
quantum
a. Department of Chemistry, Northeast Normal University, 5268 Renmin Street,
Changchun 130024, P. R. China. E‐mail: chenl686@nenu.edu.cn.
b. State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun
130022, P. R. China. E‐mail: xiez@ciac.ac.cn.
c. The First Hospital of Jilin University, Xinmin Street, Changchun, Jilin 130021, P.R. China.
E‐mail: lyy2017@ciac.ac.cn.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
fluorescence bioimaging agents with a strong competitive
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116.87, 109.46, 109.27, 97.24, 44.97, 14.61, 14.37, 12.54.
MALDI‐TOF/MS for [BSC]+: 551.26 calcd, 551.3 found. Preparation of
BDC nanoparticles and BSC nanoparticles (BDC NPs and BSC NPs)
By slowly adding, a 3 mL BDC‐THF solution was dropped into a rapid
stirring 10 ml water in 20 min. After addition, the mixed solution was
further stirred at room temperature for another 16 hours, with the
intention that THF in the mixture was diffused to the external
environment, and the nanoparticles were uniformly and stably
present in aqueous solution. With the same program, acetone was
more suitable as a solvent for BSC to prepare a more concentrated
BSC NPs solution. Photothermal Activity and Photothermal
Conversion Efficiency
Scheme 1 Schematic illustration of BDC NPs and BSC NPs for their synthesis and different
applications.
The BDC NPs and BSC NPs (65.0 μM, 200 μL) were under the
irradiation of an 808 nm laser at the intensity of 0.70 W cm‐2 for a
period of 300 s with DI water as a control. To affirm the promising
photothermal activity, we measured the different concentrations of
BDC NPs were adopted and irradiated by an 808 nm laser (0.70 W
cm−2) for 300 s, and the 65.0 μM BDC NPs were under the irradiation
of the 808 nm laser with different power densities for 300 s. The
concentrations of BDC NPs we chose were 16.3 μM, 32.5 μM, 65.0
μM, the chose different power densities of 808 nm laser were 0.23 W
cm−2, 0.48 W cm−2 and 0.70 W cm−2, the used amount of BDC NPs for
each sample was 200 μL. The temperature recorder probe was hired
to monitor the temperature changes and recorded 10 s a period. The
808 nm laser was in employment to irradiate the 70.0 μM BDC NPs
until the temperature formed a plateau, then insulated the laser,
recorded the whole temperature interval 10 s a count until the
temperature restored to room temperature again. Then calculated
photothermal conversion efficiency. The main text of the article
should appear here with headings as
yield, which provided the precondition for BDC NPs to be applied in
PTT tumor cells.
Experimental section
Synthesis of BDC and BSC
To increase the absorbance wavelength of BODIPY, we designed and
synthesized the coumarin‐BODIPY conjugate compounds. The
synthesis routes of BDC and BSC were shown in Scheme 1, and the
compounds 1 and 2 were detailed in Scheme S1, and S2 (ESI†). We
confirmed BDC and BSC by 1H NMR spectroscopy, 13
NMR spectroscopy, and MS in Fig. S1 and S2 (ESI†).
BDC
C
Compound 1 (184 mg, 0.5 mM) and compound 2 (382 mg, 1.5 mM)
were stirred in a solution of 30 mL acetonitrile, 1.0 mL piperidine and
0.8mL glacial acetic with a Dean‐Stark apparatus at 90 oC for 8h under
an argon atmosphere. Cooled, filtered, and washed with methanol
then the solid was subjected to column chromatography through a
silica gel column and eluted with DCM: EtOAc (100: 1) and finally
dried in vacuum, a brown solid was afforded. Yield = 32% (124.5 mg,
0.16 mM); 1H NMR (400 MHz, CHCl3) δ 8.04 (s, 2H), 7.70 (d, J = 16.4
Hz, 2H), 7.49 ‐ 7.46 (m, 4H), 7.43 (s, 1H), 7.39 (d, J = 9.2 Hz, 2H), 7.32
‐ 7.29 (m, 2H),
appropriate.
Cell Imaging and Flow Cytometry
The HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM; GIBCO) which composed by 10% fetal bovine serum (FBS)
and 100X Penicillin/Streptomycin Solution under a mixed humidified
atmosphere which 5% CO2 contained at 37 oC. After a 12h culture, the
HeLa cells were incubated with the nanoparticles (20.0 μM) for
different time periods. Flow cytometry was hired with a flow
cytometer by the method of
6.67 (s, 2H), 6.60 (dd, J = 8.8, 2.4 Hz, 2H), 6.48 (d, J = 2.4 Hz, 2H), 3.43
(q, J = 7.2 Hz, 8H), 1.41 (s, 6H), 1.22 (t, J = 7.0 Hz, 12H). 13C NMR (500
MHz, CHCl3) δ 161.81, 156.25, 152.42, 150.97, 141.97, 137.49, 133.76,
129.69, 129.54, 129.07, 128.97, 128.53, 118.48, 118.44, 117.02,
109.41, 109.39, 97.27, 44.99, 14.62,
cell imaging treatment process. MTT
assay
The HeLa cells were planted at a density of 105 cells per well in 96‐well
plates then cultured in DMEM for 12 hours to allow cell adhesion. By
engaging 200 µL of DMEM contained various concentrations BDC NPs
instead of the medium, 6 hours later, a NIR 808 nm laser (0.70 W cm‐2)
12.56. MALDI‐TOF/MS for [BDC]+: 778.35 calcd, 778.4 found. BSC
As a by‐product of BDC, BSC was present in the filtrate solution of
BDC. Then collected and dried in vacuum, the mixture was subjected
to column chromatography through a silica gel column and eluted
with DCM, a deep green crystal was afforded. Yield = 8% (22 mg, 0.04
o
was hired to irradiate the cells for 5 min at 37 C and culture for an
additional 24 h. Then 20 μL MTT (5 mg mL‐1) assays were added to
cells in each well and incubated for another 4 hours. Finally, removed
the medium cleanly, and DMSO (150 μL) was added. The
characteristic absorption at λ = 490 nm was determined and recorded
by a
1
mM); H NMR (400 MHz, CHCl3) δ 7.99 (s, 1H), 7.62 (d, J = 16.6 Hz,
2H), 7.49 ‐ 7.43 (m, 4H), 7.35 (d, J = 8.8 Hz, 2H), 7.30 ‐ 7.27 (m, 4H),
6.65 (s, 1H), 6.60 (dd, J = 8.8, 2.4 Hz, 2H), 6.47 (d, J = 2.0 Hz, 1H), 5.99
(s, 1H), 3.41 (q, J = 7.2 Hz, 4H), 2.61 (s, 3H), 1.38 (d, J = 9.2 Hz, 6H),
1.22 (t, J = 7.2 Hz, 6H). 13C NMR (500 MHz, CHCl3) δ 161.87, 156.27,
154.90, 152.89, 151.00, 142.63, 140.10, 137.36, 135.08, 131.91,
129.66, 129.62, 129.11, 128.97, 128.22, 121.24, 118.28, 118.19,
microplate analyzer. Live−Dead
Cell Staining To more directly
demonstrate the efficiency of
BDC NPs in photothermal toxicity
for HeLa cells, calcein AM and
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DOI: 10.1039/C9TB01165J
propidium iodide (PI) were
employed to identify live (green)
and dead (red) cells, respectively.
The HeLa cells were treated with
the same method in MTT assays,
while the untreated cells as a
control. The cells were incubated
orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)
energy levels of BDC and BSC (Fig. 1c).
Table 1 Photophysical data of BDC and BSC (a) measured in CHCl3, (b) measured for the
nanoparticles of BDC and BSC in water.
a λmax, Abs a λmax, Em
a Δλ
(nm)
40
b λmax, Abs
Comp.
a Φf
(nm)
(nm)
732
623
(nm)
772
665
BDC
BSC
0.037
0.322
759
635
under
a humidified 5% CO2
42
contained mixed atmosphere at
37 oC for 24 h. Subsequently, the
calceinAM/PI
solution
was
For BDC, we could unearth the charges distributed on HOMO and
LUMO were different, and they had an apparent charge transfer
tendency from the 7‐diethylaminocoumarin moieties to the BODIPY
core, which confirmed BDC possessed an ICT process. While the
charge of BSC was regularly distributed on HOMO and LUMO, which
indicated that the BSC molecule dominated a rarely ICT tendency.
Evidenced by transmission electron microscopy (TEM), the
self‐assembled BDC NPs and BSC NPs were confirmed as uniform solid
spheres, and their sizes were about 160 and 100 nm respectively (Fig.
2a, S3 and S4). Dynamic light scattering (DLS) was usually used to
support the results of TEM, the average hydrodynamic diameter and
the polydispersity index (PDI) of BDC NPs were about 232 nm and
0.129 for each, and BSC NPs owned 197 nm and 0.134, respectively
(Fig. S3 and S4). The stability in aqueous solution is one of the
essential indicators for nanoparticles. In Fig. S3, BDC NPs and BSC NPs
were given eight days tested of hydrodynamic diameter and PDI in
aqueous solution, and they did not exhibit a significant amplitude.
After characterizing the morphology of BDC NPs and BSC NPs, we
determined their absorption spectra (Fig. 2b). Compared with the
absorption spectra of their respective molecules in chloroform, the
absorption peak shape of nanoparticles was
employed to stain the cells for 40
min at room temperature.
Finally, the cell staining results
were
acquired by a fluorescence microscope. Cell
apoptosis test
To investigate the anticancer mechanism of BDC NPs, flow cytometry
experiments were performed to demonstrate the process of
apoptosis, in this experiment 5×103 cells were collected for each
sample. In 6‐well plates, the HeLa cells were cultured in 1.5 mL
medium for 12 h to obtain 3×105 cells per well. With the same
method of live‐dead cell staining, after a 24 h incubation discarded
the medium in each well. The cells were washed by 1 mL PBS, then
0.5 mL trypsin was treated to disperse the cells. The harvested
suspended cells were centrifuged at 2000 rpm for 5 min, discarded
the supernatant and washed by PBS. Then repeated centrifugation
and discarding, Annexin V‐FITC and PI were employed to co‐stain the
cells in dark for 20 min. Finally, the cells were collected and evaluated
by a flow cytometer.
Results and discussion
By employing UV‐vis spectrophotometer and fluorescence
spectrophotometer, the photophysical properties of BDC and BSC
were recorded. BDC and BSC were synthesized from BODIPY and
7‐diethylaminocoumarin via Knoevenagel condensation reaction.
The newly generated π‐π double bond and the one at the 3,4‐position
of 7‐diethylaminocoumarin moiety formed conjugated double bonds,
generated the conjugate effect, increased the degree of conjugation
and caused red‐shift of the maximum absorption wavelength. As
exhibited in Fig. 1a, the, absorption spectrum of BDC in CHCl3
disclosed two main peaks at 490 and 732 nm, as well as a weak hillock
shape peak at 392 nm, while BSC owned two main peaks at 623 and
443 nm. In the fluorescence spectra, BDC and BSC possessed a 772
and 665 nm emission wavelength (Fig. 1b), and the relative
fluorescence quantum yield (Φf) of BSC was 0.322, almost ten times
higher than that of BDC (Φf = 0.037). The Stock shift (Δλ) of BDC and
BSC was 40 and 42 nm, respectively. The photophysical data of BDC
and BSC measured in CHCl3 were
presented in Table 1.
Both coumarin and BODIPY can achieve intramolecular charge Fig. 1 The normalized absorption spectra (a) and fluorescence spectra (b) of BDC and BSC
in CHCl3 with the built‐in images of BDC and BSC in CHCl 3 under daylight (a) and upon
excitation by 365 nm lamp (b). (c) Electron density contours and orbital energies
calculated for the HOMO and LUMO of BDC and BSC at the Beck3LYP/DZP level in
vacuum.
transfer (ICT) by changing the substituents at different sites. In order
to apprehend the charges transfer in BDC and BSC, we appointed the
density functional theory (DFT) with B3LYP/6‐31G(d) level of the
Gaussian 09 program to calculate of the highest occupied molecular
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dependent. To measure the photothermal conversion efficiency of
BDC NPs, a 300 s heating curve was collected from the nanoparticles
(70.0 μM) under 808 nm laser irradiation at a power density of 0.70
W cm‐2 (Fig. 3d). Then we removed the irradiation and recorded a
cooling time plot versus negative natural logarithm of the
temperature as revealed in Fig. 3e (τ= 183 s) to evaluate the
photothermal conversion efficiency (η), and the calculated value was
25%. After five cycles of heating and cooling, BDC NPs still had an
apparent photothermal effect, which represented good
photothermal stability.
The cellular uptake for BDC NPs and BSC NPs was explored by
confocal laser scanning microscopy (CLSM). HeLa cells were
incubated with BDC NPs and BSC NPs with different time at 37 oC. As
revealed in Fig. 4a and 5a, the green and red fluorescence were found
in the cytoplasm around the nuclei of the cells under 488 nm (green
channel) and 555 nm (red channel) laser
Fig. 2 The Basic physical properties of BDC NPs and BSC NPs. (a) The TEM images of BDC
NPs and BSC NPs. (b) The normalized absorbance spectra of BDC NPs and BSC NPs.
basically the same. However, the maximum absorption wavelengths
of BDC NPs and BSC NPs had a specific red shift than the molecular
ones. The λmax of BDC NPs was 759 nm, a 27 nm red shift than BDC in
chloroform, in contrast, the red shift degree of BSC NPs was much
lower as a 12 nm red shift (635 nm). The longer maximum absorption
wavelength of BDC NPs forecasted a higher absorption intensity in
the near‐infrared region.
To investigate the photothermal properties of BDC NPs and BSC
NPs, firstly, we recorded and compared the photothermal heating
curves of 65.0 μM BDC NPs, BSC NPs and water with an 808 nm laser
(0.70 W cm‐2) irradiation for 300 s (Fig. 3a). Only BDC NPs were
prompted a remarkable temperature change (ΔT: 35.6 oC), while BSC
NPs and water had little temperature changes, which might be
determined by whether the absorption wavelength located at 808
nm. Then the photothermal performance of BDC NPs with different
laser power intensities and concentrations was tested. In Fig. 3b and
3c, we investigated the heating effect of BDC NPs under 808 nm laser
irradiation for 300 s and the result showed that the heating effect was
not only laser‐power intensity dependent but also concentration
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excitation after the incubation with BDC NPs and BSC NPs, which
confirmed BDC NPs and BSC NPs could be gradually endocytosed and
enriched in the cytoplasm. By comparing with the fluorescence
intensities under red channel laser excitation in different time
periods, a clear tendency of enhancement could be observed from
the cells incubated with BDC NPs from 1 to 6 h, signifying that the
BDC NPs could be continuous internalized by HeLa cells (Fig. 4b and
4c). Surprisingly, with the same concentration of BDC NPs, BSC NPs
only needed 1 minute incubation with the HeLa cells to show a similar
fluorescence intensity as BDC NPs incubated with the cells for 6 hours
(Fig. 4b, 4c, 5b and 5c). This phenomenon meant BSC NPs could
quickly stain the HeLa cells in a very short time (1 minute). With the
incubation time of BSC NPs going to 10 minutes, the fluorescence
intensity in HeLa cells was times higher than the incubation of 1
minute (Fig. 5b and 5c). It probably because BSC NPs owned a small
size and superior photophysical properties. With the same
conditions, the flow cytometry results could also demonstrate that
both BDC NPs and BSC NPs could have a tendency to accumulate in
HeLa cells (Fig. 4d and 5d). The consequences established that BSC
NPs could not only be
Fig. 5 The CLSM images of (a) HeLa cells incubated with BSC (20.0 μM) for 10 min under
the excitation of different channels, Scale bars: 20 μm. (b) HeLa cells incubated with BSC
(20.0 μM) for different periods in the red channel, Scale bars: 20 μm. (c) Relative
fluorescence intensity of HeLa cells incubated with BSC NPs under the same condition as
the CLSM images for different periods in the red channel. (d) The flow cytometry analysis
of the HeLa cells incubated with BSC NPs under the same condition of the CLSM images
for different periods with control of untreated cells by flow cytometry.
internalized by cells in a very short time but also be supported as a
rapid cell imaging agent.
To appraise the phototherapy efficacy of BDC NPs for cancer and the
biocompatibility of BSC NPs for bioimaging, the cytotoxicity of BDC
NPs and BSC NPs toward HeLa and L929 cells were
measured
by
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide (MTT) assays. No cytotoxicity was
observed from HeLa and L929 cells incubated with BDC NPs and BSC
NPs in the dark (Fig. 6a and S5), which demonstrated that BDC NPs
and BSC NPs were biocompatible. Compared with the
biocompatibility in dark, BDC NPs possessed a concentration
dependent cytotoxicity toward HeLa and L929 cells under 808 nm
laser irradiation (0.70 W cm−2, 5 min). The concentration of 26.0 μM
BDC NPs was sufficient to eradicate the vast majority of the
co‐incubated HeLa cells under the irradiation. By calculation, a half
maximal inhibitory concentration (IC50) value of BDC NPs was 14.4
μM. Such good biocompatibility and superior phototoxicity proved
BDC NPs had great potential as a photothermal agent to be applied
in the field of cancer photothermal clinical treatment.
Fig. 4 The CLSM images of (a) HeLa cells incubated with BDC NPs (20.0 μM) for 6 h under
the excitation of different channels, Scale bars: 20 μm. (b) HeLa cells incubated with BDC
(20.0 μM) for different periods in the red channel, Scale bars: 20 μm. (c) Relative
fluorescence intensity of HeLa cells incubated with BDC NPs under the same condition
as the CLSM images for different periods in the red channel. (d) The flow cytometry
analysis of the HeLa cells incubated with BDC NPs under the same condition as the CLSM
images for different periods by flow cytometry.
To intuitively define the phototoxicity of BDC NPs toward the HeLa
cells, the live‐dead cell staining experiment was established. For
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authenticating BDC NPs and laser irradiation was the triggering of the cell death, calcein‐acetoxymethyl (AM)
nanoparticles. BDC NPs and BSC NPs exhibited good
a
biocompatibility in dark. BDC NPs demonstrated the phototoxicity by
the irradiation of 808 nm laser toward the HeLa cells and BSC NPs
could be employed as a rapid cell imaging agent. Consequently, we
can assert that BDC NPs could be applied as a photothermal agent in
cancer treatment and BSC NPs could be a rapid bioimaging agent.
Conflicts of interest There
are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (Project No. 21474012, 51273037).
Fig. 6 Cytotoxicity against HeLa cells. (a) The viability of the cells exposed under the
irradiation of 808 nm laser (0.70 W cm ‐2) for 5 min. (b) The live (green) and dead (red )
fluorescence images of the cells co‐stained with calcein‐AM and PI after different
treatments, Scale bars: 100 μm. (c) The flow cytometry apoptosis analysis of the HeLa
cells treated with different conditions; the four regions represent different stages, Q1:
cell necrosis, Q2: late apoptosis, Q3: early apoptosis, and Q4: survival.
Notes and references
1. Y. Jiang, J. Li, X. Zhen, C. Xie and K. Pu, Adv. Mater., 2018, 30,
1705980.
2. H. S. Jung, P. Verwilst, A. Sharma, J. Shin, J. L. Sessler and J. S. Kim,
Chem. Soc. Rev., 2018, 47, 2280‐2297.
and propidium iodide (PI) were imposed on making a distinction
between the living cells with green fluorescence and the dead ones
with red fluorescence (Fig. 6b). The cells treated with laser irradiation
or BDC NPs in dark could maintain the normal morphology, which
indicated they were as healthy as the ones in the control group with
apparent green fluorescence. Contrariwise, the cells incubated with
BDC NPs and irradiated with laser were demonstrated a garish red
fluorescence, and the cell morphology was irreversibly destroyed.
To ascertain the evolution of the cell death, flow cytometry
apoptosis analysis was taken advantage to clarify the approach which
BDC NPs and the 808 nm laser involved in cancer treatment. The
fluoresceine isothiocyanate (Annexin V‐FITC) and PI were utilized in
double‐labeled HeLa cells to single out the process of the cells such
as early apoptotic cells, late apoptotic cells, necrotic cells, and live
cells. In Fig. 6c, a safe rate of cell apoptosis (Q1+Q2+Q3) exhibited in
the control group, the cells incubated with BDC NPs group and the
cells under 808 nm laser irradiation group. The group of the cells
treated with BDC NPs and laser irradiation demonstrated a perfect
necrotic cell rate (Q1) of 89.3%, which explained the collaboration of
BDC NPs and laser irradiation produced heat and caused the necrosis
of cells. As expected, the flow cytometry analysis results were
practically consistent with the MTT consequences and validated that
the progress of BDC NPs and laser irradiation anticancer was
achieved by photothermal toxicity caused cell necrosis.
3. X. Li, H. Bai, Y. Yang, J. Yoon, S. Wang and X. Zhang, Adv. Mater.,
2019, 31, 1805092.
4. Y. Liu, P. Bhattarai, Z. Dai and X. Chen, Chem. Soc. Rev., 2019, 48,
2053‐2108.
5. L. Y. Zhao, Y. M. Liu, R. Chang, R. R. Xing and X. H. Yan, Adv. Funct.
Mater., 2019, 29, 1806877.
6. H. S. Jung, J. H. Lee, K. Kim, S. Koo, P. Verwilst, J. L. Sessler,
C. Kang and J. S. Kim, J. Am. Chem. Soc., 2017, 139, 9972‐
9978.
7. N. Shao, Y. Qi, H. Lu, D. He, B. Li and Y. Huang, ACS Biomater. Sci.
Eng., 2018, 5, 795‐804.
8. Y. Yan, J. Chen, Z. Yang, X. Zhang, Z. Liu and J. Hua, J. Mater. Chem.
B, 2018, 6, 7420‐7426.
9. Y. Cai, W. Si, W. Huang, P. Chen, J. Shao and X. Dong, Small, 2018,
14, 1704247.
10. Y. Lyu, J. Zeng, Y. Jiang, X. Zhen, T. Wang, S. Qiu, X. Lou, M. Gao
and K. Pu, ACS Nano, 2018, 12, 1801‐1810.
11. J. Nam, S. Son, L. J. Ochyl, R. Kuai, A. Schwendeman and J. J.
Moon, Nat. Commun., 2018, 9, 1074.
12. H. Wang, J. Chang, M. Shi, W. Pan, N. Li and B. Tang, Angew.
Chem. Int. Ed. Engl., 2019, 58, 1057‐1061.
13.Z. D. Hood, K. P. Kubelick, K. D. Gilroy, D. Vanderlaan, X. Yang, M.
Yang, M. Chi, S. Y. Emelianov and Y. Xia, Nanoscale, 2019, 11,
3013‐3020.
14. N. Khlebtsov and L. Dykman, Chem. Soc. Rev., 2011, 40,
1647‐1671.
15. L. Dykman and N. Khlebtsov, Chem. Soc. Rev., 2012, 41,
2256‐2282.
Conclusions
In summary, we designed and synthesized
a pair of novel
nearinfrared coumarin‐BODIPY dyes, which could self‐assemble into
organic nanoparticles. BDC and BSC presented different conjugation
degree, absorption wavelength, and fluorescence characteristics.
The differences between BDC and BSC were expanded on their
16. W. Zhu, Y. Liu, Z. Yang, L. Zhang, L. J. Xiao, P. Liu, J. Wang, C.
F. Yi, Z. S. Xu and J. H. Ren, J. Mater. Chem. B, 2018, 6, 265‐
276.
6 | J. Name., 2012, 00, 1‐3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 9
Journal of Materials Chemistry B
Please do not adjust margins
View Article Online
Journal Name
ARTICLE
17. X. H. Zheng, L. Wang, M. Liu, P. P. Lei, F. Liu and Z. G. Xie, Chem. 40. G. Bell, G. Balasundaram, A. B. E. Attia, F.DMOaI:n1d0i.n10o3,9M/C.9OTlBiv0o11a6n5dJ
Mater., 2018, 30, 6867‐6876. I. P. Parkin, J. Mater. Chem. B, 2019, 7, 2212‐2219.
18. Y. Li, N. Xu, J. Zhou, W. Zhu, L. Li, M. Dong, H. Yu, L. Wang, W. Liu 41. N. Adonai, N. Adonai, K. N. Nguyen, J. Walsh, M. Iyer, T. Toyokuni,
and Z. Xie, Biomater. Sci., 2018, 6, 2918‐2924.
19. J. Feng, Z. Xu, P. Dong, W. Q. Yu, F. Liu, Q. Y. Jiang, F. Wang and X.
Q. Liu, J. Mater. Chem. B, 2019, 7, 994‐1004.
M. E. Phelps, T. McCarthy, D. W. McCarthy and S.
S. Gambhir, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 3030‐
3035.
20. X. H. Zheng, L. Wang, S. Liu, W. Zhang, F. G. Liu and Z. G. Xie, Adv.
Funct. Mater., 2018, 28, 1706507.
21. R. Xing, Q. Zou, C. Yuan, L. Zhao, R. Chang and X. Yan, Adv. Mater.,
2019, DOI: 10.1002/adma.201900822, 1900822.
22. X. Li, C. Y. Kim, S. Lee, D. Lee, H. M. Chung, G. Kim, S. H. Heo, C.
Kim, K. S. Hong and J. Yoon, J. Am. Chem. Soc., 2017, 139,
10880‐10886.
23. Y. Deng, L. Huang, H. Yang, H. Ke, H. He, Z. Guo, T. Yang, A. Zhu,
H. Wu and H. Chen, Small, 2017, 13, 1602747.
24. J. X. Zhang, S. Liu, X. L. Hu, Z. G. Xie and X. B. Jing, ACS Biomater.
Sci. Eng., 2016, 2, 1942‐1950.
25. C. Gao, X. Liang, S. Mo, N. Zhang, D. Sun and Z. Dai, ACS Appl.
Mater. Interfaces, 2018, 10, 3219‐3228.
26. N. Song, L. Fu, Y. Liu, Y. Y. Li, L. Chen, X. Y. Wang, S. Liu and Z. G.
Xie, Dyes Pigm., 2019, 162, 295‐302.
27. X. Wang, W. Lin, W. Zhang, C. Li, T. Sun, G. Chen and Z. Xie, J.
Colloid Interface Sci., 2019, 536, 208‐214.
28. W. Sun, X. Zhao, J. Fan, J. Du and X. Peng, Small, 2019, DOI:
10.1002/smll.201804927, 1804927.
29. X. Chen, Q. Wang, L. Liu, T. Sun, W. Zhou, Q. Chen, Y. Lu, X. He, Y.
Zhang, Y. Zhang, C. Ruan, Q. Guo, C. Li and C. Jiang, Biomaterials,
2018, 183, 258‐267.
30. D. Chen, J. Zhang, Y. Tang, X. Huang, J. Shao, W. Si, J. Ji, Q. Zhang,
W. Huang and X. Dong, J. Mater. Chem. B, 2018, 6, 4522‐4530.
31. D. Chen, Q. Tang, J. Zou, X. Yang, W. Huang, Q. Zhang, J. Shao and
X. Dong, Adv. Healthcare Mater., 2018, 7, 1701272.
42. Y. Sun, X. Zeng, Y. Xiao, C. Liu, H. Zhu, H. Zhou, Z. Chen, F. Xu, J.
Wang, M. Zhu, J. Wu, M. Tian, H. Zhang, Z. Deng, Z. Cheng and X.
Hong, Chem. Sci., 2018, 9, 2092‐2097.
43. Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014, 43,
16‐29.
44. G. Hong, A. L. Antaris and H. Dai, Nat. Biomed. Eng., 2017, 1, 0010.
45. H. He, X. Zheng, S. Liu, M. Zheng, Z. Xie, Y. Wang, M. Yu and
X. Shuai, Nanoscale, 2018, 10, 10991‐10998.
46.L. Qiao, T. Sun, X. Zheng, M. Zheng and Z. Xie, Mater. Sci. Eng. C
Mater. Biol. Appl., 2018, 85, 1‐6.
47. M. Zheng, S. Ruan, S. Liu, T. Sun, D. Qu, H. Zhao, Z. Xie, H. Gao, X.
Jing and Z. Sun, ACS Nano, 2015, 9, 11455‐11461.
48. J. Zhang, J. Qian, Q. Mei, L. Yang, L. He, S. Liu, C. Zhang and K.
Zhang, Biosens. Bioelectron., 2019, 128, 61‐67.
49. Y. Liu, C. H. Wolstenholme, G. C. Carter, H. Liu, H. Hu, L. S.
Grainger, K. Miao, M. Fares, C. A. Hoelzel, H. P. Yennawar, G.
Ning, M. Du, L. Bai, X. Li and X. Zhang, J. Am. Chem. Soc., 2018,
140, 7381‐7384.
50. N. Coquelle, M. Sliwa, J. Woodhouse, G. Schiro, V. Adam, A.
Aquila, T. R. M. Barends, S. Boutet, M. Byrdin, S. Carbajo, E. De la
Mora, R. B. Doak, M. Feliks, F. Fieschi, L. Foucar, V. Guillon, M.
Hilpert, M. S. Hunter, S. Jakobs, J. E. Koglin, G. Kovacsova, T. J.
Lane, B. Levy, M. Liang, K. Nass, J. Ridard, J. S. Robinson, C. M.
Roome, C. Ruckebusch, M. Seaberg, M. Thepaut, M. Cammarata,
I. Demachy, M. Field, R. L. Shoeman, D. Bourgeois, J. P. Colletier,
I. Schlichting and M. Weik, Nat. Chem., 2018, 10, 31‐37.
32. Y. Wang, K. Zhou, G. Huang, C. Hensley, X. Huang, X. Ma, T. Zhao, 51. X. Yang, N. N. Shi, L. Bai, Y. Ni, J. W. Li, W. L. Si, L. Li, J. J. Shao, W.
B. D. Sumer, R. J. DeBerardinis and J. Gao, Nat. Mater., 2014, 13,
204‐212.
33. D. E. Lee, H. Koo, I. C. Sun, J. H. Ryu, K. Kim and I. C. Kwon, Chem.
Soc. Rev., 2012, 41, 2656‐2672.
Huang and X. C. Dong, Dyes Pigm., 2018, 157, 396‐404.
52. H. Wan, J. Yue, S. Zhu, T. Uno, X. Zhang, Q. Yang, K. Yu, G. Hong,
J. Wang, L. Li, Z. Ma, H. Gao, Y. Zhong, J. Su, A. L. Antaris, Y. Xia, J.
Luo, Y. Liang and H. Dai, Nat. Commun.,
34. W. Tang, Z. Yang, S. Wang, Z. Wang, J. Song, G. Yu, W. Fan,
Y. Dai, J. Wang, L. Shan, G. Niu, Q. Fan and X. Chen, ACS Nano,
2018, 12, 2610‐2622.
35. Y. Wang, G. Sui, D. Teng, Q. Wang, J. Qu, L. Zhu, H. Ran, Z.
Wang, C. Jin and H. Wang, Biomater. Sci., 2018, 7, 196‐210.
36. H. Zhang, Q. Zhang, C. Liu and B. Han, Biomater. Sci., 2019, 7,
1696‐1704.
2018, 9, 1171.
53. Q. Miao and K. Pu, Adv. Mater., 2018, 30, 1801778.
54. B. Li, L. Lu, M. Zhao, Z. Lei and F. Zhang, Angew. Chem. Int. Ed.
Engl., 2018, 57, 7483‐7487.
55. W. Fan, H. Deng, L. Zhu, C. Tu, Y. Su, L. Shi, J. Yang, L. Zhou,
L.
Xu
and
2019,
X.
Zhu,
Biomater.
Sci.,
DOI: 10.1039/c9bm00255c.
56. A. Turksoy, D. Yildiz and E. U. Akkaya, Coord. Chem. Rev., 2019,
379, 47‐64.
37. D. L. Kraitchman, M. Tatsumi, W. D. Gilson, T. Ishimori, D.
Kedziorek, P. Walczak, W. P. Segars, H. H. Chen, D. Fritzges, I.
Izbudak, R. G. Young, M. Marcelino, M. F. Pittenger, M. 57. J. Zou, P. Wang, Y. Wang, G. Liu, Y. Zhang, Q. Zhang, J. Shao,
Solaiyappan, R. C. Boston, B. M. Tsui, R. L. Wahl and J. W. Bulte,
Circulation, 2005, 112, 1451‐1461.
W. Si, W. Huang and X. Dong, Chem. Sci., 2019, 10, 268‐276.
58. D. K. Sharma, S. T. Adams, Jr., K. L. Liebmann, A. Choi and S. C.
Miller, Org. Lett., 2019, 21, 1641‐1644.
59. Y. Bian, Z. Wei, Z. Wang, Z. Tu, L. Zheng, W. Wang, X. Leng and Y.
Li, J. Mater. Chem. B, 2019, 7, 2261‐2276.
60. S. Feng, D. Liu and G. Feng, Anal. Chim. Acta, 2019, 1054, 137‐144.
61. X. Xia, Y. Qian and B. Shen, J. Mater. Chem. B, 2018, 6, 30233029.
62. M. H. Chua, T. Kim, Z. L. Lim, T. Y. Gopalakrishna, Y. Ni, J. Xu, D.
Kim and J. Wu, Chem. Eur. J., 2018, 24, 2232‐2241.
38. P. Vemuri, T. G. Lesnick, S. A. Przybelski, J. Graff‐Radford, R.
I. Reid, V. J. Lowe, S. M. Zuk, M. L. Senjem, C. G. Schwarz, J. L.
Gunter, K. Kantarci, M. M. Machulda, M. M. Mielke, R. C.
Petersen, D. S. Knopman and C. R. Jack, Jr., Ann. Neurol., 2018,
84, 705‐716.
39. F. Liu, X. Li, Y. Li, Y. Qi, H. Yuan, J. He, W. Li and M. Zhou, Biomater.
Sci., 2019, DOI: 10.1039/c9bm00056a.
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Journal Name
63. S. Zhu, J. Zhang, G. K. Vegesna, R. Pandey, F.‐T. Luo, S. A. Green
and H. Liu, Chem. Commun., 2011, 47, 3508‐3510.
64. K. B. Zheng, H. Chen, S. R. Fang and Y. Wang, Sens. Actuators, B,
2016, 233, 193‐198.
65. Y. Qian, B. Yang, Y. Shen, Q. Du, L. Lin, J. Lin, H. J. S. Zhu and
A. B. Chemical, Sensors Actuators B: Chem., 2013, 182, 498‐
503.
66. H. Lee, Z. Yang, Y. Wi, T. W. Kim, P. Verwilst, Y. H. Lee, G. I. Han,
C. Kang and J. S. Kim, Bioconjugate Chem., 2015, 26,
2474‐2480.
67. X. Cao, W. Lin, Q. Yu and J. Wang, Org. Lett., 2011, 13, 60986101.
8 | J. Name., 2012, 00, 1‐3
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Graphic Abstract
Herein, two novel NIR Coumarin BODIPYs with different conjugation degrees were
comparatively investigated for photothermal therapy and fluorescent bioimaging.