An aza-BODIPY photosensitizer for photoacoustic and photothermal imaging guided dual modal cancer phototherapy

Article abstract of DOI:10.1039/c6tb02979e

Developing biocompatible, near infrared absorbing, and multi-functional photosensitizers is crucial for effective cancer phototherapy. In this contribution, a BF₂ chelate of [4-iodo-5-(4-bromophenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl][4-iodo-5

Full text of DOI:10.1039/c6tb02979e

View Article Online
View Journal
Journal of
Materials Chemistry B
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Q. Tang, W. Si, C.
Huang, K. Ding, W. Huang, P. Chen, Q. Zhang and X. Dong, J. Mater. Chem. B, 2017, DOI:
10.1039/C6TB02979E.
This is an Accepted Manuscript, which has been through the
Volume
4 Number 1 7 January 2016 Pages 1–178
Royal Society of Chemistry peer review process and has been
accepted for publication.
Journal of
Materials Chemistry B
Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Please do not adjust margins
Journal of Materials Chemistry B
Page 1 of 9
View Article Online
DOI: 10.1039/C6TB02979E
Journal Name
ARTICLE
Aza-Bodipy Photosensitizer for Photoacoustic and Photothermal
Imaging Guided Dual Modal Cancer Phototherapy†
Qianyun Tang,a Weili Si,a Chuhan Huang,a Kaikai Ding,a Wei Huang,a Peng Chen,*b Qi Zhang,*c
Xiaochen Dong*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Developing biocompatible, near infrared absorbing, and multi-functional photosensizers is critical for effective cancer
phototherapy. In this contribution, a BF2 chelate of [4-iodo-5-(4-bromophenyl)-3-(4-methoxyphenyl)-1H-pyrrol-2-yl][4-
iodo- 5-(4-bromophenyl)-3-(4- methoxyphenyl)pyrrol-2-ylidene]amine (IABDP) with high singlet oxygen generation
efficiency (~92%) has been designed and synthesized. The soluble and near infrared adsorbing nanoparticles (NPs) can be
simply obtained from self-assembly of IABDP molecules, which has a high photothermal conversion efficiency (~37.9%).
Under irradiation of a broadband Xenon lamp, IABBDP NPs are able to serve as the common photosensitizer for
photothermal imaging (PTI) and photoacoustic imaging (PAI) guided simultaneous photodynamic therapy (PDT) and
photothermal therapy (PTT). Comparing to the usual combined strategies that require two distinct photosensitizers and
two excitation sources, IABBDP NP based approach is much simplified, hence more convenient, reliable, and cost effective.
Both in vitro and in vivo studies confirm the good biosafety and prominent anti-tumor phototoxicity of IABDP NPs. Finally,
we demonstrate that the imaging guided synergistic dual modal phototherapy enabled by IABDP NPs can essentially inhibit
tumor growth (87.2% inhibition) in mice without causing appreciable side-effects, testifying the great potential of this
www.rsc.org/
multi-functional
organic
photosensizer
for
clinical
use.
is known as the transparent window for deep tissue
penetration.4 However, the remnant cells may swiftly develop
heat resisting property, leading to cancer relapse and
metastasis.5 The efficacy of phototherapy should be greatly
enhanced by synergistic combination of PDT and PTT. But the
key towards this ambition is to devise a biocompatible
photosensitizer which can produce ROS and heat
simultaneously upon NIR irradiation. Moreover, mild
hyperthermia induced by PTT can improve vascular perfusion
whereby relieving the hypoxic condition in tumor and
enhancing extravasation of therapeutic nanoparticles into the
tumor interstitium.6
Introduction
Phototherapy for cancers, including photodynamic therapy
(PDT) and photothermal therapy (PTT), has attracted
tremendous research effort because of some unique
advantages over the conventional treatments, such as low
toxicity, high selectivity, minimal invasiveness, no issue of drug
tolerance.1 PDT relies on the photosensitizer to generate
reactive oxygen species (ROS, such as singlet oxygen 1O2,
superoxide anion radical O2-, and hydroxyl radical OH) under
-
irradiation, which, in turn, destroy tumor cells and tumor
blood vessels.2 However, the efficacy of PDT is severely
compromised by the inherent hypoxic microenvironment in
tumor tissues and the poor light penetration depth because
most PDT photosensitizers are activated by ultraviolet or
visible light.3 PTT is a therapeutic strategy in which photon
energy is converted into heat to burn tumor cells. Most
current photosensitizers (or photothermal agents) for PTT
absorb light in near-infrared (NIR, 700-1100 nm) region, which
Recently, efforts have been made to develop imaging-guided
cancer therapy in order to improve the effectiveness and
reduce the side-effects through precisely locating tumor sites,
optimizing treatment time, and monitoring treatment
progress.7 Photothermal imaging (PTI) is often applied in
conjunction with PTT.8 Although offering good temperature
sensitivity and possibility of real-time monitoring, PTI however
provides poor spatial resolution.9 Photoacoustic imaging (PAI)
based on the thermal wave generated by the photosensitizer
may be employed, which is a new non-ionizing and non-
invasive imaging modality with high optical contrast, good
penetration depth and high spatial resolution.10
Some inorganic nanomaterials (e.g., gold,11 carbon,12 metal
oxide,4, 13 or metal sulfide nanoparticles14) have been utilized
to realize imaging-guided synergistic phototherapy. But the
practice use of these methods is largely hindered by
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 2 of 9
View Article Online
DOI: 10.1039/C6TB02979E
ARTICLE
Journal Name
cytotoxicity of the inorganic photosensitizers,15 requirement
for dual-wavelength laser irradiation, requirement for multiple
functional components, or tedious synthesis process. In
addition, the laser sources are bulky and expensive; laser may
cause discomfort, erythema, crusting, blistering, and
dyspigmentation;16 and the narrow bandwidth of lasers
restricts their use to specific photosensitisers only.17 In
comparison with lasers, lamps are cheap broad-band light
sources that can be easily handled and maintained in clinical
settings.18 Therefore, it is highly desirable to design
a
biocompatible photosensitizer which can simultaneously
provide PTI, PAI, PDT and PTT using a single broad-band light
source.
phosphoethanolamine- N-[methoxy(polyethylene glycol)-
2000] (DSPE- mPEG2000). The morphology of the nanoparticles
can be controlled by the ultrasonic power and the
concentrations of IABDP and DSPE-mPEG2000, ranging from
spherical to cubic NPs. As revealed by transmission electron
microscopy (TEM), the average size of the cubic and spherical
Herein,
a BF2 Chelate of [4-iodo-5-(4-bromophenyl)-3-(4-
methoxyphenyl)-1H-pyrrol-2-yl] [4-iodo-5-(4-bromophenyl)-3-
(4-methoxyphenyl)pyrrol-2-ylidene]amine (IABDP) has been
synthesized. Owing to the exceptional photophysical
properties,19 such as large molar absorption coefficient for
visible light, high fluorescence quantum yield, narrow emission
band and good photostability, aza-Bodipy was chosen as the
triplet photosensitizer. Iodo-substituents as well as bromine-
substituents were introduced to increase the singlet oxygen
NPs is about 150 nm (Fig. 1a) and 60 nm (Fig. S1
respectively.
†),
As shown in Fig. 1b, the absorption peak of the spherical IABDP
NPs is observed at ~624 nm, which has a slight blue shift as
compared with free IABDP molecules (at ~658 nm). In contrast,
the adsorption of cubic IABDP NPs is greatly red-shifted with
two prominent peaks at ~721 and 772 nm. Similar shape-
dependent adsorption has also been reported by Liu et al.21
Conceivably, this phenomenon can be attributed to the
structure specific intermolecular interaction and charge
transfer. Free IABDP molecules weakly fluoresce (peaking at
~714 nm upon 658 nm excitation) because of the quenching
quantum yield (the synthesis routes shown in Scheme S1†).
And the biocompatible and water soluble nanoparticles (NPs)
self-assembled from IABDP molecules are employed as the
common photosensitizer for PTI and PAI guided dual modal
phototherapy (PDT and PTT) (Scheme 1). IABDP NPs can
passively yet selectively target the tumor because of the
enhanced permeability and retention (EPR) effect.20 The IABDP
NPs with strong absorption in the NIR region shows high
singlet oxygen quantum yield, excellent photothermal
conversion efficiency and outstanding photoacoustic (PA)
response. Both in vitro and in vivo studies demonstrate the
good biosafety and high anti-tumor efficiency of IABDP NPs
under irradiation of a broad-band Xenon Lamp.
effect induced by the heavy atoms (Fig. S2†). At 624 nm
excitation, the spherical IABDP NPs give two broad emission
peaks at ~694 and ~733 nm and a narrower peak at ~829 nm.
In comparison, the cubic NPs have two narrow emission peaks
at 791 and 831 nm, under 721 nm excitation. To achieve deep
tissue penetration in the NIR window, the cubic NPs were
employed in the following experiments.
Results and discussion
The singlet oxygen quantum yield of IABDP, which is the key to
PDT efficiency, was assessed by monitoring oxidation-caused
adsorption decrease of 1, 3-diphenylisobenzofuran (DPBF) at
414 nm in dichloromethane under 660 nm laser irradiation
Synthesis and characterization of IABDP NPs
As shown in Scheme S1 (see ESI†), water-soluble IABDP NPs
were prepared through a simple self-assembly strategy with
the assistance of amphiphilic 1,2-distearoyl-sn-glycero-3-
(Fig. S3a†). With reference to methylene blue trihydrate (0.57
in dichloromethane) as the standard,19 IABDP demonstrates an
outstanding singlet oxygen generation efficiency (~0.92, Fig.
S3b†). As compared in Table S1 (see ESI†), the singlet oxygen
quantum yield of IABDP is much higher than the previously
reported photosensitizers. Presumably, the high singlet oxygen
quantum yield is due to the two iodo-substituents attached at
the π-core of Aza-BODIPY because these heavy atoms are able
to greatly enhance the intersystem crossing of the excited
electrons.22 Under broadband irradiation from a Xenon lamp,
IABDP NPs in the physiological solution (PBS with pH 7.4) are
also able to efficiently produce singlet oxygen as evidenced by
the decrease of DPBF adsorption in an irradiation duration
dependent manner (Fig. 2a).
In addition to the prominent photodynamic activity, IABDP NPs
also possess high photothermal conversion ability. The
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 3 of 9
View Article Online
DOI: 10.1039/C6TB02979E
Journal Name
ARTICLE
viability of ~80% at high concentration range (about >40 μM).
Such good biocompatibility and high photo-toxicity of IABDP
NPs make them desirable for selective cancer therapy.
Since the fluorescence of IABDP is largely quenching due to its
heavy atoms (especially the two iodo-substituents) and the
adjacent IABDP molecules tightly packed together in the
nanoparticle, the cellular uptake of IABDP NPs cannot be
visually confirmed by confocal microscopy. Alternatively, the
abundant and homogenous uptake of IABDP NPs into HeLa
cells was clearly evidenced by the strong photoacoustic (PA)
signal collected from the cells pre-incubated with the
nanoparticles but not those untreated cells (Fig. 3b).
Using 2’,7’-dichlorofluorescin diacetate (DCFH-DA) as the
probe, generation of ROS by IABDP NPs can be visualized in
living cells because ROS converts non-fluorescent DCFH-DA
into fluorescent 2’,7’-dichlorofluorescein (DCF) after
deacetylate by on lamp, strong green fluorescence distributed
throughout the HeLa cells treated with IABDP NPs and DCFH-
DA molecules, especially in the periphery of the nuclei (stained
wiesterase.23 Fig. 3c shows that, after illumination with a
Xenth
blue
fluorescent
4',6-diamidino-2-phenylindole
molecules, DAPI). In the control experiment, the same
irradiation but without IABDP NPs did not cause any green
fluorescence, indicating no ROS generation. This result certifies
the ability of IABDP NPs to intracellularly generate ROS under
Xenon lamp irradiation.
photothermal conversion efficiency of ~37.9% was obtained
under the irradiation of 730 nm laser (Fig. S4
than that of the previously reported photothermal agents (see
comparison in Table S2 ). Almost no photodegradation of
†), which is higher
Dual-modal in vivo imaging
Photoacoustic imaging was used to monitor in real-time the
dynamic accumulation and metabolism of IABDP NPs in mouse
tumor (Fig. 4a). Before injection of IABDP NPs, weak PA signal
from the blood vessels was observed owing to the endogenous
light absorbing hemoglobin molecules in the blood. One hour
later after tail intravenous injection of IABDP NPs, PA signal
increased greatly (particularly near the blood vessels),
†
IABDP NPs aqueous solution after irradiation of 730 nm laser
can be observed, confirming the excellent photostability of
IABDP NPs (Fig. S5†). As shown in Fig. 2b, under the irradiation
of 730 nm laser, the temperature of IABDP NPs solution rapidly
o
increased ~20 C within a short time (~3 min). In the control
experiment, the temperature of the saline solution essentially
remained the same, implying the unwanted heating of
biofluids can be avoided. Although the temperature increase
kinetics of IABDP NP solution is slower under Xenon lamp, an
o
increase of 28.3 C was attained after 20 min, which is even
higher than the maximum temperature increase induced by
o
730 nm laser (23.7 C). In contrast, the same irradiation under
Xenon lamp only caused very slow and mild temperature
increase of the saline solution. These observations indicate
that broadband Xenon lamp irradiation is advantageous over
the commonly used laser irradiation. In addition to the PDT
and PTT effects, IABDP NPs also exhibit bright photoacoustic
signal in saline (Fig. 2c), which is linearly proportional to their
concentration (Fig. 2d).
In vitro cell experiments
To examine the synergistic phototherapeutic effects of IABDP
NPs on cells in vitro, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay was carried out. As
demonstrated in Fig. 3a, the viability of the cell incubated with
various concentrations of IABDP NPs significantly decreased
under Xenon lamp irradiation in a dose-dependent manner
with a half-maximal inhibitory concentration (IC50) of ~47 µM,
whereas the untreated cells have small dark toxicity with cell
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 4 of 9
View Article Online
DOI: 10.1039/C6TB02979E
ARTICLE
Journal Name
indicating that IABDP NPs just entered the tumor tissue the tumor temperature gradually escalated to ~43 °C within 10
through so-called enhanced permeability and retention (EPR) min (Fig. 4b and c), which is high enough to eliminate tumor
effect. Over time, IABDP NPs spread throughout the tumor cells but not too high to cause unwanted tissue damages.24
tissue, reaching the strongest PA intensity at 4th hour. Noteworthy, the cell membrane permeability increases at such
Subsequently, PA signal started to decrease, suggesting that moderately high temperature (43-45 °C), and consequently,
IABDP NPs were metabolized. As shown by PA imaging, IABDP the cellular uptake of the nanoparticles (hence the
NPs are able to preferentially target on tumors. It implies that effectiveness of phototherapy) is enhanced.25 Additionally, the
the size of IABDP NPs is especially suitable for their mild hyperthermia promotes blood circulation into the tumor
penetration through the defective blood vessels and and thus alleviates the hypoxia condition in the tumor
subsequent retention in tumor tissue. Furthermore, PAI assists microenvironment,26 which is greatly helpful for improving
to accurately locate and irradiate the tumor site but not the PDT efficacy. On the contrary, the tumor temperature of the
surrounding normal tissues.
mice with saline injection showed no appreciable change
Notably, IABDP NPs have excellent photothermal conversion under Xeon lamp irradiation (Fig. 4b and c). Taken together,
ability even under irradiation of Xenon lamp. Fig. 4b shows the both PAI and PTI provide important guidance for phototherapy
PT images of the tumor site at various time points during the and demonstrate the unique capability of IABDP NPs as the
continuous irradiation of Xenon lamp, confirming the tumor common and effective photosensitizer for in vivo dual modal
accumulation of IABDP NPs and revealing the photothermal phototherapy.
conversion kinetics. For those mice with IABDP NPs injection, In vivo synergistic phototherapy
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 5 of 9
View Article Online
DOI: 10.1039/C6TB02979E
Journal Name
ARTICLE
To examine the PDT and PTT synergistic therapeutic effects of investigation. The photographs of these tumors corroborate
IABDP NPs in vivo, HeLa tumor-bearing nude mice with initial the outstanding efficacy of the phototherapy and the similarly
tumor volumes of 100-300 mm3 were randomly divided into 3 darker color for both comparison and treatment groups also
groups (5 mice/group): (i) the treatment group with tail confirms the accumulation of IABDP NPs in tumor. The
intravenous injection of IABDP NPs and broadband irradiation difference of tumor weight between the 3 groups (Fig. S6†) is
by a Xeon lamp. (ii) the comparison group with nanoparticle consistent with the observed volume change (Fig. 5a). The
injection but not irradiation. (iii) the control group with obvious body weight gain of the mice in the comparison group
injection of saline solution and lamp irradiation. As implies that the injected IABDP NPs are non-toxic without
demonstrated in Fig. 5a and b, the tumor volume steadily photo irradiation, while the slight body weight increase of both
expanded over time (28 days) for both control and comparison control and treatment groups suggests the good tolerance of
groups and no significant difference was noticed between the the phototherapy and insignificance of photo-induced side
two groups. In contrast, 14 times of phototherapy treatments effects (Fig. S7†).
during the course greatly inhibited the tumor growth (87.2% To further understand the therapeutic effects down to the
inhibition as compared to the control group). This is superior cellular level, hematoxylin and eosin (H&E) staining as well as
to the previously reported methods based on single Ki67 immuno-staining were employed to reveal the
phototherapy mode (PDT or PTT),27 testifying the advantage of morphology and proliferation of the tumor cells. As shown in
synergistic therapy. All the mice were sacrificed after in vivo Fig. 5c, the H&E staining indicates that the tumor cells of the
experiments and their tumor tissues were collected for further control group are plump and arranged closely with the nuclei
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 6 of 9
View Article Online
DOI: 10.1039/C6TB02979E
ARTICLE
Journal Name
in good condition. Only a small fraction of the tumor cells in dark. After removing the culture media including IABDP NPs,
the comparison group appear damaged by the nanoparticles. these two 96-well plates were washed with phosphate
In the stark contrast, the tumor cells of the treated group buffered saline (PBS) solution and added 200 µL fresh culture
shrink largely, leaving large gaps between adjacent cells. Such medium. Then one of these 96 well plates was irradiated
high level of necrosis and apoptosis evidences the under a Xenon lamp (> 600 nm, 20 mW cm-2) at room
effectiveness of phototherapy with IABDP NPs. The Ki67 temperature for 10 min. After illumination, these 96-well
immuno-staining assay shows that Ki67 proteins (cellular plates were incubated for additional 12 h at 37 °C. Finally, MTT
marker for proliferation) expressed at low level in the solution in PBS (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
treatment group and high level in the control group, indicating tetrazolium bromide, 5 mg mL-1 , 20
IABDP NP’s ability to potently inhibit the proliferation of tumor well and incubated for 4 h. After removing the medium, 150
cells. Fig. S8 (in ESI ) presents the H&E staining of the major of DMSO was added into each well to dissolve formazan, and
µL) was added to each
µ
L
†
organs (heart, lung, liver, spleen, and kidney). Compared with the absorbance was measured at 490 nm using an enzyme-
the control group, no obvious histological alterations can be labeled instrument. The cell viability values were calculated by
identified for the other two groups injected with IABDP NPs, the following formula:
further confirming the in vivo biocompatibility of our Cell viability (%) = (mean of absorbance value of treatment
nanoparticles. The main text of the article should appear here group)/(mean of absorbance value of control group) ×100.
with headings as appropriate.
ROS detection in vitro. HeLa cells were seeded into a glass
bottom Petri dish and incubated in the culture media (2 mL) at
37 °C under 5% CO2 for 24 h, followed by incubation in media
Experimental Section
containing IABDP NPs (50 µM, 2 mL) for additional 24 h. In the
control group, cells were just incubated in the culture media
without IABDP NPs. After removing these media, the cells were
washed thrice with PBS solution, and further incubated with
DCFH-DA for 20 min and DAPI for 3 min in darkness,
respectively. Then the cells were rinsed thrice with PBS
solution and illuminated upon a Xenon lamp (> 600 nm, 20
mW cm-2) for 10 min. After that, the fluorescence images of
the cells were obtained using a confocal laser scanning
microscopy (Olympus IX 70 inverted microscope). For DCF
detection, 488 nm laser was used for excitation, and the
fluorescence from 500 nm to 600 nm was collected. For DAPI
detection, 405 nm laser was used for excitation, and the
fluorescence from 420 nm to 500 nm was collected.
Preparation of cubic IABDP NPs. IABDP (2.0 mg) in 2 mL THF
was swiftly dropped into DSPE-mPEG2000 aqueous solution
(20.0 mg, 10 mL H2O) under sonication (250 W). THF was then
removed under reduced pressure. A royal blue aqueous
solution was obtained. The aqueous solution was further
filtered through 0.45 µm filter and washed with deionized
water, which was freeze-dried to obtain royal blue cubic NPs
powder.
Preparation of sphere IABDP NPs. IABDP (1.0 mg) in 2 mL THF
was swiftly dropped into DSPE-mPEG2000 aqueous solution
(10.0 mg, 10 mL H2O) under sonication (175 W). THF was then
removed under reduced pressure. A royal blue aqueous
solution was obtained. The aqueous solution was further
Animals and tumor model. Female athymic nude mice (4-
week old, about 20 g, Permit number: SCXK(Su)2012-0004)
used in this study were bought from Comparative Medicine
Centre of Yangzhou University. All animal experiments
conformed to the NIH guidelines for the care and use of
laboratory animals are approved by School of Pharmaceutical
Science, Nanjing Tech University. The subcutaneous xenograft
model of cervical cancer was built by injecting 3×106 HeLa cells
filtered through 0.45 µm filter and washed with deionized
water, which was freeze-dried to give sphere as royal blue
powder.
Singlet oxygen detection of IABDP NPs. The singlet oxygen
generation of IABDP NPs was detected with DPBF as 1O2
fluorescent probe in PBS solution at pH 7.4. Briefly, 20
µM
DPBF was added to IABDP NPs solution with the absorbance
around 0.2-0.3. Then the mixture was irradiated with a Xenon
lamp (> 600 nm, 20 mW cm-2) for 180 s. The oxidation of DPBF
(at 418 nm) vs. irradiation time was monitored by UV-VIS-NIR
spectrophotometer.
in 100 µL PBS solution subcutaneously into the left flank near
the front armpit of the mice. After the tumor volumes were
about 100-300 mm3, mice were employed randomly in dual-
modal imaging and phototherapy.
Cell culture. The human cervical cancer HeLa cell line used in
this study was provided by the Institute of Biochemistry and
Cell Biology, SIBS, CAS (China). HeLa cells were cultured in
Dulbecco’s Modified Eagle Medium (DMEM High Glucose,
Gibco) supplemented with 1% (v/v) antibiotics ( penicillin and
streptomycin, GENMED) and 10% (v/v) fetal bovine serum
(FBS) at 37 °C under humidified atmosphere of 5% CO2.
MTT assays. To study the toxicity of IABDP NPs, HeLa cells
were seeded into two 96-well plates at a density of 5000 cells
In vivo imaging. 100 µL IABDP NPs solution (0.5 mM) in saline
was given into HeLa tumor-bearing nude mice through tail
intravenous injection. At different time points after tail
intravenous injection of IABDP NPs, in vivo PA images were
conducted using the Endra Nexus128 small animal
photoacoustic imaging system at 721 nm. The temperature
was also monitored through a FLIR thermal camera under
irradiation of Xenon lamp (20 mW cm-2) for 10 min at 4 hours
post-injection. The mice injected with saline were evaluated as
control.
per well and incubated in the culture media (200
under 5% CO2 atmosphere for 24 h, and further incubation
with various concentrations (4.7, 9.4, 18.8, 47, 94 M) of
L/well) for another 24 h in
µL) at 37 °C
In vivo synergistic therapy. Nude mice bearing HeLa tumor
were randomly divided into 3 groups (5 mice per group). 4 h
µ
IABDP NPs in DMEM media (200
µ
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 7 of 9
View Article Online
DOI: 10.1039/C6TB02979E
Journal Name
ARTICLE
after injection, the control group and the treatment group
were irradiated with a Xenon lamp (20 mW cm-2) for 10 min.
The body weights and tumor sizes of mice were monitored
every two days and tumor volumes were computed by the
equation: volume = length×width2/2. The inhibition rate was
computed by the equation:
Notes and references
1
(a) J. Tian, L. Ding, H. Ju, Y. Yang, X. Li, Z. Shen, Z. Zhu, J.S. Yu,
C.J. Yang, Angew. Chem. Int. Ed. Engl., 2014, 53, 9544-9549;
(b) J. Mou, T. Lin, F. Huang, H. Chen, J. Shi, Biomaterials,
2016, 84, 13-24; (c) J. Cao, H. An, X. Huang, G. Fu, R. Zhuang,
L. Zhu, J. Xie, F. Zhang, Nanoscale, 2016,
J. Han, H. Xia, Y. Wu, S.N. Kong, A. Deivasigamani, R. Xu, K.M.
Hui, Y. Kang, Nanoscale, 2016, , 7861-7865; (e) G. Wang, F.
Zhang, R. Tian, L. Zhang, G. Fu, L. Yang, L. Zhu, Acs Appl.
Mater. Interfaces, 2016, , 5608-5617; (f) Y. Cai, Q. Tang, X.
Wu, W. Si, Q. Zhang, W. Huang, X. Dong, Acs Appl. Mater.
Interfaces, 2016, , 10737-10742.
8, 10152-10159; (d)
Inhibition rate (%) =100-(mean of final tumor volumes of
treatment group-mean of initial tumor volumes of treatment
group)/(mean of final tumor volumes of control group-mean of
initial tumor volumes of control group) ×100.
8
8
8
All mice were sacrificed after 14 times of treatment. Tumors
and the major organs (heart, lung, liver, spleen, and kidney) of
three groups were fixed in 10% formalin and embedded in
paraffin wax for further histomorphological analysis.
2
3
(a) Y. Liu, Y. Liu, W. Bu, C. Cheng, C. Zuo, Q. Xiao, Y. Sun, D.
Ni, C. Zhang, J. Liu, J. Shi, Angew. Chem. Int. Ed. Engl. , 2015,
54, 8105-8109; (b) H. Wu, F. Zeng, H. Zhang, J. Xu, J. Qiu, S.
Wu, Adv. Sci., 2016,
(a) C.S. Jin, J.F. Lovell, J. Chen, G. Zheng, Acs Nano, 2013,
3, 1500254.
7
,
Histological examination and immunohistochemical analysis.
The hematoxylin and eosin (H&E) staining and Ki67
immunostaining assays were carried out to stain tissue slices.
Both the tumor tissues and the major organs (heart, lung, liver,
spleen, and kidney) were stained with H&E for histological
examination. Additionally, the tumor tissues were stained with
anti-human Ki-67 to reflect the proliferation of tumor cells.
2541-2550; (b) C. Zhang, K. Zhao, W. Bu, D. Ni, Y. Liu, J. Feng,
J. Shi, Angew. Chem. Int. Ed. Engl., 2015, 54, 1770-1774.
K. Deng, Z. Hou, X. Deng, P. Yang, C. Li, J. Lin, Adv. Funct.
Mater., 2015, 25, 7280-7290.
L. Li, C. Chen, H. Liu, C. Fu, L. Tan, S. Wang, S. Fu, X. Liu, X.
Meng, H. Liu, Adv. Funct. Mater., 2016, DOI:
10.1002/adfm.201600985.
4
5
6
7
8
D. Luo, K.A. Carter, D. Miranda, J.F. Lovell, Adv. Sci., 2016,
DOI: 10.1002/advs.201600106.
X. Song, H. Gong, S. Yin, L. Cheng, C. Wang, Z. Li, Y. Li, X.
Wang, G. Liu, Z. Liu, Adv. Funct. Mater., 2014, 24, 1194-1201.
G. Song, Q. Wang, Y. Wang, G. Lv, C. Li, R. Zou, Z. Chen, Z.
Qin, K. Huo, R. Hu, J. Hu, Adv. Funct. Mater., 2013, 23, 4281-
4292.
Conclusions
In summary, a novel aza-bodipy photosensitizer (IABDP) with
high yield of singlet oxygen and excellent photothermal
conversion efficiency has been successfully designed and
synthesized. We demonstrate that the NIR absorbing IABDP
nanoparticles (NPs) are able to serve as the common agent for
photoacoustic (PA) and photothermal (PT) imaging guided
photothermal/photodynamic therapy under broadband
irradiation by a Xeon lamp. The simplicity of this platform is
highly desirable for the convenient and reproducible practical
use. The synergistic dual modal phototherapy renders the high
efficacy of the method. The dual modal imaging enables
accurate localization of tumor site in order to avoid unwanted
tissue damages, real-time monitoring of the photosensitizer
distribution and its accumulation/metabolism kinetics, and
real-time monitoring of temperature change at the tumor site
in order to determine the optimal irradiation protocol. In
comparison to inorganic nanomaterial based photosensizers,
IABDP NPs can rapidly and selectively target to the tumor site
and can be readily metabolized instead of being retained for
too long. Both in vitro and in vivo studies demonstrate the
negligible dark toxicity yet remarkable phototoxicity of IABDP
NPs. The in vivo experiments show that these nanoparticles
can potently inhibit tumor growth without causing noticeable
side-effects.
9
F. He, G. Yang, P. Yang, Y. Yu, R. Lv, C. Li, Y. Dai, S. Gai, J. Lin,
Adv. Funct. Mater., 2015, 25, 3966-3976.
10 L.V. Wang, S. Hu, Science, 2012, 335, 1458-1462.
11 (a) M. Yu, F. Guo, J. Wang, F. Tan, N. Li, Biomaterials, 2016,
79, 25-35; (b) U.S. Chung, J.-H. Kim, B. Kim, E. Kim, W.-D.
Jang, W.-G. Koh, Chem. Commun., 2016, 52, 1258-1261; (c)
C.-K. Chu, Y.-C. Tu, J.-H. Hsiao, J.-H. Yu, C.-K. Yu, S.-Y. Chen,
P.-H. Tseng, S. Chen, Y.-W. Kiang, C.C. Yang, Nanotechnology,
2016, 27, 115102; (d) G.-F. Luo, W.-H. Chen, Q. Lei, W.-X.
Qiu, Y.-X. Liu, Y.-J. Cheng, X.-Z. Zhang, Adv. Funct. Mater.,
2016, DOI: 10.1002/adfm.201505175.
12 (a) I. Marangon, C. Menard-Moyon, A.K.A. Silva, A. Bianco, N.
Luciani, F. Gazeau, Carbon, 2016, 97, 110-123; (b) H. Zhang,
X. Jiao, Q. Chen, Y. Ji, X. Zhang, X. Zhu, Z. Zhang,
Nanotechnology, 2016, 27, 085104; (c) B.-P. Jiang, L.-F. Hu,
X.-C. Shen, S.-C. Ji, Z. Shi, C.-J. Liu, L. Zhang, H. Liang, Acs
Appl. Mater. Interfaces, 2014,
H. Huang, J. Huang, H. Chen, J. Wang, K. Qiu, D. Zhao, L. Ji, H.
Chao, Acs Appl. Mater. Interfaces, 2015, , 23278-23290.
13 J. Qiu, Q. Xiao, X. Zheng, L. Zhang, H. Xing, D. Ni, Y. Liu, S.
Zhang, Q. Ren, Y. Hua, K. Zhao, W. Bu, Nano Res., 2015, 8,
3580-3590.
14 (a) Y. Yong, L. Zhou, Z. Gu, L. Yan, G. Tian, X. Zheng, X. Liu, X.
Zhang, J. Shi, W. Cong, W. Yin, Y. Zhao, Nanoscale, 2014,
6, 18008-18017; (d) P. Zhang,
7
6
,
10394-10403; (b) H. Bi, Y. Dai, R. Lv, C. Zhong, F. He, S. Gai, A.
Gulzar, G. Yang, P. Yang, Dalton Trans., 2016, 45, 5101-5110;
(c) C. Wu, A. Zhu, D. Li, L. Wang, H. Yang, H. Zeng, Y. Liu,
Expert Opin. Drug Del., 2016, 13, 155-165; (d) J. Hao, G.
Song, T. Liu, X. Yi, K. Yang, L. Cheng, Z. Liu, Adv. Sci., 2016,
DOI: 10.1002/advs.201600160; (e) Y. Chen, L. Wang, J. Shi,
Nano Today, 2016, 11, 292-308; (f) S. Wang, Y. Chen, X. Li, W.
Gao, L. Zhang, J. Liu, Y. Zheng, H. Chen, J. Shi, Adv. Mater.,
2015, 27, 7117-7122.
Acknowledgements
The work was supported by NNSF of China (61525402,
21275076), Key University Science Research Project of Jiangsu
Province (15KJA430006), QingLan Project.
15 (a) H. Gong, Z. Dong, Y. Liu, S. Yin, L. Cheng, W. Xi, J. Xiang, K.
Liu, Y. Li, Z. Liu, Adv. Funct. Mater. 2014, 24, 6492-6502; (b)
E.B. Ehlerding, F. Chen, W. Cai, Adv. Sci., 2016, 3, 1500223.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 8 of 9
View Article Online
DOI: 10.1039/C6TB02979E
ARTICLE
Journal Name
16 M. Alexiades-Armenakas, Clin. Dermatol., 2006, 24, 16-25.
17 B. Javaid, P. Watt, N. Krasner, Laser. Med. Sci., 2002, 17, 51-
56.
18 L. Brancaleon, H. Moseley, Laser. Med. Sci., 2002, 17, 173-
186.
19 S. Guo, L. Ma, J. Zhao, B. Kucukoz, A. Karatay, M. Hayvali,
H.G. Yaglioglu, A. Elmali, Chem. Sci., 2014, 5, 489-500.
20 (a) M. Zhen, C. Shu, J. Li, G. Zhang, T. Wang, Y. Luo, T. Zou, R.
Deng, F. Fang, H. Lei, C. Wang, C. Bai, Science China
Materials, 2015, 58, 799-810; (b) Y. Li, Science China
Materials, 2015, 58, 851-851; (c) H. Maeda, J. Wu, T. Sawa, Y.
Matsumura, K. Hori, J. Control. Release, 2000, 65, 271-284.
21 D. Liu, C. Li, F. Zhou, T. Zhang, H. Zhang, X. Li, G. Duan, W.
Cai, Y. Li, Sci. Rep., 2015, 5, 7686.
22 A. Gorman, J. Killoran, C. O'Shea, T. Kenna, W.M. Gallagher,
D.F. O'Shea, J. Am. Chem. Soc., 2004, 126, 10619-10631.
23 (a) K. Han, W.-Y. Zhang, J. Zhang, Q. Lei, S.-B. Wang, J.-W. Liu,
X.-Z. Zhang, H.-Y. Han, Adv. Funct. Mater., 2016, DOI:
10.1002/adfm.201600170; (b) K. Han, Q. Lei, S.-B. Wang, J.-J.
Hu, W.-X. Qiu, J.-Y. Zhu, W.-N. Yin, X. Luo, X.-Z. Zhang, Adv.
Funct. Mater., 2015, 25, 2961-2971; (c) H. Shi, W. Sun, C. Liu,
G. Gu, B. Ma, W. Si, N. Fu, Q. Zhang, W. Huang, X. Dong, J.
Mater. Chem. B, 2016,
Jiang, K.Y. Zhang, S. Liu, T. Yang, Q. Zhao, L. Yang, W. Lv, Q.
Yu, W. Huang, Adv. Sci. 2016, , 1500155; (e) Y. Cai, Q. Tang,
X. Wu, W. Si, W. Huang, Q. Zhang, X. Dong, ChemistrySelect,
2016, , 3071-3074.
24 A. Gulzar, S. Gai, P. Yang, C. Li, M.B. Ansari, J. Lin, J. Mater.
Chem. B, 2015, , 8599-8622.
25 (a) T. Liu, C. Wang, W. Cui, H. Gong, C. Liang, X. Shi, Z. Li, B.
Sun, Z. Liu, Nanoscale, 2014, , 11219-11225; (b) S.P.
4, 113-120; (d) X. Zhou, H. Liang, P.
3
1
3
6
Sherlock, S.M. Tabakman, L. Xie, H. Dai, Acs Nano, 2011, 5,
1505-1512.
26 X. Yi, K. Yang, C. Liang, X. Zhong, P. Ning, G. Song, D. Wang, C.
Ge, C. Chen, Z. Chai, Z. Liu, Adv. Funct. Mater., 2015, 25
4689-4699.
,
27 (a) Y. Li, J. Tang, D.-X. Pan, L.-D. Sun, C. Chen, Y. Liu, Y.-F.
Wang, S. Shi, C.-H. Yan, Acs Nano, 2016, 10, 2766-2773; (b) Z.
L. Li, Y. Hu, K. A. Howard, T. T. Jiang, X. L. Fan, Z. H. Miao, Y.
Sun, F. Besenbacher, M. Yu, Acs Nano, 2016, 10, 984-997.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 9
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C6TB02979E
1
A novel NIR absorbing aza-bodipy photosensitizer with high O2 quantum yield and
excellent photothermal conversion efficiency were designed for synergistic phototherapy.