An anthracene functionalized BODIPY derivative with singlet oxygen storage ability for photothermal and continuous photodynamic synergistic therapy

Article abstract of DOI:10.1039/c9tb00180h

Singlet oxygen (¹O₂) generated from the photosensitization process within tumor tissues during photodynamic therapy (PDT) is self-limiting. Fractional delivery of light tends to be a better method for PDT. Herein, BDPIA with a high ¹O₂ quantum yield (QY) of 70% in DCM has an additional anthracene module for capturing ¹O₂, which would be useful in PDT. In the light cycle, the endoperoxide of anthracene is generated with singlet oxygen while in the dark cycle, the endoperoxide undergoes thermal cycloreversion to produce ¹O₂, regenerating the anthracene module. Therefore, the photodynamic process can continue in the dark period and the light cycles, promising their continuous phototherapy efficacy. In addition, BDPIA NPs in water exhibit a high photothermal conversion efficiency (η = 38.9%) for effective photothermal therapy. In vitro MTT assay shows that BDPIA NPs have a low half inhibitory concentration (IC₅₀) of 6.293 μg mL^-1 on HeLa cells. Furthermore, lentiviral vector transfected HeLa cell lines with red fluorescent proteins were used to perform real time in vivo fluorescence imaging guided phototherapy. The model can monitor the real-time uptake of BDPIA NPs. The results indicate that BDPIA NPs are capable of inhibiting cell proliferation even at a low dosage (0.2 mg kg^-1) while the normal tissues (heart, liver, spleen, lung and kidney) suffer from no side effects. Our results demonstrate that BDPIA NPs with high phototoxicity, low dark toxicity and excellent bio-compatibility can be used as a potential photosensitizer for continuous photodynamic and photothermal synergistic therapy.

Full text of DOI:10.1039/c9tb00180h

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Regulating the stemness of mesenchymal stem cells by tuning
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Page 1 of 9
J Po lue ran sae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
View Article Online
DOI: 10.1039/C9TB00180H
Journal of Materials Chemistry B
ARTICLE
Anthracene functionalized BODIPY derivative with singlet oxygen
storage ability for photothermal and continuous photodynamic
synergistic therapy
Received 00th January 2018,
Accepted 00th January 2018
ab
b
a
a
b
a
Jianwei Zhu , Jianhua Zou , Jie Zhang , Yang Sun , Xiaochen Dong *, Qi Zhang *
DOI: 10.1039/x0xx00000x
www.rsc.org/
1
Singlet oxygen ( O
2
) generated from the photosensitized process within tumor tissues during
photodynamic therapy (PDT) is self-limiting. Fractional delivery of light tends to be a better
1
method for PDT. Herein, BDPIA with a high O
2
quantum yield (QY) of 70% in DCM has an
, which would be useful in PDT. In the light
1
additional anthracene module for capturing O
2
cycle, the endoperoxide of anthracene is generated with singlet oxygen while in the dark cycle,
1
the endoperoxide undergoes thermal cycloreversion to produce O
2
, regenerating the
anthracene module. Therefore, the photodynamic process can continue in the dark period as
well as the light cycles, promising their continuous phototherapy efficacy. In addition, BDPIA
NPs in water exhibit a high photothermal conversion efficiency (η = 38.9%) for effective
photothermal therapy. In vitro MTT assay shows that BDPIA NPs have a low half inhibitory
concentration (IC50) of 6.293 μg/mL on HeLa cells. Furthermore, lentiviral vectors transfected
HeLa cell lines with red fluorescent proteins was used to perform the real time in vivo
fluorescence imaging guided phototherapy. The model can monitor the real-time uptake of
BDPIA NPs. The results indicate that BDPIA NPs are capable of inhibiting cell proliferation even
at a low dosage (0.2 mg/kg) while the normal tissues (heart, liver, spleen, lung and kidney)
suffer from no side effects. Our results demonstrate that BDPIA NPs with high phototoxicity,
low dark toxicity and excellent bio-compatibility can be used a potential photosensitizer for
continuous photodynamic and photothermal synergistic therapy.
Over the past decades, photodynamic therapy (known as
PDT) has been universally acknowledged as a promising
Introduction
1-13
approach to treat various cancers.
The therapeutic
With the increase in the incidence and mortality of cancer
in modern society, traditional treatments, including
surgery, chemotherapy and radiotherapy, usually have
inherent limitations such as resistance, side effects, low
targeting and lack of specificity, hence the need for
effective and innovative cancer treatments is still urgent.
procedure usually involves the photosensitized generation
1
of singlet oxygen ( O
2
), and destroys tumor cells and tumor
blood vessels. However, PDT itself creates acute hypoxia by
rapidly consuming intracellular oxygen reservation, and
tumor tissues usually have an inherent hypoxic
microenvironment, which resulting in the limit of the
14
photosensitizer’s treatment effect. Many studies suggest
that intermittent delivery of light might be a better
6
approach to PDT. Since the photothermal effect of the
tumor can promote the absorption of deep oxygen in the
tumor, the photosensitizer combined with photodynamic
therapy and photothermal therapy can overcome the
dependence of oxygen to some extent. At the same time,
the assistance of photothermal effect is advantage to
better transportation and uptake in the tumor for drugs.
Thus, the therapeutic effect would be enhanced by
a.
School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South
Puzhu Road, Nanjing 211800, China. *E-mail: zhangqi@njtech.edu.cn
b.
Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),
Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing, 211800, China, *E-
mail: iamxcdong@njtech.edu.cn
†Electronic Supplementary Information (ESI) available.
1

J Po lue ran sae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
Page 2 of 9
ARTICLE
Journal of Materials Chemistry B
combining photodynamic therapy with photothermal with good bio-compatibility, low dark toxicity and high
View Article Online
DOI: 10.1039/C9TB00180H
therapy. Considering the fact that singlet oxygen is the phototoxicity are promising for PTT and continuous PDT.
cytotoxic agent required for effective PDT, while the
characteristics, such as short lifetime and the generation
only during the irradiation periods, impeded its effect on
14
tumor cells.
Thus, the application of chemically
generated singlet oxygen for the dark period is highly
desirable for fractional PDT, in which photosensitized
generation is impossible. In fact, the combination of a
photosensitizer and a chemical source of single molecule
would be a wiser approach.15
The endoperoxides of anthracene and its derivatives have
been acknowledged as reliable chemical sources of singlet
oxygen, because they tend to undergo clean
cycloreversion reactions without side reactions to release
singlet oxygen with very high yields. So far, relevant
investigations have been already reported that thermal
decomposition of anthracene endoperoxides can produce
singlet oxygen, leading to cell death by a process
resembling apoptosis. 6
Scheme 1. Illustration of BDPIA NPs for PDT/PTT synergistic
1
therapy and the mechanism of reversible O
2
capture and
delivery.
, 10-12
Dipyrromethene, known as BODIPY, are well recognized for
Experimental
Materials and apparatus
All the chemicals were purchased from Sigma (Shanghai
their high photostability, and high fluorescence etc, which
makes them potential candidates for PDT. 1
6-29
However,
their singlet oxygen generation ability is usually
disappointingly low. Conjugating heavy atoms can
1
Co.ltd) and used without further purification. The H NMR
13
and C NMR spectra were measured on Bruker DRX NMR
spectrometer (500 MHz) in CDCl solution at 298 K as the
internal standard with solvent residual (CDCl , δ = 7.26
ppm). UV-vis spectra were recorded on
effectively improve the spin obit crossing (SOC) and
3
1
therefore the singlet oxygen generation quantum yield ( O
2
3
QY) can be dramatically elevated. Inspired by these
observations, one anthracene grafting BODIPY compound,
namely, BDPIA (3,7-bis((E)-2-(anthracen-9-yl)vinyl)- 5,5-
a
spectrophotometer (UV-3600 UV-Vis-NIR, Shimadzu,
Japan). The fluorescence spectra were measured on an F-
difluoro-2,8-diiodo-
1,9-dimethyl-10-
phenyl-5H-
4
600 spectrometer (HITACHI, Japan).
dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide)
has been designed and prepared. The compound has a high
1
O
2
1
Synthesis of BDPIA
capturing O
While in the dark cycle, the endoperoxide undergoes
A mixture of BDPI (0.575 g, 0.1 mmol), anthracene-9-
carbaldehyde (0.412 g, 0.2 mmol) was dissolved in DMF (3
mL), which was heated to 150 C for 4 h under the catalysis
of acetic acid (0.4 mL) and piperidine (0.4 mL). Then the
mixture was poured into 150 mL water and extracted with
dichloromethane. The organic layer was washed with brine
and dried with anhydrous sodium sulfate. The solvent was
removed by rotary evaporation, and the crude product was
1
thermal cycloreversion to produce O
anthracene module. The reversible
2
, regenerating the
1
O
2
storage and
o
delivery can be achieved by changing illumination or not.
Also, nanoprecipitation was used to prepare the
nanoparticles (NPs) of BDPIA. Similarly, these NPs are able
1
to show reversible O
2
trapping and releasing ability by
using singlet oxygen sensor green (SOSG) as a detecting
probe, which promising their potential for continuous PDT
therapy. In vitro MTT assay indicates that such NPs have a
low half inhibitory concentration (IC50) of 0.3715 μg/mL.
Furthermore, in vivo fluorescence imaging guided
phototherapy suggests that BDPIA NPs are capable of
inhibiting the growth of tumor under laser irradiation while
cause no side effects on normal tissues (heart, kidney, liver,
spleen and lung). Our results demonstrate that BDPIA NPs
purified by column chromatography (silica gel, DCM:PE =
1
1
8
7
:2, v/v). H NMR (CDCl
3
, 400 MHz): δ (ppm) 8.44 (1H, s),
.39-8.35 (2H, d), 8.27-8.21 (1H, d), 8.05-8.00 (2H, t), 7.56-
.53 (4H, m), 7.52-7.47 (3H, d), 7.35-7.36 (2H, m), 6.92 (1H,
s), 260 (3H, s), 1.52 (3H, s), 1.42 (3H, s). C NMR (75 MHz,
CDCl ): δ (ppm) 137.7, 131.6, 129.3, 128.4, 120.0, 117.5,
13.6, 127.2, 111.2, 44.2, 31.5, 29.9, 29.4, 14.4, 13.9, 12.4.
MS: calcd. m/z = 764.02; found m/z = 764.10.
13
3
1
2
|J. Mater. Chem. B., 2018, 00, 1-3
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Page 3 of 9
J Po lue ran sae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
Journal of Materials Chemistry B
ARTICLE
Singlet oxygen detection in DCM and water
system time constant, and m
capacity of the deionized water used as the solvent.
i
and C
i
are the maVi
s
e
s
w A
a
r
n
tic
d
le O
heat
n
li
ne
DOI: 10.1039/C9TB00180H
Singlet oxygen generation ability was measured by DPBF
1
(
1,3-diphenylisobenzofuran) as the
O
indicator. The Lentiviral vectors with red fluorescent proteins for
absorbance of DPBF was adjusted around 1.0 while that of transfection of HeLa cell lines
2
BDPIA was around 0.2 to 0.3. After exposing the mixture of
HeLa cell lines (Institute of Biochemistry and Cell Biology,
SIBS, CAS (China)) were cultured in a regular growth
medium consisting of Dulbecco’s modified Eagle’s medium
BDPIA and DPBF in DCM to laser, the spectra were
recorded. To investigate the singlet oxygen generation
without laser irradiation, the mixture of BDPIA and DPBF in
2
(DMEM, Gibco), supplemented with 10% fetal bovine
DCM was irradiated by laser (660 nm, 0.5 W/cm ), and the
o
serum under an atmosphere of 5% CO
(
2
at 37 C. Lentivirus
absorbance was recorded. The singlet oxygen quantum
yield is calculated according to equation (1)
9
with red fluorescent proteins, 10 TU/mL) was purchased
from Genepharma (Shanghai, China). Puromycin
Dihydrochloride was obtained from Beyotime
푆푠푎푚
푆푠푡푑
퐹푠푡푑
훷∆(푠푎푚) = 훷∆(푠푡푑)
×
×
(1)
퐹
푠푎푚
Biotechnology (Shanghai, China). Cells were seeded into
4
2
4-well plates with 5×10 /well in 500 μL culture medium.
where sam and std designate the sample and standard
substance, respectively. S stands for the slope of plot of the
absorbance of DPBF (at 418 nm) versus irradiation time,
The cells were incubated for 24 h and then exposed to
Lentivirus at a series concentration (The values of
Multiplicity of Infection were 10, 20, 30, 50, 70, 100). Then
the 24-well plate was incubated for another 48 h.
Thereafter the previous medium was removed, and added
new fresh culture medium containing 5 μg/mL Puromycin
Dihydrochloride, the 24-well plate was continuously
incubated for another 24 h to remove the cells which were
failed to be transfected. The fluorescence images are
observed by an Olympus IX 70 inverted microscope to
evaluate the transfection efficiency, and the Optimized
MOI value was 50. The stable HeLa cell lines with Red
fluorescent proteins were established for the following
experiments in this study.
-
OD
and F can be calculated by F= 1-10 (OD represents the
optical density of sample and MB at 660 nm).
After being stored in the dark for 5 min, the absorbance
was recorded again. The singlet oxygen generation ability
of BDPIA NPs in water was investigated with SOSG with a
probe. The fluorescence was recorded after laser
irradiation. The spectrum was recorded again after 5 min.
The procedure was repeated for three times.
Preparation of BDPIA nanoparticles
MTT assay
Nanoprecipitation was used to prepare the nanoparticles
of BDPIA. DSPE-PEG-2000 (1 mg) and BDPIA (5 mg) was
dissolved in THF (1 mL). Then 200 μL of BDPIA (5 mg/mL)
was added into distilled water (5 mL) under vigorous
stirring at room temperature. Then the mixture was stirred
for 20 min, THF was removed by nitrogen bubbling. BDPIA
NPs in the solution were obtained by centrifugation. The
photothermal conversion efficiency was calculated
according to equation (2).
Cell viability assay of the nanoparticles was done. First the
NPs were dissolved in distilled water and diluted with
DMEM to various concentration and put in the 96-well
plate, respectively. Then the 96-well plate was irradiated
with laser (660 nm, 0.5 W/cm ) for 5 minutes. Cell viability
was determined by colorimetric MTT assay. A solution of 3-
2
(
4,5-Dimethylthiazol-
2-yl)-2,5-
diphenyltetrazolium
bromide (MTT) in distilled water (5 mg/mL, 20 μL) was
added to each well, followed by incubation for 3.5 h. Then
the medium was discarded and 200 μL DMSO was added at
ambient temperature. The optical absorbance was
recorded at 492 nm by a Thermo Multiskan Mk3
Microplate Reader. The cell growth inhibitory effects were
calculated by the following equation: cell viability (%) =
ℎꢀ(푇푚푎푥 − 푇푎푚푏) − 푄퐷푖푠
휂 =
(2)
퐼(1 − 10ꢁ퐴66ꢂ
)
푇 − 푇푎푚푏
ꢃ휃
∑ ꢅ 퐶
푖
푖
푝,푖
휃 =
ꢃꢄ = −휏ꢀ
τ푠 =
ꢄ = −휏ꢀ ln( 휃)
푇푀푎푥 − 푇푎푚푏
휃
ℎꢀ
where h is the heat transfer coefficient, S is the surface area (Atreatment / Acontrol) × 100 %. A cell growth inhibition
of the container, and the value of hs is obtained from the
Figure 2d. The Tmax is the temperature change of BDPIA NPs
aqueous solution at the maximum steady-state temperature,
I is the laser power, A660 is the absorbance of the BDPIA NPs at
curve was generated by plotting cell growth inhibition
against drug concentration, and the half-maximal
inhibitory concentration (IC50) was determined using
GraphPad Prism 6 software (GraphPad Software, Inc., La
Jolla, CA, USA).
6
60 nm, and QDis expresses the heat associated with light
absorption by the solvent. The variable τ is the sample-
s
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J Po lue ran sae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
Page 4 of 9
ARTICLE
Journal of Materials Chemistry B
Annexin V-FITC/ propidium iodide (PI) staining
above was repeated until the tumor disappears. The tumor
volume and body weight of mice were recorded every two
View Article Online
DOI: 10.1039/C9TB00180H
The Apoptosis was analyzed using Annexin V-
FITC/propidium iodide (PI) dual staining. HeLa cells were
plated in 6-well plates and assigned into three groups
days. Finally, these nude mice were sacrificed, followed by
the histology analysis. In details, the tumor and major
organs (heart, liver, spleen, lung, kidney) from each mouse
were isolated and fixed in 4% formaldehyde solution. After
dehydration, the tissues were embedded in paraffin
cassettes and stained with hematoxylin and eosin (H&E),
the images were recorded on a microscope. The bioimages
of tumor, heart, liver, spleen, liver and kidney were
recorded on PerkinElmer IVIS Lumina K.
(
control group, illumination group and without illumination
group). After treatment with nanoparticles at
concentration of 1 μg/mL, illumination group was
2
irradiated with laser (660 nm, 0.5 W/cm ) for 5 minutes
and incubated for anther 24h, Finally, all the HeLa cells
were harvested and stained with Annexin V-FITC/PI Cell
Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China)
according to the manufacturer’s protocol. The apoptosis
rates of the cells were then analyzed by a flow cytometer
Results and Discussion
(
BD Biosciences, San Jose, CA, USA).
Synthesis, characterization of BDPIA and its NPs
The synthetic route of BDPIA was proposed in Figure S1
(
supporting information), which has been fully structurally
Cellular uptake and fluorescence imaging of cellular ROS
1
13
characterized by HNMR, CNMR and mass spectrometry.
Generally, reactions of benzoyl chloride with 2,4-
dimethylporrole in DCM, followed by the addition of
triethylamine and BF
treated with NIS (N-iodosuccinimide) in a mixture of
chloroform and acetic acid (CHCl /HOAc V/V 3:1) to
produce BDPI. Eventually, BDPIA could be obtained by
treating BDPIA and anthracene-9-carbaldehyde through a
knoevenagel condensation using acetic and piperidine as
catalyst in moderate yield. As shown in Figure 1a, the
absorbance of BDPIA in THF shows the maximum intensity
at 628 nm while red shift of 55 nm of DSPE-PEG-2000 coated
BDPIA NPs was observed, this can be explained by either
the solvent effect or the aggregation of BDPIA.
HeLa cells were incubated with BDPIA NPs (6 μg/mL, 2 mL)
in a confocal dish for 24 h in dark, the previous medium
was discarded, followed by the addition of 1 mL
polyoxymethylene for 25 min. Then polyoxymethylene was
discarded and the cells were washed with PBS three times.
The sample with BDPIA NPs for 24 h was further incubated
with 10 µM of 2,7-dichlorodihydrofluorescein diacetate
3
·OEt give BDP. Then, BDP was
2
3
(
DCF-DA) for another 3 min, which was washed with 1 mL
PBS three times. This sample was irradiated by laser (660
nm, 0.5 W/cm ) for 1 minutes. The fluorescence images of
2
were observed by Olympus IX 70 inverted microscope. For
the cellular uptake samples，they were excited at the
wavelength of 633 nm and collected fluorescence from 650
to 750 nm. While the samples for fluorescence imaging of
cellular ROS, they were excited with 488 nm laser and
collected fluorescence from 490 to 600 nm.
Singlet oxygen detection with or without irradiation
High singlet oxygen generation ability of photosensitizer is
crucial to photodynamic therapy. DPBF was used as the
singlet oxygen probe to evaluate the singlet oxygen
quantum yield of BDPIA using methylene blue (MB) as the
standard substance (Φ
2
Histology examination and bioimage in vivo tumor
treatment
Δ
= 57% in DCM). As shown in Figure
b, 2c, DPBF exhibits a quite high degradation speed under
the presence of BDPIA with laser irradiation (660 nm, 0.5
W/cm ), compared with that of MB in DCM (Figure S2). The
singlet oxygen is calculated to be 70%, which can be
explained by the so-called heavy atom effect.
The animal ethic approval was obtained from Animal
Center of Nanjing Medical University (NJMU, Nanjing,
China). 12 nude mice were purchased and then injected
with HeLa cells into the armpit as the tumor source. When
2
3
the tumor volume reached about 80 mm , the mice were
1
Anthracene is an interesting group that can be used as O
2
divided into 3 groups randomly. For the illumination and
without illumination group, the mice were intravenously
injected with BDPIA NPs (60 μg/mL, 100 μL) in PBS solution,
while for the control group, saline was injected. 4 hours
after administration, the tumors of the illumination group
were irradiated for 8 minutes while the mice in the other
two groups were not additionally irradiated. The process
detector. The mechanism of this process usually contains
1
the O
2
addition to the anthracene to form anthracene
endoperoxides with light irradiation. While in the dark
1
condition, the O
2
can be released from the anthracene
endoperoxides to form anthracene, which is a reversible
6, 12, 14
1
process.
To investigate the O storage and release
2
ability of BDPIA in DCM with or without irradiation, the
4
|J. Mater. Chem. B., 2018, 00, 1-3
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J Po lue ran sae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
Journal of Materials Chemistry B
degradation of DPBF is also reported. As shown in Figure NPs, the fluorescence spectra was recorded
ARTICLE
V
u
ie
s
w
i
A
n
r
g
ticle
S
O
O
n
S
line
G
DOI: 10.1039/C9TB00180H
1
d, the absorbance of DPBF decreases with laser (singlet oxygen sensor green). Figure 2b shows that the
irradiation, further degradation was also observed when fluorescence intensity of SOSG was greatly enhanced with
the solution was stored in dark for another 5 min, showing laser irradiation. When the solution was put in dark for
1
1
the continuous O
2
release ability of such compound. The another 5 min, the intensity keeps rising, indicating the O
2
process was conducted for 4 cycles, obvious degradation release from BDPIA NPs. Similarly, the absorbance
could be always detected, indicating the repeatability of increases when the process was repeated three times,
1
1
such process. Since the O
2
quantum yield of BDPIA is as indicating the continuous O
2
release, which is consistent
1
high as 70%, it can effectively generate O
irradiation. It is assumed that part of the O can be
2
with laser with the results of BDPIA in DCM mentioned above.
1
2
From the point view of synergistic therapy, combining
photodynamic therapy and photothermal therapy is a wise
strategy for cancer treatment. When the NPs was
prepared, BDPIA will suffer from aggregation caused
quenching (ACQ). The NPs with NIR absorbance can
convert energy to heat by non-radiative transition upon
light irradiation, thus lead to the photothermal ability. To
evaluate the photothermal conversion ability of BDPIA
NPs, photothermal conversion efficiency was calculated by
recording the temperature elevation with irradiation and
cooling process without irradiation of BDPIA NPs. As
captured by the addition reaction with anthracene to form
1
endoperoxides (Scheme 1.). These O
2
can be released
again without irradiation, promising the continuous
photodynamic efficacy of such compound.
o
illustrated in Figure 2c, a temperature elevation of 15.4 C
could be observed with low power laser irradiation (660
2
nm, 0.5 W/cm ) while water shows a negligible elevation,
o
only 3.2 C, indicating the excellent photothermal
conversion ability of such NPs. Furthermore, the
photothermal conversion efficiency (η) was calculated to
be 38.9%, which is higher than that of BDPmPh (27.3%) and
BDPbiPh (37.9%) but lower than that of BDPtriPh
60.5%)[
30]
(
Figure 1 (a) Normalized absorbance of BDPIA in THF and
NPs in water. (b) Degradation of DPBF in the presence of
BDPIA for different time. (c) Linear fitting of the
degradation of DPBF. (d) Degradation of DPBF in the
presence or absence of laser irradiation, showing the
singlet oxygen generation without irradiation.
Singlet oxygen generation and photothermal conversion
efficiency of BDPIA NPs
Nanoprecipitation with DSPE-PEG-2000 was used to
synthesize the NPs to improve the water dispersity of
BDPIA. Transmission electron microscope (TEM) and
dynamic light scattering (DLS) have been applied to
characterize the morphology and size of BDPIA NPs, as
shown in Figure 2a. BDPIA can form uniform spherical
particles with size distribution from 44 to 285 nm. And the
average diameter is 89.3 nm, promising their potential for
enhanced penetration and retention (EPR) effect. The TEM
Figure (2) DLS and TEM of BDPIA NPs in water. (b)
Fluorescence enhancement of SOSG with or without laser
irradiation. (c) Temperature elevation of water and BDPIA
2
NPs with laser irradiation (660 nm, 0.5 W/cm ). (d) Linear
result is in good agreement with that of the DLS. To
1
investigate the O
2
capture and release ability of BDPIA
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J Po lue ran sae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
Page 6 of 9
ARTICLE
Journal of Materials Chemistry B
fitting of -lnθ and time in the cooling curve, showing the
calculated photothermal conversion efficiency of 38.9%.
View Article Online
DOI: 10.1039/C9TB00180H
In vitro MTT assay, ROS generation and flow cytometry
To validate the synergistic phototherapeutic effects of
BDPIA NPs, we evaluated the inhibitory effect of BDPIA NPs
on the cell viability of HeLa cells with red fluorescent
proteins (Fig. 3c) at different concentrations. After
treatment of BDPIA NPs for 24 h, MTT assay shows that the
IC50 (the concentration of drug inhibiting 50 % of cells)
value of HeLa cells was 0.3715 μg/mL (Fig. 3b). We tried to
evaluate only the photodynamic efficacy of BDPIA NPs. We
2
used a lower power laser irradiation (660 nm, 0.2 W/cm )
Figure 3 (a) Cellular uptake of BDPIA NPs and ROS
generation with DCF-DA as a probe. (b) MTT assay of BDPIA
NPs with different conversations. (c) Confocal imaging of
HeLa Cells transfected with Lentivirus. (d) Flow cytometry
assay (e) Apoptosis rate of control, no illumination and
to reduce the amount of generated heat, and the MTT
results show that the IC50 was 15.73 μg/mL, which is much
higher than that obtained under a higher laser density (660
2
nm, 0.5 W/cm ) (Figure S3).
And then Annexin V-FITC/PI double staining assay was used illumination groups.
to confirm the pro-apoptotic effect of BDPTA NPs.
In vivo fluorescence imaging guided PDT/PTT
Compared with the control group, the apoptotic rates of
HeLa cells in illumination group were significantly
increased after treatment with BDPTA NPs (Fig. 3d and 3e),
which demonstrated that BDPTA NPs induced typical
apoptosis in HeLa cells, while the without illumination
group has not significant difference, indicating that the
BDPTA NPs have low dark toxicity.
The self-fluorescent tumor model was used to investigate
the uptake of BDPIA NPs in vivo. Double wavelengths (460
and 660 nm) were used as the excitation for different
period of time. As illustrated in Figure 4a, the tumor could
always be excited at 460 nm which is an appropriate time
for the excitation of the red fluorescent proteins. As
mentioned in Figure 1a, BDPIA NPs shows strong
absorbance around 660 nm, so it was used as the excitation
wavelength. For 0 h, no obvious fluorescence can be
detected when excited at 660 nm. 2 h after intravenous
injection BDPIA NPs, the fluorescence become stronger,
indicating the uptake of the BDPIA NPs by enhanced
penetration and retention effect (EPR). 4 h after injection,
the fluorescence reaches the peak, showing that 4 h is the
best time point for phototherapy, and the fluorescence
could still be detected 24 h after administration. The
mouse was then sacrificed and the fluorescence imaging of
tumor, heart, liver, spleen, lung and kidney were recorded.
Figure 4b also demonstrates that the fluorescence
intensity of the tumor is the highest, followed by liver and
kidney, as further confirmed by the quantified intensity of
the tissues in Figure 4c. Furthermore, the imaging excited
at 460 nm can be well merged with that at 660 nm, this
model can accurately display the accumulation process of
the BDPIA NPs at the tumor site, showing the advantages
and reliability compared with common tumor model.
Cellular uptake behavior of BDPIA NPs was studied using a
confocal live cell imaging system. The results were shown
in Fig. 3a. The high intracellular fluorescence intensity in
the cytoplasmic matrix (nuclei dyed with DAPI) can be
observed, indicating that the BDPIA NPs can be used for cell
imaging and therapy in vitro.
6
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Journal of Materials Chemistry B
ARTICLE
Furthermore, compared with control group
V
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ticle
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e
DOI: 10.1039/C9TB00180H
weight changes of the other two groups can also suggest
that the BDPIA NPs have no side effects because the weight
tend to increase during the therapy process.
After treatment, these nude mice of the three groups were
sacrificed and photographs of the tumors were illustrated
in Figure 5e. Then the H&E stained pictures of the tumors
in the control and no illumination groups were shown in
Figure 5f and 5g, the nucleus of HeLa cells remain common
and no obvious distortion was observed, suggesting the
low dark toxicity of BDPIA NPs. Furthermore, the H&E
stained pictures of the normal tissues indicate that such
NPs cause no damage to heart, liver, spleen, lung and
Figure 4 (a) Real-time fluorescence imaging of nude mice kidney, showing the low dark toxicity and good bio-
under different excitation (460 and 660 nm) for 0, 2 ,4, 8, compatibility (Figure 6). [31-37] In conclusion, BDPIA NPs with
1
2 and 24 h. (b) Fluorescence imaging of tumor, heart, high phototoxicity, excellent bio-compatibility and low
liver, spleen, lung and kidney of the sacrificed nude mice dark toxicity would be a potential candidate drug for anti-
after 24 h injection. (c) Fluorescence intensity of the tumor, cancer treatment.
heart, liver, lung and kidney.
In vivo photothermal imaging and phototherapy
Owing to the high photothermal conversion efficiency of
BDPIA NPs in water, photothermal imaging were recorded
to show the temperature elevation to evaluate the
photothermal therapy efficacy of BDPIA NPs in vivo. As
indicated by the fluorescence imaging, post-injection for 4
h is the most suitable time point for phototherapy, 4 h after
dosing, the mouse was irradiated at 0, 2, 4, 6, 8 min,
respectively. For the control group, the tumor site
o
temperature only increased by 4.2 C, which was almost
negligible. In contrast, when the mouse was injected with
BDPIA NPs, the temperature can increase to 51.3 C under
Figure 5 (a) In vivo photothermal imaging of the nude mice
after injection with PBS or BDPIA NPs under laser
o
the same condition, which is high enough to kill the HeLa
irradiation. (b) Temperature elevation of the group
cells (Figure 5a, 5b). Due to the excellent phototherapy
injected with PBS or BDPIA NPs under laser irradiation (660
efficacy of BDPIA NPs, the treatment in vivo was
performed. In order to evaluate the antitumor effect and
toxicity of this BDPIA NPs, the tumor volume and body
weight were recorded every two days. As shown in Figure
2
nm, 0.5 W/cm ). (c) Relative tumor volume (d) Body weight
change of the control, no illumination and illumination
groups. (e) Digital photograph of the tumors. H&E stained
pictures of (f) control and (g) BDPIA NPs only groups. Scale
bar:100 μm.
5
c, the tumors in the illumination group disappeared after
treatment for 6 days and no recurrence were observed
when the mice were kept for another 10 days,
demonstrating the complete ablation of the tumor. While
in the control group and without illumination group, the
eventual relative tumor volume are 12.1 and 11.8 times
bigger than that of the original one, respectively, and there
was no significant difference between the two groups,
showing the low dark toxicity of BDPIA NPs. The mice after
treatment were shown in Figure S4.
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ARTICLE
Journal of Materials Chemistry B
Notes and references
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In conclusion, BDPIA with high O
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by SOSG as a probe. Reversible O
2
storage and release
characteristics of BDPIA NPs make them potential
candidates for continuous photodynamic therapy.
Furthermore, superior phototherapy efficacy can be
obtained due to the high photothermal conversion
efficiency (38.9%) of BDPIA NPs. Such NPs can effectively
inhibit the growth of tumors and cause no adverse effects
on normal organs at a low dosage (0.2 mg/kg). The results
demonstrate that the continuous photodynamic therapy
characteristic can be achieved by appropriate molecular
design, and the BDPIA NPs with high phototoxicity,
excellent bio-compatibility and low dark toxicity achieve
good therapeutic efficiency without obvious side effects,
which make them has a great potential for photodynamic
and photothermal synergistic therapy.
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