Rational Design of Conjugated Photosensitizers with Controllable Photoconversion for Dually Cooperative Phototherapy

Article abstract of DOI:10.1002/adma.201801216

High-performance photosensitizers are highly desired for achieving selective tumor photoablation in the field of precise cancer therapy. However, photosensitizers frequently suffer from limited tumor suppression or unavoidable tumor regrowth due to the pr

Full text of DOI:10.1002/adma.201801216

CommuniCation
Conjugated Photosensitizers
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Rational Design of Conjugated Photosensitizers with
Controllable Photoconversion for Dually Cooperative
Phototherapy
Shuyue Ye, Jiaming Rao, Shihong Qiu, Jinglong Zhao, Hui He,* Ziling Yan, Tao Yang,
Yibin Deng, Hengte Ke, Hong Yang, Yuliang Zhao, Zhengqing Guo,* and Huabing Chen*
Photosensitizers have extensively explored
High-performance photosensitizers are highly desired for achieving selective
tumor photoablation in the field of precise cancer therapy. However,
as emerging versatile compounds in many
fields including photodynamic therapy
(PDT), photocatalysis, cell signaling, and
biosensors.[1] For cancer therapy, excited
photosensitizer is able to produce highly
cytotoxic reactive oxygen species (ROS)
such as singlet oxygen via intersystem
crossing (ISC)-mediated singlet-to-triplet
transition and subsequent energy transfer,
thus causing the apoptosis through the
oxidation of biologically relevant mole-
cules in mitochondria and nucleus to
cause selective suppression against malig-
nant shallow tumors.[2] PDT possesses
several distinct advantages over conven-
tional therapeutics including precise spa-
tiotemporal control, selective treatment
with minimized adverse side effect, and
negligible drug resistance.[2b,3] To date,
several types of organic photosensitizers
including boron dipyrromethene (BDP),
phthalocyanine, and porphyrin have
been extensively developed for achieving
photosensitizers frequently suffer from limited tumor suppression or unavoidable
tumor regrowth due to the presence of residual tumor cells surviving in
phototherapy. A major challenge still remains in exploring an efficient approach
to promote dramatic photoconversions of photosensitizers for maximizing the
anticancer efficiency. Here, a rational design of boron dipyrromethene (BDP)-
based conjugated photosensitizers (CPs) that can induce dually cooperative
phototherapy upon light exposure is demonstrated. The conjugated coupling of
BDP monomers into dimeric BDP (di-BDP) or trimeric BDP (tri-BDP) induces
photoconversions from fluorescence to singlet-to-triplet or nonradiative
transitions, together with distinctly redshifted absorption into the near-infrared
region. In particular, tri-BDP within nanoparticles shows preferable conversions
into both primary thermal effect and minor singlet oxygen upon near-infrared
light exposure, dramatically achieving tumor photoablation without any regrowth
through their cooperative anticancer efficiency caused by their dominant late
apoptosis and moderate early apoptosis. This rational design of CPs can serve as
a valuable paradigm for cooperative cancer phototherapy in precision medicine.
effective PDT through versatile strategies such as heavy-atom
effect, spin converter, charge recombination, exciton coupling,
suppressed photoinduced electron transfer, as well as func-
tional substitution of pH-activatable dimethylaminophenyl
group, mitochondria-targeted triphenylphosphonium bro-
mide, or antiangiogenic acetazolamide moiety.[4] Moreover,
versatile drug vehicles such as micelles, vesicles, graphene
oxide, mesoporous silica nanoparticles, and metal–organic
frameworks are frequently utilized to boost their anticancer
efficiency through enhanced singlet oxygen generation, self-
supplied oxygen, improved resistance to photobleaching, or
preferable tumor accumulation.[2b,c,3,4,5] Unfortunately, these
photosensitizers frequently suffer from limited tumor sup-
pression or unavoidable tumor regrowth due to the residual
tumor cells surviving from light irradiation, usually owing to
their several drawbacks including shallow light penetration
depth in visible region (frequently less than 650 nm), absolute
oxygen dependence, insufficient cytoplasmic drug transloca-
tion, and inadequate cell damage from singlet oxygen-mediated
apoptosis. Hence, highly potent photosensitizer with distinctly
redshifted absorption is highly desired for achieving tumor
photoablation.[2b,4b]
S. Y. Ye, Dr. S. H. Qiu, J. L. Zhao, Dr. H. He, Z. L. Yan, T. Yang,
Dr. Y. B. Deng, Dr. H. T. Ke, Prof. H. Yang, Prof. H. B. Chen
State Key Laboratory of Radiation Medicine and Protection
Jiangsu Key Laboratory of Neuropsychiatric Diseases, and
College of Pharmaceutical Sciences
Soochow University
Suzhou 215123, China
E-mail: hhe@suda.edu.cn; chenhb@suda.edu.cn
J. M. Rao, Dr. Z. Q. Guo, Prof. H. B. Chen
State Key Laboratory of Radiation Medicine and Protection
Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher
Education Institutions, and School of Radiation Medicine and Protection
Soochow University
Suzhou 215123, China
E-mail: zqguo@suda.edu.cn
Prof. Y. L. Zhao
CAS Key Laboratory for Biomedical Effects of Nanomaterials
and Nanosafety
Center of Excellence for Nanosciences
National Center for Nanoscience and Technology of China
Beijing 100190, China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adma.201801216.
DOI: 10.1002/adma.201801216
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In the past few years, many efforts have been made to ration-
ally synthesize efficient photosensitizers toward enhanced sin-
glet oxygen generation for improving their PDT efficacy.[3a–i,6]
Some emerging approaches are explored to boost the photocon-
versions of photosensitizers such as BDP into singlet oxygen
through long-lived triplet state, together with reduced radiative
transition.[4f] For instance, the intramolecular resonance energy
transfer (RET) and resonance energy transfer were exploited to
enhance the ISC process and subsequent singlet oxygen gen-
eration;[7] Radical enhanced ISC was introduced for causing
long-lived triplet state;[8] Photoinduced electron transfer pro-
cess was also applied to generate locally excited triplet state of
heavy atom-free dyes in visible absorption.[9] Although these
approaches effectively enhance the singlet oxygen generation
of photosensitizers, a facile and efficient approach is highly
desired to tune their photoconversion behaviors, thus maxi-
mizing the photocytotoxicity to cause potent phototherapy.
Previously, we utilized platinum-coordinated BDP to generate
both heavy atom effect and nonradiative transition that can
cause singlet oxygen and thermal effect for synergistic PDT and
photothermal therapy (PTT), implying a possibility to achieve
potent phototherapeutic efficacy through maximizing their
photoconversions.[10] Herein, we first report a rational design of
BDP-based conjugated photosensitizers (CPs) that can induce
dually cooperative phototherapy for achieving tumor photoab-
lation surgery upon NIR light exposure (Figure 1). The conju-
gated coupling of BDP monomers into dimeric BDP (di-BDP)
or trimeric BDP (tri-BDP) induces the photoconversions from
fluorescence to singlet-to-triplet or nonradiative transition,
together with distinctly redshifted absorption into NIR region.
In particular, tri-BDP within nanoparticles shows the preferable
conversions into both primary thermal effect and minor singlet
oxygen upon 785 nm light exposure as compared to di-BDP
with its main conversion into singlet oxygen through ISC, dra-
matically achieving the total tumor photoablation without any
regrowth.
To synthesize BDP-based CPs with tunable photoconver-
sions, the conjugated coupling of BDP monomers into di-BDP
and tri-BDP was designed as depicted in Figure 2a. Briefly, the
formylation of highly emissive BDP precursor into β-formyl
BDP (mono-BDP), followed by subsequent one-pot Knoevenagel
self-condensation reaction that finally afforded the conjugated
di-BDP and tri-BDP. The chemical structures and molecular
weights of mono-BDP, di-BDP, tri-BDP, and their precursors
were fully characterized using 1H NMR, 13C NMR, and HR-MS
analysis (Figures S1–S7, Supporting Information).
To deliver hydrophobic di-BDP and tri-BDP, they were added
into poly(ethylene glycol)114-b-poly(caprolactone)60 copolymer
in dimethyl sulfoxide (DMSO), respectively, and then dispersed
into distilled water under ultrasonication, followed by the for-
mation of di-BDP-loaded nanoparticles (di-BDP-NPs) or tri-
BDP-loaded nanoparticles (tri-BDP-NPs) at the loading level of
20% after the purification through the dialysis (Figure 1a). The
mono-BDP-loaded nanoparticles (mono-BDP-NPs) were also
prepared as the control using a similar procedure. di-BDP-NPs
and tri-BDP-NPs were observed using transmission electron
microscopy (TEM), indicating the spherical morphologies with
average diameters of 85.0 6.0 and 69.0 8.0 nm, respectively
(Figure 2b; and Figure S8, Supporting Information). Dynamic
light scattering (DLS) measurements demonstrate that they
possessed the average hydrodynamic diameters of 95.0 and
82.0 nm, respectively (Figure 2c; and Figure S8, Supporting
Information), implying their potential passive targeting ability
through enhanced permeation and retention (EPR) effect. Free
Figure 1. a) Chemical structures and nanoparticles of CPs. b) Photoconversion routes of various CPs. c) Cooperative phototherapy of tri-BDP-NPs
against tumor cells through PTT/PDT treatments under light exposure.
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Figure 2. a) Synthetic route of mono-BDP, di-BDP, and tri-BDP. i) TFA, CH2Cl2; DDQ; Diisopropylethylamine; BF3⋅Et2O; ii) POCl3, DMF; iii) HOAc,
Piperidine. b) TEM image and c) size distribution of tri-BDP-NPs.
di-BDP and tri-BDP were also dissolved in aqueous solutions
containing 0.5% DMSO as the controls (Figure S8, Supporting
Information), respectively, which show broad size distributions
due to their aggregations in aqueous solutions.
The absorption spectra of di-BDP-NPs and tri-BDP-NPs
were observed in water. As shown in Figure 3a, they displayed
the absorption peaks at 649 and 738 nm in water, respectively,
while mono-BDP-NPs showed an absorption peak at 491 nm.
Distinctly, the conjugated coupling of BDP monomers into di-
BDP or tri-BDP caused the distinctly redshifted absorption into
visible or NIR region due to the extended delocalized π system.
In particular, tri-BDP-NPs with NIR absorption might provide
deeper light penetration depth for tumor treatment. Moreover,
they also showed the broadened absorptions and displayed the
redshifts of about 20 nm in their absorption as compared to
free di-BDP and tri-BDP in DMSO, reasonably owing to the
formations of their J-type and H-type aggregates with di-BDP
or tri-BDP aggregates with intermolecular π–π stacking within
the nanoparticles.[6,10] These redshifts were further confirmed
by their redshifted emission peaks at 756 and 794 nm with
large stokes shifts (Figure 3b). Clearly, the conjugated coupling
and subsequent J-type aggregation of BDP monomers within
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Figure 3. a) Normalized absorption spectra and b) emission spectra of mono-BDP-NPs, di-BDP-NPs, and tri-BDP-NPs as compared to free mono-BDP,
di-BDP, and tri-BDP in DMSO. c,d) Calculated UV–vis absorption spectra and HOMO/LUMO of energy-minimized calculated (Gaussian) di-BDP (c)
and tri-BDP (d). Calculation was performed at B3LYP/6-311G* level with Gaussian 09W (Scaling factors are 0.9).
nanoparticles account for their apparently redshifted absorp-
tion in a controlled manner.
region. Apparently, the conjugated coupling of BDP monomers
significantly results in the redshifted absorption into NIR region,
suggesting that this type of CPs might be excited at redshifted
wavelength that depends on their conjugated conformations.
To distinguish the type of ROS from di-BDP-NPs and tri-BDP-
NPs upon light exposure, the electron spin resonance (ESR)
was applied using 2,2,6,6-tetramethylpiperide (TEMP) and
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping
adducts (Figure 4a). Both of their ESR spectra showed the char-
acteristic 1:1:1 multiplicity from TEMP-1-oxyl after 5 min light
exposure, confirming the generation of singlet oxygen from
these two nanoparticles.[12] The transient absorption spectra
show that di-BDP and tri-BDP had the triplet lifetimes (τ) of 93.6
and 70.6 µs (Figure 4b), respectively, suggesting that both of
them undergo their efficient singlet-to-triplet transition, and di-
BDP shows a more favorable capacity to produce singlet oxygen
as compared to tri-BDP.[10] The singlet oxygen generations
from di-BDP-NPs and tri-BDP-NPs were further investigated
To illustrate the electronic states of di-BDP and tri-BDP within
the nanoparticles, we carried out the time-dependent density
theory (TDDFT) calculation (Figure 3c,d). The calculated UV–vis
absorption maxima of di-BDP and tri-BDP were located at 642 and
752 nm, respectively, which accord well with their experimental
absorptions in Figure 3a. Distinctly, the S1 states in di-BDP and
tri-BDP originate from highest occupied molecular orbitals
(HOMO) to lowest unoccupied molecular orbitals (LUMO) with
the largest extinction coefficient (≈105 L mol−1 cm−1), showing a
typical π–π* characteristics. According to the frontier orbitals of
di-BDP and tri-BDP (Figure 3c,d), the electronic density is delo-
calized on the backbone of conjugated BDP and thus cause the
lower energy gap between HOMO and LUMO in moieties.[11]
Moreover, tri-BDP possess a narrower energy gap for π–π* tran-
sition due to their larger conjugated π system as compared to
di-BDP, thus causing their more intensive absorption in NIR
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Figure 4. a) ESR spectra of di-BDP-NPs and tri-BDP-NPs under 660 and 785 nm light exposures at 0.5 W cm−2, respectively. b) Decay trace of di-BDP
at 660 nm and tri-BDP at 780 nm at the excitation of 600 nm in their transient absorption. c,d) Normalized absorbance of DPBF at 410 nm in the
solutions of di-BDP-NPs (c) and tri-BDP-NPs (d) at different concentrations under 660 or 785 nm light exposure at 0.5 W cm−2 for 3 min. e,f) Tempera-
ture elevations of di-BDP-NPs (e) and tri-BDP-NPs (f) under 660 or 785 nm light exposure at 0.5 W cm−2 for 5 min.
using 1,3-diphenyliso-benzofuran (DPBF) as a probe under
light exposure. di-BDP-NPs exhibited the distinct concen-
tration-dependent singlet oxygen generation in the range of
7.5–75.0 nmol L−1 (Figure 4c), while tri-BDP-NPs showed a sim-
ilar singlet oxygen generation in a much higher concentration
range (Figure 4d). Distinctly, di-BDP-NPs are able to generate
much more singlet oxygen under light exposure as compared
to tri-BDP-NPs. To further quantify the abilities of di-BDP-NPs
and tri-BDP-NPs to generate singlet oxygen under light expo-
sure, their singlet oxygen quantum yields were also evalu-
ated. They showed the singlet oxygen quantum yields (Φ∆) of
0.25 and 0.18 (Table 1), respectively, which are higher than those
of mono-BDP-NPs (Φ∆ = 0.09), chlorin e6-loaded nanoparticles
(Ce6-NPs, Φ∆ = 0.12), and indocyanine green-loaded nanoparti-
cles (ICG-NPs, Φ∆ = 0.1, Table S1, Supporting Information). In
contrast, free di-BDP and tri-BDP as the control had the singlet
oxygen quantum yields of 0.22 and 0.19 (Table S1, Supporting
Information), respectively. Apparently, the encapsulation of
di-BDP or tri-BDP into the nanoparticles shows no significant
influence on the singlet oxygen generation, presumably due to
the formation of well-organized J-type and H-type aggregates.
Moreover, BDP-based conjugated photosensitizers possess a
preferable capacity to generate photothermal conversion and
singlet oxygen generation as compared to mono-BDP, Ce6,
Table 1. Photoconversion parameters of mono-BDP-NPs, di-BDP-NPs,
and tri-BDP-NPs (Φ∆: singlet oxygen quantum yield; ηT: photothermal con-
version efficiency; ΦF: fluorescence quantum yield; n. d., none detected).
tri-BDP-NPs
0.18
di-BDP-NPs
0.25
mono-BDP-NPs
0.09
Φ∆
ηT
45.2%
32.2%
n. d.
ΦF
9.6 × 10−5
8.3 × 10−4
3.2 × 10−2
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and ICG, possibly due to the distinctly reduced fluorescence
quantum yield (Table 1). In addition, di-BDP-NPs possess a
preferable ability to produce singlet oxygen as compared to
tri-BDP-NPs.
free di-BDP and tri-BDP during 48 h incubation, indicating their
preferable endocytosis that is highly favorable for causing sub-
sequent photocytotoxicity. Next, their endocytic pathways were
further assessed using various pathway inhibitors (Figure 5b).
di-BDP-NPs and tri-BDP-NPs exhibited the decreases of ≈50%
in their cellular uptake in the presence of chlorpromazine, an
inhibitor against clathrin-mediated endocytosis, indicating that
both of them undergo the clathrin-mediated endocytosis.
To assess the photothermal conversion capacities of di-BDP-
NPs and tri-BDP-NPs through nonradiative transition, their
photothermal conversion efficiencies (ηT) were measured
under light exposure at 0.5 W cm−2. di-BDP-NPs and tri-BDP-
NPs exhibited the photothermal conversion efficiencies of
32.2% and 45.2% (Table 1; and Figure S9, Supporting Informa-
tion), respectively, while no obvious photothermal conversion
was observed for mono-BDP-NPs, and ICG-NPs and Ce6-NPs
only had the photothermal conversions of 26.0% and 14.0%
(Table S1, Supporting Information), respectively. Distinctly,
the coupling of BDP monomers into di-BDP or tri-BDP caused
their effective photothermal conversion. In particular, tri-BDP-
NPs exhibit a more significant photothermal conversion as
compared to di-BDP-NPs under light exposure due to their
preferable nonradiative transition and relatively shorter triplet
lifetime, and are also comparable to those of the existing photo-
thermal agents such as CuS nanoparticles.[13] Reasonably, the
enhanced triplet lifetime might be responsible for the lower
photothermal conversion of di-BDP-NPs as shown in Figure 4b.
To further evaluate the photothermal conversion, we
measured the temperature elevations of di-BDP-NPs and tri-
BDP-NPs under light exposure at 0.5 W cm−2 (Figure 4e,f).
tri-BDP-NPs exhibited the temperature elevation (∆T) of
38 °C at the concentration of 80.0 µmol L−1 in 300 s, and also
showed the concentration-dependent temperature elevations
(Figure 4f), resulting from their high photothermal conversion
efficiency and remarkable absorbance. In contrast, di-BDP-NPs
had the lower temperature elevations under light exposure as
compared to tri-BDP-NPs (Figure 4e). Thus, both di-BDP-NPs
and tri-BDP-NPs as a type of CPs afford the dual photoconver-
sions into singlet oxygen and photothermal effect that results
from their reduced radiative transition, as further evidenced by
their reduced fluorescence quantum yields (ΦF, Table 1).[10] In
particular, tri-BDP-NPs with an extendedly conjugated structure
primarily hold a preferable photothermal conversion ability
together with moderate singlet oxygen generation upon light
exposure. Oppositely, di-BDP-NPs display a primary singlet
oxygen generation accompanied with a minor photothermal
conversion due to their favorable ISC-mediated triplet.
To evaluate the photocytotoxicity, di-BDP-NPs and tri-BDP-
NPs were incubated with 4T1 cells for 24 h, followed by 660 and
785 nm light exposures at 0.5 W cm−2, respectively. In the dark-
ness, both di-BDP-NPs and tri-BDP-NPs showed no signifi-
cant photocytotoxicity, while they exhibited the IC50 values of
0.22 and 1.30 µmol L−1 upon 5 min light exposure (Figure 5c,d;
and Table S2, Supporting Information), reasonably resulting
from both singlet oxygen and photothermal effect. Moreover,
the longer light exposure (15 min) further caused the decreases
of their IC50 values (Figures S12 and S13, Supporting Informa-
tion), indicating their dependence on light exposure as well.
Interestingly, the distinct early apoptosis was responsible for
the photocytotoxicity of di-BDP-NPs due to their favorable sin-
glet oxygen generation (Figure 5e), while the late apoptosis pri-
marily contributed to that of tri-BDP-NPs presumably owing to
their preferable photothermal effect-mediated hyperthermia,
although both of them have dual photoconversions into both
singlet oxygen and photothermal effect. Afterward, to differ-
entiate the photothermal cell damage, the photocytotoxicity of
di-BDP-NPs and tri-BDP-NPs were further investigated against
4T1 cells that were incubated with ROS scavenger Vitamin C
(Vc). They only had the IC50 values of ≈20.0 and ≈12.0 µmol L−1
under 660 and 785 nm light exposures (Figure 5c,d), respec-
tively. Obviously, the PTT damage alone causes the distinctly
reduced photocytotoxicity in the absence of singlet oxygen,[10,15]
and simultaneously tri-BDP-NPs possess a preferable photo-
thermal cytotoxicity as compared to di-BDP-NPs owing to their
favorable photothermal effect. To further distinguish the role
of photodynamic damage in the photocytotoxicity of di-BDP-
NPs and tri-BDP-NPs, we temporarily incubated 4T1 cells at
≈4 °C during light exposure to avoid temperature elevation
(Figures S14 and S15, Supporting Information). They showed
the IC50 values of 0.53 and 34.8 µmol L−1 that were caused by
the PDT damage alone, respectively. Distinctly, di-BDP-NPs
show more favorable photodynamic cytotoxicity as compared
to tri-BDP-NPs. Subsequently, the cooperative index (CI) of di-
BDP-NPs and tri-BDP-NPs were calculated to be 0.42 and 0.15,
respectively. It suggests that both of them have the apparently
cooperative effect between their PDT and PTT efficiencies (CI
<0.8 is considered as cooperative effect),[16] and tri-BDP-NPs
possess a stronger cooperative phototherapeutic efficiency as
compared to di-BDP-NPs, owing to their primary late apoptosis
and moderate early apoptosis.
The resistances of di-BDP-NPs and tri-BDP-NPs to photo-
bleaching were observed due to their key role in light treatment.
di-BDP-NPs and tri-BDP-NPs only showed the subtle changes
in their absorbance during 16 min under light exposure
(Figure S10, Supporting Information), indicating a preferable
resistance to photobleaching when compared with free di-BDP,
free tri-BDP, Ce6-NPs, and ICG-NPs as the controls. Clearly, the
encapsulation within nanoparticles protects their unsaturated
bonds from the damage of radicals in solution for suppressing
the photo-oxidation.[14] Moreover, we observed the chemical
stability of di-BDP-NPs and tri-BDP-NPs in aqueous solutions
(Figure S11, Supporting Information), suggesting their good
chemical stability without any degradation during 48 h.
To further explore the cooperative mechanism of their photo-
cytotoxicity, the confocal laser scanning microscopy (CLSM)
was employed to observe their cytoplasmic translocation behav-
iors of di-BDP-NPs and tri-BDP-NPs under light exposure. In
the darkness, they showed the colocalization percentages of
93.1% and 94.5% with the lysosomes after 0.5 h incubation due
to their effective endocytosis (Figure 5f; and Figure S16, Sup-
porting Information). However, after 5 min light exposure at
The cellular uptakes of di-BDP-NPs and tri-BDP-NPs were
investigated on 4T1 tumor cells (Figure 5a). They exhibited
remarkable increases in their cellular uptakes as compared to
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Figure 5. a) Cellular uptake of di-BDP-NPs and tri-BDP-NPs at the dose of 80.0 µmol L−1 by 4T1 cells after 6, 24, and 48 h incubation, respectively.
b) Normalized absorbance of di-BDP-NPs and tri-BDP-NPs internalized by 4T1 tumor cells treated with PBS, amiloride, flipin, and chlorpromazine at
37 °C, and PBS at 4 °C, respectively. c) Relative cell viability of 4T1 tumor cells treated with di-BDP-NPs at various doses under 660 nm light exposure at
0.5 W cm−2 for 5 min. d) Relative cell viability of 4T1 tumor cells treated with tri-BDP-NPs at various doses under 785 nm light exposure at 0.5 W cm−2
for 5 min. e) Apoptosis level of 4T1 cells treated with di-BDP-NPs and tri-BDP-NPs at the dose of 1.0 µmol L−1 for 24 h incubation, followed by 660 or
785 nm light exposure at 0.5 W cm−2 for 5 min using Annexin V-FITC/PI Apoptosis Detection Kit (Student’s t-test, **p < 0.01). f) CLSM images of 4T1
cells stained by Lysotracker Green DND 26 and Hoechst 33342 after 0.5 h incubation with tri-BDP-NPs under 785 nm light exposure at 0.5 W cm−2
for 5 min (scale bar, 10 µm).
0.5 W cm−2, their colocalizations were respectively reduced to
71.9% and 65.2%, probably owing to the ROS-mediated lyso-
somal rupture through photochemical internalization effect, as
evidenced by their green fluorescence caused by the lysosomal
rupture in the acridine orange (AO) staining (Figure S17, Sup-
porting Information).[10] Apparently, this type of CPs undergo
an effective cytoplasmic translocation from the lysosome to
cytoplasm in several minutes under light exposure, which
might favorably promote their accessibility to nucleus and
mitochondria for cooperative photocytotoxicity.[17]
To demonstrate the capacities of di-BDP-NPs and tri-BDP-NPs
to accumulate at tumor sites, their biodistributions were studied
in the mice bearing 4T1 tumors at the dose of 8.0 mg kg−1. Both
of them exhibited distinctly improved tumor accumulations at
24 h postinjection when compared with free di-BDP and tri-BDP
(Figure 6a), indicating that the nanoparticles resulted in the
passive tumor targeting through their EPR effect. To evaluate
the in vivo hyperthermia at tumor, di-BDP-NPs and tri-BDP-
NPs were intravenously administrated into the mice bearing
4T1 tumor at the dose of 8.0 mg kg−1, respectively, monitored
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Figure 6. a) Biodistribution of di-BDP-NPs and tri-BDP-NPs on the mice at 24 h postinjection, respectively. b) Temperature elevation at the tumors of
the mice injected with di-BDP-NPs and tri-BDP-NPs at the dose of 8.0 mg kg−1 at 24 h postinjection under 660 or 785 nm light exposure at 0.5 W cm−2
for 5 min. c) DHE staining of the tumor sections of the mice treated with di-BDP-NPs and tri-BDP-NPs at dose of 8.0 mg kg−1 in the presence or absence
of Vc at 24 h postinjection under 660 or 785 nm light exposure at 0.5 W cm−2 for 5 min (Scale bar, 100 µm). d) Tumor growth profile of the mice treated
with tri-BDP-NPs at the dose of 8.0 mg kg−1 in the presence or absence of Vc under 785 nm light exposure at 0.5 W cm−2 for 5 min (Student’s t-test,
*p < 0.05, and **p < 0.01), and e) their tumor photo at the end of the experiment.
by the infrared thermography under 5 min light exposure at
0.5 W cm−2 at 24 h postinjection. di-BDP-NPs and tri-BDP-NPs
caused the temperature elevations of 10.2 and 18.0 °C under
light exposure, respectively (Figure 6b; and Figure S18, Sup-
porting Information), while free di-BDP and tri-BDP as the
control only resulted in the negligible temperature elevations.
Distinctly, tri-BDP-NPs possess a preferable ability to generate
potent hyperthermia for causing PTT treatment at 785 nm as
compared to di-BDP-NPs, reasonably owing to their preferable
photothermal conversion ability.[18] Moreover, the in vivo singlet
oxygen generation was also observed from di-BDP-NPs and tri-
BDP-NPs at the tumors using dihydroethidium (DHE) staining,
respectively. Free di-BDP and tri-BDP showed no fluorescence
under light irradiation, suggesting the absence of singlet oxygen
at the tumors (Figure 6c). Interestingly, di-BDP-NPs had a dis-
tinct red fluorescence that was further scavenged by Vc at tumor,
while tri-BDP-NPs caused a relatively weak red fluorescence
(Figure 6c). Clearly, di-BDP-NPs effectively produce more intra-
cellular singlet oxygen at tumor site as compared to tri-BDP-NPs
under light exposure. Thus, di-BDP-NPs preferably cause more
singlet oxygen at tumor, and tri-BDP-NPs primarily result in
stronger in vivo hyperthermia, although both of them possess
both singlet oxygen and photothermal effect.
To evaluate the in vivo phototherapeutic efficacy, di-BDP-
NPs and tri-BDP-NPs were intravenously injected into the
tumor-bearing mice at a single dose of 8.0 mg kg−1, respec-
tively, and then suffered from 660 or 785 nm light irradiation
at 0.5 W cm−2 for 5 min at 24 h postinjection. To distinguish
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the role of PDT, Vc as a ROS scavenger was also intratumorally
injected into the tumor before light exposure. The tumor vol-
umes were monitored during subsequent 30 d. As depicted in
Figure 6d; and Figure S19 (Supporting Information), phosphate
buffer saline (PBS) as a control caused the significant increases
of more than ≈30-folds in their tumor volumes despite of
light exposure, indicating that the light exposure itself has no
obvious influence on the tumor growth. di-BDP-NPs and tri-
BDP-NPs also displayed the similar tumor growth profiles to
PBS in the lack of light exposure, suggesting both of them have
no significant dark cytotoxicity (Figure 6d; and Figure S19,
Supporting Information). Importantly, tri-BDP-NPs effectively
resulted in the total tumor ablation without any regrowth
during 30 d (Figure 6e), while di-BDP-NPs also effectively led
to the tumor ablation, accompanying with tumor regrowth
(Figure S19, Supporting Information). Apparently, tri-BDP-NPs
possess a preferable ability to totally ablate the tumors upon
NIR light exposure when compared with di-BDP-NPs. Possibly,
the NIR absorption, favorable hyperthermia, moderate singlet
oxygen, and cytoplasmic translocation cooperatively contribute
to their total tumor ablation through their primary late apop-
tosis and moderate early apoptosis. Interestingly, both of them
also displayed more obvious tumor regrowths in the presence
of ROS scavenger Vc, suggesting that the PTT treatment alone
causes the survivals of tumor cells due to the absence of PDT
treatment. The intracellular singlet oxygen at tumor sites plays
a key role in severely injuring the residual tumor cells surviving
from the hyperthermia-mediated PTT damage. Obviously, tri-
BDP-NPs show a distinct cooperative PTT/PDT anticancer
efficacy that accounts for the total tumor photoablation. In addi-
tion, free di-BDP and tri-BDP showed a similar tumor growth
including enhanced cellular uptake, effective cytoplasmic translo-
cation, and preferable tumor accumulation. In particular, di-BDP-
NPs might behave as an effective CPs with enhanced triplet state
that displays a primary singlet oxygen generation under 660 nm
light exposure, while tri-BDP-NPs with extendedly conjugated
structure primarily hold a preferable photothermal effect under
785 nm light exposure together with moderate singlet oxygen
generation, thereby achieving the tumor photoablation surgery
without any regrowth through both primary late apoptosis and
moderate early apoptosis. This therapeutic benefit is possibly
caused by several advantages of CPs including deeper light pen-
etration depth due to distinctly redshifted absorption, improved
resistance to photobleaching, reduced oxygen depletion, cyto-
plasmic drug translocation, as well as combination of late and
early apoptosis. Moreover, this approach might avoid the use of
heavy atoms such as I, Br, and Pt in conventional strategies that
might potentially cause safety concern,[4f] and also suggests a
preferably concise regime to synthesize potent CPs with tunable
photoconversions, as compared to sophisticated design of donor–
acceptor-type conjugated macromolecules with phototherapy.
This type of CPs shows an emerging potential to fabricate highly
potent photosensitizers with distinctly redshifted absorption, and
we believe that this rational design can serve as a valuable para-
digm for cooperative cancer phototherapy in precision medicine.
Experimental Section
Detailed experimental materials and methods can be found in the
Supporting Information.
profiles to PBS, suggesting their poor anticancer efficacy due Supporting Information
to the absence of tumor accumulation. Thus, extendedly con-
jugated tri-BDP-NPs effectively achieve the cooperative photo-
therapy with tumor ablation under NIR light exposure.
Supporting Information is available from the Wiley Online Library or
from the author.
To confirm the in vivo photocytotoxicity of di-BDP-NPs and
tri-BDP-NPs, the hematoxylin & eosin (H&E) staining was
applied to observe their in vivo injuries against the tumors at the
dose of 8.0 mg kg−1 at 6 h postirradiation. Both of them resulted
in destructive necrosis of tumor cells, while free di-BDP and
tri-BDP only caused the negligible injuries on the tumors
(Figure S20, Supporting Information). Distinctly, both di-BDP-NPs
and tri-BDP-NPs are able to cause potent in vivo damage on the
tumor cells through their hyperthermia and singlet oxygen, indi-
cating that this type of CPs can act as potent photosensitizers for
highly efficient phototherapy. In addition, both of them showed no
obvious damage against the normal tissues including heart, liver,
spleen, lung, and kidney (Figure S21, Supporting Information),
confirming their ability to act as a selective therapeutic modality.
In summary, we first demonstrated a rational design of BDP-
based CPs that could produce dually cooperative phototherapy
for achieving tumor photoablation surgery upon NIR light expo-
sure. The conjugated coupling of BDP monomers into di-BDP
or tri-BDP caused the photoconversion from fluorescence to
intersystem crossing or nonradiative transition, together with
distinctly reduced radiative transition and redshifted absorp-
tion into NIR region. Upon encapsulation within nanoparticles,
both di-BDP-NPs and tri-BDP-NPs also possessed the collective
characteristics for achieving efficient cancer phototherapy
Acknowledgements
S.Y.Y., J.M.R., S.H.Q., and J.L.Z. contributed equally to this work.
This work was supported by National Basic Research Program
(No. 2014CB931900), National Natural Science Foundation of China
(Nos. 51473109, 31671016, 31771089, 21401136, and 81402779), 2017
National Level of Innovation and Entrepreneurship Training Program for
College Students (No. 201710285046Z), and Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD). The
animal protocols used in this study were approved by Dr. Xuechu Zhen,
chairman of the College Review Committees of Soochow University for
animal use and welfare.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
boron dipyrromethene, conjugated photosensitizers, near-infrared
absorption, photodynamic therapy, photothermal therapy
Received: February 24, 2018
Revised: April 19, 2018
Published online:
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