BODIPY-Based Photodynamic Agents for Exclusively Generating Superoxide Radical over Singlet Oxygen

Article abstract of DOI:10.1002/anie.202106748

Developing Type-I photosensitizers is considered as an efficient approach to overcome the deficiency of traditional photodynamic therapy (PDT) for hypoxic tumors. However, it remains a challenge to design photosensitizers for generating reactive oxygen species by the Type-I process. Herein, we report a series of α,β-linked BODIPY dimers and a trimer that exclusively generate superoxide radical (O₂^?.) by the Type-I process upon light irradiation. The triplet formation originates from an effective excited-state relaxation from the initially populated singlet (S₁) to triplet (T₁) states via an intermediate triplet (T₂) state. The low reduction potential and ultralong lifetime of the T₁ state facilitate the efficient generation of O₂^?. by inter-molecular charge transfer to molecular oxygen. The energy gap of T₁-S₀ is smaller than that between ³O₂ and ¹O₂ thereby precluding the generation of singlet oxygen by the Type-II process. The trimer exhibits superior PDT performance under the hypoxic environment.

Full text of DOI:10.1002/anie.202106748

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How to cite:
International Edition: doi.org/10.1002/anie.202106748
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Photodynamic Therapy
Hot Paper
BODIPY-Based Photodynamic Agents for Exclusively Generating
Superoxide Radical over Singlet Oxygen
Kun-Xu Teng+, Wen-Kai Chen+, Li-Ya Niu, Wei-Hai Fang, Ganglong Cui,* and Qing-
Zheng Yang*
Abstract: Developing Type-I photosensitizers is considered as
an efficient approach to overcome the deficiency of traditional
photodynamic therapy (PDT) for hypoxic tumors. However, it
remains a challenge to design photosensitizers for generating
reactive oxygen species by the Type-I process. Herein, we
report a series of a,b-linked BODIPY dimers and a trimer that
among 3PS*, adjacent substrates and molecular oxygen
ꢀ
produce superoxide (O2 C), peroxide (O22ꢀ), hydroxyl (OHC)
radicals or others; while, in the Type-II process, singlet oxygen
(1O2) is produced by triplet-triplet energy transfer between
3PS* and oxygen.[17]
Up to now, most PSs produce 1O2 by the Type-II process.
However, the hypoxic microenvironment of solid tumor
(pO2 < 5 mmHg) severely reduces the therapeutic effect of
Type-II PSs in PDT because such treatment is heavily
dependent on O2 concentration.[18–21] This has been recog-
nized as a major bottleneck of PDT in the clinical trans-
formation.[22] Unfortunately, rapid O2 consumption and
vascular damage during the type-II PDT further worsen its
shortage.[23–25] It is reported that Type-I PSs can lower oxygen
dependence by avoiding direct and fast O2 depletion in PDT
to solve the hypoxic problem.[26–29] More importantly, formed
ꢀ
exclusively generate superoxide radical (O2 C) by the Type-I
process upon light irradiation. The triplet formation originates
from an effective excited-state relaxation from the initially
populated singlet (S1) to triplet (T1) states via an intermediate
triplet (T2) state. The low reduction potential and ultralong
ꢀ
lifetime of the T1 state facilitate the efficient generation of O2 C
by inter-molecular charge transfer to molecular oxygen. The
3
1
energy gap of T1-S0 is smaller than that between O2 and O2
thereby precluding the generation of singlet oxygen by the
Type-II process. The trimer exhibits superior PDT perfor-
mance under the hypoxic environment.
ꢀ
O2 C species by the Type-I process not only serve as oxidants
to kill tumor cells, but also participate in superoxide
dismutase-triggered catalytic cascades to form highly cyto-
toxic OHC species and simultaneously produce O2 for
recycling.[30]
Introduction
Photodynamic therapy (PDT) has attracted significantly
increasing attention in the treatment of various cancer
diseases over the past decades because of its minimal
invasion, low systemic toxicity, negligible drug resistance,
and high spatiotemporal selectivity.[1–7] PDT usually involves
exposing photosensitizers (PSs) to specific-wavelength light
in conjunction with molecular oxygen to generate reactive
oxygen species (ROS), which cause tumor cell apoptosis and/
or necrosis.[8–12] ROS are dominantly generated from oxygen
through two distinct mechanisms called Type-I and Type-
II.[13,14] First, photoirradiation excites PSs to a specific excited
singlet state (1PS*) which is followed by efficient intersystem
crossing (ISC) to an excited triplet state (3PS*).[15,16] In the
Type-I process, a cascade of electron and/or proton transfers
However, to our best knowledge, only one selective Type-
I organic photosensitizer has been reported by Peng et al.,
ꢀ
with emphasis on revealing the action mechanism of O2 C
under hypoxia, leaving molecular design of Type-I PSs
unexplored.[30] Although a few groups reported some metal
complexes capable of producing ROS through the Type-I
process, their short-wavelength absorption, complicated me-
tabolism, and dark toxicity severely limit the clinical appli-
cation.[31,32] Heavy-metal-free PSs have shown great potential
in clinical applications. Some organic PSs that can generate
ROS through combined Type-I and -II processes were also
reported, as enumerated in Table S1, but rapid O2 consump-
tion by the Type-II process to certain extent reduces the
therapeutic effect.[26,33,34] To maximize the PDT efficiency
under hypoxic environment, developing organic Type-I PSs is
highly desirable albeit it remains a challenge owing to the lack
of general design strategy.[35–37] To generalize molecular
design principle for pure Type-I PSs, we propose that several
requirements should be fulfilled simultaneously. First, there
exist efficient ISC processes from initially populated singlet to
triplet excited states, usually the T1 state. Second, the T1 state
should have a long lifetime for subsequent reactions to
produce ROS. Third, the T1 energy should be lower than that
required to produce 1O2 by excitation energy transfer so that
the Type-II process is inhibited.[38,39] Fourth, an appropriate
redox potential is requested to facilitate electron transfer to
[*] K.-X. Teng,[+] L.-Y. Niu, Q.-Z. Yang
Institution Key Laboratory of Radiopharmaceuticals
College of Chemistry, Beijing Normal University
Beijing 100875 (P. R. China)
E-mail: qzyang@bnu.edu.cn
W.-K. Chen,[+] W.-H. Fang, G. Cui
Key Laboratory of Theoretical and Computational Photochemistry,
Ministry of Education, College of Chemistry
Beijing Normal University, Beijing 100875 (P. R. China)
E-mail: ganglong.cui@bnu.edu.cn
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202106748.
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Figure 1. a) Previously reported BODIPY dimers. b) Illustration of structure of a,b-linked BODIPYs and photo-induced exclusive generation of O2ꢀC
radical over 1O2.
ꢀ
yield O2 C. Because of these demanding conditions, the Type-I
metal-free PSs have been rarely reported until now.[40]
Herein we report a series of a,b-linked boron dipyrrome-
thene (BODIPY) dimers (1a–g) and a trimer (2), which
BODIPY derivatives, as sister compounds of porphyrin,
have gained considerable attention as PSs in PDT since they
possess ideal photosensitizer characteristics: high extinction
coefficients, environmental insensitivity, as well as excellent
photostability and biocompatibility.[41–45] BODIPY-based PSs
are considered most likely to be approved for clinical trials.[46]
They are commonly obtained by the introduction of heavy
atoms (e.g. transition metals or halogens) into structures to
enhance singlet-to-triplet ISC efficiency as a result of heavy-
atom effects.[47–50] However, introducing heavy atoms often
brings unavoidable disadvantages, such as increased “dark
toxicity” and cost, decreased photostability and solubility, as
well as reduced triplet-state lifetime.[28,51] BODIPY dimers as
heavy-atom-free PSs have attracted increasing attention since
the first report of b,meso-BODIPY dimer to produce 1O2.[52,53]
To date, a,a-, b,b-, meso,meso-, b,meso-, a,meso- and a,g-
linked BODIPY dimers have been reported (Figure 1a).[54–59]
Generally, the co-planar BODIPY dimers, that is, a,a- and
b,b-dimer, manifest absorption and emission in near-infrared
region (NIR), but cannot generate ROS. Orthogonal BOD-
IPY dimers, such as meso,meso-, b,meso-, a,meso- and a,g-
ꢀ
generate O2 C exclusively by the Type-I process upon near-
infrared light irradiation (Figure 1b). The efficient triplet
formation originates from the initially populated singlet (S1)
to triplet (T1) states mediated by an intermediate triplet (T2)
state. The ultralong lifetime of the T1 state up to microsecond
timescale and the low reduction potential facilitate efficient
ꢀ
O2 C generation, whereas the low T1 energy closes the Type-II
process down. The T1-S0 energy gap of compound 1a is
slightly lower than the energy needed to produce 1O2 by
excitation energy transfer (1.05 versus 1.12 eV at CASPT2/
PCM level). Compound 2 manifests strong absorption at
740 nm (e = 6.0 ꢀ 104 Mꢀ1 cmꢀ1) and ultralong triplet lifetime
(1060 ms). It exhibits superior PDT performance under the
hypoxic environment of tumor cells. It shows excellent
photocytotoxicity in HepG2 cells (2% O2) with a half-
maximal inhibitory concentration (IC50) of 0.56 mM. Note that
it is 23-folds lower than the commercial PS Ce6 at identical
conditions. The distinct solid tumor ablation in vivo was also
achieved when compound 2 was applied for PDT in tumor
treatment of mouse models.
1
dimer, are used as heavy-atom-free PSs to generate O2.[60]
However, these BODIPY dimers as potential PSs suffer from
two drawbacks: 1) none of them operate in NIR due to the
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nonplanar structure, which limits their clinical applications
and 2) all of them generate ROS through the Type-II process
and their therapeutic effects are thus severely restricted by
hypoxia. In this work, a,b-linked BODIPYs exhibit strong
absorption in NIR. As pure Type-I PSs, they exclusively
generate O2 C over 1O2, which maintains high PDT efficiency
ꢀ
in hypoxic tumors. To our best knowledge, this is the first
example of a,b-linked BODIPYs and the first case of
BODIPY-based PSs that generate ROS exclusively by the
Type-I process.
Results and Discussion
Based on the properties of reported BODIPY dimers, we
speculate that a,b-linked BODIPYs would possess excellent
photophysical properties as potential PSs for PDT.[61–63] First,
a,b-linked BODIPYs with much smaller steric hindrance
between two BODIPY units than that of orthogonal dimers
could allow the conjugation of two BODIPY units to make
absorption red-shifted. Second, electron-deficient a site con-
nected with electron-rich b site might tune electron inter-
action between the two BODIPY moieties to increase the ISC
efficiency and to tune the reduction potential. Keeping this
idea in mind, we have designed and synthesized a series of
a,b-linked BODIPY dimers and a trimer (Scheme S1).
Compound S2 was obtained from the reaction of S1 with N-
Bromosuccinimide (NBS) at room temperature. Target
molecule 1a was obtained by Suzuki–Miyaura cross-coupling
of S4 with S3, which was synthesized through Pd-catalyzed
borylation of compound S2 with bis(pinacolato)diboron
(B2pin2). To further expand the conjugation, compound 2
was synthesized from S6 through borylation and Suzuki–
Miyaura cross-coupling with S4. Compound 3 was obtained
through 1,3,5,7-methyl-substituted BODIPY by similar syn-
thetic routes. Compound 1b was prepared by the same
method and it was easily modified to obtain different a,b-
linked BODIPY dimers through a one-step coupling reaction
or substitution reaction. All new compounds were fully
characterized by 1H-NMR, 13C-NMR and high-resolution
mass spectrometry (HRMS). Furthermore, crystallography
analysis of single crystals of 1a and 3, grown from a mixed
solvent of n-hexane and dichloromethane, further confirms
their molecular structures (Figure 2c and d).
Figure 2. Structures of a) 1a and b) 3. Single-crystal structures of
c) 1a and d) 3. Structures with ellipsoids at 50% probability level.
e) Absorption and f) fluorescence spectra of compound S1, 1a and 3
in DMSO (compound was S1 excited at 500 nm; compound 1a was
excited at 628 nm; compound 3 was excited at 532 nm).
The low fluorescence quantum yield of compound 1a
gives a hint that the ISC process from singlet to triplet excited
states might be efficient (Table S3 and Figure S4). To verify
this point, we have checked the triplet-state formation
through evaluating the ROS generation of compound 1a by
2’,7’-dichlorodihydrofluorescein (DCFH), a commercial in-
dicator for any general types of ROS. As shown in Figure 3a,
the solution of DCFH in the presence of 1a shows about 65-
fold fluorescence enhancement at 522 nm upon light-irradi-
ation (400–800 nm white LED light, 20 mWcmꢀ2) for 3
minutes (Figure S5), indicating the efficient generation of
ROS. The fluorescence intensity for 3 does not increase at the
identical condition thereby indicating no ROS generation.
Then, 9,10-anthracenediyl-bis(methylene)-dimalonic acid
(ABDA) as a singlet oxygen scavenger was used to detect
1O2. When ABDA solution in the presence of 1a was exposed
to light-irradiation for 6 minutes, the absorbance of ABDA
We first chose the simple a,b-linked BODIPY dimer 1a to
study its photophysical properties. As shown in Figure 2e,
compound 1a exhibits intense absorption at 628 nm (e = 4.7 ꢀ
1
changes negligibly (Figure S6), indicating no O2 generated.
104 Mꢀ1 cmꢀ1) and very weak fluorescence at 746 nm (FPL
=
We have further detected the O2ꢀC production by the O2 C
ꢀ
0.8%). The optical spectra of 1a shows a significant bath-
ochromic shift compared with that of BODIPY monomer S1
and compound 3 with steric hindrance groups. As shown in
Figure 2c, crystallography analysis of single-crystal structure
indicates that compound 1a shows planar structure with small
dihedral angle of 19.708 for the two BODIPY units (Fig-
ure 2c). By contrast, compound 3 exhibits nonplanar con-
formation with a large dihedral angel of 56.428 in its crystal
structure due to the steric hindrance groups (Figure 2d).
Therefore, the red-shifted spectra of 1a is attributed to the
expanded p-conjugation.
indicator dihydrorhodamine 123 (DHR 123). As shown in
Figure 3b, obvious fluorescence enhancement of DHR 123
was observed in the presence of 1a under illumination,
ꢀ
indicating the O2 C generation (Figure S7). Droethidium
ꢀ
(DHE) was also employed to detect the O2 C generation and
the same results were obtained (Figure S9). Electron spin
resonance (ESR) spectroscopy was employed to further
ꢀ
confirm the O2 C generation by sensitization of 1a (Figure 3c).
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as
ꢀ
a spin-trap agent for O2 C and 2,2,6,6-tetramethylpiperidine
1
(TEMP) was applied as a spin trapper to identify O2. Upon
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triplet states of 1a is 1514 ms measured by laser flash
photolysis (Figures 3e and f). The ultralong lifetime of triplet
states is important for electron transfer from substrate to PS
thereby improving the O2ꢀC generation efficiency.
To explore excited-state properties and photophysics of
compound 1a, we carried out theoretical calculations.
Ground-state structures were optimized with the B3LYP/6-
31G* method while excited-state structures are done with the
TD-B3LYP/6-31G* method (see supporting information for
computational details).[66–70] Further single-point energies are
refined by the much accurate multi-reference complete active
space self-consistent field method with second-order pertur-
bation (CASPT2) approach.[71,72] The solvent effects are
implicitly considered by the polarizable continuum model
(PCM).[73]
The first electronically excited singlet state that is, S1 is of
pp* character and thus was firstly populated at the Franck-
Condon point, that is, the S0 stable structure, due to relevant
considerable transition dipole moments. This singlet state
corresponds to exciting an electron from HOMO to LUMO.
Because HOMO and LUMO are mostly distributed on the
two BODIPY units, the S1 state is of partial charge-transfer
character. Below the bright S1 state, there are two triplet
states available, that is, T2 and T1. Both triplet states are of
pp* character, but their electronic characters remain differ-
ent. The T1 state is mainly caused by moving an electron from
HOMO to LUMO, which is the same as that of the S1 state.
Furthermore, both the S1 and T1 states have typical single-
reference character. In contrast, the T2 state is primarily
composed of two comparable electronic configurations, that
is, HOMO-1 to LUMO and HOMO to LUMO + 1 (see
Figure 4).
In terms of optimized minima and computed linearly
interpolated internal coordinate paths for compound 1a, an
efficient excited-state relaxation pathway from the initially
populated S1 to T1 states is uncovered as shown in Figure 5a.
Upon photoexcitation at the Franck–Condon point, com-
pound 1a will relax quickly to its S1 minimum, wherefrom it
will further decay to an intermediate T2 state through an
intersystem crossing process. This process is efficient because
of a small S1-T2 energy gap of 0.13 eV at the S1 minimum. In
Figure 3. a) ROS and b) O2ꢀC generation of 1a and 3. Both of them
were measured in DMSO (10 mM) after irradiation by white LED light
source (400–800 nm, 20 mWcmꢀ2). DCFH as a ROS probe (50 mM,
lex =504 nm, lem =522 nm), DHR123 as a O2ꢀC probe (60 mM,
lex =500 nm, lem =526 nm). c) ESR spectra to detect O2ꢀC generated
by 1a (0.5 mM) under illumination, using DMPO as a spin trapper.
d) Cyclic voltammogram of 1a and 3 in DCM with 0.1 M (n-
Bu)4N+PF6ꢀ as a supporting electrolyte, Ag/Ag+ as a reference elec-
trode, platinum-carbon compound electrode as a working electrode, Pt
wire as a counter electrode, and (n-Bu)4N+PF6ꢀ (0.1 M) as supporting
electrolyte, and a scan rate of 20 mVsꢀ1. Fc/Fc+ was used as an
external reference. e) Time-resolved transient difference absorption of
1a in DMSO. f) Decay trace of 1a at 954 nm.
light-irradiation of the aerated solution of 1a and DMPO,
a characteristic paramagnetic adduct was observed and
ꢀ
ꢀ
matched with the O2 C signal thereby confirming the O2 C
production. No detectable ESR signals was observed when
irradiating solution of 1a and TEMP (Figure S10), which
1
further confirms no generation of O2. Based on the above
results, we reasonably speculate that 1a undergoes ISC upon
light-irradiation to generate triplet excited states followed by
ꢀ
the O2 C generation through charge transfer.
We further studied electrochemical properties of 1a to
ꢀ
help us understand why 1a are prone to generating O2 C via
the Type-I process. Cyclic voltammetry was used to inves-
tigate redox properties of 1a and 3. As shown in Figure 3d,
the reduction event of 1a0/ꢀ at ꢀ1.21 V (vs. Fc/Fc+) is 260 mV
less negative than ꢀ1.47 V (vs. Fc/Fc+) that observed for 30/ꢀ
.
The anodic shifts of 1a compared to 3 originates from its
lower LUMO energy (see Table S6 and Figure S49). The
anodic shift of 1a facilitates it to accept electrons, which
endows 1a with the potential to produce O2ꢀC by the Type-I
ꢀ
process.[16,33,64,65] Another key factor for PSs to generate O2 C
Figure 4. TD-B3LYP/PCM calculated frontier orbitals relevant to the
is the lifetime of triplet states. Specifically, the lifetime of
involved excited singlet and triplet states (isosurface value: 0.03).
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Table 1: Photophysical properties of compound 1.
Compd[a] labs [nm] (e [ꢁ10ꢀ3 Mꢀ1 cmꢀ1 [b]
Toluene DCM DMSO
]
lPL [nm][c] FPL [%][d]
Toluene
1a
1b
1c
1d
1e
1 f
1g
1h
1i
657 (58.3) 628 (46.6) 628 (46.7) 705
7.8
5.3
3.3
4.9
1.8
2.1
2.1
54.9
7.6
650 (47.0) 630 (45.4) 633 (40.2) 707
679 (40.6) 665 (39.3) 690 (36.3) 759
696 (36.3) 676(34.4) 678 (32.5) 752
683 (30.0) 679 (31.5) 676 (28.9) 782
677 (30.7) 662 (28.1) 665 (31.3) 770
651 (34.7) 739 (31.9) 642 (30.1) 748
640 (53.7) 625 (46.5) 628 (42.8) 676
742 (22.3) 721 (24.0) 744 (22.2) 818
Figure 5. a) Suggested excited-state relaxation pathway from the ini-
tially populated S1 to T1 states of compound 1a mediated by an
intermediate T2 state. b) Energy profile for the Type-II energy transfer
from the T1 species of 1a to 3O2. Also shown are relevant energies (in
eV) calculated at the CASPT2/PCM level.
[a] 10 mM. [b] Absorption maxima. [c] Fluorescence maxima. [d] Absolute
fluorescence quantum yields in toluene.
ꢀ
compound 1b–1g are able to sensitize oxygen to produce O2 C
under light-irradiation. While no 1O2 was detected by
1
the T2 state, the system will first relax to its minimum and then
continue to decay to the T1 state followed by a T2!T1 internal
conversion process in the vicinity of the T2 minimum where
the T2-T1 energy gap is estimated to be 0.08 eV at the
CASPT2/PCM level. In the T1 state, the system will be
trapped for a while because of a very large T1-S0 energy gap of
1.05 eV at the CASPT2/PCM level. Moreover, the present
calculations demonstrate that excitation energy transfer from
the T1 state of compound 1a to 3O2 is not favorable in the view
of energetics because this process is subtle endothermic. The
vertical T1!S0 emission energy of compound 1a is calculated
at its T1 minimum to be 1.05 eV at the CASPT2/PCM level,
which is a little smaller than that required to excite 3O2 to its
utilization of ABDA as a specific indicator for O2 for all
these BODIPY dimers (Figure S22). We further calculated
the T1-S0 energy gaps of compounds 1b, 1c, 1 f and 1g, which
are also smaller than the 3O2-1O2 energy difference (Ta-
ble S7). The reason for no O2ꢀC generation by 1h is probably
the introduction of amino groups that affect electronic
structures. Two large substituents attached on both 3 and 5
positions probably destroyed the quasi-planar structure so
that no ROS is generated for 1i.
To obtain photosensitizers with strong absorption in NIR,
we further synthesized a,b-linked BODIPY trimer 2 to
expand the conjugation (Figure 6a). Its central BODIPY unit
is connected with the two BODIPY groups in b positions and
these two groups are far from each other. As expected,
compound 2 manifests red-shifted absorption compared with
1a at 740 nm in DMSO with large molar extinction coefficient
of 6.0 ꢀ 104 Mꢀ1 cmꢀ1 (Figure 6b). It exhibits weak fluores-
cence in various solvents (Table S3 and Figure S4). DHR 123,
1
singlet excited state producing O2 (Figure 5b). The similar
situation is also seen for compound 3 (see Figure S50).
Therefore, the Type-II process should be not as efficient as the
Type-I process observed in the experiments.
To understand the structure-property relationship of a,b-
linked BODIPYs, we further designed and synthesized
a series of 1a derivatives with different substituents. Struc-
tures of 1a derivatives are shown in Scheme 1. Their photo-
physical properties including absorption and fluorescence
maxima, molar extinction coefficients (e) and absolute
photoluminescence quantum yields (FPL) are summarized in
Table 1 (Figures S12–S19). Cyclic voltammetry was used to
investigate redox properties of 1b–1g (Figure S20). We have
ꢀ
1
ABDA and DHE were used as indicators for O2 C and O2,
respectively, to determine the ability of 2 to generate ROS.
The results confirm that 2 exclusively produces O2 C over 1O2.
ꢀ
As shown in Figures 6c and S9, in the presence of compound
2, the fluorescence of DHR 123 and DHE are significantly
enhanced, while the absorption of ABDA changes negligibly
under light-irradiation (Figure S8). The ESR experiments
ꢀ
further confirm the generation of O2 C without 1O2 for
ꢀ
checked their capabilities for O2 C generation by using DHR
compound 2 under NIR irradiation (Figures 6d and S10).
Furthermore, the laser flash photolysis data show that 2 also
possesses an ultra-long triplet state lifetime up to 1060 ms
(Figures 6e and f). The above results provide solid evidence
that compound 2 is an excellent pure Type-I PS for PDT.
ꢀ
123 as an O2 C indicator (Figure S21). The results indicate that
ꢀ
Encouraged by both efficient O2 C generation and strong
absorption ability of compound 2 in near-infrared region with
advantages for biological applications, we continue to study
its PDT activity in vitro under both normoxic and hypoxic
conditions (Figures S23 and S24). The ROS probe, 2’,7’-
dichlorodihydrofluorescein diacetate (DCFH-DA), was used
to evaluate the cellular ROS during PDT. As shown in
Figure 7a, after light-irradiation, bright green fluorescence
was observed in 2-treated HepG2 cells by confocal laser
scanning microscopy (CLSM), suggesting effective ROS
generation. No 1O2 signal was detected at identical conditions
Scheme 1. Structures of compound 1a derivatives.
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light-irradiation. Viable cells were stained by calcein-AM to
emit fluorescence in the green channel and apoptotic cells
were stained by PI to emit fluorescence in the red channel. It
can be seen in Figure 7g that HepG2 cells treated with
0.16 mM 2 showed obvious fluorescence in the green and red
channels after illumination indicating partial cell death. When
the 2 concentration increased to 0.64 mM, only the red
channel had signal, indicating that all the HepG2 cells were
dead. By contrast, the cells without 2 only exhibited
fluorescence in the green channel under identical conditions,
indicating no cell death. Furthermore, an Annexin V-FITC/PI
apoptosis detection kit was used to investigate the possible
death mechanism by flow cytometry experiments. The results
in Figure 7h demonstrated that the cell toxicity was mainly
associated with apoptosis. These results prove that 2 effec-
tively induces tumor cell apoptosis under illumination.
We chose a block copolymer, Pluronic F127, as the
encapsulation matrix to co-assemble with 2 to form nano-
particles for therapy in vivo. The nanoparticles were charac-
terized as well-dispersed nanoparticles by dynamic light
scattering (DLS) and scanning electron microscope (SEM)
(Figure S26). The absorption spectrum, fluorescence spec-
trum and superoxide anion radical generation of nanoparti-
cles was detected in water to confirm that they can absorb
Figure 6. a) Structure of compound 2. b) Absorption and fluorescence
spectra of compound 2 in DMSO. c) The O2ꢀC generation of 2 (10 mM)
in DMSO, using DHR 123 as O2ꢀC probe (60 mM, lex =500 nm,
lem =526 nm). d) The ESR spectra to detect O2ꢀC generated by 2
(0.5 mM) under illumination, using DMPO as spin trapper. e) Time-
resolved transient difference absorption of 2 in DMSO. f) Decay trace
of 2 at 937 nm.
ꢀ
NIR light to generate O2 C after assembly (Figures S27–S29).
The absorbance of nanoparticles showed almost no change
after 60 minutes of irradiation (Figure S30), indicating their
high photostability. Furthermore, absorption of nanoparticles
for a constant 48 h in fetal calf serum, indicated that
nanoparticles of compound 2 are remarkably stable under
physiological conditions (Figure S30). Then we evaluated the
tumor enrichment effect of the nanoparticles on immunode-
ficient mouse models by subcutaneous tumor model of human
liver cancer HepG2 cells in BALB/c mice. As shown in
Figure S31, obvious fluorescence was detected in the tumor
position after 3 hours for intravenous injection due to the
enhanced permeability and retention (EPR) effect.[74] At
24 hours after the injection, the ex vivo biodistribution of the
photosensitizer was evaluated (Figure S31c), and the fluores-
cence in the tumor was stronger than that in other organs. We
further investigated the antitumor efficiency of PDT by
compound 2 in vivo for HepG-2 tumor-bearing immunocom-
petent BALB/c mice (primary tumor volume: ꢁ 100 mm3).
The nanoparticles of compound 2 were injected into the mice
by tail vein injection, followed by irradiation (730 nm LED
light, 120 mWcmꢀ2) at 12 hours and 24 hours post injection.
Then, the body weights (Figure 8a) and tumor volumes
(Figure 8b) were recorded during the subsequent 12 days.
The weight of the mice increased slightly, suggesting the
negligible systemic cytotoxicity of 2 during PDT. For the
group treated with 2 with light, the tumor of mice disappeared
on the sixth day after irradiation and did not relapse,
indicating that 2 effectively suppressed the tumor. The group
treated with 2 without irradiation exhibited similar tumor
growth rate to the PBS-treated group, suggesting that 2 was
nontoxic in the absence of light. The mice were sacrificed on
the 12th day and all the tumor tissues were peeled and
weighed (Figures 8c and d). The hematoxylin & eosin (H&E)
staining was applied to examine tumor damage of 2 (Fig-
when singlet oxygen sensor green reagent (SOSG, a commer-
1
cial O2 probe) was employed (iodine-bearing BODIPY as
a reference, Figure S25). DHE as a O2ꢀC probe was employed
to evaluate the cellular O2ꢀC species in normoxic environ-
ments (21% O2) and hypoxic environment (2% O2). As
shown in Figure 7b, bright red fluorescence was detected in 2-
treated HepG2 cells under both normoxic and hypoxic
environment after illumination, suggesting 2 could generate
ꢀ
O2 C even under hypoxic environment. Then, we evaluated
the PDT effects of 2 to HepG2 cells by cell counting kit-8
(CCK-8) assays. Under irradiation with LED light (730 nm,
50 mWcmꢀ2) for 10 min, compound 2 shows obvious cytotox-
icity to HepG2 cells in normoxic environments, with a half-
maximal inhibitory concentration (IC50) of 0.39 mM (Fig-
ure 7c). More importantly, compound 2 also manifests good
anti-tumor effects even under hypoxic conditions with an IC50
of 0.56 mM. In the absence of light-irradiation, 2 shows no
toxicity to HepG2 cells under both normoxia and hypoxia
conditions (Figure 7d). As shown in Figures 7e and f, when
commercial PS Ce6 was exposed to LED light-irradiation
(660 nm, 50 mWcmꢀ2), IC50 was 6.0 mM under normoxia
which is 15.3-fold higher than that of 2, and it was 12.9 mM
under hypoxia, 23.2-fold higher than that of 2. These results
indicate that 2, as a pure Type-I PS, is less dependent on
oxygen concentration, and is still able to generate ROS to
inhibit tumor proliferation even under hypoxic conditions.
Next, a calcein-AM and propidium iodide (PI) assay was
employed to evaluate the inhibition of tumor cells by 2 with
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Figure 7. a) Detection of ROS and 1O2 in HepG2 cells with DCFH-DA and SOSG. The scale bar represents 50 mm. b) O2ꢀC detection in HepG2
cells under normoxia (21% O2) and hypoxia (2% O2) conditions by using DHE. The scale bar represents 50 mm. Cell viability of HepG2 cells
subjected to a range of 2 concentrations in the c) presence and d) absence of light-irradiation under normoxia or hypoxia conditions. e) Cell
viability of HepG2 cells subjected to a range of Ce6 concentration in the presence of light-irradiation under normoxia (21% O2) or hypoxia (2%
O2). *P<0.05 (one-way ANOVA) f) The half-maximal inhibitory concentration of 2 and Ce6 under normoxia and hypoxia. g) CLSM images of
calcein AM/PI-stained HepG2 cells. The scale bar represents 100 mm. h) Apoptosis analysis of HepG2 cells treated with 2 at various doses. (the
parameter of CLSM: green channel: 500–550 nm, excited at 487 nm; red channel: 570–620 nm, excited at 562 nm; NIR channel: 663–738 nm,
excited at 638 nm).
ure S32) and the results showed that 2 could destroy tumor
tissue.
from the initially populated S1 to T1 states, in which a multi-
reference intermediate T2 state plays as a relay to regulate
relevant inter-state nonradiative electronic transitions. It is
also found that the inter-molecular energy transfer process
from the T1 state of compound 1a to O2 by the Type-II process
is precluded due to the energy gap of T1-S0 being narrower
than that between 3O2 and 1O2. In contrast, the Type-I process
Conclusion
In this work we have reported a series of heavy-atom-free
superoxide radical species generators based on a,b-linked
BODIPYs, which exhibit excellent potential as specific Type-I
ꢀ
that generates superoxide O2 C radical becomes efficient
because of the low reduction potential and the long lived T1
state up to microsecond. Consequently, these BODIPY-based
dimers and trimer solely produce superoxide radical species
by the Type-I photosensitization, which coincides with the
present experimental measurement.
Furthermore, in vitro studies demonstrate that a,b-linked
BODIPY trimer 2 have excellent anti-tumor effects even
under environments with severe O2 shortage, and its PDT
effects are evidently superior to that of the commercial Ce6.
The further in vivo studies reveal that compound 2 shows
irreversible cytotoxicity to tumor tissue resulting in distinct
ꢀ
photosensitizers in PDT. They exclusively produce O2 C by the
Type-I process upon NIR light illumination and have large
molar extinction coefficients in NIR, ultra-long triplet life-
times up to microsecond timescale, low dark toxicity, and
great phototoxicity.
The quasi-planar conformational structure of these a,b-
linked BODIPYs plays an important role in red-shifted
absorption band and lowering their reduction potential.
Subsequent theoretical studies on compound 1a uncovered
an efficient excited-state radiationless relaxation pathway
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Angewandte
Research Articles
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Keywords: BODIPY · hypoxia · photodynamic therapy ·
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Manuscript received: May 19, 2021
Revised manuscript received: June 20, 2021
Accepted manuscript online: July 6, 2021
Version of record online: && &&
,
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Research Articles
Photodynamic Therapy
Heavy-atom-free boron dipyrromethene
(BODIPY)-based photosensitizers gen-
erate ROS exclusively by the Type-I pro-
cess upon near-infrared light illumination
for tumor ablation.
K.-X. Teng, W.-K. Chen, L.-Y. Niu,
W.-H. Fang, G. Cui,*
Q.-Z. Yang*
&&&& — &&&&
BODIPY-Based Photodynamic Agents for
Exclusively Generating Superoxide
Radical over Singlet Oxygen
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These are not the final page numbers!