A Photosensitive Polymeric Carrier with a Renewable Singlet Oxygen Reservoir Regulated by Two NIR Beams for Enhanced Antitumor Phototherapy

Article abstract of DOI:10.1002/smll.202101180

Photodynamic therapy (PDT), which utilizes photosensitizer to convert molecular oxygen into singlet oxygen (¹O₂) upon laser irradiation to ablate tumors, will exacerbate the already oxygen shortage of most solid tumors and is thus se

Full text of DOI:10.1002/smll.202101180

ReseaRch aRticle
www.small-journal.com
A Photosensitive Polymeric Carrier with a Renewable Singlet
Oxygen Reservoir Regulated by Two NIR Beams for Enhanced
Antitumor Phototherapy
Chun Yang, Meihui Su, Pei Luo, Yanan Liu, Feng Yang, and Changhua Li*
tumors result in severe O2 deficiency,[10–18]
Photodynamic therapy (PDT), which utilizes photosensitizer to convert
molecular oxygen into singlet oxygen (1O2) upon laser irradiation to ablate
tumors, will exacerbate the already oxygen shortage of most solid tumors
and is thus self-limiting. Herein, a sophisticated photosensitive polymeric
material (An-NP) that allows sustained 1O2 generation and sufficient oxygen
supply during the entire phototherapy is engineered by alternatively applying
PDT and photothermal therapy (PTT) controlled by two NIR laser beams. In
addition to a photosensitizer that generates 1O2, An-NP consists of two other
key components: a molecularly designed anthracene derivative capable of
trapping/releasing 1O2 with superior reversibility and a dye J-aggregate with
superb photothermal performance. Thus, in 655 nm laser-triggered PDT
process, An-NP generates abundant 1O2 with extra 1O2 being trapped via
the conversion into EPO-NP; while in the subsequent 785 nm laser-driven
PTT process, the converted EPO-NP undergoes thermolysis to liberate the
captured 1O2 and regenerates An-NP. The intratumoral oxygen level can
be replenished during the PTT cycle for the next round of PDT to generate
1O2. The working principle and phototherapy efficacy are preliminarily
demonstrated in living cells and tumor-bearing mice, respectively.
which substantially decreases the treat-
ment outcome of oxygen-dependent
PDT.[19–22] Moreover, the oxygen consump-
tion during PDT will further aggravate the
tumor hypoxia, probably leading to cancer
metastasis.[13,23]
Recently, fractional PDT (fPDT) with
intermittent light irradiation has been
developed to allow oxygen replenish-
ment between two PDT cycles.[24–27]
Compared with traditional PDT, fPDT
is promising to relieve photo-induced
tumor hypoxia during light treatment.
To further enhanced the fPDT efficacy,
Akkaya et al.[28] introduced a 2-pyridone
module in the PS design as a renewable
1
1O2 reservoir to capture O2 in the light
cycle and release it in the dark cycle,
1
thereby enabling sustained O2 generation
during fPDT. However, the 1O2-trapped
product of 2-pyridone, that is, endoper-
oxide (EPO), underwent a spontaneous
cycloreversion reaction to release 1O2 in an
uncontrolled manner. In contrast, remote control of the smooth
release of 1O2 from the generated EPO during the oxygen
replenishment process is expected to realize the full potential
of fPDT.[29] EPOs of anthracene were demonstrated to produce
1. Introduction
Photodynamic therapy (PDT), a clinically approved antitumor
modality, has attracted increasing attention due to its distinct
advantages including minimal invasiveness, high therapeutic
accuracy, low systemic toxicity, to name a few.[1–3] In a typical
PDT process, under light irradiation, the photosensitizer (PS)
transfers the photoexcited energy of the triplet excited state to
nearby oxygen molecules to generate singlet oxygen (1O2), which
leads to efficient ablation of malignant cells and tissues.[4–8]
Remarkably, PDT can provoke an immune response,[2,9] which
renders it truly promising. However, aggressive cellular prolif-
eration and insufficient neovascularization in fast-growing solid
1
1O2 upon heating[29–37] and thus have been applied as O2 car-
1
riers to achieve remote light-controlled O2 release by incor-
poration with photothermal agents (PTAs).[38,39] However, due
to the lack of a suitable anthracene molecule that is capable of
proceeding both a highly efficient cycloaddition reaction to trap
1O2 to generate EPO and an efficient and clean thermo-induced
1
cycloreversion reaction of EPO to liberate O2, anthracene has
not been used as a renewable 1O2 reservoir for fPDT.
Herein, through rational molecular design, we success-
fully obtained a novel anthracene molecule (An-3) that pre-
sents reversible and clean switching between An and EPO
states accompanied by 1O2 trapping and releasing. In addi-
tion to the thermo-induced renewable 1O2 reservoir with
high quality, we also introduced a pure organic PTA based on
BODIPY J-aggregate (JBDP), which displays significant pho-
tothermal performance including red-shifted absorbance into
the NIR region (785 nm), high photothermal conversion capa-
bility (PCE = 59.8%), and significant photobleaching resist-
C. Yang, Dr. M. Su, P. Luo, Y. Liu, F. Yang, Prof. C. Li
State Key Laboratory of Medicinal Chemical Biology
College of Pharmacy
Key Laboratory of Functional Polymer Materials of Ministry of Education
Nankai University
Tianjin 300071, P. R. China
E-mail: chli@nankai.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.202101180.
1
ance, to realize remote light-controlled O2 release based on
photothermy. By co-assembling with an amphiphilic diblock
DOI: 10.1002/smll.202101180
2101180 (1 of 12)
Small 2021, 17, 2101180
© 2021 Wiley-VCH GmbH

www.advancedsciencenews.com
www.small-journal.com
Scheme 1. Schematic illustration of the photosensitive polymeric nanoparticle (An-NP) with a renewable singlet oxygen (1O2) reservoir controlled by
two laser beams for enhanced fractional PDT (fPDT). PS and PTA represent photosensitizer and photothermal agent, respectively.
copolymer, three key components, that is, a 1O2 reservoir
2. Results and Discussion
(An-3), a PTA (JBDP), and a PS (I-BDP), were orchestrated in
a single nanosystem to construct a sophisticated photosensi-
tive nanomaterial (An-NP) for antitumor fPDT (Scheme 1). As
detailed below, upon NIR laser-1 irradiation (655 nm), An-NP
generates abundant 1O2 with excessive 1O2 being stored by
forming EPO-NP; while after NIR laser-2 irradiation (785 nm),
the converted EPO-NP undergoes thermolysis to release the
2.1. Molecular Design of Anthracene Derivatives
for 1O2 Reservation
1
A renewable chemical reservoir of O2 is the key to achieving
1
1
sustained release of O2 in the fPDT process. An ideal O2 res-
1
ervoir should store O2 generated in PDT cycle and release it
1
trapped O2 and regenerates An-NP. The oxygen level in the
in a controlled manner during the interval between two PDT
cycles. Anthracene (An) derivatives have been recognized
tumor should be replenished in the PTT process for the next
1
1
1
round of PDT to generate O2. For the first time, there is a
as reliable O2 carriers as they react with O2 to form EPOs
which can undergo cycloreversion reactions when heated to
1
single nanosystem that enables sustained O2 generation and
1
sufficient oxygen supply during the fPDT process by alter-
natively applying PDT and PTT regulated by two NIR laser
beams. Dual-light irradiation can provide more controllability
and has recently been widely used in multiple biological and
biomedical fields, such as cancer treatment,[21] antibacterial,[40]
photopharmacology,[41] optogenetics,[42] and optical-control of
protein function and signaling,[43–45] etc. Moreover, the replace-
ment of the dark cycle of conventional fPDT with PTT further
enhanced the overall outcome of the anticancer phototherapy
due to the synergistic effect of PDT and PTT (Scheme 2).[46–50]
release the stored O2 (Figure 1a). It has been reported that
the substituents in the 9,10-positions of anthracene could sub-
stantially affect both cycloaddition and cycloreversion reac-
tions in organic solvents.[37] However, the substituent-effect of
anthracene in aqueous media has yet been studied. Aiming
1
at obtaining a reliable anthracene-based O2 reservoir that has
to work in aqueous environment of living system, a set of
anthracene derivatives (An-1, An-2, and An-3) with different
9,10-substituents were designed and successfully synthesized as
outlined in Scheme 3. Then, an amphiphilic diblock copolymer,
Small 2021, 17, 2101180
2101180 (2 of 12)
© 2021 Wiley-VCH GmbH

www.advancedsciencenews.com
www.small-journal.com
Scheme 2. Conventional fractional PDT (fPDT) with intermittent light irradiation allowing oxygen replenishment between two PDT cycles; the designed
enhanced-fPDT with sustained 1O2 generation and additional PTT between two PDT cycles.
Figure 1. a) Cycloaddition reaction between anthracene (An) with the photogenerated singlet oxygen (1O2) yields EPO, which undergoes thermo-
induced cycloreversion to restore An and release 1O2. b–d) UV–vis absorption spectra and e) the absorbance at absorption maximum (Abs @ λmax)
recorded for An-1 (b), An-2 (c), and An-3 (d) before and after sequential 1O2 and thermal treatments; insets of (b) and (c) show an enlarged view of the
area marked with a blue rectangle. 1O2 was generated by I-BDP (2.3 µm) upon laser-1 irradiation. Laser-1: 655 nm, 10 mW cm−2
.
2101180 (3 of 12)
© 2021 Wiley-VCH GmbH
Small 2021, 17, 2101180

www.advancedsciencenews.com
www.small-journal.com
Scheme 3. Chemical structures of three anthracene (An) derivatives and the corresponding EPO.
poly(ethylene glycol)-block-poly(ε−caprolactone) (denoted as
PEG-PCL) was employed to stabilize the hydrophobic anthra-
cene derivatives via forming water-stable micelles. In the hydro-
phobic cores of the three formed micelles (An-1 micelle, An-2
micelle, and An-3 micelle), a PS (I-BDP) and different anthra-
cene derivatives were embedded. The hydrodynamic sizes
(Figure S1, Supporting Information) and the An-loading con-
tents (Figure 1b–d) of the three micelles have been investigated
by dynamic light scattering (DLS) test and UV–vis spectroscopy,
respectively.
clean cycloreversion of EPO to release the trapped 1O2. As expected,
the respective introduction of a phenyl and a phenylethynyl sub-
stituent at the 9,10 positions of anthracene scaffold endowed the
derivative, An-3, with both favorable cycloaddition and cyclorever-
sion activities (Figure 1d,e). Moreover, compared with its organic
solution, An-3 loaded in polymeric micelle displayed stronger
1O2 trapping capability (Figure S5, Supporting Information). This
should be attributed to the closer proximity between An-3 and
1
I-BDP in the micelle core, which allows more O2 to be trapped
by An-3. The thermo-controlled cycloreversion of its EPO (EPO-3)
was further studied. As shown in Figure S6, Supporting Informa-
tion, inappreciable change in the absorption spectra of the aqueous
EPO-3 solution at room temperature was observed over 24 h, indi-
cating the stability of EPO-3 in water. Upon gradually elevating the
temperature (37–90 °C), absorbance of the thermo-converted An-3
at 408 nm increased more abruptly (Figure S7, Supporting Infor-
1
We further evaluated the O2 trapping and releasing capa-
bility of the three anthracene derivatives, that is, An-1, An-2,
and An-3, in aqueous media using UV–vis absorption spec-
troscopy. Upon laser-1 irradiation (655 nm; 10 mW cm−2),
1
the embedded I-BDP will generate O2, and then reacts with
anthracene to form EPO, resulting in the decrease in the
absorption band of anthracene. Upon heating, the formed EPO
1
mation). Given the striking O2-capturing and thermo-controlled
1
1
can undergo cycloreversion to release the trapped O2 and con-
O2-releasing performance, An-3 was selected as the O2 reservoir
vert back to anthracene, leading to an increase in the absorp-
tion band. As shown in Figure 1b, with two phenyl moieties at
9,10 positions, An-1 displayed active cycloaddition reaction with
1O2, while the formed EPO (EPO-1) showed negligible cyclor-
eversion activity of releasing 1O2 (Figure 1b,e). In stark contrast,
An-2 substituted with two phenylethynyl groups showed much
to construct the final nanosystem.
2.2. Stable J-Aggregation of BODIPY as the PTA
PTA can absorb light energy and convert it into heat, thereby
inducing the thermolysis of EPO to release singlet oxygen. We
then decided to utilize the concept of light-to-heat conversion of
PTAs to realize the photo-controlled release of singlet oxygen. In
contrast to inorganic PTAs which possess potential long-term in
vivo toxicity,[51,52] biocompatible organic PTAs are more suitable
for in vivo applications. However, several weaknesses of most
organic PTAs, such as low PCE and weak resistance against
photobleaching, substantially hampered their applications in
1
weaker cycloaddition activity with O2, but the EPO-2 product
had almost quantitative cycloreversion reactivity (Figure 1c).
Notably, the consumed and restored amounts of anthracene
derivatives during irradiation and heat process, respectively,
were calculated and summarized in Table 1.
The above results clearly indicated that phenyl substituent effec-
tively promotes the cycloaddition process of anthracene to capture
1O2, while phenylethynyl substituent is beneficial to achieving the
Table 1. The optical properties and 1O2 trapping/releasing capabilities of anthracene derivatives.
Compounda)
An-1
λAbsa)/nm
376
εa)/cm−1 M−1
15500
An-to-EPOb)/µmol
EPO-to-Anc)/µmol
16.1
0.5
6.3
0.2
0.4
4.7
An-2
465
44 700
An-3
408
23900
a)The compound was embedded in PEG-PCL micelle and data were collected in water; b)The compound and I-BDP were embedded in PEG-PCL micelle which was exposed
to laser-1 irradiation for 2 min. An-to-EPO represents the amount of the produced EPO estimated by UV–vis spectroscopy. Laser-1: 655 nm, 10 mW cm−2 c)The sample was
;
subjected to heating at 80 °C for 30 min. EPO-to-An represents the amount of the restored An estimated by UV–vis spectroscopy.
Small 2021, 17, 2101180
2101180 (4 of 12)
© 2021 Wiley-VCH GmbH

www.advancedsciencenews.com
www.small-journal.com
Figure 2. a) Schematic illustration of the preparation of a water-stable polymeric nanoparticle (BDP-J790) with Br-BDP J-aggregates embedded in the
hydrophobic core. b) Normalized UV–vis absorption spectra of Br-BDP in DMF and BDP-J790 in water. c) Size distribution and d) representative TEM
image of BDP-J790 nanoparticle. e) Temperature elevations recorded for BDP-J790 (3.5 µm Br-BDP) upon laser-2 irradiation. f) IR thermal images of i)
BDP-J790 and ii) water after 10 min laser-2 irradiation in the same conditions as (e). g) Relative absorbance changes recorded for aqueous solutions of
BDP-J790 (3.5 µm Br-BDP) and ICG (with a same absorbance at 785 nm) after 30 min laser-2 irradiation. Laser-2: 785 nm, 1.8 W cm−2
.
living system.[51–55] In our previous work, we reported that the
J-aggregation of BODIPY dyes yielded a pure organic PTA with
striking performance including red-shifted absorbance into the
NIR region, high PCE, and significant resistance against pho-
tobleaching.[56–58] We thus employed BODIPY J-aggregates as
the photothermal component of the nanosystem (Figure 2a).
Possessing two bromine atoms at the 2,6-positions, the Br-BDP
can readily form slip-stacked J-aggregate through π–π stacking
and halogen-bonding interactions,[58,59] as evidenced by the
drastic 117 nm redshift of the absorption maximum (Figure 2b).
An amphiphilic PEG-PCL was then used to stabilize the
Br-BDP J-aggregate to obtain a water-stable nanoparticle (BDP-
J790) with controlled size of 80 nm (Figure 2c,d; see Supporting
Information).
We then evaluated the photothermal performance of BDP-
J790 by using an IR thermal camera to record the solution
temperature. Upon IR thermal imaging, the photo-induced
temperature elevation of BDP-J790 dispersion (3.5 µm Br-BDP)
2101180 (5 of 12)
© 2021 Wiley-VCH GmbH
Small 2021, 17, 2101180

www.advancedsciencenews.com
www.small-journal.com
after laser-2 irradiation (785 nm; 1.8 W cm−2; 10 min) can be
visualized (Figure 2f). The quantitative temperature eleva-
tion versus irradiation time was also recorded, and it dem-
onstrated that the solution temperature can rise to 70.9 °C at
10 min (Figure 2e), which is high enough to trigger the ther-
molysis of EPO-3 (Figure S7d, Supporting Information). By
contrast, the temperature of pure water upon laser irradiation
in the same condition was elevated only to 28.5 °C (Figure 2e).
The PCE of BDP-J790 was calculated to be ≈59.8% (Figure S8,
Supporting Information),[60] which is relatively high among
reported organic PTAs.[61–66] In addition to photothermal effi-
ciency, photothermal stability is another concern in our design
photosensitivity. Considering that the spectral overlap between
QDPBF and An-3 of An-NP may affect the O2 determination,
1
we then prepared I-BDP micelle (see the Supporting Infor-
mation) as the An-NP mimic to evaluate the photosensitivity
of I-BDP. As shown in Figure 3f and Figure S11, Supporting
Information, QDPBF was significantly consumed upon laser-1
irradiation (3 mW cm−2), indicating the efficient photoactivity
1
of An-NP in generation of O2 due to the I-BDP component.
After laser-2 irradiation (1.8 W cm−2), An-NP displayed excellent
photothermal conversion ability (PCE = 59.3%) due to the JBDP
component (Figure 3g,h; Figures S12 and S13, Supporting
Information). Moreover, upon 5 cycling of photo-mediated
heating and cooling, negligible changes in the photothermal
performance (Figure S14a, Supporting Information), nanopar-
ticle size (Figure S14c, Supporting Information), and absorp-
tion spectra (Figure S14b, Supporting Information) were found,
indicating that the multiple photothermal processes did not
affect the structure and performance of the An-NP material.
1
to ensure constant photo-release of trapped O2. We collected
the UV–vis absorption spectra (Figure 2g and Figure S9b,
Supporting Information) and size distribution (Figure S9a,
Supporting Information) of BDP-J790 before and after 30 min
laser-2 irradiation. Gratifyingly, negligible changes were
observed for the BDP-J790, in stark contrast to FDA approved
ICG dye, which suffered severe photobleaching (Figure 2g and
Figure S9c, Supporting Information). Collectively, the above
results have demonstrated the efficient photothermal property
of BDP-J790 containing J-aggregated BODIPY dyes.
2.4. Remotely Controlled 1O2 Trapping/Releasing Through Two
NIR Beams In Vitro
1
Having established the efficient photogeneration of O2 during
2.3. Construction of the Target Photosensitive Polymeric Carrier
laser-1-inducted photosensitizing process of the I-BDP com-
ponent of An-NP and significant heat generation during laser-
J
Encouraged by the above findings, we moved to prepare the
desired photosensitive polymeric carrier with a sustained
1O2-production regulated by two NIR beams for enhanced anti-
tumor phototherapy. To achieving such a sophisticated func-
tion, three key components, that is, a PS (I-BDP) to generate
2-triggered photothermal process of the BDP component, we
1
then investigated the two NIR beams-controlled O2 trapping/
releasing performance given by the An-3 component (Figure 4a).
Upon laser-1 irradiation, An-3 component trapped the I-BDP-
1
generated abundant O2 to form EPO-3 as demonstrated by the
1
1O2, a reservoir to store O2, and a PTA to release the stored
time-dependent attenuation of the absorption of An-3 (Figure 4b).
Subsequently, upon laser-2 irradiation, the heat generated by the
JBDP component triggered the thermolysis of EPO-3 of EPO-NP,
1O2, should be orchestrated in one single nanosystem. As
depicted in Figure 3a, based on the strategy of co-assembly
with an amphiphilic PEG-PCL, the desired polymeric nanopar-
ticle (An-NP) embedded with a PS (I-BDP), a reservoir (An-3),
1
releasing the stored O2 and recovering An-3 (Figure 4c), which
1
will work in the next O2 storing/releasing cycle. After demon-
J
and a PTA (J-aggregated Br-BDP; denoted as BDP) within the
strating the renewability of the An-3 component and the photo-
1
hydrophobic core was successfully obtained. Transmission elec-
tron microscopy (TEM) revealed a nanoparticle morphology of
120 nm in diameter (Figure 3b), and DLS measurement gave an
intensity-average hydrodynamic diameter of 140 nm (Figure 3c).
It is worth of noting that the size of 140 nm should benefit the
antitumor application of An-NP in vivo as it meets the cri-
teria for the EPR (enhanced permeability and retention) effect,
which endows nanoparticle with the ability to passively target
tumor issue.[67] Moreover, the UV–vis absorption spectrum of
An-NP showed the characteristic absorption bands of An-3,
mediated reversible O2 trapping/releasing properties of An-NP
in solution (Figure 4d and Figure S15, Supporting Information),
we continued to apply it in living cells.
HeLa cells were incubated with An-NP for 4 h, and the effi-
cient cellular uptake of An-NP was demonstrated by the fluo-
rescence of An-3 in cells through confocal imaging (Figure S16,
Supporting Information). DCF-DA was used as a fluorescence
1
1
indicator of O2 to estimate the photo-controlled release of O2
in living cells.[57,68,69] HeLa cells were divided into three groups
for different treatments as outlined in Figure 5a (also see Sup-
porting Information). In group 1, the cells were incubated with
An-NP and DCF-DA, then exposed to laser-1 irradiation (10 mW
cm−2) for 5 min. Intense green fluorescence of the oxidized DCF
was observed (Figure 5b i), indicating the effective 1O2 generation
during the laser-1-induced PDT process. On the contrary, HeLa
J
I-BDP, and BDP (Figure 3d), indicating the encapsulation of
the three key components and the stability of the J-aggregation
of Br-BDP. The loading content and encapsulation efficiency of
the three components in An-NP have been estimated (Table S1,
Supporting Information). In addition, inappreciable changes in
the size of An-NP in PBS, cell-culture medium, or fetal bovine
serum were observed (Figure 3e and Figure S10, Supporting
Information), suggesting its significant colloidal stability.
We further assessed the photodynamic and photothermal
performance of the final An-NP, which were controlled by
two NIR beams (laser-1 and laser-2), respectively. A water
1
cells pretreated with sodium azide (NaN3; O2 scavenger[70]) dis-
played undetectable fluorescence upon laser-1 irradiation, further
verifying the photosensitized 1O2 generation of An-NP (Figure 5c
and Figure S17, Supporting Information). In group 2, the cells
were incubated with EPO-NP (for its preparation, see the Sup-
porting Information) and DCF-DA, then subjected to laser-2
irradiation (1.0 W cm−2) or incubated in dark for 10 min before
1
soluble O2 indicator (QDPBF)[57] was adopted to evaluate the
Small 2021, 17, 2101180
2101180 (6 of 12)
© 2021 Wiley-VCH GmbH

www.advancedsciencenews.com
www.small-journal.com
Figure 3. a) Schematic illustration of the preparation of the target water-stable photosensitive polymeric nanoparticle (An-NP) with An-3, I-BDP, and
Br-BDP J-aggregates embedded in the hydrophobic core. b) Typical TEM image, c) size distribution, and d) UV–vis absorption spectra of An-NP. e)
Time-evolution of the particle size of An-NP in PBS, cell culture media, or fetal bovine serum. Error bars represent mean SD (n = 3). f) Time-dependent
UV–vis absorption spectra of QDPBF within the aqueous solution of I-BDP micelle upon laser-1 irradiation. g) Temperature elevations recorded for
BDP-J790 at different concentrations of Br-BDP upon laser-2 irradiation. h) IR thermal images of BDP-J790 at different concentrations of Br-BDP after
10 min laser irradiation in the same conditions as (e). Laser-1: 655 nm, 3 mW cm−2; laser-2: 785 nm, 1.8 W cm−2
.
imaging. Compared with undetectable fluorescence in the cells
without laser treatment (Figure 5d and Figure S18, Supporting
Information), the DCF fluorescence within the cells after laser-2
irradiation was strong (Figure 5b ii). Moreover, pretreatment
with sodium azide could substantially reduce the DCF fluores-
cence (Figure 5d and Figure S18, Supporting Information). The
above results clearly indicated that laser-2 irradiation could effi-
ciently induce the 1O2 release from EPO-NP in living cells owing
to the photoconverted heat which triggered the cycloreversion of
EPO-3 embedded in EPO-NP.
In group 3, the cells were incubated with An-NP and exposed
to laser-1 irradiation (10 mW cm−2) for 10 min, then stained
with DCF-DA, followed by laser-2 irradiation (1.0 W cm−2)
for 10 min. No detectable DCF fluorescence was observed
before laser-2 irradiation (Figure 5e and Figure S19, Supporting
Information), and a clear DCF fluorescence was observed after
laser-2 irradiation (Figure 5b iii and Figure S19, Supporting
Information). The above findings demonstrated that in living
cells, the internalized An-NP can be converted into EPO-NP by
1
capturing O2 during laser-1-induced PDT process, which can
2101180 (7 of 12)
© 2021 Wiley-VCH GmbH
Small 2021, 17, 2101180

www.advancedsciencenews.com
www.small-journal.com
Figure 4. a) Schematic illustration of the interconversion between An-NP and EPO-NP regulated by two NIR laser beams. b) Evaluation of the laser-
1-triggered An-NP-to-EPO-NP transition and c) laser-2-induced EPO-NP-to-An-NP conversion by UV–vis spectroscopy. d) Absorbance changes of An-3
embedded in An-NP upon repeated cycles of laser-1 irradiation for 5 min and laser-2 irradiation for 35 min. Laser-1: 655 nm, 10 mW cm−2; laser-2:
785 nm, 1.8 W cm−2
.
1
readily release the trapped O2 in the laser-2-driven PTT pro-
cess. Moreover, we prepared a control nanoparticle (-An)-NP
without An-3 being encapsulated (see the Supporting Infor-
then exposed to laser-2 irradiation (1.0 W cm−2) for 10 min. As
shown in Figure 6, PTT effect of the BDP component within
J
An-NP resulted in 40.5% inhibition of cell viability. While
for EPO-NP, laser-2 irradiation in the same condition caused
69.4% inhibition of cell viability (Figure 6). Considering that
An-3 to EPO-3 conversion within the nanoparticle did not
affect the J-aggregation of Br-BDP which contribute the photo-
thermal effect (Figure 4b), we speculated that the cytotoxicity
induced by the PTT effect of EPO-NP should be close to that
induced by the PTT effect of An-NP. Thereby, compared with
the photocytotoxicity of An-NP, the extra 28.9% viability inhi-
bition caused by EPO-NP should be attributed to the 785 nm
laser-released 1O2. Pretreatment of 1O2 scavenger, that is, NaN3,
effectively reduced the cytotoxicity of EPO-NP upon laser-2 irra-
diation, with the inhibition of cell viability decreased to 44.6%
1
mation), and compared its PDT-induced intracellular O2 gen-
eration ability and in vitro photocytotoxicity with An-NP. As
expected, both (-An)-NP and An-NP micelles produced abun-
dant O2 in living cells upon laser-1 irradiation, and no detect-
able changes in the photogenerated O2 amount (Figure S20,
Supporting Information) and photocytotoxicity (Figure S21,
Supporting Information) were observed, indicating that An-3
did not affect the in vitro treatment outcome ascribed to the
generated 1O2 during PDT process. Notably, all HeLa cells in dif-
ferent experimental groups without nanoparticles or laser treat-
ment showed inappreciable fluorescence of DCF (Figure 5c–e
and Figures S17–S19, Supporting Information), suggesting that
the nanoparticles and laser beams did not interfere with the 1O2
1
1
1
(Figure 6). This finding further demonstrated the efficient O2
1
detection. The sustained O2 generation capability of An-NP
release from EPO-NP during laser-2 irradiation, which induced
distinct cytotoxicity to cancer cells.
enables the 1O2 stored in PDT process to be constantly released
in the interval between two PDT processes which is used for
the oxygen replenishment.
1
In addition to controlling the release of O2 from EPO-NP
2.5. In Vivo Antitumor Phototherapy
in the interval of PDT processes, laser-2 can induce the PTT
effect of An-NP and/or EPO-NP, might further enhance the
overall outcome of the anticancer phototherapy. We then inves-
tigated the laser-2-mediated in vitro photocytotoxicity resulted
from the released O2 and PTT, respectively, by MTT assay.
HeLa cells were incubated with An-NP or EPO-NP for 4 h,
We further studied the antitumor phototherapeutic efficacy of
An-NP in vivo. First, low-dose An-NP (3.0 mg/kg BDP) was
injected intravenously into 4T1 tumor-bearing Balb/c mice to
assess the tumor accumulation of the nanoagent by recording
the PTT-induced temperature elevations. IR thermal images
J
1
Small 2021, 17, 2101180
2101180 (8 of 12)
© 2021 Wiley-VCH GmbH

www.advancedsciencenews.com
www.small-journal.com
Figure 5. a) Summary and b) typical fluorescence images of the DCF-DA staining experiments to determine the intracellular 1O2 produced via the pho-
tosensitizing process of An-NP (group 1) or via the photo-releasing process of EPO-NP (groups 2 and 3). Group 1: laser-1 irradiation for 5 min; Group
2: laser-2 irradiation for 10 min; Group 3: pre-irradiated by laser-1 for 10 min and laser-2 irradiation for 10 min. c–e) Normalized mean fluorescence
intensity (Norm. MFI) calculated from DCF-DA staining fluorescence images of HeLa cells after different treatments as indicated. Laser-1: 655 nm,
10 mW cm−2; laser-2: 785 nm, 1.0 W cm−2
.
were recorded after irradiation of laser−2 (0.5 W cm−2; 5 min)
at different time points post intravenous injection, and the
time-dependent thermal images of the mice were shown in
Figure S23a, Supporting Information. After administration of
An-NP, obvious PTT-induced temperature elevations were found
at the tumor site and reached a maximum post 8 h of injection
(Figure S23b, Supporting Information). The observation indicated
that An-NP was stable during the blood circulation, and could be
accumulated at the tumor site owing to EPR effect.[67] For mice
treated with higher dose of An-NP (6.0 mg/kg JBDP) for 8 h, the
mean temperature at the tumor sites rose from 38.1 to 50.7 °C
(Figure 7a,b), allowing thermolysis of EPO to release 1O2.
The antitumor phototherapy of An-NP controlled by two
NIR laser beams was further investigated. After the xenograft
4T1 tumor grew to ≈100 mm3, the mice were randomly divided
into four groups for different treatments: i) treated with PBS
(200 µL) only, ii) treated with PBS and lasers irradiation (laser-1
for 10 min, followed by laser-2 for 10 min), iii) treated with
An-NP (200 µL, 6.0 mg/kg) only, and iv) treated with An-NP
and lasers irradiation (laser-1 for 10 min, followed by laser-2 for
10 min). After 8 h of the intravenous injection of PBS or An-NP
(6.0 mg/kg BDP), the tumors were exposed to laser-1 irradia-
J
tion (10 mW cm−2; 10 min) and subsequent laser-2 irradiation
(0.5 W cm−2; 10 min). The changes in the tumor volume were
recorded to evaluate the phototherapy efficacies. For group iv),
the tumors regressed and eliminated 2 and 4 days after the laser
treatment, respectively (Figure 7c,d and Figure S24, Supporting
Information), and no tumor recurrence was observed. On
the contrary, similar to PBS-treatment, treatment solely with
An-NP or laser irradiation did not inhibit the tumor growth
as reflected by the observation of continuous tumor growth
(Figure 7c). After two weeks of observation, all the mice in dif-
ferent groups were sacrificed and the xenograft tumors were
collected if still have (Figure 7d and Figure S25, Supporting
Information), showing that the tumors completely disappeared
in group (iv). Taken together, the above results demonstrated
an efficient antitumor phototherapy of An-NP nanoagent con-
trolled by two NIR beams.
2101180 (9 of 12)
© 2021 Wiley-VCH GmbH
Small 2021, 17, 2101180

www.advancedsciencenews.com
www.small-journal.com
Figure 6. a) Cell viability of An-NP- or EPO-NP-treated HeLa cells after different treatments as indicated (n = 4). b) Summary of the treatment modali-
ties as well as the cell-inhibition efficiencies, which were calculated by the formula: (1 – cell viability) × 100%.
Notably, during two weeks of observation, no appreci-
able loss in body weight was found for all mice in dif-
ferent groups (Figure S26, Supporting Information), indi-
cating that An-NP is biocompatible. Moreover, to further
evaluate the biosafety of An-NP, the main organs (heart,
liver, spleen, lung, and kidney) of the sacrificed mice were
collected for histological hematoxylin and eosin (H&E)
analysis (Figure 7e). As expected, no obvious pathological
changes were observed from all H&E sections. The above
results confirmed the biocompatibility of An-NP nanoagent
Figure 7. a) Typical IR thermal images and b) mean temperatures of the tumor regions recorded for xenograft tumor-bearing mice treated without
J
or with An-NP (6.0 mg/kg BDP) under laser-2 irradiation. c) Time-dependent changes in the tumor volume of mice in different groups. d) Optical
photographs of stark tumors collected after 2-week observation. e) H&E costaining images of major organs. Scale bar: 200 µm. Data represents mean
SD (n = 4), ***P < 0.001 (one-way ANOVA). Laser-2: 785 nm, 0.5 W cm−2
.
Small 2021, 17, 2101180
2101180 (10 of 12)
© 2021 Wiley-VCH GmbH

www.advancedsciencenews.com
www.small-journal.com
in antitumor phototherapy. As controls, the antitumor per-
formances of single PDT of An-NP, single PTT of An-NP,
and combinational PDT/PTT of (-An)-NP were studied. As
shown in Figure S27, Supporting Information, single PDT of
An-NP, single PTT of An-NP, and combinational PDT/PTT
of (-An)-NP inhibited tumor growth. However, none of them
achieved tumor elimination as synergistic phototherapy of
(An)-NP did, demonstrating the unique synergistic anti-
tumor phototherapy efficacy of An-NP.
Keywords
photoactive, photodynamic therapy, photothermal therapy, polymeric
nanoparticles, singlet oxygen
Received: February 26, 2021
Revised: April 15, 2021
Published online: June 18, 2021
[1] X. S. Li, S. Y. Lee, J. Yoon, Chem. Soc. Rev. 2018, 47, 1174.
[2] A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer 2006, 6,
535.
3. Conclusions
[3] T. F. Zhang, Y. Y. Li, Z. Zheng, R. Q. Ye, Y. R. Zhang, R. T. K. Kwok,
J. W. Y. Lam, B. Z. Tang, J. Am. Chem. Soc. 2019, 141, 5612.
[4] D. van Straten, V. Mashayekhi, H. S. de Bruijn, S. Oliveira,
D. J. Robinson, Cancers 2017, 9, 19.
[5] Á. Juarranz, P. Jaén, F. Sanz-Rodríguez, J. Cuevas, S. González,
Clin. Transl. Oncol. 2008, 10, 148.
In this work, we have successfully prepared a novel photo-
sensitive polymeric carrier with a sustained 1O2-production
regulated by two NIR beams for enhanced antitumor pho-
totherapy. By co-assembling with an amphiphilic diblock
copolymer, three key components, that is, a PS (I-BDP) to
generate 1O2, a reservoir (An-3) to store 1O2, and a PTA (JBDP)
[6] Y. S. Dong, Y. L. Tu, K. W. Wang, C. F. Xu, Y. Y. Yuan, J. Wang,
Angew. Chem., Int. Ed. 2020, 59, 7168.
1
to release the stored O2, were orchestrated in a single nano-
[7] Y. Cai, D. Q. Ni, W. Y. Cheng, C. D. Ji, Y. L. Wang, K. Müllen,
Z. Q. Su, Y. Liu, C. Y. Chen, M. Z. Yin, Angew. Chem., Int. Ed. 2020,
59, 14014.
[8] Y. Yang, W. J. Zhu, L. Cheng, R. Cai, X. Yi, J. X. He, X. S. Pan,
L. Yang, K. Yang, Z. Liu, W. H. Tan, M. W. Chen, Biomaterials 2020,
246, 119971.
[9] P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti,
S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D. Kessel,
M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B. C. Wilson,
J. Golab, Ca-Cancer J. Clin. 2011, 61, 250.
system to yield the sophisticated nanomaterial (An-NP). We
demonstrated that in both solution and living cells, upon
655 nm laser irradiation, the PDT process of An-NP occurred
1
1
to generate abundant O2 with extra O2 being stored via the
formation of EPO-NP; while in the subsequent 785 nm laser-
driven PTT process, the converted EPO-NP underwent ther-
1
molysis to release the trapped O2 and recover An-NP. The
oxygen level can be replenished in the PTT process for the
1
next round of PDT to generate O2. The presented photosen-
[10] D. Hanahan, R. A. Weinberg, Cell 2000, 100, 57.
[11] H. Harada, M. Inoue, S. Itasaka, K. Hirota, A. Morinibu,
K. Shinomiya, L. H. Zeng, G. F. Ou, Y. X. Zhu, M. Yoshimura,
W. G. McKenna, R. J. Muschel, M. Hiraoka, Nat. Commun. 2012, 3,
783.
[12] K. Kiyose, K. Hanaoka, D. Oushiki, T. Nakamura, M. Kajimura,
M. Suematsu, H. Nishimatsu, T. Yamane, T. Terai, Y. Hirata,
T. Nagano, J. Am. Chem. Soc. 2010, 132, 15846.
sitive nanomaterial allows alternative application of PDT and
PTT controlled by two NIR laser beams, thus enabling suf-
ficient oxygen supply and sustained 1O2 generation during the
entire synergistic phototherapy. The nanomaterial was further
applied to antitumor phototherapy in vivo and demonstrated
striking efficacy.
[13] J. Pouysségur, F. Dayan, N. M. Mazure, Nature 2006, 441, 437.
[14] S. S. Wang, H. Liu, J. Mack, J. W. Tian, B. Zou, H. Lu, Z. F. Li,
J. X. Jiang, Z. Shen, Chem. Commun. 2015, 51, 13389.
[15] J. Xu, S. B. Sun, Q. Li, Y. Yue, Y. D. Li, S. J. Shao, Analyst 2015, 140,
574.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
[16] G. Q. Zhang, G. M. Palmer, M. W. Dewhirst, C. L. Fraser,
Nat. Mater. 2009, 8, 747.
[17] X. C. Zheng, X. Wang, H. Mao, W. Wu, B. R. Liu, X. Q. Jiang,
Nat. Commun. 2015, 6, 5834.
Acknowledgements
Financial support from National Natural Science Foundation of
China (Grant 51673101) and the Fundamental Research Funds for
Central Universities (Nankai University, Grant 92022030) is gratefully
acknowledged. All animal experiments were carried out in compliance
with the guide for the care and use of laboratory animals of Tianjin
Science and Technology Commission.
[18] K. Y. Zhang, P. L. Gao, G. L. Sun, T. W. Zhang, X. L. Li, S. J. Liu,
Q. Zhao, K. K.-W. Lo, W. Huang, J. Am. Chem. Soc. 2018, 140, 7827.
[19] P. Vaupel, O. Thews, M. Hoeckel, Med. Oncol. 2001, 18, 243.
[20] Z. J. Zhou, J. B. Song, L. M. Nie, X. Y. Chen, Chem. Soc. Rev. 2016,
45, 6597.
[21] H. He, S. S. Ji, Y. He, A. J. Zhu, Y. L. Zou, Y. B. Deng, H. T. Ke,
H. Yang, Y. L. Zhao, Z. Q. Guo, H. B. Chen, Adv. Mater. 2017, 29,
1606690.
[22] X. Z. Zhao, S. R. Long, M. L. Li, J. F. Cao, Y. C. Li, L. Y. Guo, W. Sun,
J. J. Du, J. L. Fan, X. J. Peng, J. Am. Chem. Soc. 2020, 142, 1510.
[23] R. Sullivan, C. H. Graham, Cancer Metastasis Rev. 2007, 26, 319.
[24] A. L. R. de Souza, K. Marra, J. Gunn, K. S. Samkoe, S. C. Kanick,
S. C. Davis, M. S. Chapman, E. V. Maytin, T. Hasan, B. W. Pogue,
Br. J. Cancer 2016, 115, 805.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
[25] Z. W. Xiao, S. Halls, D. Dickey, J. Tulip, R. B. Moore, Clin. Cancer
Res. 2007, 13, 7496.
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
2101180 (11 of 12)
© 2021 Wiley-VCH GmbH
Small 2021, 17, 2101180

www.advancedsciencenews.com
www.small-journal.com
[26] L. Y. Yang, Y. C. Wei, D. Xing, Q. Chen, Lasers Surg. Med. 2010, 42,
671.
[27] C. Li, X. C. Zheng, W. Z. Chen, S. L. Ji, Y. Yuan, X. Q. Jiang,
Nano Lett. 2020, 20, 6526.
[28] I. S. Turan, D. Yildiz, A. Turksoy, G. Gunaydin, E. U. Akkaya,
Angew. Chem., Int. Ed. 2016, 55, 2875.
[49] Y. X. Xu, X. L. Liang, P. Bhattarai, Y. Sun, Y. M. Zhou, S. M. Wang,
W. Chen, H. Y. Ge, J. R. Wang, L. G. Cui, Z. F. Dai, Adv. Funct. Mater.
2017, 27, 1704096.
[50] C. Y. Yang, Y. D. Chen, W. Guo, Y. Gao, C. Q. Song, Q. Zhang,
N. N. Zheng, X. J. Han, C. S. Guo, Adv. Funct. Mater. 2018, 28,
1706827.
[29] Z. Yuan, S. Yu, F. Y. Cao, Z. W. Mao, C. Y. Gao, J. Ling, Polym. Chem.
2018, 9, 2124.
[30] S. Martins, J. P. S. Farinha, C. Baleizao, M. N. Berberan-Santos,
Chem. Commun. 2014, 50, 3317.
[31] L. Slavetinska, J. Mosinger, P. Kubat, J. Photochem. Photobiol., A
2008, 195, 1.
[32] J. M. Aubry, C. Pierlot, J. Rigaudy, R. Schmidt, Acc. Chem. Res. 2003,
36, 668.
[51] S. C. Zhang, Q. Z. Li, N. Yang, Y. H. Shi, W. Ge, W. J. Wang,
W. Huang, X. J. Song, X. C. Dong, Adv. Funct. Mater. 2019, 29,
1906805.
[52] Y. J. Liu, P. Bhattarai, Z. F. Dai, X. Y. Chen, Chem. Soc. Rev. 2019, 48,
2053.
[53] X. T. Gao, S. S. Jiang, C. Y. Li, Y. z. Chen, Y. F. Zhang, P. Huang,
J. Lin, Biomaterials 2021, 267, 120454.
[54] L. Y. Zhao, Y. M. Liu, R. Chang, R. R. Xing, X. H. Yan, Adv. Funct.
Mater. 2019, 29, 1806877.
[33] W. Fudickar, T. Linker, Chem. - Eur. J. 2011, 17, 13661.
[34] D. Zehm, W. Fudickar, M. Hans, U. Schilde, A. Kelling, T. Linker,
Chem. - Eur. J. 2008, 14, 11429.
[55] H. S. Jung, P. Verwilst, A. Sharma, J. Shin, J. L. Sessler, J. S. Kim,
Chem. Soc. Rev. 2018, 47, 2280.
[35] L. Slavetinska, J. Mosinger, M. Dracinsky, M. Posta, J. Inclusion
Phenom. Macrocyclic Chem. 2008, 61, 241.
[56] X. S. Yan, M. H. Su, Y. N. Liu, Y. K. Zhang, H. Zhang, C. H. Li,
Adv. Funct. Mater. 2021, 31, 2008406.
[36] W. Fudickar, T. Linker, J. Org. Chem. 2017, 82, 9258.
[37] W. Fudickar, T. Linker, J. Am. Chem. Soc. 2012, 134, 15071.
[38] S. Kolemen, T. Ozdemir, D. Lee, G. M. Kim, T. Karatas, J. Yoon,
E. U. Akkaya, Angew. Chem., Int. Ed. 2016, 55, 3606.
[39] A. M. Asadirad, Z. Erno, N. R. Branda, Chem. Commun. 2013, 49, 5639.
[40] Y. Li, X. M. Liu, L. Tan, Z. D. Cui, X. J. Yang, Y. F. Zheng,
K. W. K. Yeung, P. K. Chu, S. L. Wu, Adv. Funct. Mater. 2018, 28,
1800299.
[57] M. H. Su, S. X. Li, H. Zhang, J. Q. Zhang, H. L. Chen, C. H. Li,
J. Am. Chem. Soc. 2019, 141, 402.
[58] M. H. Su, Q. J. Han, X. S. Yan, Y. N. Liu, P. Luo, W. H. Zhai,
Q. Z. Zhang, L. Y. Li, C. H. Li, ACS Nano 2021, 15, 5032.
[59] M. H. Su, X. S. Yan, X. Guo, Q. W. Li, Y. S. Zhang, C. H. Li,
Chem. - Eur. J. 2020, 26, 4505.
[60] Y. Lyu, C. Xie, S. A. Chechetka, E. Miyako, K. Pu, J. Am. Chem. Soc.
2016, 138, 9049.
[41] V. Arkhipova, H. Fu, M. W. H. Hoorens, G. Trinco, L. N. Lameijer,
E. Marin, B. L. Feringa, G. J. Poelarends, W. Szymanski,
D. J. Slotboom, A. Guskov, J. Am. Chem. Soc. 2021, 143, 1513.
[42] P. Y. Lam, A. R. Thawani, E. Balderas, A. J. P. White, D. Chaudhuri,
M. J. Fuchter, R. T. Peterson, J. Am. Chem. Soc. 2020, 142, 17457.
[43] K. Aggarwal, T. P. Kuka, M. Banik, B. P. Medellin, C. Q. Ngo, D. Xie,
Y. Fernandes, T. L. Dangerfield, E. Ye, B. Bouley, K. A. Johnson,
Y. J. Zhang, J. K. Eberhart, E. L. Que, J. Am. Chem. Soc. 2020, 142,
14522.
[44] J. Morstein, M. A. Dacheux, D. D. Norman, A. Shemet,
P. C. Donthamsetti, M. Citir, J. A. Frank, C. Schultz, E. Y. Isacoff,
A. L. Parrill, G. J. Tigyi, D. Trauner, J. Am. Chem. Soc. 2020, 142,
10612.
[61] Y. Jiang, P. K. Upputuri, C. Xie, Z. L. Zeng, A. Sharma, X. Zhen,
J. C. Li, J. G. Huang, M. Pramanik, K. Y. Pu, Adv. Mater. 2019, 31,
1808166.
[62] X. X. Yao, P. Yang, Z. K. Jin, Q. Jiang, R. R. Guo, R. H. Xie, Q. J. He,
W. L. Yang, Biomaterials 2019, 197, 268.
[63] S. Y. Ye, J. M. Rao, S. H. Qiu, J. L. Zhao, H. He, Z. L. Yan, T. Yang,
Y. B. Deng, H. T. Ke, H. Yang, Y. L. Zhao, Z. Q. Guo, H. B. Chen,
Adv. Mater. 2018, 30, 1801216.
[64] Q. L. Zou, M. Abbas, L. Y. Zhao, S. K. Li, G. Z. Shen, X. H. Yan,
J. Am. Chem. Soc. 2017, 139, 1921.
[65] X. Cai, A. Bandla, C. K. Chuan, G. Magarajah, L.-D. Liao,
D. B. L. Teh, B. K. Kennedy, N. V. Thakor, B. Liu, Mater. Horiz. 2019,
6, 311.
[45] R. C. Sarott, A. E. G. Viray, P. Pfaff, A. Sadybekov, G. Rajic,
V. Katritch, E. M. Carreira, J. A. Frank, J. Am. Chem. Soc. 2021, 143,
736.
[66] Z. B. Zha, X. L. Yue, Q. S. Ren, Z. F. Dai, Adv. Mater. 2013, 25, 777.
[67] J. Fang, H. Nakamura, H. Maeda, Adv. Drug Delivery Rev. 2011, 63,
136.
[46] K. R. Deng, Z. Y. Hou, X. R. Deng, P. P. Yang, C. X. Li, J. Lin,
Adv. Funct. Mater. 2015, 25, 7280.
[47] K. Hayashi, M. Nakamura, H. Miki, S. Ozaki, M. Abe,
T. Matsumoto, T. Kori, K. Ishimura, Adv. Funct. Mater. 2014, 24, 503.
[48] Z. He, L. L. Zhao, Q. Zhang, M. J. Chang, C. X. Li, H. S. Zhang,
Y. Lu, Y. S. Chen, Adv. Funct. Mater. 2020, 30, 1910301.
[68] Y. H. Cheng, H. Cheng, C. X. Jiang, X. F. Qiu, K. K. Wang, W. Huan,
A. H. Yuan, J. H. Wu, Y. Q. Hu, Nat. Commun. 2015, 6, 8785.
[69] N. M. Idris, M. K. Gnanasammandhan, J. Zhang, P. C. Ho,
R. Mahendran, Y. Zhang, Nat. Med. 2012, 18, 1580.
[70] W. H. Zhai, Y. K. Zhang, M. Liu, H. Zhang, J. Q. Zhang, C. H. Li,
Angew. Chem., Int. Ed. 2019, 58, 16601.
Small 2021, 17, 2101180
2101180 (12 of 12)
© 2021 Wiley-VCH GmbH