A Lattice-Oxygen-Involved Reaction Pathway to Boost Urea Oxidation

Article abstract of DOI:10.1002/anie.201909832

The electrocatalytic urea oxidation reaction (UOR) provides more economic electrons than water oxidation for various renewable energy-related systems owing to its lower thermodynamic barriers. However, it is limited by sluggish reaction kinetics, especially by CO₂ desorption steps, masking its energetic advantage compared with water oxidation. Now, a lattice-oxygen-involved UOR mechanism on Ni⁴⁺ active sites is reported that has significantly faster reaction kinetics than the conventional UOR mechanisms. Combined DFT, ¹⁸O isotope-labeling mass spectrometry, and in situ IR spectroscopy show that lattice oxygen is directly involved in transforming *CO to CO₂ and accelerating the UOR rate. The resultant Ni⁴⁺ catalyst on a glassy carbon electrode exhibits a high current density (264 mA cm^?2 at 1.6 V versus RHE), outperforming the state-of-the-art catalysts, and the turnover frequency of Ni⁴⁺ active sites towards UOR is 5 times higher than that of Ni³⁺ active sites.

Full text of DOI:10.1002/anie.201909832

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Biological and Medical Applications of Materials and Interfaces
Intrinsically Cancer-Mitochondria-Targeted Thermally
Activated Delayed Fluorescence Nanoparticles for Two-Photon
Activated Fluorescence Imaging and Photodynamic Therapy
Jinfeng Zhang, Fang Fang, Bin Liu, Ji-Hua Tan, Wen-Cheng Chen, Zelin Zhu, Yi Yuan, Yingpeng
Wan, Xiao Cui, Shengliang Li, Qing-Xiao Tong, Junfang Zhao, Xiangmin Meng, and Chun-Sing Lee
ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b14552 • Publication Date (Web): 11 Oct 2019
Downloaded from pubs.acs.org on October 22, 2019
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Physics and Chemistry; City University of Hong Kong, Joint Laboratory of
Nano-organic Functional Materials and Devices (TIPC and CityU)
Meng, Xiangmin; Chinese Academy of Sciences, Technical Institute of
Physics and Chemistry; City University of Hong Kong, Joint Laboratory of
Nano-organic Functional Materials and Devices (TIPC and CityU)
Lee, Chun-Sing; City University of Hong Kong, Center of Super-Diamond
and Advanced Films (COSDAF) & Department of Chemistry; City
University of Hong Kong, Joint Laboratory of Nano-organic Functional
Materials and Devices (TIPC and CityU)
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Intrinsically Cancer-Mitochondria-Targeted Thermally Activated
Delayed Fluorescence Nanoparticles for Two-Photon Activated
Fluorescence Imaging and Photodynamic Therapy
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†
†
‡,§
#,‖
#,‖
#,‖
#,‖
Jinfeng Zhang , Fang Fang , Bin Liu , Ji-Hua Tan , Wen-Cheng Chen , Zelin Zhu , Yi Yuan ,
#
,‖
#,‖
#,‖,
§
Δ,‖
Yingpeng Wan , Xiao Cui , Shengliang Li *, Qing-Xiao Tong , Junfang Zhao , Xiang-Min
Meng Δ,‖ and Chun-Sing Lee #,,‖*
†
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Sciences, Beijing Institute
of Technology, Beijing, P. R. China
‡
School of Science, Westlake Institute for Advanced Study, Westlake University, 18 Shilongshan
Road, Hangzhou, P. R. China, Department of Physics, Fudan University, Shanghai, P. R. China
§
Department of Chemistry and Key Laboratory for Preparation and Application of Ordered
Structural Materials of Guangdong Province, Shantou University, 243 University Road, Shantou,
Guangdong 515063, P.R. China
#
Center of Super-Diamond and Advanced Films (COSDAF) & Department of Chemistry, City
University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, P. R. China
E-mail: lishengliang@iccas.ac.cn; apcslee@cityu.edu.hk
‖
Joint Laboratory of Nano-organic Functional Materials and Devices (TIPC and CityU), City
University of Hong Kong, Hong Kong SAR, P. R. China
Δ
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190,
China
KEYWORDS: cancer-mitochondria-targeted, thermally activated delayed fluorescence (TADF),
two-photon activated, fluorescence imaging, photodynamic therapy,
ABSTRACT: A recent breakthrough in the discovery of thermally activated delayed fluorescence
(TADF) emitters which characterized with small single-triplet energy offsets (ΔE ) offers a wealth
ST
of new opportunities to exploit high-performance metal-free PSs. In this report, two intrinsically
cancer-mitochondria-targeted TADF emitters based nanoparticles (TADF NPs) have been developed
for two-photon activated photodynamic therapy (PDT) and fluorescence imaging. The as-prepared
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TADF NPs integrate the merits of 1) high O quantum yield of 52%; 2) sufficient near-infrared
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(
NIR) light penetration depth due to two-photon activation; 3) excellent structure-inherent
mitochondria-targeting capabilities without extra chemical or physical modifications, inducing
remarkable endogenous mitochondria-specific ROS production and excellent cancer-cell-killing
ability upon an ultralow light irradiance. We believe that the development of such intrinsically
multifunctional TADF NPs stemming from a single molecule will provide new insights into
exploration of novel PDT agents with strong photosensitizing ability for various biomedical
applications.
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. INTRODUCTION
Photodynamic therapy (PDT) is one of the most promising modalities against cancer owing to its
minimal invasiveness, good specificity, negligible drug resistance as well as excellent repeatability
with relatively mild side effects.1–9 In PDT, an optically-excited photosensitizer (PS) transfers its
obtained energy to surrounding oxygen molecules to generate reactive oxygen species (ROSs),
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typically singlet oxygen ( O ). Upon photoexcitation, the PS will first form a singlet excited state as
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governed by the requirement of spin conservation. Before passing its energy to the surrounding
oxygen, the singlet-excited PS has to go through an intersystem crossing (ISC) to shape into a
1
triplet-excited state. Therefore, an efficient ISC process is critical for the formation of O and
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improvement of PDT performance.10–13 Currently, a commonly used strategy is to introduce
heavy-metal atoms into the PS molecules for enhancing spin-orbit coupling and thus ISC.14–16
However, the inherent cytotoxicity and unsatisfactory degradability originating from these
heavy-metal-containing PSs would hinder their further wide clinical translations.10,17,18
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Fortunately, a recent breakthrough in the discovery of thermally activated delayed fluorescence
TADF) emitters in the area of organic light-emitting device (OLED) 15–22 offers a new possibility of
(
getting high performance metal-free PSs with strong photosensitizing ability. TADF emitters are
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pure organic fluorescent molecules characterized with small single-triplet energy offsets (ΔEST).
Particularly, such small ΔE will lead to easily energy conversion between singlet and triplet PS and
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thus enabling efficient ISC. In fact, while these TADF emitters exhibit many fascinating optical
properties, there have been so far only few reports on their biomedical applications which only
exploit their delayed fluorescence for time-resolved luminescence imaging.23–25 As a proof of
principle, we have shown for the first time that TADF emitters can indeed be used as novel
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metal-free PSs for O generation. While this new concept for developing metal-free PDT agents is
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promising, O quantum yield of these TADF PDT agents are so far below 15%. This value is
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clearly much lower than the new concept should suggest in principle. On the other hand, there are
still limitations in most reported PDT agents which hindered their wide clinical applications. In
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addition to O quantum yield, enhancements in (i) optical penetration depth in body tissue, (ii)
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targeting to specific subcellular organelles and (iii) image-guided theranostic capability are highly
desirable. More importantly, to the best of our knowledge, there has been no any single TADF based
agent that can be able to concurrently address all of the above issues, which is attractive for PDT
treatment and future clinical application.
In this work, we developed two intrinsically cancer-mitochondria-targeted TADF based organic
nanoparticles (TADF NPs) for two-photon activated PDT and fluorescence imaging. These two
1
TADF nanophotosensitizers combine the merits of high O quantum yields, sufficient near-infrared
2
(NIR) light penetration depth, good in vitro biocompatibility, two-photon activated cellular
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fluorescence imaging, endogenous mitochondria-specific ROS production and excellent
cancer-cell-killing ability. We believe that the development of such intrinsically multifunctional
TADF based nanostructures will provide new insights into exploration of novel PDT agents for
various biomedical applications.
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. EXPERIMENTAL SECTION
2.1 Materials. 9,10-anthracenedipropionicacid (ADPA), 2,7-dichlorofluorescein diacetate
DCFH-DA), 4, 6-diamidino-2-phenylindole (DAPI), Rose bengal, 4, 5-Dimethylthiazol-2-yl)-2,
-diphenyl tetrazolium bromide (MTT) were purchased from Sigma. Dulbecco’s modified Eagle’s
medium (DMEM, high glucose), trypsin-EDTA, Dulbecco’s phosphate buffered saline (PBS, 10X,
(
5
pH
7.4),
penicillin-streptomycin,
fetal
bovine
serum
iodide (JC-1)
(FBS),
were
5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidoazolylcarbocyanino
purchased from ThermoFisher Scientific.
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.2 Synthesis of An-TPA and An-Cz-Ph. 2-Bromoanthraquinone (2.67 mmol, 765 mg),
-(diphenylamino)phenylboronic acid (2.67 mmol, 772 mg) or 9-phenyl-9H-carbazol-3-ylboronic
4
acid (2.67 mmol, 765 mg), tetrakis-(triphenylphosphine)-palladium(0) (0.13 mmol 150 mg), 8 mL
aqueous K CO (2M), 8 mL ethanol were added into toluene (30 mL). The mixture was stirred at 120
2
3
°
C for 24 h under argon atmosphere. After cooling to room temperature, the organic phase was
washed with 20 mL water and extracted with dichloromethane, then was dried with anhydrous
Na SO and concentrated by rotary evaporation. Finally, column chromatography (petroleum ether:
2
4
CH Cl = 4: 1) on silica gel was used to purify the residue to obtain a red powder (An-TPA: 0.94
2
2
g/78%, An-Cz-Ph: 0.88 g/73%)
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Characterization of An-TPA: H NMR (600 MHz, CDCl ) δ 8.66 (s, 1H), 8.51 (s, 1H), 8.39 (d, J =
3
8
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.1 Hz, 1H), 8.37-8.31 (m, 2H), 8.23 (d, J = 7.7 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.80 (dt, J = 14.7,
.0 Hz, 3H), 7.64 (t, J = 7.7 Hz, 2H), 7.59 (d, J = 7.7 Hz, 2H), 7.50 (t, J = 7.8 Hz, 2H), 7.47-7.40 (m,
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2H), 7.34 (t, J = 7.1 Hz, 1H). C NMR (151 MHz, CDCl ) δ [ppm]: 183.5, 183.0, 147.6, 141.3,
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37.3, 134.1, 133.7, 132.2, 131.4, 130.8, 130.0, 128.1, 127.8, 127.2, 127.0, 126.5, 125.3, 124.1,
123.2, 120.5, 119.3, 110.4, 110.1. MS (ESI): m/z = 449.1 [M]+.
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Characterization of An-Cz-Ph: H NMR (600 MHz, CDCl ) δ 8.50 (s, 1H), 8.33 (dd, J = 10.7, 6.5 Hz,
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H), 7.98 (dd, J = 8.1, 1.4 Hz, 1H), 7.83-7.77 (m, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.29 (t, J = 7.8 Hz,
1
3
4H), 7.15 (dd, J = 8.4, 2.8 Hz, 6H), 7.08 (t, J = 7.3 Hz, 2H). C NMR (151 MHz, CDCl ) δ [ppm]:
3
1
83.4, 182.8, 148.8, 147.2, 146.3, 145.7, 134.1, 133.9, 133.7, 133.6, 131.9, 131.5, 129.4, 128.1,
128.0, 127.2, 127.2, 125.0, 123.6, 122.9. MS (ESI): m/z = 452.2 [M + H]+.
.3 Preparation of the An-TPA NPs and An-Cz-Ph NPs. An-TPA NPs and An-Cz-Ph NPs were
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prepared using the traditional nanoprecipitation method, in which An-TPA and An-Cz-Ph were
respectively dissolved in THF with 1 mg/mL concentration. 200 µL of the above solution was
quickly added into 5 mL of DI water drop by drop under energetic stirring for 10 min. Another 10
min of sonication was applied to the above-obtained solutions in order to ensuring total THF
evaporation and then dispersions of the An-TPA NPs or An-Cz-Ph NPs were finally collected for
further use.
2.4 Characterization of the An-TPA NPs and An-Cz-Ph NPs. Sizes and morphologies of the
as-fabricated TADF NPs were characterized with both scanning electron microscopy (SEM, Philips
XL-30 FEG) and transmission electron microscopy (TEM, Hitachi H-7700). Dynamic light
scattering (DLS) and polydispersity index (PDI) measurements were determined by using a Malvern
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Zetasizer. Optical absorption and emission spectra were individually measured with a UV-vis-NIR
spectrophotometer (PE Lamda 19) and a fluorescence spectrophotometer (Cary Eclipse).
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.5 SPE and TPE cellular imaging. All cells including two cancer cell lines (A549 or Hela cancer
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cells) and two normal cell lines (NIH 3T3 or MCR-5 normal cells) were cultured with standard
DMEM culture medium which contained 10% FBS and 1% penicillin/streptomycin in a humidified
5% CO -incubator at 37 °C. For SPE cellular imaging, the assigned cells were first seeded on 6-well
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plates for 24 h and then the TADF NPs dispersions with final concentration of 8 g/mL were added
to each plate. After incubation for another 4 h, the treated cells were washed thoroughly and then
stained with nuclei-dye DAPI for 5 min. Finally, SPE fluorescent images of the treated cells were
captured by using a Nikon ECLIPSE 80i fluorescent microscope. For TPE cellular imaging, the cells
were seeded on 35 mm Laser confocal petri dishes for 24 h and the TADF NPs dispersions with final
concentration of 8 g/mL were individually added to each plate. After incubation for 4 h, TPE
fluorescent images of the treated cells were recorded by using a confocal laser scanning microscopy
(CLSM, Leica TCS SP5) with 800 or 880 nm two-photon fs excitation.
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.6 O quantum yield measurements. Production of O was determined by using the disodium
2 2
1
salt of ADPA which absorption at 378 nm would be degraded upon exposure to O . Rose bengal
2
1
was selected as a calibration standard with O quantum yield of 75% in water. 60 μL of ADPA
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water solution (1 mg/mL) was added into 2 mL of the TADF NPs dispersions. The mixed solutions
were irradiated with a xenon lamp only passing light from 400 to 700 nm by equipping with a filter.
The maximal absorption of all samples was adjusted to 0.15-0.3 OD and recorded every 30 s to
1
obtain the decay rate of the photosensitizing process. O quantum yield of the NP samples were
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calculated using the following formula:
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Фsample=Фrose bengal *Ksample*Arose bengal /(Krose bengal *Asample); where Ksample and Krose bengal are the
decomposition rate constants of the disodium salt of ADPA by the TADF NPs and Rose bengal,
respectively. Arose bengal and Asample represent integral areas of the optical absorption bands between
400 and 700 nm by the Rose bengal and TADF NPs, respectively.
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.7 Colocalization of TADF NPs with the MitoTracker Green or LysoTracker Green. Two
cancer cell lines (A549 or Hela cancer cells) and two normal cell lines (NIH 3T3 or MCR-5 normal
cells) were seeded on 35 mm Laser confocal petri dishes and incubated with TADF NPs with final
concentration of 8 μg/mL for 4 h. Then the treated cells were co-incubated with commercial
mitochondrion-staining agent MitoTracker Green (100 nM) or lysosome-staining agent LysoTracker
Green (100 nM) for 30 min. The live cells were then visualized by using a confocal laser scanning
microscopy (CLSM, Leica TCS SP5)
2
.8 Cytotoxicity by MTT assay. Firstly, the A549 cells were cultured in 96-well plates with
standard DMEM culture medium in a humidified 5% CO -incubator at 37 °C. After growing
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overnight, original medium were removed. Then 200 μl of DMEM medium containing TADF NPs
with given concentration were added to the plates where the final concentration of these TADF NPs
in the designated wells ranged from 1.25 to 20 μg/mL. After 24 h dark incubation, the cells incubated
with these NPs were irradiated for 5 min with white light (400 to 700 nm) at a power density of 28
2
mW/cm . The irradiated cells were then incubated darkly for another 24 h. Subsequently, removing
the original medium in each well. Afterwards, 200 μL of DMEM-MTT solution (10 % MTT stock
solution) in the absence of FBS were added in each well and incubated darkly for 4 hours. After
completely removing the MTT-containing medium, 200 μL of DMSO was added into each well. Cell
viabilities were finally determined by using a microplate reader (BioTek Powerwave XS).
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. RESULTS AND DISCUSSION
.1. Synthesis and Characterization of TADF Molecules. The two TADF molecules,
2-(4-(diphenylamino)phenyl)anthracene (An-TPA) and
-(9-phenyl-9H-carbazol-6-yl)anthracene-9,10-dione (An-Cz-Ph) are prepared by typical Suzuki
coupling reactions as shown in Figure 1a. Chemical structures of the An-TPA and the An-Cz-Ph
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were individually confirmed with H/ C NMR and mass spectrometric analysis. Optimized
molecular geometries and frontier molecular orbital distribution calculated via DFT approach using
B3LYP/6-31G(d) basis set with the Gaussian 09 program are displayed in Figure 1b. It can be found
that both molecules have nonplanar conformations which will reduce intermolecular π-π stacking in
solid and enable the TADF materials to exhibit good fluorescence in solid state. As shown in Figure
S1, intense red emissions from the An-TPA and the An-Cz-Ph powders can be clearly observed
while the fluorescence of a commercial green dye FITC is nearly quenched due to
aggregation-caused quenching effect, which is beneficial for potential imaging application.
Optimization of the singlet and triplet excited states was performed by the time-dependent
DFT/B3LYP/6-31G(d) method. Figure 1b also displays that both molecules have well-separated
HOMO and LUMO distributions which are essential for getting small ΔEST (0.17 and 0.24 eV for
An-TPA and An-Cz-Ph) needed for TADF and efficient ISC for high performance PDT. It is worth
noting that triphenylamine (TPA) is a more electron-rich group with strong intramolecular
charge-transfer ability than N-phenylcarbazole (Cz-Ph) group. So An-TPA exhibited smaller ΔEST
due to much more separated HOMO/LUMO distribution.
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Figure 1. a) Synthetic routes for An-TPA and An-Cz-Ph; b) Molecular geometries and HOMO-LUMO
distributions of An-TPA and An-Cz-Ph, which were optimized by DFT calculations.
To investigate TADF properties of An-TPA and An-Cz-Ph, spectroscopic characteristics of these
molecules dissolved in 2-methyltetrahydrofuran and doped in a bis[2-(diphenylphosphino)phenyl]
ether oxide (DPEPO) solid film have been measured. Figure S2 presents the steady-state
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fluorescence (measured at room temperature) and phosphorescence spectra (measured at 77 K) of the
An-TPA and the An-Cz-Ph dissolved in solution. According to the difference in the onset
wavelengths of fluorescence and phosphorescence spectra, ΔEST values of An-TPA and An-Cz-Ph
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were determined to be 0.11 eV and 0.23 eV, respectively. These experimental ΔE values are well
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consistent with the calculated ones shown in Figure 1b. Furthermore, transient photoluminescence
decay curves of the An-TPA and the An-Cz-Ph doped solid films (Figures S2 c, d), clearly show
short and long decay components which are individually ascribed to the prompt fluorescence and
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delayed TADF components. Besides, temperature-dependent transient photoluminescence spectra
of the An-TPA and An-Cz-Ph samples further confirm the distinct TADF characteristics of An-TPA
and An-Cz-Ph (Figure S3).
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.2. Preparation and Characterization of TADF NPs. Due to the poor water solubility of TADF
molecules, to improve their bioavailability, water-dispersible TADF NPs were fabricated via a
traditional nanoprecipitation method. As illustrated in Figure 2a, An-TPA NPs and An-Cz-Ph NPs
were prepared by self-assembly for two-photon activated cellular fluorescence imaging (TPA
imaging) and mitochondria-specific ROS production. Figures 2b, f and 2c, g display respectively
SEM and TEM images of the An-TPA NPs and the An-Cz-Ph NPs, uncovering their monodispersed
and well-defined spherical morphologies of ~ 80-90 nm in diameters. Corresponding hydrodynamic
diameters of the An-TPA NPs and the An-Cz-Ph NPs were determined to be 78.1 and 93.3 nm by
DLS measurements (Figures 2d, h). The small size below 100 nm of the two TADF NPs is not only
beneficial for their uptake in tumor cells, but also in favor of the efficient accumulation at the tumor
sites due to the enhanced permeability and retention (EPR) effect in future in vivo study, which
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would greatly boost their practical PDT and fluorescence imaging. Both samples have PDI values
below 0.2 suggesting their excellent monodispersibility. Besides, the as-prepared NPs also show
good water-dispersibility (Insets of Figures 2d, h).
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We also measured absorption and emission spectra of the free TADF molecules dissolved in
tetrahydrofuran (THF) and the TADF NPs dispersed in deionized water respectively. As depicted in
Figures 2e, i, the normalized absorbance profiles (dotted lines) of both TADF NPs exhibit a range of
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00-600 nm with a red-shift of ~20-30 nm at the absorption edges comparing to that of their
corresponding free TADF molecules, which are attributed to the strong intermolecular π-π
interactions within the TADF NPs. Insets of Figures 2e, i are respectively photographs of the
An-TPA or An-Cz-Ph molecules dissolved in THF (left) and the An-TPA or An-Cz-Ph NPs
dispersed in deionized water under UV light (right), showing that TADF NPs display an obvious red
or orange emission. Interestingly, compare with the An-Cz-Ph, the An-TPA is a typical
aggregation-induced emission (AIE) based molecule with a flexible TPA group, leading to strong
non-radiative transition via molecular vibration in solvent. Therefore, the An-TPA compound is
non-emissive in THF but become highly emissive upon NP formation because the restriction of
intramolecular rotations, which is in favor of the bioimaging application of the as-prepared TADF
NPs. Moreover, the photoluminescence quantum yield (PLQY) of the An-TPA and An-Cz-Ph NPs
dispersed in water were respectively determined to be ~3.7% and ~0.8% by an absolute method
using a fluorescence spectrometer (FLS920P, Edinburgh Instruments) equipped with an integrating
sphere. The higher PLQY of the An-TPA NPs might contribute to a lower singlet oxygen quantum
yield since fluorescence emission competes with the singlet oxygen generation through intersystem
crossing upon photo-excitation.
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Figure 2. a) Schematic illustration of preparing the TADF NPs by self-assembly for two-photon activated cellular
fluorescence imaging (TPA imaging) and mitochondria-specific ROS production; SEM images and corresponding
TEM images of the An-TPA NPs (b, c) and An-Cz-Ph NPs (f, g); Dynamic light scattering (DLS) and
polydispersity index (PDI) measurement of the d) An-TPA NPs and h) An-Cz-Ph NPs (The insets are photographs
of the TADF NPs dispersed in deionized water under room light); e) Normalized absorbance and fluorescence
spectra of free An-TPA molecules in THF and An-TPA NPs dispersion in water respectively (The insets are
corresponding photographs of the An-TPA molecules (left) and the An-TPA NPs dispersion under UV light (right));
i) Normalized absorbance and fluorescence spectra of free An-Cz-Ph molecules in THF and An-Cz-Ph NPs
dispersion in water respectively (The insets are corresponding photographs of the An-Cz-Ph molecules (left) and
the An-Cz-Ph NPs dispersion under UV light (right))
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.3. Single-photon excited (SPE) and two-photon excited (TPE) cellular imaging of the
TADF NPs. Two-photon activated PDT has been recently proposed to expand the PDT’s biomedical
applications, wherein two NIR photons instead of one visible photon are applied for PS
excitation.28–31 Penetration depth of the commonly applied visible light is very low while two-photon
activated PDT exhibits not only better penetration depth but also spatial selectivity. To investigate
the cellular imaging capability of the TADF NPs, both single-photon excited (SPE) and two-photon
excited (TPE) fluorescence imaging were conducted individually with fluorescence microscopy as
well as confocal laser scanning microscopy (CLSM) in A549 cell. For the SPE fluorescence imaging
experiments, TADF NPs were excited at 380-420 nm. As exhibited in Figure 3, intense red
fluorescence from both An-TPA and An-Cz-Ph NPs in cytoplasm were clearly detected. Additionally,
CLSM Z-stack scanning mode images from the top to the bottom of A549 cell incubated with these
NPs as shown in Figure S4, demonstrating that the TADF NPs can be successfully taken up into the
cells rather than just being attached on the surfaces of cancer cells.
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Figure 3. Single-photon excited (SPE) cellular imaging of the TADF NPs in A549 human lung cancer cells,
detected by fluorescence microscopy. (a) Bright field channel; (b) DAPI channel (excitation wavelength: 330-380
nm); (c) TADF NPs channel (excitation wavelength: 380-420 nm); (d) Merge channel. Scale bar is 50 μm. Upper:
An-TPA NPs group; Lower: the An-Cz-Ph NPs group.
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Next, before the TPE cellular imaging experiments, two-photon optical properties of the
as-prepared TADF NPs were evaluated by using a femtosecond (fs) laser pulse. The log-log plots of
the detected TPE fluorescence emission intensity of the An-TPA NPs (Figure 4a) and the An-Cz-Ph
NPs (Figure 4c) versus the excitation laser power intensity displayed slopes of 1.85 and 1.93
respectively. These near quadratic relationship between TPE emission intensity and input 800 nm
excitation laser power firstly certified the TPE nature of the TADF NPs. Furthermore, the
two-photon processes were also confirmed by the strong two-photon fluorescence of the TADF NPs
at an excitation wavelength of 800 nm. TPE fluorescence spectra of the An-TPA NPs (Figure 4b)
and the An-Cz-Ph NPs (Figure 4d) are respectively similar to that of their corresponding SPE
fluorescence spectra (Figures 2e, i). Apart from 800 nm fs laser excitation, the two-photon
characteristics of the TADF NPs were also demonstrated under excitation with an 880 nm fs laser
(Figure S5).
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Figure 4. Input excitation power dependence curves of two-photon excited (TPE) emission intensity at a) 653 nm
of An-TPA NPs and c) 612 nm of An-Cz-Ph NPs under 800 nm fs excitation; The corresponding normalized TPE
emission spectra of b) An-TPA NPs and d) An-Cz-Ph NPs under 800 nm fs excitation; e) Cellular TPE CLSM
images of the TADF NPs, monitored with excitation wavelength of 800 nm in A549 human lung cancer cells; f)
SPE and TPE Z-stack CLSM images of A549 3D multicellular tumor spheroids (MCTSs) treated with An-TPA
NPs and An-Cz-Ph NPs.
After confirming their two-photon properties, cellular TPE CLSM images of the TADF NPs were
acquired under excitation at 880 nm fs laser in A549 cells. As exhibited in Figure 4e, strong red
signals can be detected in cells after incubation with both An-TPA NPs and the An-Cz-Ph NPs. To
further prove the superiority of the TPE fluorescence imaging, SPE and TPE Z-stack CLSM images
of A549 3D multicellular tumor spheroids (MCTSs) respectively treated with An-TPA NPs and
An-Cz-Ph NPs were captured. It has been well-documented that the heterogeneous 3D MCTSs,
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exhibiting similar histological characteristics to the solid tumor nidus, have commonly been used to
assess the performance of nano-biomaterials. As shown in Figure 4f, compared with the SPE images,
the TPE-based Z-stack imaging modes displayed deeper penetration depths in A549 MCTS
incubated with either An-TPA NPs or An-Cz-Ph NPs, owing to the long two-photon excitation
wavelength at 800 nm. While in the case of the SPE modes, only the periphery of the MCTS
exhibited relatively strong fluorescence at a much lower penetration depth. The corresponding
quantifications of TADF fluorescence intensities further confirmed these results (Figure S6).
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water-dispersible TADF NPs, we employed the disodium salt of 9,10-anthracenedipropionicacid
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(ADPA) as a water-soluble O sensor by recording the absorption degradation at 378 nm upon
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exposure to O . Time-dependent loss of ADPA absorption caused by the An-TPA NPs, the
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An-Cz-Ph NPs and DI water were respectively illustrated in Figures 5a-c. Figure 5d summarizes
the bleaching results in ADPA absorption at 378 nm of different groups as a function of the light
exposure time. Both the An-TPA NPs and the An-Cz-Ph NPs clearly displays that as the irradiation
duration increases, absorption intensities of ADPA decrease significantly while the control group
(only ADPA incubated in DI water without TADF NPs) shows negligible bleaching of ADPA. These
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results prove that the two TADF NPs are potential PS for PDT. Subsequently, we measured the O
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Rose Bengal ( O quantum yield = 75% in water) as the standard reference. As presented in Figure
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S7, the An-TPA NPs and the An-Cz-Ph NPs dispersions exhibited high O quantum yield which
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determined to be 22 % and 52 %, respectively. These yields are much higher than that of our
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Figure S2). Therefore, the exciton populating time in triplet-excited state of An-TPA is much longer
than that in An-Cz-Ph, implying that the oxygen molecules at group state have higher probability to
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interact with triplet exciton of An-TPA for higher singlet oxygen quantum yield.
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images of live A549 cells treated with different groups, where 2,7-dichlorofluorescein diacetate
(DCFH-DA) was used as the ROS indicator which would emit the green fluorescence in the presence
of endogenous ROS (Figure 5e). Live A549 cells were pretreated with the An-TPA NPs or the
An-Cz-Ph NPs and incubated with DCFH-DA. Before light irradiation, the cells show only red
fluorescence from the NPs and green signal from ROS can only be observed after irradiation
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confirming intracellular generation of O .
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Intracellular ROS analysis by CLSM images of live A549 human lung cancer cells treated with different groups
Ex: 488 nm; Em: 640-660 nm for TADF NPs; 510-525 nm for ROS indicator).
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.5. Cancer-mitochondria-targeting capabilities. To optimize the efficacy of PDT, subcellular
targeting strategy has drawn extensive attention in PDT treatment since it enables the PSs to target
specific subcellular organelles such as nuclei,32,33 lysosomes,34,35 plasma membrane36,37 and
mitochondria38–41 which are more vulnerable to ROSs. Most particularly, mitochondria which are the
“powerhouse” of the cell, have been recognized as a promising therapeutic target for highly efficient
PDT cancer treatment. As one of the most crucial subcellular organelles in organisms, mitochondria
undertake various critical biological functions in cells, including energy supply, redox status
maintenance and molecular metabolism.40 Mitochondrial dysfunction could induce
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To demonstrate the efficient mitochondria-targeting capabilities of the as-prepared TADF NPs, we
firstly investigated the intracellular colocalization of the two TADF NPs with a commercial
mitochondrion-staining agent MitoTracker Green and a lysosome-staining agent LysoTracker Green
in A549 cancer cells, respectively. As shown in Figure 6, in the merge channels, it is found that the
red signals from the TADF NPs merges well with the green fluorescence from MitoTracker.
However, in the case of lysosome co-localization images, these two signals exhibited poor overlap.
Furthermore, the Pearson’s colocalization coefficients which is used to assess the overlap efficiency
between the TADF NPs’ red emission and MitoTracker’s or LysoTracker’s green emission, are
calculated to be above 0.9 (0.93 for the An-TPA NPs, 0.95 for the An-Cz-Ph NPs) and 0.5 (0.55 for
the An-TPA NPs, 0.53 for the An-Cz-Ph NPs), respectively. The corresponding fluorescence
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topographic profiles displaying the fluorescence intensity along the white lines marked in TADF NP
channel (red) and organelle-tracker channel (green), further indicate that fluorescence of the TADF
NPs overlap well with that of MitoTracker instead of LysoTracker. Meanwhile, we propose the
possible reason why the TADF NPs show preferable mitochondria-targeting properties is probably
because of the high lipophilicity stemming from the five phenyl groups in their molecular
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Figure 6. a) Representative mitochondria co-localization images of the An-TPA NPs and the An-Cz-Ph NPs by
CLSM in A549 human lung cancer cells, where r indicates Pearson’s colocalization coefficient; d) Representative
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lysosome co-localization images of the An-TPA NPs and the An-Cz-Ph NPs by CLSM in A549 cells, where rp
indicates Pearson’s colocalization coefficient. Fluorescence topographic profiles display fluorescence intensity
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Subsequently, to further investigate their cancer-mitochondria-targeting capabilities, we compared
the intracellular mitochondrial localization of the TADF NPs in two cancer cell lines (A549 or Hela
cancer cells) and two normal cell lines (NIH 3T3 or MCR-5 normal cells). As exhibited in Figure 7,
for both A549 and Hela cells, confocal microscopy analysis indicates that the red fluorescence
signals are well-overlapped with the green signals from the MitoTracker Green. It is interesting, the
mitochondrial targeting characteristic of the two NPs are much less obvious in the two normal cell
lines (Figures 7c and d). Particularly, we also observed that while almost all NPs are successfully
internalized into the cancer cells, the uptake in normal cells appears to be much less effective as
indicated by the red fluorescence outside the cells (marked with white dashed regions in figures 7c
and d). Therefore, the mitochondria targeting ability of the TADF NPs is dependent on cell type in
which the TADF NPs exhibit better selectivity for mitochondria in cancer cells compared to normal
cells, showing great promise as a powerful tool for PDT in cancer treatments. Meanwhile, the
cancer-mitochondria-targeting mechanisms of these TADF NPs should be further systematically
studied and clarified in future works.
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Figure 7. Representative mitochondria colocalization images of the An-TPA NPs and the An-Cz-Ph NPs by CLSM
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in cancer cells (A549 or Hela cancer cells) and normal cells (NIH 3T3 or MCR-5 normal cells).
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that PDT effect on mitochondria would cause mitochondrial dysfunction as well as lead to cell
apoptosis. Loss of mitochondrial membrane potential (MMP) is a typical characteristic of
mitochondrial damage. Therefore, to monitor the mitochondrial dysfunction in cancer cells, a
membrane-permeable JC-1 dye was selected to evaluate the MMP changes. As described in Figure
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a, JC-1 dye can undergo a reversible fluorescence change between its aggregate (red; healthy cells
with high MMP) and monomer (green; apoptotic cells with low MMP) states to observe the
mitochondrial depolarization process. Representative CLSM images of JC-1 stained A549 cells after
different treatments are shown in Figure 8b. Obviously, in the TADF NPs incubated cells, the green
fluorescence signals increased while the red fluorescence signals decreased upon light irradiation,
demonstrating that the generated ROS would induce the loss of MMP and dysfunction of
mitochondria. In contrast, only light irradiation without NPs as well as the uptake of TADF NPs in
mitochondria darkly do not depolarize the mitochondrial membrane, as proved by the weak green
signals and intense red signals of JC-1 dye. These results further suggested the mitochondria
targeting ability of the as-fabricated TADF NPs which can induce mitochondria-mediated apoptosis.
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Figure 8. a) Schematic illustration of a membrane-permeable JC-1 dye to observe mitochondrial depolarization
where JC-1 dye exhibits reversible fluorescence between its aggregate (red; healthy cells) and monomer (green;
apoptotic cells) states; b) CLSM images of JC-1 stained A549 cells after different treatments. The increase of green
fluorescence in the light irradiated cells added TADF NPs indicated a loss of mitochondrial membrane potential
(MMP).
Finally, we measured the in vitro cytotoxicities of the An-TPA NPs and the An-Cz-Ph NPs with or
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without an ultralow intensity white light irradiation (28 mW/cm for 5 min) by using MTT assay. As
presented in Figures 9a, b, the two TADF NPs show dose-dependent cytotoxicities while both NPs
without light (pink and blue columns) and the only light group exhibit low toxicities. The excellent
cancer-cell-killing abilities at such low concertation and upon such ultralow irradiance are attributed
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to the high O quantum yield as well as good mitochondria-specific ROS production. In addition, to
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further track the cytotoxic effect induced by TPE irradiation, morphology changes of live A549 cells
were observed by CLSM under TPE excitation with an 880 nm fs laser. As shown in Figure 9c, with
the irradiation time extended from 0 to 15 min, the morphology of live A549 cells pretreated with the
TADF NPs changed dramatically where cell shrinkage and numerous blebs were notably found. For
comparison, the cells without NPs exhibit negligible change under the same laser irradiation.
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Figure 9. In vitro cytotoxicity of a) the An-TPA NPs and b) the An-Cz-Ph NPs with or without white light
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irradiation (28 mW/cm for 5 min); c) TPE fluorescence images of A549 cells with or without TADF NPs, and the
corresponding bright field, time-dependent (0-15 min) CLSM images. The red dotted arrows indicate membrane
blebs.
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. CONCLUSION
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In conclusion, for the first time, we developed two intrinsically cancer-mitochondria-targeted
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TADF NPs for two-photon activated fluorescence imaging and PDT. The as-prepared TADF NPs
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provided following merits: 1) satisfactory O quantum yield from 22% to 52%, which are much
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higher than that of previously reported TADF NPs (10-15%); 2) sufficient light penetration depth
due to two-photon activation to boost the fluorescence imaging as well as PDT performances; 3)
excellent intrinsically mitochondria targeting capabilities without the need of extra chemical or
physical modifications, induced a mitochondria-mediated apoptosis; meanwhile, these two TADF
NPs also exhibited better selectivity for mitochondria in cancer cells compared to normal cells,
showing great promise as a powerful tool for PDT; 4) remarkable cancer-cell-killing abilities upon
an ultralow white light irradiance as well as two-photon activated excitation with an 880 nm fs laser.
Besides, since the TADF NPs can cause mitochondria dysfunction which would be potentially used
to induce immunogenic cell death, the TADF NPs would also be explored to achieve synergistic
therapeutic outcome integrating PDT with cancer immunotherapy in our future study. Nevertheless,
the intrinsically cancer-mitochondria-targeting mechanisms as well as surface modification for
further functionalization should be further systematically studied in our future works. All in all, we
believe such multifunctional TADF NPs based on a single molecule will open new opportunities for
the development of novel PDT agents for cancer diagnosis and therapy.
ASSOCIATED CONTENT
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Supporting Information. Description of materials and methods and Supporting Figures:
Figure S1) Photographs of the An-TPA powder, An-Cz-Ph powder and the FITC powder taken
(
under room light (upper row) and UV light (lower row), (Figure S2) Steady-state fluorescence
spectra, phosphorescence spectra and fluorescence lifetime of An-TPA and An-Cz-Ph, (Figure S3)
Steady-state fluorescence spectra, phosphorescence spectra and fluorescence lifetime of An-TPA and
An-Cz-Ph, (Figure S4) CLSM Z-stack scanning mode images of A549 cell incubated with the
An-TPA NPs and An-Cz-Ph NPs, (Figure S5) Input excitation power dependence of TPE emission
intensity and the normalized TPE emission spectra of An-TPA NPs and An-Cz-Ph NPs under 880
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nm fs excitation, (Figure S6) The mean fluorescence intensities of SPE and TPE Z-stack CLSM
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images of A549 3D MCTSs, (Figure S7) Chemical trapping measurements of the O quantum yield.
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This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
E-mail: lishengliang@iccas.ac.cn
* E-mail: apcslee@cityu.edu.hk
ORCID
Chun-Sing Lee: 0000-0001-6557-453X
Shengliang Li: 0000-0002-3890-8482
Jinfeng Zhang: 0000-0002-1899-888X
Wen-Cheng Chen: 0000-0003-3788-3516
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
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C.S.L. would like to thank financial support by City University of Hong Kong (CityU Internal Funds
for External Grant Schemes Project No. 9678157 and CityU Applied Research Grant Project no.
9667160). J.Z. would like to acknowledge financial support by Beijing Institute of Technology
Research Fund Program for Young Scholars and the National Natural Science Foundation of China
(Project No. 91859123).
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REFERENCES
[
1] Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic Therapy and Anti-Tumour Immunity.
Nat. Rev. Cancer 2006, 6, 535–545.
[
2] Lovell, J. F.; Liu, T. W. B.; Chen, J.; Zheng, G. Activatable Photosensitizers for Imaging and
Therapy. Chem. Rev. 2010, 110, 2839–2857.
[3] Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015,
15, 1990–2042.
1
[
4] Zhang, J.; Liang, Y.-C.; Lin, X.; Zhu, X.; Yan, L.; Li, S.; Yang, X.; Zhu, G.; Rogach, A. L.; Yu,
P. K. N.; Shi, P.; Tu, L. C.; Chang, C. C.; Zhang, X.; Chen, X.; Zhang, W.; Lee, C. S.
Self-Monitoring and Self-Delivery of Photosensitizer-Doped Nanoparticles for Highly Effective
Combination Cancer Therapy in Vitro and in Vivo. ACS Nano 2015, 9, 9741–9756.
5] Li J.; Pu K. Development of Organic Semiconducting Materials for Deep-Tissue Optical
Imaging, Phototherapy and Photoactivation. Chem. Soc. Rev. 2019, 48, 38-71.
6] Jiang Y.; Pu K. Multimodal Biophotonics of Semiconducting Polymer Nanoparticles. Acc. Chem.
Res. 2018, 51, 1840-1849.
[
[
2
6
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Page 28 of 32
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1
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2
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3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[7] Cui D.; Huang J.; Zhen X.; Li J.; Jiang Y.; Pu K. A Semiconducting Polymer Nano-prodrug for
Hypoxia-Activated Photodynamic Cancer Therapy. Angew. Chem. Inter. Ed. 2019, 58, 5920.
[
[
[
8] Zhu H.; Li J.; Qi X.; Chen P.; Pu K. Oxygenic Hybrid Semiconducting Nanoparticles for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Enhanced Photodynamic Therapy. Nano Lett. 2018, 18, 586-594.
9] Li, J.; Huang, J.; Lyu, Y.; Huang, J.; Jiang,Y.; Xie, C.; Pu. K. Photoactivatable Organic
Semiconducting Pro-nanoenzymes. J. Am. Chem. Soc. 2019, 141, 4073-4079.
10]Xu, S.; Yuan, Y.; Cai, X.; Zhang, C.-J.; Hu, F.; Liang, J.; Zhang, G.; Zhang, D.; Liu, B. Tuning
the Singlet-Triplet Energy Gap: A Unique Approach to Efficient Photosensitizers with
Aggregation-Induced Emission (AIE) Characteristics. Chem. Sci. 2015, 6, 5824–5830.
11]Guo, Z.; Zou, Y.; He, H.; Rao, J.; Ji, S.; Cui, X.; Ke, H.; Deng, Y.; Yang, H.; Chen, C.; Zhao, Y.;
Chen, H. Bifunctional Platinated Nanoparticles for Photoinduced Tumor Ablation. Adv. Mater.
[
2
016, 28, 10155–10164.
[
12]Wang, D.; Lee, M. M. S.; Shan, G.; Kwok, R. T. K.; Lam, J. W. Y.; Su, H.; Cai, Y.; Tang, B. Z.
Highly Efficient Photosensitizers with Far-Red/Near-Infrared Aggregation-Induced Emission for
In Vitro and In Vivo Cancer Theranostics. Adv. Mater. 2018, 30, 1802105.
[13]Wu, W.; Mao, D.; Xu, S.; Kenry; Hu, F.; Li, X.; Kong, D.; Liu, B. Polymerization-Enhanced
Photosensitization. Chem 2018, 4, 1937–1951.
[
[
[
14]Chi, Y.; Chou, P.-T. Transition-Metal Phosphors with Cyclometalating Ligands: Fundamentals
and Applications. Chem. Soc. Rev. 2010, 39, 638–655.
15]Zhao, J.; Wu, W.; Sun, J.; Guo, S. Triplet Photosensitizers: From Molecular Design to
Applications. Chem. Soc. Rev. 2013, 42, 5323–5351.
16]Jiang, X.; Peng, J.; Wang, J.; Guo, X.; Zhao, D.; Ma, Y. Iridium-Based High-Sensitivity Oxygen
2
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Page 29 of 32
ACS Applied Materials & Interfaces
1
2
3
4
5
6
7
8
9
Sensors and Photosensitizers with Ultralong Triplet Lifetimes. ACS Appl. Mater. Interfaces 2016,
8
, 3591–3600.
[
17]Stohs, S. J.; Bagchi, D. Oxidative Mechanisms in the Toxicity of Metal Ions. Free Radic. Biol.
Med. 1995, 18, 321–336.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
18]Lim, S. H.; Thivierge, C.; Nowak-Sliwinska, P.; Han, J.; van den Bergh, H.; Wagnières, G.;
Burgess, K.; Lee, H. B. In Vitro and in Vivo Photocytotoxicity of Boron Dipyrromethene
Derivatives for Photodynamic Therapy. J. Med. Chem. 2010, 53, 2865–2874.
[
19]Uoyama, H.; Goushi, K.; Shizu, K.; Nomura, H.; Adachi, C. Highly Efficient Organic
Light-Emitting Diodes from Delayed Fluorescence. Nature 2012, 492, 234–238.
20]Goushi, K.; Yoshida, K.; Sato, K.; Adachi, C. Organic Light-Emitting Diodes Employing
Efficient Reverse Intersystem Crossing for Triplet-to-Singlet State Conversion. Nat. Photonics
[
2
012, 6, 253–258.
[
21]Liu, X.-K.; Chen, Z.; Zheng, C.-J.; Liu, C.-L.; Lee, C.-S.; Li, F.; Ou, X.-M.; Zhang, X.-H.
Prediction and Design of Efficient Exciplex Emitters for High-Efficiency, Thermally Activated
Delayed-Fluorescence Organic Light-Emitting Diodes. Adv. Mater. 2015, 27, 2378–2383.
[22]Liu, X.-K.; Chen, Z.; Qing, J.; Zhang, W.-J.; Wu, B.; Tam, H. L.; Zhu, F.; Zhang, X.-H.; Lee,
C.-S. Remanagement of Singlet and Triplet Excitons in Single-Emissive-Layer Hybrid White
Organic Light-Emitting Devices Using Thermally Activated Delayed Fluorescent Blue Exciplex.
Adv. Mater. 2015, 27, 7079–7085.
[
23]Xiong, X.; Song, F.; Wang, J.; Zhang, Y.; Xue, Y.; Sun, L.; Jiang, N.; Gao, P.; Tian, L.; Peng, X.
Thermally Activated Delayed Fluorescence of Fluorescein Derivative for Time-Resolved and
Confocal Fluorescence Imaging. J. Am. Chem. Soc. 2014, 136, 9590–9597.
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