Two birds with one stone: a NIR fluorescent probe for mitochondrial protein imaging and its application in photodynamic therapy

Article abstract of DOI:10.1039/d1tb00881a

Mitochondrial proteins, most of which are encoded in the nucleus and the rest of which are regulated by the mitochondrial genome, play pivotal roles in essential cellular functions. However, fluorescent probes that can be used for monitoring mitochondrial proteins have not yet been widely developed, thereby severely limiting the exploration of the functions of proteins in mitochondria. Towards this end, here we propose a near-infrared (NIR) fluorescence probeMPPto effectively illuminate the dynamic changes in mitochondrial proteins in live cells under oxidative stress, with excellent temporal and spatial resolution. Of particular importance,MPPextends the study of the pharmacology involved in apoptosis induced by anti-cancer drugs (hydroxycamptothecin (HCPT), epirubicin (Epi) and cyclophosphamide (CPA)) for the first time. Furthermore, employing a protein-activatable strategy, this probe could serve as an excellent phototherapeutic agent in photodynamic therapy (PDT). Finally,in vivoexperiments suggest that this versatile probe can be used to image tumors in HeLa tumor-bearing mice for 24 h, which demonstrates that our probe could play a dual role as a robust phototherapeutic and imaging agent.

Full text of DOI:10.1039/d1tb00881a

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Two birds with one stone: a NIR fluorescent
probe for mitochondrial protein imaging and
its application in photodynamic therapy†
Cite this: J. Mater. Chem. B, 2021,
9, 6068
Ya-Lin Qi,‡ Long Guo,‡ Li-Li Chen, Dan-Dan Yuan, Hai-Rong Wang, Yu-Yao Cao,
Yu-Shun Yang* and Hai-Liang Zhu
*
Mitochondrial proteins, most of which are encoded in the nucleus and the rest of which are regulated
by the mitochondrial genome, play pivotal roles in essential cellular functions. However, fluorescent
probes that can be used for monitoring mitochondrial proteins have not yet been widely developed,
thereby severely limiting the exploration of the functions of proteins in mitochondria. Towards this end,
here we propose a near-infrared (NIR) fluorescence probe MPP to effectively illuminate the dynamic
changes in mitochondrial proteins in live cells under oxidative stress, with excellent temporal and spatial
resolution. Of particular importance, MPP extends the study of the pharmacology involved in apoptosis
induced by anti-cancer drugs (hydroxycamptothecin (HCPT), epirubicin (Epi) and cyclophosphamide
(CPA)) for the first time. Furthermore, employing a protein-activatable strategy, this probe could serve as
an excellent phototherapeutic agent in photodynamic therapy (PDT). Finally, in vivo experiments suggest
that this versatile probe can be used to image tumors in HeLa tumor-bearing mice for 24 h, which
demonstrates that our probe could play a dual role as a robust phototherapeutic and imaging agent.
Received 19th April 2021,
Accepted 6th July 2021
DOI: 10.1039/d1tb00881a
rsc.li/materials-b
oxidation, the urea cycle, the antioxidant response system, etc.7 In
particular, mutated mitochondrial proteins could lead to damage to
Introduction
It is well known that mitochondria play vital roles in generating the mitochondria.8 Furthermore, there is increasing evidence to
adenosine triphosphate (ATP) to support the entire lifecycle of demonstrate that mitochondrial proteins are closely associated with
biological cells and are crucial for cellular life, death, aging and maternally inherited disorders, aging and neurodegenerative
differentiation.1 Furthermore, they also generate reactive oxi- diseases.9 On the other hand, it is noteworthy that at least 70% to
dative species (ROS) to actively trigger intracellular signaling 80% of drugs exert their efficacy by acting on these membrane/
pathways.2 In the meantime, mitochondrial dysfunction and hydrophobic proteins.10,11 Therefore, understanding the dynamic
damage are tightly linked to more than 50 types of serious behavior of mitochondrial proteins and monitoring their related
diseases, including neonatal fatalities, cancer and type II physiological procedures inside living cells is essential in cell
diabetes.3 Thus, it is of great significance to unfold the complex biology, physiology and pharmacology. Up to now, several
roles mitochondria play in living cells.
approaches have been developed for detecting mitochondrial pro-
On the other hand, mitochondria are organelles with a double- teins, involving immunoblot assay, mass spectrometry, NMR
layer membrane structure.4 Mitochondrial biogenesis and function spectroscopy and immunofluorescence.1,11,12 Nonetheless, these
are closely linked to membrane proteins with strong hydrophobicity typical methods are inconvenient, costly, and incapable of providing
integrated on the inner and outer membranes.5 Mitochondria may real-time information regarding mitochondrial proteins in live cells.
contain up to 1500 distinct proteins, 13 kinds of which are encoded Thus, an efficient approach for precisely and vividly detecting
by the mitochondrial genome, the rest of which must be imported mitochondrial protein dynamics is urgently needed.
after being synthesized as precursors on cytosolic ribosomes.1,6
With operational simplicity and high spatiotemporality,
These proteins are crucial in oxidative phosphorylation, fatty acid fluorescence imaging has attracted great interest in imaging
small biomolecules in vitro and in vivo.13 Recently, Zhang et al.
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
reported a versatile fluorescent probe for visualizing mitochon-
drial nucleoprotein dynamics via super-resolution imaging.14
Nanjing University, Nanjing, 210023, P. R. China. E-mail: ys_yang@nju.edu.cn,
zhuhl@nju.edu.cn
Nevertheless, the functions of mitochondrial proteins in their work
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
was not further explored. On the other hand, with great biocom-
patibility, optimal biosafety, desirable therapeutic efficiency and so
d1tb00881a
‡ These authors contributed equally to this work.
6068 | J. Mater. Chem. B, 2021, 9, 6068–6075
This journal is © The Royal Society of Chemistry 2021

View Article Online
Paper
Journal of Materials Chemistry B
on, organic small molecules have been recognized as promising albumin (HSA) could serve as a model for the mitochondrial
agents in photodynamic therapy (PDT).15,16 However, only a very proteins.14 The test solutions of MPP (10 mM) containing
few protein-activatable PDT theranostic molecules have been different concentrations of HSA in phosphate-buffered saline
proposed.17,18
(PBS) buffer (10 mM, pH 7.4, 1% MeCN) were kept at 37 1C for
Herein, we propose a novel NIR fluorescent probe (named 2 min.
MPP) to visualize the dynamic changes in mitochondrial pro-
teins and then participate in PDT. This versatile fluorescent
Results and discussion
Molecular design and synthesis
probe was successfully applied for deciphering the crosstalk
between H2O2 and mitochondrial proteins in live cells. More-
over, facilitated by MPP, the pharmacological mechanisms
between the three anti-cancer drugs (HCPT, Epi and CPA) and
the corresponding proteins have been directly elucidated for
the first time. Significantly, this mitochondrial protein respon-
sive theranostic probe shows good potential in PDT. Finally,
MPP possesses remarkable tumor retention properties in vivo.
Accordingly, our work presents a novel multifunctional fluor-
escent probe which not only enabled highly accurate monitor-
ing of mitochondrial proteins, but also led to image-guided
photodynamic cancer cell ablation.
In the probe MPP, a triphenylamine (TPA) moiety was selected
as an electron-donating (D) unit and this hydrophobic part
rendered a fluorescent probe with an extended p conjugation
structure.19,20 Additionally, the TPA group tended to rotate in
the excitation state which endowed MPP with prominent
twisted intramolecular charge transfer (TICT) properties21 and
provided a large potential hydrophobic binding site for the
hydrophobic protein.22 In particular, a cationic quinolinium
group, allowing MPP to accumulate in mitochondria, was
selected as an electron-accepting (A) unit. In addition, a lipo-
philic p-conjugated thiophene group23 and a carbon–carbon
double bond worked as a p-bridge, which increased the
conjugation length of the platform. With its positive charge,
1-ethylquinolin-1-ium could significantly increase the localiza-
Experimental
Materials and equipment
All chemicals were available commercially and the solvents tion in mitochondria.24 Therefore, we hypothesized that MPP,
used in the spectral measurement are of chromatographic as a multifunctional probe, could be applied for imaging the
grade. The 1H and 13C NMR spectra were determined using mitochondrial proteins based on TICT effects and act as a
Bruker DRX-600 and 400 instruments. Fluorescence measure- robust PS in situ. The synthetic route for MPP is depicted in
ments and UV-vis spectra were performed on a Hitachi Fluores- Scheme 1. The chemical structures of MPP and the intermedi-
cence Spectrophotometer F-7000 and Shimadzu UV-2550 ates were fully confirmed by 1H NMR, 13C NMR, and HRMS
spectrometer, respectively. The mass spectra were obtained (Fig. S1–S7, ESI†).
from the Mass Spectrometry Facility at Nanjing University.
Optical properties of MPP and mechanism
In vitro and in vivo imaging were obtained by the live imager
of small animals (IVIS Lumina XR, Nanjing University). Mito-
tracker (MitoTrackers Deep Red FM (MTDR)), Lyso-tracker
(LysoBlue), ER-tracker (ER-Tracker Blue-White DPX) and
Nucleus-tracker (Hoechst 33342) were available commercially.
The imaging experiments were carried out with a confocal
fluorescence microscope (Zeiss LSM 980 with Airyscan 2,
Zeiss LSM 880 with Airyscan and Olympus FV1000, Nanjing
University).
As expected, MPP (10 ꢀ 10ꢁ6 M) was initially non-fluorescent
when excited at 526 nm owing to the TICT effect, which
remarkably quenched its fluorescent emission (Fig. 1A). How-
ever, after the addition of HSA (8 mg mLꢁ1) to the above
solution, an obviously red emission peak at 659 nm was
observed. Meanwhile, there was an absorption band shift from
495 to 515 nm, with a very large Stokes shift (Dl = 144 nm),
which ensured minimum interference induced by the excita-
tion light (Fig. 1B). In addition, the fluorescence quantum yield
(F) of MPP was determined to be 0.0034; after incubation with
Preparation of the test solution
A stock solution of the probe MPP (1.0 ꢀ 10ꢁ3 M) was prepared HSA (8 mg mLꢁ1) the value rose to 0.024. The fluorescent
in acetonitrile. With hydrophobic pockets, human serum response over various pH buffers showed that MPP was clearly
Scheme 1 The synthesis route of MPP.
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 6068–6075 | 6069

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 1 Response of MPP toward HSA. (A) The fluorescence spectra of MPP (10 ꢀ 10ꢁ6 M) incubated with or without HSA (8 mg mLꢁ1). (B) The absorption
spectra of MPP (10 ꢀ 10ꢁ6 M) incubated with or without HSA (8 mg mLꢁ1). (C) Fluorescence emission spectra of MPP (10 ꢀ 10ꢁ6 M) incubated with
increasing concentrations of HSA (0–8 mg mLꢁ1) for 2 min. (D) The linear relationship between the fluorescence intensity and the concentration of
HSA (0–1.0 mg mLꢁ1). (E) Fluorescence intensity of MPP (10 ꢀ 10ꢁ6 M) at 659 nm incubated with various biological molecules (2–26 (10 ꢀ 10ꢁ4 M), 27–43
(8 mg mLꢁ1)): (1) = blank, (2) = Al3+, (3) = Ca2+, (4) = Cr3+, (5) = Cu2+, (6) = Cu+, (7) = Fe2+, (8) = Fe3+, (9) = K+, (10) = Mg2+, (11) = Mn2+, (12) = Zn2+
,
,
(13) = CH3COOꢁ, (14) = Clꢁ, (15) = CO32ꢁ, (16) = Fꢁ, (17) = HCO3ꢁ, (18) = HSO3ꢁ, (19) = HSO4ꢁ, (20) = NO3ꢁ, (21) = S2O32ꢁ, (22) = SCNꢁ, (23) = SO4
2ꢁ
(24) = ClOꢁ, (25) = H2O2, (26) = ONOOꢁ, (27) = Tyr, (28) = His, (29) = Glu, (30) = Pro, (31) = Asp, (32) = Arg, (33) = Ser, (34) = Cys, (35) = GSH, (36) = pepsin,
(37) = trypsin, (38) = lysozyme, (39) = hemoglobin, (40) = RNA, (41) = DNA, (42) = BSA, (43) = HSA. (F) Tests for the binding stoichiometry between MPP
and HSA based on fluorescence intensity at 659 nm. MPP and HSA were mixed at different molar ratios while keeping the total concentration constant
(10 mM). The error bars are ꢂstandard deviation (SD) from the average of three independent tests.
constant under physiological conditions (Fig. S8, ESI†). As All the above results implied that MPP had great potential for
shown in Fig. S9 (ESI†), a marked enhancement in fluorescence imaging mitochondrial proteins in living cells, with a rapid
recorded at 659 nm could be clearly obtained after 60 s of response, excellent specificity, high sensitivity and NIR report-
incubation, and the enhancement reached a plateau within ing signals.
200 s. Furthermore, the fluorescence emission intensity with
In order to explore the switch-on mechanism of MPP
increasing concentrations of HSA (0–10 mg mLꢁ1) was also towards HSA, a binding competition experiment was carried
examined (Fig. 1C and Fig. S10, ESI†). Notably, there was a out with phenylbutazone and ibuprofen, two classic molecules
good linear relationship (R2 = 0.9973) with HSA concentrations widely utilized to investigate the interaction with HSA.26,27 The
(0–1.0 mg mLꢁ1) (Fig. 1D). The limit of detection (LOD) of MPP fluorescence emission of MPP decreased when treated with the
was as low as 34.95 mg mLꢁ1 (ca. 514.3 nM) based on the Site I binding drug phenylbutazone, whereas no obvious signal
3s/slope rule.25 These results suggested that MPP shows a fast change at 659 nm was obtained when incubated with the Site II
response to HSA and is highly sensitive toward low concentra- binding drug ibuprofen (Fig. S12, ESI†). These results con-
tions of HSA.
firmed that MPP was included in Site I of HSA. As shown in
In a selectivity test, the strongest response for albumin the Job’s plot analysis, MPP could form a complex with HSA in a
proteins and almost no fluorescence changes for other species, 1 : 1 (molar ratio) binding stoichiometrically (Fig. 1F). Owing to
including inorganic salts, ROS, and amino acids, were the fact that increasing solvent viscosity can greatly impede the
observed, which demonstrated the high specificity of MPP TICT effect,28 the sensing mechanism of probe MPP toward
toward albumin proteins (Fig. 1E). In particular, there is little HSA was examined in methanol–glycerol mixed solvents of
interference from proteins without exposed hydrophobic pock- variable viscosities to verify the existence of the TICT process
ets. The selectivity of MPP toward albumin proteins affected by (Fig. S13, ESI†). With increasing viscosity of the medium, the
inorganic salts, ROS, amino acids and so on was validated in red fluorescence of MPP greatly rose, which implied a strong
the presence of these interference materials, and the results TICT effect. Thus, due to the tight binding of MPP into the
implied that MPP had strong anti-interference ability (Fig. S11, hydrophobic pocket of HSA, the intramolecular rotation was
ESI†). In addition, the hydrophobicity of MPP (log P = 2.129) inhibited and the TICT effect was disrupted within MPP upon
allowed the probe to bind to hydrophobic pockets in proteins. chelation, which finally led to the obvious turn-on red signal.
6070 | J. Mater. Chem. B, 2021, 9, 6068–6075
This journal is © The Royal Society of Chemistry 2021

View Article Online
Paper
Journal of Materials Chemistry B
In vitro cell imaging
first time via the probe MPP. Obviously, when HeLa cells were
pretreated with Epi and HCPT for 60 min and then incubated with
MPP (4 mM) for 60 min, remarkable fluorescence enhancement
was observed compared with the control group (Fig. 4). Therefore,
these results directly suggested that the two agents stimulated the
production of mitochondrial proteins during the process of
apoptosis. However, CPA led to no significant signal change,
which implied that its pharmacological mechanism might be
different from the two above-mentioned drugs. As a whole, this
study provided abundant and important information about the
dynamic changes in the relative expression of mitochondrial
proteins before and after apoptosis induced by various antitumor
agents. More importantly, it lays a solid foundation for further
elucidating the complex roles acted by anti-cancer agents during
apoptosis in proteomics research, and paves a new way for
understanding the pharmacology of anti-cancer drugs in the
future.
Owing to the fact that dithiothreitol (DTT) can degrade protein
structures in live cells, negative experiments were first per-
formed employing DTT.14 The fluorescence of cells sequentially
treated with DTT and MPP was obviously reduced compared
with the control group (Fig. 2). Undoubtedly, the red fluores-
cence came from the proteins in mitochondria.
Next, we further investigated the dynamic changes in mito-
chondrial proteins under oxidative stress. When HeLa cells
were treated with H2O2 (25 mM) for 60 min and then incubated
with MPP (4 mM) for 60 min, the red fluorescence obviously
decreased compared with the control group (Fig. 3). Previous
studies demonstrated that rotenone (Rot) which served as an
inhibitor of the oxidative phosphorylation chains in mitochon-
drial respiration could cause the excess production of
H2O2.29,30 With the stimulation of Rot (20 mM), the red signals
of MPP remarkably deceased, as expected. In contrast, when the
cells were pretreated with Rot for 60 min, then with an ROS
scavenger N-acetyl cysteine (NAC, 5 ꢀ 10ꢁ4 M) for 60 min, and
MPP for 60 min, a relatively low change in fluorescence signal
was obtained compared with a control group which was incu-
bated with MPP alone. The results demonstrated that MPP was
capable of monitoring the dynamic changes in mitochondrial
proteins in living cells and proved that the biosynthesis of
mitochondrial proteins was tightly correlated with oxidative
stress.
In order to evaluate whether MPP could still stain mitochon-
dria with decreased mitochondrial membrane potential (MMP),
a
mitochondrial uncoupling experiment was performed.
Carbonyl cyanide m-chlorophenylhydrazone (CCCP) is widely
utilized to induce rapid acidification of mitochondria to uncou-
ple MMP.36 Live HeLa cells were sequentially incubated with
CCCP and MPP or in the reverse treatment sequence (MPP then
CCCP), and, interestingly, no obvious fluorescence change from
the control group was obtained (Fig. S14, ESI†). Therefore, this
phenomenon clearly confirmed the mitochondria-targeting
ability of MPP and might be illuminated via the good lipid
solubility of MPP as mentioned above and its strong interaction
with mitochondrial proteins. Furthermore, we concluded that
MPP was able to monitor the state of mitochondria under
various MMP concentrations.
It is well known that HCPT, Epi and CPA have been commonly
used as antitumor drugs for a variety of cancers,31–35 but the
relationship between them and mitochondrial proteins at the
molecular and protein level have not yet been clarified. Thus,
herein we made an effort to explore the molecular mechanism
involved in apoptosis induced by those anti-tumor drugs for the
Fig. 2 (A) Negative control adding dithiothreitol DTT (50 mM or 100 mM) to HeLa cells. HeLa cells were first treated with DTT (25 mM and 100 mM) for
60 min, then incubated with MPP (4 mM) for 60 min. (B) Quantification of imaging data. lex = 543 nm, lem = 600–750 nm. The error bars are ꢂstandard
deviation (SD) from the average of five independent tests. Statistical analyses were employed with a two-tailed Student’s t-test with unequal variance,
*p-value o0.05. Scale bar: 20 mm.
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 6068–6075 | 6071

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 3 (A) Fluorescent signals upon the addition of endogenous and exogenous H2O2. (B) Quantification of imaging data. lex = 543 nm, lem = 600–750 nm.
The error bars are ꢂstandard deviation (SD) from the average of five independent tests. Statistical analyses were employed with a two-tailed Student’s t-test
with unequal variance, *p-value o0.05, ***p-value o0.001. Scale bar: 20 mm.
Fig. 4 (A) Characterization of MPP for imaging the effect of anti-tumor drugs on mitochondrial proteins in living HeLa cells. HeLa cells were first treated
with Epi (50 mM or 100 mM), CPA (50 mM or 100 mM) or HCPT (50 mM or 100 mM) for 60 min separately, then incubated with MPP (4 mM) for 60 min.
(B) Quantification of imaging data. lex = 543 nm, lem = 600–750 nm. The error bars are ꢂstandard deviation (SD) from the average of five independent
tests. Statistical analyses were employed with a two-tailed Student’s t-test with unequal variance, *p-value o0.05, **p-value o0.01. Scale bar: 20 mm.
To characterize the intracellular distribution of MPP, HeLa orbital (HOMO) and lowest unoccupied molecular orbital
cells were co-incubated with MPP and various commercial
organelle-targeting probes. As expected, the fluorescence images
from cells treated with MPP mainly overlapped with those of the
Mito-Tracker over Lyso- or ER- or Nucleus-Tracker (Pearson’s
correlation coefficients were 0.78, 0.52, 0.38 and 0.26, respec-
tively), suggesting the major distribution of MPP in mitochondria
(Fig. S15, ESI†). In particular, the Pearson’s correlation coefficient
of MPP in mitochondria not being very high might be caused by
MPP showing granular fluorescent spots in mitochondria.
As shown in Fig. S16 (ESI†), the photostability of MPP was
investigated in living cells. Indeed, MPP performed with even
better photostability than the commercial mitochondria dye
MTDR within 150 s, which implied that the former showed
higher resistance to photobleaching.
(LUMO) distribution and reduction of singlet–triplet energy
gaps (DEST), which effectively promotes ROS generation.37
Furthermore, spin–orbit coupling (SOC) and DEST are key
factors in promoting intersystem crossing (ISC) to increase ROS
production.38 Hence, the SOC effect and DEST were calculated
by time-dependent density functional theory (TD-DFT) at the
B3LYP/6-311G(d) level (Table S1, ESI†). As illustrated in Fig. S17
(ESI†), DES1T2 was determined to be 0.298 eV with an SOC of
0.21 cmꢁ1, which indicated that the ISC process was possible
before T2 to T1 internal conversion (IC) took place. Thus, it was
safe to conclude that MPP was capable of generating ROS with
high efficiency. Then, we evaluated the ROS generation cap-
ability of MPP with 20,70-dichlorodihydrofluorescein diacetate
(H2DCF-DA) upon light illumination (17 W mꢁ2) for 0 min,
5 min and 10 min in PBS for its ability to generate fluorescence
at 525 nm after being oxidized by ROS.39 As shown in
Fig. 5A, solutions of H2DCF-DA (10 mM) containing HSA
(8 mg mLꢁ1) and MPP (10 mM) respectively exhibited no
Evaluation of PDT efficacy
Typically, the strong D–A effect can greatly promote NIR emis-
sion, leading to separation of the highest occupied molecular
6072 | J. Mater. Chem. B, 2021, 9, 6068–6075
This journal is © The Royal Society of Chemistry 2021

View Article Online
Paper
Journal of Materials Chemistry B
2.757 and 20.187 mM to MCF-7 and 293T cells, respectively.
Meanwhile, the values were 8.449 and 22.438 mM toward the
two above-mentioned cell lines in the dark, separately. It was
particularly noteworthy that MPP possessed selective toxicity to
normal cells (293T) and cancer cells (HeLa and MCF-7 cells).
According to these results, we rationally hypothesized that this
result was attributable to the fact that levels of mitochondrial
proteins in HeLa cells might be higher than those in MCF-7
cells and normal cells. Besides, previous reports showed
that carcinoma cells exhibit more negative mitochondrial
membrane potential than normal cells.42,43 Furthermore, we
believed that HeLa cells might have higher negative mitochon-
drial membrane potential than MCF-7 cells. Collectively, owing
to both the high levels of mitochondrial proteins and negative
mitochondrial membrane potential in HeLa cells, MPP pos-
sessed specific phototoxicity toward this type of cells. Thus, this
feature equipped the MPP with desirable biocompatibility and
biosafety as a promising theranostic agent for in vivo applica-
tions. Next, we compared the phototoxicity of MPP and RB to
HeLa cells. As shown in Fig. S20 (ESI†), no obvious phototoxi-
city was observed in HeLa cells after incubation with RB.
Moreover, the IC50 values of RB under light and in the dark
were calculated to be 6.601 and 7.081 mM to HeLa cells,
respectively. Thus, MPP presented higher PDT efficiency
than RB.
Additionally, flow cytometry analysis by Annexin V-FITC and
PI was used to determine cell apoptosis. The percentages of
HeLa cells treated by MPP (10 mM) in the apoptotic region for
0 min, 5 min and 10 min were 5.2%, 10.3% and 39%, respec-
tively (Fig. 6A). In particular, upon light irradiation (17 W mꢁ2),
the apoptotic percentages of HeLa cells incubated with MPP
(4 mM) for 10 min were about 8-fold larger than those of the
control group (Fig. 6B). Furthermore, intracellular ROS detec-
tion was performed by using H2DCF-DA as an indicator.
A strong green signal was obtained in the group incubated
with H2DCF-DA (10 mM) and MPP (4 mM) under light illumina-
tion for 5 min, whereas no noticeable fluorescence signals were
obtained in other groups (Fig. S21, ESI†). As a whole, these data
Fig. 5 ROS generation upon light illumination and the PDT assessment of
MPP. (A) Relative change in fluorescence intensity (I/I0) at 525 nm of
H2DCF-DA, mixtures of H2DCF-DA and MPP, mixtures of H2DCF-DA and
HSA, and mixtures of H2DCF-DA, HSA and MPP in PBS upon light
illumination for various times (0–10 min). Concentrations: 4 mM (MPP)
and 10 mM (H2DCF-DA). (B) Survival rates of HeLa cells stained with various
concentrations of MPP with or without green light illumination for 10 min.
(C) Survival rates of MCF-7 cells incubated with various concentrations of
MPP with or without green light illumination for 10 min. (D) Survival rates of
293T cells incubated with various concentrations of MPP with or without
green light illumination for 10 min. Light power: 17 W mꢁ2. The error bars
are ꢂstandard deviation (SD) from the average of five independent tests.
Statistical analyses were employed with a two-tailed Student’s t-test with
unequal variance, *p-value o0.05, **p-value o0.01, ***p-value o0.001.
apparent fluorescence enhancement, while co-incubation of
HSA and MPP led to a 12-fold enhancement within 6 min,
which indicated that the ROS generation ability of MPP could
be activated by HSA, as we expected, and the ROS generation
was efficient. In addition, the singlet oxygen (1O2) generation
ability of MPP was analysed with 1,3-diphenylisobenzofuran
(DPBF) as the 1O2 trapping agent.40 In the presence of MPP and
HSA under irradiation, the absorbance of DPBF at 425 nm
decreased remarkably, while no significant decline in absor-
bance was found in the presence of HSA (Fig. S18, ESI†).
The singlet oxygen quantum yield (F) of MPP in MeCN/PBS
(v/v = 1/199) was calculated to be 0.52 with Rose Bengal (RB) as
the standard (F = 0.75 in water)41 (Fig. S19, ESI†).
Inspired by the remarkable performance of MPP for ROS
production, we tested three different cell lines, HeLa, MCF-7
and 293T, by standard MTT assay to further test the photo-
therapy efficacy of MPP. As reflected in Fig. 5B and C,
MPP showed substantial phototoxicity upon light illumination
(17 W mꢁ2) to HeLa cells, while slightly lower phototoxicity was
generated to MCF-7 cells. Evidently, negligible biotoxicity and
phototoxicity to 293T normal cells was found with or without Fig. 6 The PDT study of MPP (4 mM) by flow cytometry analysis.
(A) Apoptosis analysis by Annexin V-FITC/PI double staining after light
illumination for different times (0 min, 5 min and 10 min). (B) Relative
green light irradiation (Fig. 5D). Notably, under green light
illumination, the IC50 value (inhibitory concentration of 50%
cell death) of the probe toward HeLa cells was 0.163 mM, which
apoptosis percentage in (A). The error bars were ꢂstandard deviation (SD)
from the average of three independent tests. Statistical analyses were
was 19-fold lower than that of the probe in the dark (3.142 mM).
employed with
a two-tailed Student’s t-test with unequal variance,
And the IC50 values of MPP under light were determined to be ***p-value o0.001.
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 6068–6075 | 6073

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 7 In vivo imaging and cytotoxicity test. (A) Biodistribution of MPP in HeLa tumor-bearing mice after intratumoral injection of MPP (10 mM, 50 mL)
at various times. (B) The radiant efficiency of HeLa tumor-bearing mice after intratumoral injection of MPP (10 mM, 50 mL) at various times in (A).
(C) H&E-staining analysis of organ slices from mice in (A). The mice were sacrificed at 24 h post-injection. Excitation filter: 535 nm; emission filter: DsRed,
575–650 nm. The error bars are ꢂstandard deviation (SD) from the average of three independent tests.
demonstrated that MPP produced a large number of ROS under might pave a new way for the design of novel fluorescent probes
light illumination and showed great potential as a robust agent to unveil the essential roles of mitochondrial proteins in cell
for PDT.
biology and physiology, and provide a promising versatile tool
for imaging-guided precision therapy.
In vivo imaging experiment
Aiming to achieve a versatile probe with integrated functional-
ities of phototheranostics and imaging-guided precision sur-
gery, the ability of tumor imaging by MPP was investigated
in vivo by an imaging experiment. As depicted in Fig. 7A and B,
fluorescence signals reached a peak at 9 h after the injection of
MPP (10 mM), which suggested ideal tumor retention proper-
ties. Moreover, MPP could accumulate at the tumour region
with good specificity at 24 h postinjection (Fig. S22, ESI†). And
H&E-stained organ slices proved the negligible toxicity to
organisms of MPP (Fig. 7C).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors gratefully acknowledge the financial support from
the Major Science and Technology Program for Water Pollution
Control and Treatment, China (No. 2017ZX07602-002). The first
author Ya-Lin Qi is very grateful for the help from Master Li-Li
Chen and Master Hai-Rong Wang.
Conclusions
In conclusion, we have reported a versatile NIR fluorescence
probe, MPP, to track mitochondrial protein activity in live cells
and to participate in PDT. This multifunctional probe was able
to monitor the dynamic changes in mitochondrial proteins
during oxidative stress. Furthermore, using this probe, for the
first time a study of the pharmacology involved in apoptosis
induced by anti-cancer drugs (HCPT, Epi and CPA) was con-
ducted. The results disclosed that HCPT and Epi could promote
the upregulation of mitochondrial proteins, while no obvious
changes were generated by CPA during apoptosis. Meanwhile,
MPP showed great potential as a robust PS with excellent ROS
generation ability. Finally, we successfully applied MPP for
tumor imaging after about 24 h, attesting to its dual function
as an eminent phototherapeutic and imaging agent. This work
References
1 D. J. Pagliarini, S. E. Calvo, B. Chang, S. A. Sheth, S. B. Vafai,
S. E. Ong, G. A. Walford, C. Sugiana, A. Boneh, W. K. Chen,
D. E. Hill, M. Vidal, J. G. Evans, D. R. Thorburn, S. A. Carr
and V. K. Mootha, Cell, 2008, 134, 112–123.
2 J. K. Andersen, Nat. Med., 2004, 10, S18–S25.
3 N. Zhu, G. Xu, R. Wang, T. Zhu, J. Tan, X. Gu and C. Zhao,
Chem. Commun., 2020, 56, 7761–7764.
4 D. Tomasek, S. Rawson, J. Lee, J. S. Wzorek, S. C. Harrison,
Z. Li and D. Kahne, Nature, 2020, 583, 473–478.
5 C. U. Mårtensson, C. Priesnitz, J. Song, L. Ellenrieder,
K. N. Doan, F. Boos, A. Floerchinger, N. Zufall, S. Oeljeklaus,
B. Warscheid and T. Becker, Nature, 2019, 569, 679–683.
6074
|
J. Mater. Chem. B, 2021, 9, 6068–6075
This journal is © The Royal Society of Chemistry 2021

View Article Online
Paper
Journal of Materials Chemistry B
6 R. Di Maio, P. J. Barrett, E. K. Hoffman, C. W. Barrett, 25 Y.-L. Qi, L.-L. Chen, L. Guo, C. Shao, Y. Liu, Y.-S. Yang, Z.-X.
A. Zharikov, A. Borah, X. Hu, J. McCoy, C. T. Chu, He and H.-L. Zhu, Chem. – Asian J., 2020, 15, 3551–3557.
E. A. Burton, T. G. Hastings and J. T. Greenamyre, Sci. 26 X. Chai, H. H. Han, A. C. Sedgwick, N. Li, Y. Zang,
Transl. Med., 2016, 8, 1–15.
7 A. S. Hebert, K. E. Dittenhafer-Reed, W. Yu, D. J. Bailey,
E. S. Selen, M. D. Boersma, J. J. Carson, M. Tonelli, A. J.
T. D. James, J. Zhang, X. Le Hu, Y. Yu, Y. Li, Y. Wang,
J. Li, X. P. He and H. Tian, J. Am. Chem. Soc., 2020, 142,
18005–18013.
Balloon, A. J. Higbee, M. S. Westphall, D. J. Pagliarini, 27 L. Yang, J. Zheng, Z. Zou, H. Cai, P. Qi, Z. Qing, Q. Yan,
T. A. Prolla, F. Assadi-Porter, S. Roy, J. M. Denu and
J. J. Coon, Mol. Cell, 2013, 49, 186–199.
L. Qiu, W. Tan and R. Yang, Chem. Commun., 2020, 56,
1843–1846.
8 S. M. Yoo and Y. K. Jung, Mol. Cells, 2018, 41, 18–26.
9 L. Ruan, C. Zhou, E. Jin, A. Kucharavy, Y. Zhang, Z. Wen,
L. Florens and R. Li, Nature, 2017, 543, 443–446.
28 Y. Zhang, C. Yan, C. Wang, Z. Guo, X. Liu and W.-H. Zhu,
Angew. Chem., Int. Ed., 2020, 59, 9059–9066.
29 M. Tian, J. Sun, B. Dong and W. Lin, Angew. Chem., Int. Ed.,
2018, 57, 16506–16510.
10 S. Da Cruz, I. Xenarios, J. Langridge, F. Vilbois, P. A. Parone
and J. C. Martinou, J. Biol. Chem., 2003, 278, 41566–41571. 30 Y. Lu, B. Peng, X. Qiu, X. Li, Z. Li, D. Zhang, W. Ji, B. Fang,
11 B. Devreese, F. Vanrobaeys, J. Smet, J. V. Beeumen and
Q. Wu, C. Zhang, L. Li and W. Huang, Mater. Adv., 2020, 1,
R. V. Coster, Electrophoresis, 2002, 23, 2525–2533.
1448–1454.
12 M. J. Berardi, W. M. Shih, S. C. Harrison and J. J. Chou, 31 A. V. Patterson, D. M. Ferry, S. J. Edmunds, Y. Gu,
Nature, 2011, 476, 109–114.
13 J.-B. Li, H.-W. Liu, T. Fu, R. Wang, X.-B. Zhang and W. Tan,
Trends Chem., 2019, 1, 224–234.
R. S. Singleton, K. Patel, S. M. Pullen, K. O. Hicks,
S. P. Syddall, G. J. Atwell, S. Yang, W. A. Denny and
W. R. Wilson, Clin. Cancer Res., 2007, 13, 3922–3932.
14 G. Yang, Z. Liu, R. Zhang, X. Tian, J. Chen, G. Han, B. Liu, 32 Y. Mao, E. T. Keller, D. H. Garfield, K. Shen and J. Wang,
X. Han, Y. Fu, Z. Hu and Z. Zhang, Angew. Chem., Int. Ed.,
2020, 59, 16154–16160.
15 L. Wu, J. Liu, P. Li, B. Tang and T. D. James, Chem. Soc. Rev.,
2021, 50, 702–734.
Cancer Metastasis Rev., 2013, 32, 303–315.
33 Z. Guo, C. Yan and W. Zhu, Angew. Chem., Int. Ed., 2020, 59,
9812–9825.
34 Y. R. Fu, Z. J. Yi, Y. R. Yan and Z. Y. Qiu, Mitochondrion,
2006, 6, 211–217.
16 M. Li, J. Xia, R. Tian, J. Wang, J. Fan, J. Du, S. Long, X. Song,
J. W. Foley and X. Peng, J. Am. Chem. Soc., 2018, 140, 35 Y. R. Fu, Z. J. Yi, Y. R. Yan and Z. Y. Qiu, Anticancer Drugs,
14851–14859. 2007, 18, 1045–1052.
17 V. N. Nguyen, S. Qi, S. Kim, N. Kwon, G. Kim, Y. Yim, S. Park 36 R. Zhang, G. Niu, X. Li, L. Guo, H. Zhang, R. Yang, Y. Chen,
and J. Yoon, J. Am. Chem. Soc., 2019, 141, 16243–16248. X. Yu and B. Z. Tang, Chem. Sci., 2019, 10, 1994–2000.
18 X. Li, C. Y. Kim, S. Lee, D. Lee, H. M. Chung, G. Kim, 37 Y. Dai, F. He, H. Ji, X. Zhao, S. Misal and Z. Qi, ACS Sens.,
S. H. Heo, C. Kim, K. S. Hong and J. Yoon, J. Am. Chem. Soc.,
2017, 139, 10880–10886.
19 J. Yin, M. Peng, Y. Ma, R. Guo and W. Lin, Chem. Commun.,
2018, 54, 12093–12096.
2020, 5, 225–233.
38 Z. Liu, H. Zou, Z. Zhao, P. Zhang, G. G. Shan, R. T. K. Kwok,
J. W. Y. Lam, L. Zheng and B. Z. Tang, ACS Nano, 2019, 13,
11283–11293.
20 Y. Wu, P. Jin, K. Gu, C. Shi, Z. Guo, Z.-Q. Yu and W.-H. Zhu, 39 Z. Zhou, J. Liu, J. Huang, T. W. Rees, Y. Wang, H. Wang,
Chem. Commun., 2019, 55, 4087–4090.
21 R. Zhang, G. Niu, Z. Liu, J. H. C. Chau, H. Su, M. M. S. Lee,
X. Li, H. Chao and P. J. Stang, Proc. Natl. Acad. Sci. U. S. A.,
2019, 116, 20296–20302.
Y. Gu, R. T. K. Kwok, J. W. Y. Lam and B. Z. Tang, 40 Q. Zhang, S. Xu, F. Lai, Y. Wang, N. Zhang, M. Nazare and
Biomaterials, 2020, 242, 119924. H. Y. Hu, Org. Lett., 2019, 21, 2121–2125.
22 S. Samanta, M. Huang, F. Lin, P. Das, B. Chen, W. Yan, 41 D. Wang, H. Su, R. T. K. Kwok, X. Hu, H. Zou, Q. Luo,
J.-J. Chen, K. Ji, L. Liu, J. Qu and Z. Yang, Anal. Chem., 2020,
92, 1541–1548.
M. M. S. Lee, W. Xu, J. W. Y. Lam and B. Z. Tang, Chem. Sci.,
2018, 9, 3685–3693.
23 S. Ma, G. Chen, J. Xu, Y. Liu, G. Li, T. Chen, Y. Li and 42 Q. Hu, M. Gao, G. Feng and B. Liu, Angew. Chem., Int. Ed.,
T. D. James, Coord. Chem. Rev., 2021, 427, 213553. 2014, 53, 14225–14229.
24 D. Cheng, J. Peng, Y. Lv, D. Su, D. Liu, M. Chen, L. Yuan and 43 J. Dai, X. Wu, S. Ding, X. Lou, F. Xia, S. Wang and Y. Hong,
X. Zhang, J. Am. Chem. Soc., 2019, 141, 6352–6361.
J. Med. Chem., 2020, 63, 1996–2012.
This journal is © The Royal Society of Chemistry 2021
J. Mater. Chem. B, 2021, 9, 6068–6075 | 6075