Article

A Two-Photon Zn(II) Complex Photosensitizer with pH/Viscosity

Dual Response for Enhanced Tumor Therapy
Yu Zhang, Shao-Qi Guan, Ya-Ping Wang and Mei Pan *

MOE Laboratory of Bioinorganic and Synthetic Chemistry, Lehn Institute of Functional Materials, IGCME,
GBRCE for Functional Molecular Engineering, School of Chemistry, Sun Yat-sen University,
Guangzhou 510006, China; [email protected] (Y.Z.); [email protected] (S.-Q.G.);
[email protected] (Y.-P.W.)
* Correspondence: [email protected]

Abstract: In recent years, an increasing number of studies have shown that novel metal
complexes with bio-imaging capabilities could enhance precision oncology, particularly
through optimized photosensitizer (PS) design for subcellular organelle targeting pho-
todynamic therapy (PDT). Based on this, we successfully developed a two-photon (TP)
fluorescent Zn(II) complex, LIFM–ZY–3, characterized by the specifical targeting capability
of lysosome. This complex was capable of monitoring dual changes in pH and viscosity.
Additionally, our findings indicated that the complex could generate multiple reactive
oxygen species (ROS), including singlet oxygen (1 O2 ), hydroxyl radicals (•OH), and super-
oxide anion radicals (O2 − ) under white light irradiation in vivo and in vitro. These findings
underscored the remarkable versatility of LIFM–ZY–3 as an advanced multifunctional PS
for both microenvironment monitoring and tumor therapy.

Keywords: Zn(II) complex; TP fluorescence; dual monitoring; ROS; PDT

1. Introduction
Recent advancements in tumor therapy and pathogenesis research have highlighted
Academic Editor: Athanassios targeted subcellular organelle therapy for its ability to enhance therapeutic specificity [1–3].
C. Tsipis
Lysosomes are the “digestive system” within cells, responsible for degrading and recycling
Received: 16 April 2025 intracellular and extracellular waste and damaged molecules [4]. In tumor cells, lysosomal
Revised: 4 May 2025
function is often abnormal, making them a potential target for cancer therapy [5]. In recent
Accepted: 14 May 2025
years, significant progress has been achieved in the application of lysosome-targeted ther-
Published: 31 May 2025
apy for tumor treatment [6]. A notable advancement is lysosome-targeted photodynamic
Citation: Zhang, Y.; Guan, S.-Q.;
therapy (LTPDT), which utilizes photosensitizers (PS) to specifically localize within lyso-
Wang, Y.-P.; Pan, M. A Two-Photon
Zn(II) Complex Photosensitizer with
somes [7]. Upon light activation, these photosensitizers generate reactive oxygen species
pH/Viscosity Dual Response for (ROS), leading to lysosomal membrane permeabilization and subsequent induction of cell
Enhanced Tumor Therapy. Molecules death. This approach enhances the precision and efficacy of tumor treatment by selectively
2025, 30, 2430. https://doi.org/ targeting lysosomal pathways in cancer cells.
10.3390/molecules30112430
The tumor microenvironment (TME) is acidic (pH 6.0–7.0), with lysosomal pH even
Copyright: © 2025 by the authors. lower (4.5–5.5) [8]. Acidic conditions influence PS properties, altering absorption spectra
Licensee MDPI, Basel, Switzerland. and reducing ROS generation efficiency [9]. Low pH also enhances lysosomal membrane
This article is an open access article
sensitivity to ROS, promoting lysosomal membrane permeabilization (LMP), which triggers
distributed under the terms and
hydrolytic enzyme release, leading to apoptosis or necrosis and improving PDT efficacy.
conditions of the Creative Commons
Attribution (CC BY) license
Furthermore, ROS stabilization under acidic conditions may contribute to the enhanced ac-
(https://creativecommons.org/ tivity of pH-sensitive PSs [10]. Elevated viscosity, another key TME feature [11], synergizes
licenses/by/4.0/). with low pH to enhance tumor invasion and metastasis [12]. Since pH and viscosity are

Molecules 2025, 30, 2430 https://doi.org/10.3390/molecules30112430

Molecules 2025, 30, 2430 2 of 13

interconnected yet independent, dual monitoring is essential for accurate tumor diagnosis
and staging [13]. Developing novel fluorescent probes and multimodal imaging technolo-
gies for dual monitoring can advance precision oncology, particularly in optimizing PS
design for subcellular organelle-targeted tumor therapy.
Fluorescent Zn(II) complexes exhibit significant potential as PSs in PDT and biological
imaging [14] due to their unique photophysical properties, excellent biocompatibility, and
tunable photosensitizing performance, making them ideal candidates for tumor treatment,
antibacterial therapy, and photodynamic diagnosis. The integration of metal Zn(II) com-
plexes with two-photon (TP) property can optimize their photophysical properties with
fluorescence modulation capabilities in deeper tissue penetration and high spatial resolu-
tion, and PDT efficacy [15]. The naphthalene imide derivatives with TP property, [16] as
multifunctional ligands, improve the optical properties, TME responsiveness, and targeted
therapeutic effects of Zn(II) complexes. By combining PDT with real-time TME sensing,
Zn(II) complexes open new avenues for advancing cancer treatment strategies.
Building on these foundations, we designed and synthesized a lysosome-targeted
Zn(II) complex by integrating naphthalene imide derivatives with the metal zinc ion. The
complex had pH and viscosity dual-responsive TP emission, and it exhibited superior
LTPDT efficacy compared to the ligand alone. Notably, TP lysosome-targeted PS combined
with Zn(II) complexes for detecting dual pH/viscosity remained rare, positioning our
work as a significant contribution to the field. We anticipated that Zn(II) complexes could
be employed for self-reporting dynamics PDT by taking the exceptional tumor micro-
environment. Through further optimization and clinical translation, Zn(II) complexes
were poised to become essential tools in precision medicine, offering new solutions for
cancer treatment.

2. Results and Discussion

2.1. Optical Properties
The lysosome-targeted TP fluorescent Zn(II) complex, LIFM–ZY–3, was designed to
exhibit dual responsiveness of pH and viscosity. Also, it was constructed by linking a
naphthalene imide derivative with a zinc ion via coordination bonding, as illustrated in
Scheme 1. The viscosity response mechanism of LIFM–ZY–3 was governed by the twisted
intramolecular charge transfer (TICT) mechanism with a rotatable benzene ring [17]. In
contrast, the pH response mechanism of LIFM–ZY–3 relied on the reversible protonation
and deprotonation of the morpholine moiety, which modulates the N lone pair of electrons
on the ligand [18]. In addition, the morpholine group had superior targeting lysosome
property, [19], and it was widely utilized in bio-imaging applications due to its versatility
in biology. The synthetic routes of ligand L3 and complex LIFM–ZY–3 were detailed in
Scheme S1, and the structures of L3 and LIFM–ZY–3 were fully characterized using 1 H
NMR, 13 C NMR, and HRMS in Figures S1–S7.
We initially investigated the optical properties of the complex LIFM–ZY–3. All spec-
troscopic measurements were performed in a mixed ethanol–glycerol solvent system to
simulate and establish an in vitro viscous environment. As shown in Figure S8A, the
absorption spectra of LIFM–ZY–3 in various solvents exhibited characteristic absorption
peaks at 350 nm. Compared to other solvents, the fluorescence intensity of LIFM–ZY–3
displayed apparent enhancement in glycerol in Figure S8B, indicating that the complex
could detect viscosity changes. Subsequently, we further analyzed the fluorescence changes
in LIFM–ZY–3 in solutions with varying viscosities. As depicted in Figure 1A, the fluores-
cence intensity of LIFM–ZY–3 exhibited a significant enhancement, increasing up to 5-fold,
in response to elevated viscosity. Also, there was a strong linear correlation between the
log of viscosity changes and the log of fluorescence intensity at 438 nm in Figure S9. These
Molecules 2025, 30, 2430 3 of 13

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results demonstrated that the complex LIFM–ZY–3 was capable of quantitatively detecting
and measuring changes in viscosity.

Scheme 1.
Scheme (A) The
1. (A) The design
design of
of tetrahedrally–coordinated
tetrahedrally–coordinated zinc(II)
zinc(II) complex
complex LIFM–ZY–3.
LIFM–ZY–3. (B)(B) The
The
complex LIFM–ZY–3 can monitor the viscosity and pH dual changes in vitro. (C) The PDT efficacy
complex LIFM–ZY–3 can monitor the viscosity and pH dual changes in vitro. (C) The PDT efficacy of
LIFM–ZY–3 was evaluated at the cellular level.
of LIFM–ZY–3 was evaluated at the cellular level.
Considering the broad biological applicability of Zn(II) complexes in responding to
We initially investigated the optical properties of the complex LIFM–ZY–3. All
TME and cell imaging, we further explored the TP properties of LIFM–ZY–3 to gain deeper
spectroscopic measurements were performed in a mixed ethanol–glycerol solvent system
insights into its potential for high-resolution, deep-tissue imaging and its interactions with
to simulate and
complicated establish
cellular an in vitro As
environments. viscous
shown environment. As shown
in Figure S10A,B, in Figureexhibited
LIFM–ZY–3 S8A, the
absorption
a maximumspectra of LIFM–ZY–3
TP absorption in various
cross-section solvents
of 75 GM at exhibited characteristic absorption
670 nm. Furthermore, a linear re-
peaks at 350 nm. Compared to other solvents, the fluorescence intensity
lationship was observed between the log of the emitted fluorescence intensity and the of LIFM–ZY–3
displayed apparent enhancement
log of the excitation power, with ainslope
glycerol
of 2 in
in Figure
Figure S8B, indicating
S10C,D, that the
confirming thecomplex
TP fluo-
could detect viscosity changes. Subsequently, we further analyzed the
rescence characteristics of Zn(II) complex LIFM–ZY–3. Additionally, the TP fluorescence fluorescence
changes
intensityinofLIFM–ZY–3
LIFM–ZY–3inexhibited
solutionsawith varying
17-fold viscosities.
increase As depicted
in response in Figure
to elevated 1A, the
viscosity in
fluorescence intensity of LIFM–ZY–3 exhibited a significant enhancement,
Figures 1B and S11. It demonstrated that LIFM–ZY–3 is capable of achieving both OP increasing up
to
and5-fold,
highlyinefficient
responseTPtoresponses
elevated viscosity.
to viscosity Also, there underscoring
changes, was a strong linear correlation
its significant po-
between the log of viscosity changes and the log of fluorescence intensity
tential for deep-tissue biological imaging applications. Consistent with previous at 438studies,
nm in
Figure S9. These results demonstrated
most viscosity-responsive that the
fluorescent probes have complex LIFM–ZY–3
been reported was changes
to exhibit capable inof
quantitatively detecting and measuring changes in viscosity.
fluorescence lifetime in response to viscosity variations [20]. Then, we investigated the
fluorescence lifetime changes in LIFM–ZY–3 at different viscous solvents. As shown in
Figure 1C, the fluorescence lifetime of LIFM–ZY–3 showed a significant increase in response
to elevated viscosity. Moreover, a strong linear correlation between the fluorescence lifetime
of LIFM–ZY–3 and viscosity were observed in Figure S12, demonstrating the capability of
LIFM–ZY–3 to qualitatively detect and quantitatively measure viscosity changes through
changing fluorescence lifetime.
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30, 2430
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emission spectrum
Figure 1. (A) The fluorescence emission spectrum of of LIFM–ZY–3 (5 µM) μM) in the changed changed viscosity
media of the EtOH/glycerol (v/v) mixtures. (B) The TP induced
media of the EtOH/glycerol (v/v) mixtures. (B) The TP induced emission spectrum of emission spectrum of LIFM–ZY–3
LIFM–ZY–3
(10 µM) in the changed viscosity media of the EtOH/glycerol (v/v) mixtures
(10 μM) in the changed viscosity media of the EtOH/glycerol (v/v) mixtures at 690 nm. (C) The at 690 nm. (C)time–
The
time–resolved emission
resolved emission decays
decays of LIFM–ZY–3
of LIFM–ZY–3 (10(10 µM)atatdifferent
μM) differentviscosities
viscosities inin EtOH/glycerol
EtOH/glycerol (v/v) (v/v)
mixtures. (D) The pH–responsive fluorescence intensity changes in the complex LIFM–ZY–3 (5 µM)
mixtures. (D) The pH–responsive fluorescence intensity changes in the complex LIFM–ZY–3 (5 μM)
in different viscosity systems. (E) The TP fluorescence intensity of LIFM–ZY–3 (10 µM) at changed
in different viscosity systems. (E) The TP fluorescence intensity of LIFM–ZY–3 (10 μM) at changed
pH environments at 460 nm. (F) The changes in fluorescence intensity of LIFM–ZY–3 (5 µM) treated
pH environments at 460 nm. (F) The changes in fluorescence intensity of LIFM–ZY–3 (5 μM) treated
with different interfering substances 1–27 (Fe3+ , Co2+ , Zn2+ , Ca2+ , Cu2+ , Mg2+ , Mn2+ , Cu+ , K+ , Na+ ,
with different
Cysteine, GSH, interfering
H2 O2 , OCl−substances
, CH3 COO− 1–27, NO(Fe −3+, Co2+ −, Zn2+−, Ca2+, Cu − 2+, Mg−2+, Mn22+ −, Cu − +, K−+, Na+,
−
2 , NO3 , HS , HSO3 , HSO4 , SO4 , F , Cl , Br ,
Cysteine,
− GSH, H 2O 2 , OCl
I , and glycerol) at 450 nm.
−, CH 3COO − , NO 2 − , NO 3 − , HS − , HSO 3 − , HSO 4 − , SO 4 2− , F − , Cl −, Br −, I −, and

glycerol) at 450 nm.

Given the acidic nature of TME in biological system, [21] we investigated the pH–
Considering
dependent behavior theofbroad biological
LIFM–ZY–3 applicability
in Figure 1D. Theoffluorescence
Zn(II) complexes
intensityin responding
of LIFM–ZY–3 to
TME and acell
exhibited imaging,drop
significant we further exploredpH
with increasing theinTPbothproperties of LIFM–ZY–3
low–viscosity (PBS) andtohigh–
gain
deeper insights
viscosity into its= 1/1)
(PBS/glycerol potential for high-resolution,
conditions. Subsequently, we deep-tissue imaging and
further investigated its
the TP
interactions with
fluorescence complicated
intensity cellularunder
of LIFM–ZY–3 environments.
varying pH As conditions.
shown in FigureThe TP S10A,B, LIFM–
fluorescence
ZY–3 exhibited
intensity a maximum
of LIFM–ZY–3 TP absorption
gradually decreased cross-section of 75pH
with increasing GMconditions
at 670 nm.in Furthermore,
Figures S13
a linear
and 1E. relationship
And the quantum was observed
yield (QY) between the log ofhas
of LIFM–ZY–3 the been
emitted fluorescence
measured intensity
at various pH
and the
levels inlog of the
Figure excitation
S14A, the QYpower, with a slope
of LIFM–ZY–3 of 2 inwith
decreased Figure S10C,D,pH
increasing confirming
conditions,theand
TP
afluorescence characteristics
linear relationship was observedof Zn(II)
between complex
the QY andLIFM–ZY–3.
the different Additionally, the TP
fractions of glycerol,
fluorescence
with intensity
0.95 in Figure S14B.of LIFM–ZY–3
Collectively, exhibited
these resultsa 17-fold increase that
demonstrated in response
the complexto elevated
LIFM–
viscosity
ZY–3 couldin effectively
Figures 1Bmonitor
and S11.pH It and
demonstrated that LIFM–ZY–3
viscosity changes under bothisOP capable
and TP of excitation,
achieving
both OP and
highlighting its highly
potential efficient TP responses
for TME-sensitive to viscosity
applications changes,
in biological underscoring its
systems.
significant potential
To eliminate for deep-tissue
potential interference biological
from solventimaging applications.
polarity Consistent
on the viscosity with
response,
previous
we examinedstudies, most viscosity-responsive
the fluorescence fluorescent under
spectrum of LIFM–ZY–3 probesvarying
have been reported
polarities. Intoa
tetrahydrofuran–water mixed system, the complex LIFM–ZY–3 displayed
exhibit changes in fluorescence lifetime in response to viscosity variations [20]. Then, we 50 nm red shifts
with increasing
investigated polarity in Figure
the fluorescence lifetime S15; however,
changes fluorescence
in LIFM–ZY–3 intensityviscous
at different of LIFM–ZY–3
solvents.
was notably lower compared to that observed in a glycerol
As shown in Figure 1C, the fluorescence lifetime of LIFM–ZY–3 showed a significant solvent. Subsequently, we
evaluated
increase inthe selectivity
response of LIFM–ZY–3
to elevated viscosity. against various
Moreover, potential
a strong interfering
linear correlation substances,
between
including 1–27 (Fe 3+ , Co2+ , Zn2+ , Ca2+ , Cu2+ , Mg2+ , Mn2+ , Cu+ , K+ , Na+ , Cysteine, GSH,
the fluorescence lifetime of LIFM–ZY–3 and viscosity were observed in Figure S12,
H − − , NO − , NO − , HS− , HSO − , HSO − , SO 2− , F− , Cl− , Br− , I− ,
2 O2 , OCl , CH
demonstrating 3 COO
the capability 2of LIFM–ZY–3
3 to qualitatively
3 4 detect 4 and quantitatively
and glycerol). As shown in Figure 1F, the fluorescence
measure viscosity changes through changing fluorescence lifetime. intensity of LIFM–ZY–3 at 450 nm
Molecules 2025, 30, 2430 5 of 13

remained largely unchanged in the presence of these interfering substances, demonstrating

minimal interference compared to its response in viscous environments. Additionally, we
evaluated the photostability of LIFM–ZY–3 in Figure S16. The result demonstrated that
its fluorescence intensity remained nearly constant under continuous irradiation for up
to 8 h. These findings confirmed that LIFM–ZY–3 could specifically detect viscosity and
pH changes with high stability, demonstrating its adaptability to complicated biological
systems and underscoring its strong potential for biological applications.

2.2. Quantification of ROS

Considering the MLCT phenomenon observed in fluorescent Zn(II) complexes, [22]
we further investigated the potential of LIFM–ZY–3 as a PS for PDT. Firstly, we evaluated
the ability of LIFM–ZY–3 to generate singlet oxygen (1 O2 ) using 9,10–anthracenediyl–
bis(methylene)dimalonic acid (ABDA) [23] under white light irradiation at an intensity
of 35 mW/cm2 . As shown in Figure S17A, the maximal absorption of ABDA gradually
decreased with increasing irradiation time, confirming the 1 O2 generation capability of
LIFM–ZY–3 increased steadily under 35 mW/cm2 white light irradiation. Additionally,
we evaluated the ligand L3 under identical conditions and observed that it also generated
1 O , though with significantly lower efficiency compared to LIFM–ZY–3 in Figure S17B.
2
For comparative analysis, we utilized rhodamine B (RhB), a conventional small molecule
photosensitizer, as a reference to evaluate ROS generation efficiency [24]. As shown in
Figure S17C, the maximum absorption of ABDA decreased with increasing irradiation time,
which demonstrated that commercial RB could generate 1 O2 . In contrast to the complex
LIFM–ZY–3, the 1 O2 production efficiency of RB was significantly lower than that of LIFM–
ZY–3 in Figure 2A, comparable to that of ligand L3 under identical conditions. These results
demonstrated that LIFM–ZY–3 exhibited superior 1 O2 generation efficiency compared to
both ligand L3 and RB, emphasizing its potential as an effective photosensitizer for Type II
photodynamic therapy.
To assess the potential of the complex LIFM–ZY–3 for biological applications, we
adjusted the white light irradiance to a biologically relevant level of 20 mW/cm2 and re-
evaluated the 1 O2 production rate of LIFM–ZY–3. The result demonstrated that LIFM–ZY–3
still maintained high 1 O2 production efficiency even at lower light irradiance in Figure S18,
and there was a strong linear relationship (correlation coefficient = 0.999) between the
maximum absorption of LIFM–ZY–3 and irradiation time. It indicated that LIFM–ZY–
3 could stably and predictably generate 1 O2 under light irradiation. Additionally, we
employed a fluorescence–based method using 2′ ,7′ –dichlorodihydrofluorescein diacetate
(DCFH–DA) [25] to confirm the generation of ROS by complex LIFM–ZY–3 under light
irradiation. As shown in Figure 2B and Figure S19, the enhancing fluorescence of DCFH–
DA confirmed that the complex LIFM–ZY–3 could effectively produce ROS under white
light irradiation.
Hydroxyphenyl fluorescein (HPF) selectively and dose-dependently reacts with hy-
droxyl radicals (•OH), leading to a corresponding increase in fluorescence intensity [26].
Taking advantage of this property, we evaluated the ability of the complex LIFM–ZY–3
to generate •OH under 20 mW/cm2 white light irradiation using HPF as a sensing probe.
As shown in Figure S20A, the fluorescence intensity of HPF exhibited a gradual increase
with prolonged irradiation time, demonstrating the ability of LIFM–ZY–3 to generate •OH
under 20 mW/cm2 white light irradiation. Then, the •OH generation of the ligand L3 was
examined under the same conditions in Figure S20B. The results revealed that ligand L3 , at
the same concentration as LIFM–ZY–3, could also generate •OH, though with significantly
lower efficiency compared to the complex LIFM–ZY–3. And the fluorescence intensity of
HPF had a negligible impact on the •OH generation compared to the complex LIFM–ZY–3,
Molecules 2025, 30, 2430 6 of 13

the ligand L3 , and the HPF in Figure 2C and Figure S20C. Collectively, these findings
demonstrated that the complex LIFM–ZY–3 could efficiently generate not only 1 O2 but
Molecules 2025, 30, x FOR PEER REVIEW
also
6 of 13
•OH upon white light irradiation, confirming its potential as a versatile photosensitizer for
type I PDT.

Figure 2.
Figure 2. (A) Comparison of absorption intensity of complex complex LIFM–ZY–3,
LIFM–ZY–3, ligand
ligand LL3,3, and RB
RB atat
different
different white light irradiation times and 380 nm wavelength in the same concentration.
light irradiation times and 380 nm wavelength in the same concentration. (B) At the(B) At
the
samesame concentration,
concentration, thethe fluorescence
fluorescence intensity
intensity ofofthe
thecomplex
complexLIFM–ZY–3,
LIFM–ZY–3, the
the ligand
ligand L L33,, and
and
DCFH–DA
DCFH–DA at different white light irradiation times and 530 nm wavelength were compared. (C) At
at different white light irradiation times and 530 nm wavelength were compared. (C) At
the same concentration, the fluorescence intensity of the complex LIFM–ZY–3, the ligand L3 , and
the same concentration, the fluorescence intensity of the complex LIFM–ZY–3, the ligand L3, and
HPF at different white light irradiation times and 530 nm wavelength. (D) At the same concentration,
HPF at different white light irradiation times and 530 nm wavelength. (D) At the same
the producing O2 − efficiency of the complex LIFM–ZY–3, the ligand L3, and DHE at different white
concentration, the producing O2− efficiency of the complex LIFM–ZY–3, the ligand L3, and DHE at
light irradiation times and 530 nm wavelength.
different white light irradiation times and 530 nm wavelength.
Additionally, we utilized the dihydroethidium (DHE) to detect the superoxide anions
(O2 − )To assess
[27]. the potential
As shown in FigureofS21A,
the complex LIFM–ZY–3
the fluorescence for of
intensity biological applications,
DHE gradually we
increased
adjusted
with the white
prolonged light irradiance
irradiation to a biologically
time, demonstrating relevant
the ability level of 20 mW/cm
of LIFM–ZY–3 O2 −
2 and re–
to generate
evaluated
under 20 mW/cm 2 white light
the 1O2 production rate of LIFM–ZY–3.
irradiation. The OThe − result demonstrated
capabilitythat LIFM–ZY–
2 production of the L3 was
3 still maintained
tested at the same high 1
concentrationO2 production efficiencyineven
as LIFM–ZY–3 Figure at S21B.
lower The
lightresults
irradiance in Figure
indicated that
S18, −
and there was a strong linear relationship (correlation coefficient
the O2 generation efficiency of ligand L3 was only half that of the complex LIFM–ZY–3. = 0.999) between the
maximum
As a control,absorption
we confirmed of LIFM–ZY–3
that DHE itself andhad irradiation time. impact
no noticeable It indicated
on thethat −
O2 LIFM–ZY–3
generation
could stably
abilities and predictably
of complex LIFM–ZY–3 and generate
ligand1O under
L32 in Figureslight
2D irradiation.
and S21C. These Additionally,
experimental we
employed
results a fluorescence–based
demonstrated method LIFM–ZY–3
that the complex using 2′,7′–dichlorodihydrofluorescein
could efficiently generate adiacetate variety
(DCFH–DA)
of ROS, including 1 Oconfirm
[25] to , • OH, the
and O − , uponof
generation ROS
white by
light complex
irradiationLIFM–ZY–3
within a under light
biologically
2 2
irradiation.
tolerable As shown
range. in Figures 2BROS
The multifunctional andgeneration
S19, the enhancing
capability fluorescence
demonstratedofthe DCFH–DA
potential
confirmed
of LIFM–ZY–3 thatasthe complex PS
a versatile LIFM–ZY–3
for biologicalcould effectively
targeting and produce ROS under
tumor therapy, white light
emphasizing its
irradiation.promise for advanced biomedical applications.
significant
Hydroxyphenyl
Considering the pH fluorescein
sensitivity(HPF) selectively
of LIFM–ZY–3 and dose-dependently
in optical reacts with
experiments, we investigated
hydroxyl
its radicals
ability to generate 1
(•OH), leading
O2 under to a corresponding
different pH conditionsincrease using ABDAin fluorescence intensity2
under 20 mW/cm
[26]. Taking
white advantage of
light irradiation. thisresults
The property, we evaluated
revealed the ability of
that the absorption of the
ABDAcomplex LIFM–
exhibited a
ZY–3 to generate •OH under 20 mW/cm white light irradiation using HPF as a sensing
2

probe. As shown in Figure S20A, the fluorescence intensity of HPF exhibited a gradual
increase with prolonged irradiation time, demonstrating the ability of LIFM–ZY–3 to
generate •OH under 20 mW/cm2 white light irradiation. Then, the •OH generation of the
 Molecules 2025, 30, 2430 7 of 13

gradual decrease with prolonged irradiation time, and the 1 O2 generation efficiency of
LIFM–ZY–3 was significantly enhanced at lower pH values, as illustrated in Figures 3A–C
and S22–S24. Compared to ligand L3 and RB, the complex LIFM–ZY–3 demonstrated signif-
icantly 1 O2 production efficiency across a range of pH environments, as evidenced by the
accelerated decay rate of ABDA in Figure 3D. And the 1 O2 exhibited a characteristic near–
infrared phosphorescence emission peak around 1270 nm, originating from its ¹∆g→³Σg−
transition. Based on this, we measured the spectrum of phosphorescence, it showed that the
complex LIFM–ZY–3 had higher phosphorescent intensity with decreasing pH conditions
in Figure S25. These results showed that LIFM–ZY–3 generated 1 O2 more efficiently in
s 2025, 30, x FOR PEER REVIEW acidic conditions, matching the acidic microenvironment of cancer cells, indicating that the
complex LIFM–ZY–3 could enhance PDT efficacy in cancer cells compared to the neutral
conditions of normal cells.

Figure (A) The comparison diagram

diagram of of
thethe
ability of the complex LIFM–ZY–3LIFM–ZY–3
to produce O2 to1
by produc
Figure 3. (A)3.The comparison ability of the complex
ABDA in PBS solutions with different pH (5, 6, 7, 8). (B) The comparison diagram of the ability of
ABDA in PBS
ligand L3 solutions with
to produce 1 O different pH (5, 6, 7, 8). (B) The comparison diagram of the a
2 by ABDA in PBS solutions with different pH (5, 6, 7, 8). (C) A comparative
ligand Ldiagram of the ability
3 to produce 1O2ofbydye in PBS1 Osolutions
RB to produce
ABDA 2 by ABDA with
in PBS different
solutions with
pHdifferent pH8).
(5, 6, 7, (5, (C)
6, 7, A com
8). (D) LIFM–ZY–3, L , and RB produced 1 O ability comparison diagrams by ABDA in pH = 5 PBS
diagram of the ability of3 dye RB to produce
solution, respectively.
2 1O2 by ABDA in PBS solutions with different p

7, 8). (D) LIFM–ZY–3, L3, and RB produced 1O2 ability comparison diagrams by ABDA in
PBS solution, respectively.

2.3. Subcellular Organelle Co-Localized Imaging

To further evaluate the biological suitability of LIFM–ZY–3, we initially asse
Molecules 2025, 30, 2430 8 of 13

2.3. Subcellular Organelle Co-Localized Imaging

To further evaluate the biological suitability of LIFM–ZY–3, we initially assessed its
cytotoxicity using the MTT assay. Given the PDT property of LIFM–ZY–3, we separately
evaluated its phototoxicity and dark toxicity on HeLa cells. As shown in Figure S26, under
dark conditions, the complex LIFM–ZY–3 exhibited minimal toxicity (95% cellular viability)
to live cells; however, the cellular viability dropped to less than 40% under 20 mW/cm2
white light irradiation. It indicated that LIFM–ZY–3 could effectively exert its PDT effect
in HeLa cells, making it a promising candidate for further in-depth studies in tumor
therapy. Subsequently, we conducted cellular imaging at the subcellular organelle level in
HeLa cells using the low-toxicity LIFM–ZY–3 under dark conditions. Then, we co–stained
commercially available organelle targeting probes, including lysosome targeted deep red
(LTDR), endoplasmic reticulum targeted red (ERTR), lipid droplet targeted red (LDTR), and
mitochondria targeted deep red (MTDR), with the complex LIFM–ZY–3 in HeLa cells, as
illustrated in Figure S27. The co-localization coefficient of LIFM–ZY–3 reached 0.89 when
co–stained with LTDR, while the co-localization coefficients with other commercial dyes
(MTDR, ERTR, and LDTR) were significantly lower at 0.41, 0.36, and 0.56, respectively.
It demonstrated that the complex LIFM–ZY–3 exhibited subcellular organelle–targeting
capabilities, with a specific ability to target lysosomes in HeLa cells.

2.4. Visualization of Lysosomal Viscosity Changes and Apoptosis

According to previous literature, lysosomal viscosity could significantly increase
following the stimulation of cells with dexamethasone (DXMS) [28]. HeLa cells were
pretreated with dexamethasone (DXMS) for 1 h, 2 h, and 3 h, respectively, followed by
incubation with LIFM–ZY–3 for 2 h. The complex LIFM–ZY–3 exhibited obvious fluores-
cence enhancement with increasing DXMS incubation time in both OP and TP channels in
Figures 4A,D and S28. It indicated that LIFM–ZY–3 could monitor viscosity changes in-
duced by DXMS through fluorescence enhancement. According to related literature, an
increase in cytoplasmic viscosity, often accompanied by lysosomal membrane permeabi-
lization, serves as a key marker of apoptosis [29]. Hydrogen peroxide (H2 O2 ) can induce
cells to produce elevated levels of cytotoxic ROS, and high concentrations of ROS can
cause oxidative damage in tumor cells, ultimately leading to apoptosis [30]. Based on this,
HeLa cells were pretreated with varying concentrations of H2 O2 (2 mM, 4 mM, 6 mM) for
0.5 h, followed by staining with LIFM–ZY–3 for 2 h. As shown in Figures 4B,E and S29,
LIFM–ZY–3 exhibited a clear fluorescence enhancement in both OP and TP channels with
increasing H2 O2 incubation concentration. Additionally, we induced apoptosis in cells
using the pro–apoptotic drugs rotenone [31] and paclitaxel [32]. The experimental results
demonstrated that LIFM–ZY–3 also exhibited significant fluorescence enhancement in
HeLa cells following stimulation with pro–apoptotic drugs in Figure S30. It indicated that
LIFM–ZY–3 could monitor H2 O2 -induced apoptosis through fluorescence enhancement,
serving as evidence of its ability to self–report apoptosis following PDT treatment.

2.5. Visualization of Intracellular pH Changes

Based on the optical properties of LIFM–ZY–3, we conducted intracellular pH visu-
alization experiments in HeLa cells. HeLa cells were incubated with LIFM–ZY–3 for 2 h,
washed with PBS, treated with PBS solutions of different pH for 15 min, rinsed with PBS,
and then subjected to confocal imaging. As shown in Figure 4C,F, LIFM–ZY–3 displayed
gradually decreasing fluorescence intensity in both OP and TP channels with increasing pH
conditions. Additionally, under strongly acidic conditions, the complex LIFM–ZY–3 lost
the targeting lysosome specificity, suggesting that the strong acid disrupted the integrity
of the subcellular organelles in HeLa cells, leading to their leakage into the cytoplasm
 concentrations of ROS can cause oxidative damage in tumor cells, ultimately leading
apoptosis [30]. Based on this, HeLa cells were pretreated with varying concentrations
Molecules 2025, 30, 2430 H2O2 (2 mM, 4 mM, 6 mM) for 0.5 h, followed by staining with LIFM–ZY–3 9 of 13for 2 h.

shown in Figures 4B,E and S29, LIFM–ZY–3 exhibited a clear fluorescence enhancem
in both OP and TP channels with increasing H2O2 incubation concentration. Additiona
and resulting in a rapid fluorescent enhancement. Unlike the acidic environment, the
we induced
integrity apoptosis
of subcellularin organelles
cells using in the
HeLapro–apoptotic drugs
cells was disrupted rotenone
under strongly[31] and paclita
alkaline
[32]. conditions
The experimental resultsfluorescence
as well, achieving demonstrated that ofLIFM–ZY–3
quenching LIFM–ZY–3. Thealsodifferences
exhibitedinsignific
the above results indicated that LIFM–ZY–3 could have a highly
fluorescence enhancement in HeLa cells following stimulation with pro–apoptotic sensitive response to dru
low pH in HeLa cells, particularly in cancer cells, which were characterized by an acidic
in Figure S30. It indicated that LIFM–ZY–3 could monitor H2O2-induced apopto
micro-environment. These findings demonstrated that LIFM–ZY–3 could monitor intracel-
through fluorescence
lular pH changes throughenhancement, serving asasevidence
fluorescence enhancement of itshighlighting
acidity increased, ability toitsself–rep
apoptosis following
potential PDT treatment.
for intracellular pH self-reporting after PDT in solid tumors.

Figure 4. (A)
Figure HeLa
4. (A) cells
HeLa cellswere
were incubated with
incubated with DXMS
DXMS for 1for
h, 21h,h,and
2 h, and
3 h, 3 h, respectively,
respectively, and then
and then the
incubated HeLa
incubated cells
HeLa were
cells werestained withLIFM–ZY–3
stained with LIFM–ZY–3 (5 μM):
(5 µM): (a) OP(a) OP confocal
confocal images ofimages of LIFM–ZY
LIFM–ZY–3;
(b)confocal
(b) TP TP confocal imagesof
images of LIFM–ZY–3.
LIFM–ZY–3. (B)(B)
HeLa cells were
HeLa cellsincubated with different
were incubated withconcentrations of
different concentrati
H2 O2 (2 mM, 4 mM, 6 mM) for 0.5 h, and then the incubated HeLa cells were stained with LIFM–ZY–3
(5 µM) for 2 h: (a) OP confocal images of complex LIFM–ZY–3; (b) TP confocal images of LIFM–ZY–3.
(C) HeLa cells were stained with LIFM–ZY–3 (5 µM) for 2 h, and then the incubated HeLa cells were
incubated with different pH solutions of PBS (pH= 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5) for 15 min in OP
channel (a) and TP channel (b). (D) The (A) mean intensity of LIFM–ZY–2. (E) The (B) mean intensity
of LIFM–ZY–2. (F) The (C) mean intensity of LIFM–ZY–2. Scale bar = 20 µm.
 Molecules 2025, 30, 2430 10 of 13

2.6. Photodynamic Therapy at the Cellular Level

Based on the ROS–related studies, the complex LIFM–ZY–3, compared to ligand L3 and
RB, demonstrated the ability to generate multiple types of ROS (1 O2 , •OH, and O2 − ) under
white light irradiation. These generated ROS could rapidly oxidize intracellular DNA, small
molecules, and proteins, disrupting cellular functions and ultimately inducing apoptosis
or necrosis in tumor cells [33]. Firstly, we employed the probe DCFH–DA to directly
visualize and quantify the effective ROS levels in HeLa cells. As shown in Figure 5A,D,
almost no green fluorescence of LIFM–ZY–3 was observed under the dark field condition in
HeLa cells, indicating the absence of ROS generation without light irradiation. Also, there
was no green fluorescence of DCFH–DA in the blank control group after light exposure,
confirming the biologically safe light intensity. In contrast, widespread green fluorescence
lecules 2025, 30, x FOR PEER REVIEW 11 o
was observed in HeLa cells treated with LIFM–ZY–3 after light exposure, indicating that
the complex LIFM–ZY–3 could generate ROS upon light irradiation in HeLa cells.

Figure 5. (A)
Figure HeLa
5. (A) cells
HeLa cellswere
were first incubated
first incubated with
with LIFM–ZY–3
LIFM–ZY–3 (5 µM)(5
forμM) for then
2 h, and 2 h, DCFH–DA
and then DCFH–
was added
was added forfor incubationfor
incubation for 20
20 min
minininboth dark
both and and
dark light light
conditions to obtaintoconfocal
conditions obtainimages.
confocal ima
(B) HeLa cells were first incubated with LIFM–ZY–3 (5 µM) for 2 h, and then HFP was added for
(B) HeLa cells were first incubated with LIFM–ZY–3 (5 μM) for 2 h, and then HFP was added
incubation for 20 min in both dark and light conditions to obtain confocal images. (C) HeLa cells were
incubation for 20 with
first incubated minLIFM–ZY–3
in both dark
(5 µM)and
for light conditions
2 h, and to obtain
then DHE was confocal
added for images.
incubation (C) HeLa c
for 20 min
wereinfirst
bothincubated
dark and light
withconditions
LIFM–ZY–3to obtain
(5 confocal
μM) forimages. (D) then
2 h, and The mean
DHEintensity of DCFH–DA,
was added for incubation
HFP, and DHE in both dark and light conditions. Scale bar = 20 µm.
20 min in both dark and light conditions to obtain confocal images. (D) The mean intensity
DCFH–DA, HFP,
Then, weand DHE
tested the in bothofdark
ability and light
LIFM–ZY–3 to conditions.
generate •OHScale barHPF
using = 20atμm.
the cellular
level in Figure 5B,D. The weak green fluorescence was observed in HeLa cells treated with
LIFM–ZY–3 under dark
Then, we tested the conditions, suggesting the to
ability of LIFM–ZY–3 generation
generate of •OH
a smallusing
amountHPF of •at
OHthe cellu
even in the absence of light conditions. The fluorescence intensity in the blank
level in Figure 5B,D. The weak green fluorescence was observed in HeLa cells treated w control group
without LIFM–ZY–3 under light condition matched that of cells incubated with LIFM–ZY–3
LIFM–ZY–3 under dark conditions, suggesting the generation of a small amount of •O
in the dark, indicating that the experimental light intensity induced an appropriate level of
even•OHin the absence
production in of light
blank conditions.
HeLa cells. And aThe
clearfluorescence
enhancement inintensity in intensity
fluorescence the blank con
group
waswithout
observedLIFM–ZY–3 underwith
in HeLa cells treated light condition
LIFM–ZY–3 undermatched that ofindicating
light condition, cells incubated
that w
the complex
LIFM–ZY–3 inLIFM–ZY–3
the dark,could indicating •OH, the
generate that consistent with the optical
experimental lightresults.
intensity induced
In addition to the above studies, we also evaluated the ability of LIFM–ZY–3 to
appropriate level of •OH production in blank HeLa cells. And a clear enhancement
generate O2 − in HeLa cells using the commercial probe DHE. Under dark conditions,
fluorescence intensity was observed in HeLa cells treated with LIFM–ZY–3 under li
weak green fluorescence of LIFM–ZY–3 was observed in the cytoplasm of HeLa cells in
condition, indicating that the complex LIFM–ZY–3 could generate •OH, consistent w
the optical results.
In addition to the above studies, we also evaluated the ability of LIFM–ZY–3
generate O2− in HeLa cells using the commercial probe DHE. Under dark conditions, w
Molecules 2025, 30, 2430 11 of 13

Figure 5C,D, while DHE had not yet bound to the nucleus to form ethidium bromide,
indicating minimal generation of O2 − in HeLa cells without light exposure. Under light
conditions, HeLa cells without LIFM–ZY–3 incubation served as the blank control group,
which showed negligible changes in fluorescence intensity but exhibited clear nuclear
staining, demonstrating that the experimental light intensity induced an appropriate level
of O2 − production in blank cells. It was evident that the fluorescence intensity and nuclear
staining in HeLa cells treated with LIFM–ZY–3 were significantly enhanced compared to
the blank control group under light conditions, manifesting that LIFM–ZY–3 could generate
O2 − following light treatment in HeLa cells. All the above experiments demonstrated that
the complex LIFM–ZY–3 possessed sufficient PDT capability to achieve the death of cancer
cells, providing cellular-level data to support its potential for further application in tumor
therapy and real-time monitoring.

3. Conclusions
In conclusion, we have successfully designed and synthesized LIFM–ZY–3, a TP
fluorescent Zn(II) complex with lysosome-targeting properties. The complex not only
exhibited dual responsiveness to pH and viscosity changes but also demonstrated excellent
PDT efficacy against tumor cells. The lysosome-targeting capability of LIFM–ZY–3 was
confirmed through cellular co-localization analysis. Using commercial probes as indicators,
we verified that LIFM–ZY–3 generated multiple ROS, including 1 O2 , •OH, and O2 − , thereby
achieving the integration of Type I and II PDT under white light irradiation. This study
highlighted that the fluorescent Zn(II) complexes could serve not only as potential PS but
also achieve dual lysosomal pH and viscosity sensing, providing a structural foundation
for the future design of ideal self-reporting fluorescent PS based on Zn(II) complexes. These
findings underscored the potential of LIFM–ZY–3 as a versatile tool for tumor therapy and
micro-environment monitoring, paving the way for tumor therapy.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules30112430/s1.

Author Contributions: Conceptualization, M.P. and Y.Z.; methodology, Y.Z.; software, Y.Z.; valida-
tion, Y.Z., Y.-P.W. and S.-Q.G.; formal analysis, Y.Z.; investigation, Y.Z.; resources, M.P.; data curation,
Y.Z. and S.-Q.G.; writing—original draft preparation, Y.Z.; writing—review and editing, Y.Z. and M.P.;
visualiza-tion, Y.Z. and M.P.; supervision, M.P.; project administration, M.P.; funding acquisition, M.P.
and Y.-P.W. All authors have read and agreed to the published version of the manuscript.

Funding: This work was supported by the NKRD Program of China (2021YFA1500401), National
Natural Science Foundation of China (22171291, 92261114, 92461302), and the China Postdoctoral
Science Foundation (2023M734059).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The original contributions presented in this study are included in the
article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest: The authors declare no conflicts of interest.

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