RESEARCH ARTICLE

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Remote Manipulation of TRPV1 Signaling by Near-Infrared

Light-Triggered Nitric Oxide Nanogenerators for Specific
Cancer Therapy
Shuangling Wang, Yalin Wang, Jie Lv, Chunzhe Xu, Yuxin Wei, Guiying Wang,*
and Meng Li*

cancer is still a critical public health prob-

Specific activation of transient receptor potential vanilloid member 1 (TRPV1) lem worldwide.[1] The off-target effects and
channels provides a new avenue for cancer treatment by inducing excessive low pharmacologic selectivity of the thera-
Ca2+ influx. However, controllable manipulation of TRPV1 signaling for clinical peutic agents result in unsatisfactory clin-
application has remained elusive due to the challenge in finding a mild and ical outcomes.[2,3] These issues highlight
the vital importance of developing thera-
effective method of exerting external stimulus without adverse side effects in
peutic models with high antitumor activ-
living systems. Herein, a TRPV1-targeting near-infrared (NIR) triggered nitric ity via spatiotemporal control of cell behav-
oxide (NO)-releasing nanoplatform (HCuS@PDA-TRPV1/BNN6) based on ior to achieve desired therapeutic response
polydopamine (PDA) coated hollow copper sulfide nanoparticles (HCuS NPs) only to tumor cells.
is developed for specific cancer therapy. Upon NIR irradiation, the NO donor Protein ion channels have been re-
ported to be key regulators of cellular
BNN6 encapsulated in NIR-responsive nanovehicles can locally generate NO
functions.[4] Specifically, transient recep-
to activate TRPV1 channels and induce Ca2+ influx. This NIR controlled mode tor potential vanilloid member 1 (TRPV1)
enables the nanoplatform to exert its therapeutic effects below the apoptotic which is highly expressed in several types of
threshold temperature (43°C), minimizing the photothermal damage to malignant tumors,[4,5] especially in glioblas-
normal tissue. Integrating this special NO-mediated therapy with HCuS NPs toma, has emerged as a potential target for
mediated chemodynamic therapy, the designed nanoplatform exhibits a cancer therapy. As a non-selective cation
channel, TRPV1 can be activated by nox-
boosted anticancer activity with negligible systematic toxicity. Together, this
ious heat (a threshold of ≈43°C), vanil-
study provides a promising strategy for site-specific cancer therapy by loids, acidosis and various other stimuli.[4]
spatiotemporally controlled activation of surface ion channels, thus offering a Activation of TRPV1 can induce an exces-
solution to an unmet clinical need in cancer treatment. sive Ca2+ influx, thereby affecting multi-
ple cancer-related biological processes, in-
cluding cell proliferation, migration and
1. Introduction apoptosis.[6–8] Therefore, accurate modulation of TRPV1 signal-
ing would be a promising approach for precision cancer therapy.
Despite chemotherapy as the preferred option for cancer ther- To this end, on the basis of the inherent thermosensitivity of
apy has made tremendous progress over the last few decades, TRPV1, many efforts have been devoted to designing nanoma-
terials with photothermal behavior to regulate TRPV1 signaling
S. Wang, J. Lv, C. Xu, Y. Wei, M. Li in a remote-controlled manner.[9,10] Although promising, a suf-
College of Pharmacy ficient heat dose (>43°C) to achieve therapeutic temperatures is
Key Laboratory of Innovative Drug Development and Evaluation required for activation of TRPV1, which would inevitably result
Hebei Medical University
Shijiazhuang 050017, China
in severe damage to surrounding normal tissue. Manipulation of
E-mail: limeng@hebmu.edu.cn TRPV1 signaling without adverse side effects in living systems
S. Wang remains challenging.
Department of Environmental and Chemical Engineering Nitric oxide (NO) as a biologically active signaling molecule
Hebei College of Industry and Technology plays critical roles in multiple physiological processes.[11–13] Early
Shijiazhuang 050091, China studies have shown that NO can enable the activation of TRPV1
Y. Wang, G. Wang signaling and then induce an increase of intracellular Ca2+
The Second Department of General Surgery
The Fourth Hospital of Hebei Medical University concentration,[14,15] which prompts to utilize NO as a novel stim-
Shijiazhuang 050011, China ulator to controllably activate TRPV1 signaling within the phys-
E-mail: wangguiying@hebmu.edu.cn iological temperature range for cancer specific therapy. To real-
ize this function, designing NO-releasing materials to achieve
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.202303579 controlled and localized delivery of NO with very little systemic
toxicity is highly desirable. Moreover, the appropriate releasing
DOI: 10.1002/adhm.202303579

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Figure 1. Schematic illustration of the NIR-responsive NO nanogenerator integrating CDT and photoactivation of TRPV1 channels for specific cancer
treatment.
doses of NO in a spatiotemporal manner is also greatly im-
portant for clinical applications. On the basis of this concept,
various internal or external stimuli have been explored to real-
ize on-demand NO release from NO-releasing materials.[16–19]
Among them, near-infrared (NIR) light irradiation with high
tissue-penetration capability and minimal damage to nearby nor-
mal tissues has shown an exciting possibility to trigger controlled
NO release.[19–21] Consequently, an opportunity has risen to de-
sign TRPV1 targeted NIR-triggered NO-releasing materials as
an efficient strategy for remote-controlled activation of TRPV1
signaling.
Intracellular Ca2+ overloading induced by TRPV1 activation
can cause mitochondrial dysfunction to disrupt the intracellu-
lar redox homeostasis.[22–24] Interestingly, amplifying intracel-
lular oxidative stress can in turn efficiently destroy the mito-
chondrial Ca2+ buffering capacity to induce an enhanced Ca2+ -
overloading, which would further aggravate cell apoptosis.[24,25]
Benefiting from these features, combination of reactive oxy-
gen species (ROS)-mediated therapy with modulation of TRPV1
channel function would produce synergistic effects on cancer
treatment. As a noninvasive emerging ROS-based anticancer
modality, chemodynamic therapy (CDT) which principally cat-
alyzes the decomposition of H2 O2 to produce hydroxyl radical
(•OH) via Fenton/Fenton-like reactions can result in escalated
oxidase stress inside cells.[26–28] Therefore, combination TRPV1
targeted therapy with CDT can be a feasible solution to selectively
amplify intracellular oxidative stress.
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In the present study, by integrating NO-mediated therapy and
CDT, a TRPV1 targeted NIR-triggered intelligent nanoplatform
(HCuS@PDA-TRPV1/BNN6) based on polydopamine (PDA)
coated hollow copper sulfide nanoparticles (HCuS NPs) has
been developed for specific cancer therapy (Figure 1). HCuS
NPs with the potential to convert endogenous H2 O2 into •OH
through Fenton-like reaction can exert cancer CDT. In con-
trast to Fe- and Mn-based catalysts, Cu-containing materials still
show relatively high catalytic activities in slightly acidic envi-
ronments, leading to an enhanced CDT efficiency.[29–31] Subse-
quently, PDA possessing a loose structure and high hydrophilic-
ity was coated on the surface of HCuS NPs to not only con-
trol the exposure of HCuS NPs and their derived chemody-
namic reaction but also serve as a nanocarrier to encapsulate
the NIR-responsive NO donors (BNN6, N,N′-di-sec-butyl-N,N′-
dinitroso-1,4-phenylenediamine). Finally, the monoclonal anti-
body TRPV1 peptide was covalently conjugated onto the PDA
layer (HCuS@PDA-TRPV1/BNN6) to enable specific binding to
the plasma membrane TRPV1 channels of cancer cells. The PDA
layer can absorb the NIR light and transform the photons to ac-
tive electrons, inducing NO release from BNN6 at temperatures
below the apoptotic threshold (43°C), which can then specifically
activate TRPV1 channels and simultaneously minimize the pho-
tothermal damage to normal tissue. In addition, upon being in-
ternalized into cancer cells after long-term incubation, the ex-
posed HCuS NPs could deplete cellular glutathione (GSH) to en-
hance the efficiency of CuS-mediated CDT, which could further
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Figure 2. Characterization of HCuS@PDA-TRPV1. A) The TEM image of HCuS NPs. B) The XRD patterns of HCuS NPs and HCuS@PDA. C) FTIR
spectra of HCuS NPs, HCuS@PDA, and HCuS@PDA-TRPV1. D) TEM image of HCuS@PDA.
accelerate the oxidative damage. As expected, the NIR-triggered
multifunctional nanoplatform exhibited an amplified anticancer
activity with negligible systematic toxicity both in vitro and in
vivo. To the best of our knowledge, this is the first report of de-
veloping spatiotemporally controllable NO-releasing materials as
activators of TRPV1 for site-specific cancer synergetic therapy.
2. Results and Discussion
2.1. Synthesis and Characterization of
HCuS@PDA-TRPV1/BNN6
HCuS NPs were first prepared by solvothermal method using
sacrificial Cu2 O nanospheres as hard templates.[32] The trans-
mission electron microscopy (TEM) image indicated that the
monodispersed HCuS NPs exhibited an average size of ≈100 nm
with hollow interiors (Figure 2A; Figure S1A, Supporting Infor-
mation). The X-ray powder diffraction (XRD) pattern of HCuS
NPs (Figure 2B) was well-indexed to the hexagonal phase of
CuS (JCPDS No. 06–0464).[33] Fourier translation infrared (FTIR)
spectroscopy was used to determine the chemical composition
of HCuS NPs. As displayed in Figure 2C, the peaks at 1123
cm−1 and 618 cm−1 were the characteristic stretching vibra-
tions of the C─O and Cu─S in HCuS NPs, respectively. The
N2 adsorption–desorption measurement was used to determine
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the surface area of HCuS NPs, which was calculated to be 16.19
m2 g−1 (Figure S1B, Supporting Information). Through self-
polymerization of dopamine in a slightly alkaline environment,
PDA layer was coated onto the surface of HCuS NPs.[32,34] The
broad absorbance band occurred at 3500– 3000 cm−1 in the FTIR
spectrum of HCuS@PDA was attributed to the stretching vibra-
tion of N─H and O─H groups of PDA, which authenticated the
successful fabrication of HCuS@PDA. The TEM image revealed
that at the 1:1 molar ration of HCuS NPs to PDA, PDA coating
formed a smooth layer with thickness of 15 nm on the surface
of HCuS NPs (Figure 2D) and the mean size of HCuS@PDA
was ≈ 130 nm (Figure S1C, Supporting Information). Further-
more, the surface of the nanocomposites became more negatively
charged after PDA coating, changing from −8.8 to −28.1 mV
(Figure S1D, Supporting Information). Dynamic light scatter-
ing (DLS) measurements suggested that the average sizes of
the synthesized HCuS NPs and HCuS@PDA were 199.2 ± 14.4
and 285.54 ± 7.0 nm, respectively (Figure S1E, Supporting In-
formation). Besides, the structure of HCuS NPs was not dis-
turbed by modification with PDA, which was validated by the
results of XRD analysis and UV–vis–NIR spectra (Figure 2B;
Figure S1F, Supporting Information). Subsequently, TRPV1 anti-
body was covalently conjugated onto the surface of the nanocom-
posites to realize the TRPV1 targeting capability. As indicated
in the FTIR spectrum of HCuS@PDA-TRPV1 (Figure 2C), af-
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ter conjugation of TRPV1 antibody, the characteristic absorption
band of C─O in the carboxyl groups at ≈ 1100 cm−1 occurred.
The average size obtained from DLS analysis for HCuS@PDA-
TRPV1 was 294.91 ± 8.4 nm, which was comparable to that
of HCuS@PDA (Figure S1E, Supporting Information). All
these characteristics suggested the successful synthesis of the
nanocomposites.
2.2. The CDT Efficiency
The efficiency of the nanocomposites induced CDT was in-
vestigated using colorimetric assay. To verify the ability of the
nanocomposites to generate ROS in acidic tumor microenviron-
ment (TME), 3,3′,5,5′-tetramethylbenzidine (TMB) which can be
oxidized by •OH to produce oxidant TMB (oxTMB) with a max-
imum absorption at 652 nm was selected as the probe.[35,36] As
shown in Figure S2A (Supporting Information), after incubat-
ing HCuS NPs with H2 O2 and TMB, the reaction system dis-
played a strong absorption peak at 652 nm, indicating their po-
tential as Fenton-like catalysts. Critically, compared with the con-
ventional solid CuS nanoparticles which were synthesized ac-
cording to previously reported method (Figure S2B,C, Support-
ing Information),[37] the HCuS NPs possessed a higher catalytic
activity (Figure S2A,D, Supporting Information). And the bind-
ing affinity between HCuS NPs and H2 O2 was stronger (Figure
S2E,F and Table S1, Supporting Information). All these phenom-
ena verified the superior catalytic performance of HCuS NPs with
hollow structures, which was of great significance for achieving
great anticancer efficiency. More interestingly, after coating with
PDA, HCuS@PDA still exhibited a relatively high catalytic activ-
ity in acidic or even weakly acidic conditions (Figure 3A), thereby
enabling them to sever as promising CDT agents to transform
H2 O2 to •OH.
2.3. The Photothermal Effect
To demonstrate the photothermal performance of the nanocom-
posites, the photothermal effects of the HCuS NPs, PDA
nanoparticles (Figure S3, Supporting Information) and
HCuS@PDA nanocomposites were investigated. As indi-
cated in Figure S4 (Supporting Information), a significant
increase of temperature was observed in all these samples upon
NIR irradiation. And the photothermal conversion efficiency
(ƞ) was also calculated. According to the results (Figure S4,
Supporting Information), HCuS@PDA (ƞ = 23.86%) showed
an enhanced photothermal conversion capability compared to
HCuS NPs (ƞ = 20.92%), which was probably attributed to the
high NIR absorption and excellent photothermal conversion
efficiency of PDA (ƞ = 30.40%). Therefore, the excellent pho-
tothermal performance of HCuS@PDA was originated from
both the PDA layer and HCuS NPs. Importantly, conjugation
of TRPV1 antibody onto the surface of the nanocomposites
showed no influence on their photothermal effect (Figure S5,
Supporting Information). Moreover, the photothermal effect of
the HCuS@PDA-TRPV1 showed a radiant energy- (Figure 3B)
and materials concentration-dependent fashion (Figure 3C).
Additionally, HCuS@PDA-TRPV1 achieved the same heating
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effect even after heating/cooling for five cycles, suggesting their
excellent photothermal stability (Figure 3D). To ensure complete
release of NO and minimize the thermal damage to healthy
tissues, a relatively low laser power density of 0.5 W cm−2 that
only caused a mild temperature elevation upon irradiation for
5 min was chosen for the following study (Figure S6, Supporting
Information).
2.4. NO Release in HCuS@PDA/BNN6
Encapsulation of the NO donor, BNN6, into the PDA layer
of the nanocomposites was achieved via an in situ polymer-
ization method for responsive release of NO. BNN6 was syn-
thesized according to the previous report.[38] And its success-
ful synthesis was confirmed by nuclear magnetic resonance
(NMR) (Figure S7, Supporting Information). The PDA layer in
HCuS@PDA/BNN6 is capable of absorbing NIR light and con-
verting photons into active electrons,[39,40] triggering the release
of NO from BNN6 (Figure S8, Supporting Information). In or-
der to further study the overall release of NO by NIR laser ir-
radiation, the NO release behavior of HCuS@PDA/BNN6 was
monitored under continuous laser irradiation by a commonly
used Griess assay.[41] As shown in Figure 3E, negligible NO
was released from the solutions of HCuS@PDA/BNN6 with-
out laser exposure, revealing the good stability of BNN6. In con-
trast, laser irradiation remarkably induced the NO release from
HCuS@PDA/BNN6. Moreover, the amount of NO molecules re-
leased by HCuS@PDA/BNN6 increased significantly with in-
creasing irradiation time. Critically, the release of NO can be
well switched off/on by controlling the switching of an ex vivo
NIR laser to manipulate the active drug at the appropriate dose
in vivo within the required time to produce a continuous thera-
peutic effect and minimize the risk of NO poisoning. To address
whether HCuS@PDA/BNN6 possessed a NIR controllability for
NO release, the NO released from the samples that heated di-
rectly to 60°C without NIR irradiation was also monitored. As ex-
hibited in Figure 3F, there was no significant release of NO from
HCuS@PDA/BNN6 during 10 min of direct heating at 60°C,
while NIR irradiation (0.5 W cm−2 , 10 min) did appreciably cause
NO release. All these results demonstrated that the release of NO
from HCuS@PDA/BNN6 can be efficiently controlled by NIR
laser, revealing a unique and intelligent release mode.
2.5. Optimization of the Nanocomposites
The thickness of PDA shell was critical for the therapeutic ef-
ficacy of the nanoplatform. To achieve satisfactory CDT effi-
ciency together with high sensitivity of NIR-triggered NO re-
lease, the PDA shell thickness was optimized. In this case,
three types of HCuS@PDA were synthesized by varying the
mass ratios of PDA/HCuS NPs (Figure 4A–C). As displayed in
Figure 4D and Figure S9 (Supporting Information), the change
in the PDA shell thickness largely affected the efficiency of
HCuS@PDA-mediated Fenton-like reaction in acidic condition.
Under the conditions with an equal amount of HCuS@PDA,
the catalytic activity of the nanocomposites gradually decreased
with decreasing the mass ratio of HCuS NPs to PDA from
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Figure 3. A) UV–vis spectra of TMB to probe the HCuS@PDA-mediated Fenton-like reaction under different pH values. Temperature curves of
HCuS@PDA-TRPV1 at B) power densities and C) different concentrations. D) Photothermal stability curves of HCuS@PDA-TRPV1 for five on/off ir-
radiation cycles. E) The in vitro NO release profiles of HCuS@PDA/BNN6 under different conditions. Arrows indicate the initiation of 808 nm laser
irradiation. F) The NO release profiles of nanocomposites under NIR laser irradiation (0.5 W cm−2 ) or direct heating at different temperature.

2:1 to 1:2. The absorbance of produced oxTMB in 2:1 system
showed a 1.6-fold increase compared to that in 1:2 system. Be-
sides, HCuS@PDA/BNN6 with different mass ratios of HCuS
NPs to PDA were also synthesized. The characteristic peak
of BNN6 at 252 nm was observed in the UV–vis spectra of
HCuS@PDA/BNN6, confirming the successful encapsulation
of BNN6 (Figure S10A, Supporting Information). Furthermore,
with the increase of PDA content, the absorbance intensity of
the nanocomposites at 252 nm was increased. The BNN6 load-
ing capability of HCuS@PDA/BNN6 with different mass ratios
of HCuS NPs to PDA was determined by the UV absorption of
BNN6 at 252 nm (Figure S10, Supporting Information), which
was calculated to be 96.2 μg BNN6/mg HCuS@PDA (HCuS
NPs:PDA = 2:1), 115.2 μg BNN6/mg HCuS@PDA (HCuS
NPs:PDA = 1:1) and 123.1 μg BNN6/mg HCuS@PDA (HCuS
NPs:PDA = 1:2), respectively. While, for NO release (Figure 4E),
the amount of NO released from HCuS@PDA/BNN6 with the
same concentration was increased with the increased mass ratio
of PDA in HCuS@PDA. Considering the efficiency of NO re-
lease and CDT effect, HCuS@PDA with a mass ratio of 1:1 was
selected for the subsequent in vitro and in vivo studies.
2.6. The Activation of TRPV1 Signaling
The targeting ability of the nanocomposites toward TRPV1 chan-
nels is of great significance for specific activation of TRPV1 sig-
naling by NO released from the nanosystem. As revealed in TEM
images (Figure 5A), after the first 2 h of incubation, different
from HCuS@PDA, a higher density of HCuS@PDA-TRPV1 was
firmly attached on the plasma membrane of U373 cells, the well
characterized TRPV1 positive cells. To further analyze the tar-

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Figure 4. TEM images of HCuS@PDA with different mass ratios of HCuS NPs to PDA: A) HCuS NPs:PDA = 1:2, B) HCuS NPs:PDA = 1:1, and C) HCuS
NPs:PDA = 2:1. The effects of the mass ratio of HCuS NPs to PDA on D) the catalytic activity of HCuS@PDA nanocomposites (pH 5.0) and E) their in
vitro NO release efficacy upon laser irradiation (0.5 W cm−2 ).
get ability of HCuS@PDA-TRPV1 to U373 cells, a quantitative
inductively coupled plasma-atomic emission spectrometer (ICP-
AES) analysis was also employed to determine the Cu content in
the cell lysates. As depicted in Figure S11A (Supporting Infor-
mation), after incubating with the nanocomposites for 2 h, the
amount of Cu in the cell lysates was increased with increasing
concentrations of the nanocomposites. It was worth noting that
the Cu content in HCuS@PDA-TRPV1 treated cells was signif-
icantly higher than that in the HCuS@PDA group. TRPV1 an-
tibody functionalization led to a 3.6-fold increase of Cu content
in cell lysates when the cells were treated with the nanocompos-
ites at the concentration of 10 μg mL−1 . Meanwhile, the lower
Cu amount in the cell culture supernatant of the HCuS@PDA-
TRPV1 group (Figure S11B, Supporting Information) also sug-
gested that the TRPV1 antibody modified on the surface could
endow the nanosystem with a specific and strong binding ability
with TRPV1 channels.
To ensure that the released NO was sufficient to induce the
activation of TRPV1 signaling, the intracellular Ca2+ levels were
measured using a Ca2+ -sensitive fluorescent dye, Fluo-3 AM.[23]
As depicted in Figure 5B, compared with the untreated cells, NIR
irradiation only induced a remarkable increased fluorescence sig-
nal in U373 cells pretreated with HCuS@PDA-TRPV1/BNN6.
By contrast, no apparent fluorescence change was detected in
HeLa cells, the well-known TRPV1-negative cells[4,42] (Figure 5C).
Moreover, when TRPV1 channels in U373 cells were blocked by
capsazepine (Cpz), a selective TRPV1 antagonist, the cellular flu-
orescence intensity did not increase obviously as well (Figure 5D).
Concurrently, HCuS@PDA-TRPV1/BNN6 itself without laser ir-
radiation could not induce noticeable intracellular Ca2+ influx
(Figure 5B). Hence, obvious Ca2+ influx was only initiated in
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HCuS@PDA-TRPV1/BNN6 pretreated U373 cells under laser
irradiation. To further eliminate the photothermal effect trig-
gered by NIR irradiation on TRPV1 signaling activation and min-
imize hyperthermia-induced side effects, the NIR laser irradia-
tion (0.5 W cm−2 ) was accurately controlled to keep the cell tem-
perature < 43°C (Figure S6, Supporting Information). Under this
condition, without BNN6, HCuS@PDA-TRPV1 with laser irra-
diation did not induce an altered Ca2+ response in U373 cells
(Figure 5B). Moreover, the amounts of NO released into culture
media were also measured to verify that ability of HCuS@PDA-
TRPV1/BNN6 to produce NO in the cells in the presence of
laser irradiation (Figure S12, Supporting Information). All the
above results revealed that the NIR-triggered release of NO from
HCuS@PDA-TRPV1/BNN6 was sufficient to evoke TRPV1 acti-
vation.
2.7. In Vitro Therapeutic Effect
Encouraged by the excellent properties of HCuS@PDA-
TRPV1/BNN6, the in vitro therapeutic effect of this nanocom-
posites was evaluated. As indicated in Figure 5E, after treated
U373 cells for 24 h, HCuS@PDA-TRPV1 and HCuS@PDA-
TRPV1/BNN6 showed comparable cytotoxicity in which the
cell viabilities reduced gradually with the increasing concen-
tration of the nanocomposites. The anticancer activity can be
predominantly attributed to the CDT effect of HCuS NPs.
Fluorescence images indicated that with a prolonged incuba-
tion time, cellular internalization of the nanocomposites was
observed in U373 cells but not in TRPV1-negative Hela cells
(Figure S13, Supporting Information). After internalization, the
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Figure 5. A) Representative TEM images of U373 cells incubated with HCuS@PDA or HCuS@PDA-TRPV1. Fluorescence images of B) U373 cells, C)
HeLa cells, or D) Cpz pretreated U373 cells treated with different nanocomposites before and after laser irradiation. E) The relative viability of U373
cells treated with HCuS@PDA-TRPV1 or HCuS@PDA-TRPV1/BNN6 at various concentrations with or without laser irradiation (n = 3, *** p < 0.001). F)
Fluorescence images of U373 cells after various treatments stained with DCFH-DA. G) Cell viability of U373 cells or HeLa cells after being treated with
HCuS@PDA-TRPV1 and HCuS@PDA-TRPV1/BNN6. H) MMP of U373 cells analysis using JC-1 mitochondrial membrane dye. Scale bar = 50 μm.

nanocomposites largely accumulated in lysosomes. The highly
acidic environment of lysosome could induce the peeling of PDA
coating and facilitate HCuS NPs mediated Fenton-like reaction,
thus remarkably enhancing the CDT efficiency. To confirm this,
direct visualization of intracellular ROS level was conducted
with the ROS sensitive probe 2′,7′-dichlorodihydrofluorescein
diacetate (DCFH-DA). As expected, the intracellular fluorescence
images demonstrated that the fluorescence signals observed in
HCuS@PDA-TRPV1 or HCuS@PDA-TRPV1/BNN6 treated
U373 cells were stronger than that in untreated cells (Figure 5F),

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Figure 6. The biocompatibility of nanomaterials. A) Hemolysis assays for HCuS NPs and HCuS@PDA. The concentration of these nanocomposites
varied from 1 to 100 μg mL−1 . B) The body weights from tumor-free mice after intravenous administration of saline, HCuS NPs or HCuS@PDA (5 mg
kg−1 B.W.). (n = 6). C) The H&E-stained images of main organ slices collected from mice of different groups. Scale bars = 50 μm.

indicating the generation of ROS through HCuS NPs medi-
ated Fenton reaction. Critically, under the mild photothermal
condition, the HCuS@PDA-TRPV1/BNN6 treatment showed
greatly increased cytotoxicity than HCuS@PDA-TRPV1, which
was attributed to the amplified therapeutic effect of combined
TRPV1 targeted therapy and CDT (Figure 5E). The NIR laser
irradiation induced enhanced cytotoxicity of HCuS@PDA-
TRPV1/BNN6 was also supported by the elevated ROS level
in U373 cells (Figure 5F), since the intracellular Ca2+ overload
could further amplify ROS burst. It has been reported that
CuS-based nanomaterials possess GSH depletion property to
increase the sensitivity of tumors to ROS-associated therapy.[43]
Based on this, the intracellular GSH content after treated with
nanocomposites was detected using the GSH assay kits. As
shown in Figure S14 (Supporting Information), HCuS@PDA-
TRPV1 dramatically reduced the intracellular GSH level in a
concentration dependent manner. The depletion of cellular GSH
could efficiently elevate the intercellular ROS level and enhance
the efficiency of HCuS NPs-mediated CDT.
To demonstrate the possibility of the nanoplatform to in-
duce killing activities specifically in TRPV1-positive cancer
cells, their toxicity on HeLa cells was also assessed. As
shown in Figure 5G, with the less localization on HeLa
cells membranes, both HCuS@PDA-TRPV1 and HCuS@PDA-
TRPV1/BNN6 were non-toxic to the TRPV1-negative cells even
at the concentration up to 40 μg mL−1 . Besides, NIR irradia-
tion apparently had no effect on the cytotoxity of HCuS@PDA-
TRPV1 and HCuS@PDA-TRPV1/BNN6 to HeLa cells. Taken to-
gether, these findings suggested that the specific binding of the
nanocomposites to TRPV1 channels was of a crucial importance
to trigger CDT and the photoactivation of TRPV1.
Since both the Ca2+ influx and ROS production could in-
duce mitochondrial damage, we moved on to investigate the
synergistic outcomes of our nanoplatform on the change of
mitochondrial membrane potential (MMP) to further explore
their toxic mechanism toward TRPV1-positive cancer cells. As
illustrated in Figure 5H and Figure S15A (Supporting Infor-
mation), unlike the red fluorescence observed in the control
group, a red-to-green fluorescence conversion of JC-1 was ob-
served in HCuS@PDA-TRPV1 and HCuS@PDA-TRPV1/BNN6
pretreated U373 cells, manifesting the contribution of HCuS NPs
mediated CDT to the decrease of MMP and the damage of mi-
tochondria. More importantly, with laser irradiation treatment,
HCuS@PDA-TRPV1/BNN6-incubated U373 cells showed obvi-
ous green fluorescence, which indicated the strong synergistic
effect of Ca2+ influx and CDT on the alteration of MMP. Interest-
ingly, the alteration of MMP could be significantly inhibited by
Cpz, highlighting the critical role of TRPV1 activation in mito-
chondrial dysfunction (Figure S15B, Supporting Information).
2.8. In Vivo Biocompatibility
The in vivo biocompatibility of the nanoplatform was also de-
tected to evaluate the safety of our nanoplatform in cancer treat-
ment. Hemolysis assay was employed for quantitative determi-
nation of hemoglobin to estimate the biocompatibility of HCuS
NPs and HCuS@PDA via their interactions with the red blood
cells (RBCs). Figure 6A illustrated that both HCuS NPs and
HCuS@PDA over the concentration range of 1–100 μg mL−1
only exhibited 1% of RBCs hemolysis, which was clinically ac-
ceptable according to ISO/TR 7406.[44] To further evaluate the in

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vivo biocompatibility of HCuS NPs and HCuS@PDA, the weight
fluctuation in the mice was recorded for 21 days following in-
travenous injection. As displayed in Figure 6B, treatment with
the nanocomposites even at the dose of 5 mg kg−1 body weight
(B.W.) showed no effects on the body weights gain in mice within
21 days. On day 21, all mice pre-treated with saline and differ-
ent nanomaterials were sacrificed with major organs collected
for Hematoxylin-eosin (H&E) staining (Figure 6C). Similar to
those from the control group, no obvious pathological changes
were observed in H&E-stained pathological sections of the main
organs (hearts, livers, spleens, lungs, kidneys and brains) of
the nanomaterials-treated mice. All these data revealed the good
biocompatibility and safety of the synthesized HCuS NPs and
HCuS@PDA for in vivo therapeutic applications.
2.9. In Vivo Anticancer Effect
Inspired by the excellent in vitro therapeutic effect and good bio-
compatibility, we next investigated the in vivo antitumor efficacy
on tumor mouse models. The in vivo behavior of the nanocom-
posites after intravenous injection were first systematically as-
sessed to ensure the effectiveness of the nanoplatform for in vivo
application. First, the in vivo pharmacokinetics of HCuS@PDA-
TRPV1 was investigated through detecting the Cu concentrations
in blood samples after intravenous injection of HCuS@PDA-
TRPV1 into BALB/c mice. As shown in Figure S16 (Support-
ing Information), the HCuS@PDA-TRPV1 nanocomposites per-
formed a prolonged blood circulation half-life of 2.69 h. In ad-
dition, the in vivo circulation behavior and biodistribution of
the nanocomposites were also measured (Figure S17, Support-
ing Information). Taking advantage of the excellent photother-
mal performance of HCuS@PDA-TRPV1, IR thermal imaging
was conducted after intravenously injected with HCuS@PDA-
TRPV1. As displayed in Figure S17A (Supporting Information),
the signals of tumor areas gradually strengthened post injec-
tion and reached peak values at 4 h following injection. More-
over, ICP-AES was also employed to analyze the distribution of
HCuS@PDA-TRPV1 in the main tissues of U373 tumor-bearing
mice at 4 h post intravenous injection. The accumulation of
HCuS@PDA-TRPV1 in tumor tissues was 10.00 ± 1.52% ID/g
(Figure S17B, Supporting Information). All these results demon-
strated the passive accumulation of HCuS@PDA-TRPV1 in tu-
mor areas.
To evaluate the in vivo synergistic therapeutic activities of
HCuS@PDA-TRPV1/BNN6, U373 tumor-bearing mice were di-
vided into 6 groups randomly: 1) saline (control group), 2) saline
+ laser irradiation, 3) HCuS@PDA-TRPV1, 4) HCuS@PDA-
TRPV1 + laser irradiation, 5) HCuS@PDA-TRPV1/BNN6,
and 6) HCuS@PDA-TRPV1/BNN6 + laser irradiation. The
tumor-bearing mice were intravenously injected with different
nanocomposites (2.8 mg kg−1 B.W.) on day 0. For the photother-
apeutic groups, the laser treatment process was conducted under
strict control to keep the temperature at tumor sites lower than
43°C (Figure S18, Supporting Information). In this condition,
to avoid the burn injury by hyperthermia and simultaneously
achieve satisfactory therapeutic effect, 0.4 W cm−2 , the human
skin-sustainable maximum value permitted by the American Na-
tional Standards Institute,[45] was chosen as the laser power den-
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sity in the in vivo therapy experiments. The average tumor vol-
umes of mice in all groups were recorded as a function of time af-
ter treatment. As represented in Figure 7A, the tumor size of the
control group increased rapidly throughout the study. However,
due to the CDT activity induced by HCuS NPs, administration
of HCuS@PDA-TRPV1 and HCuS@PDA-TRPV1/BNN6 could
partially reduce tumor growth, with tumor growth inhibition
(TGI) values of 40.62% and 43.03%, respectively. Laser irradiation
itself showed almost no effect on the control and HCuS@PDA-
TRPV1 treated group. By contrast, the tumor size was suppressed
obviously in mice treated with HCuS@PDA-TRPV1/BNN6 plus
laser irradiation, with a TGI value of 82.15%, which highlighted
the synergistic effect of the CDT and photo-activation of TRPV1
channels. These findings were further supported by the images
of the nude mice bearing U373 tumors (Figure 7B).
To further evaluate the synergistic results, the H&E-stained
tumor sections were collected for measuring cell necrosis of
each group (Figure 7C). Among all the groups, HCuS@PDA-
TRPV1/BNN6 with laser irradiation induced the most noticeable
cell apoptosis in tumor tissues, which was consistent with their
highest TGI value. Importantly, no significant difference was ob-
served in the body weights among all the experimental mice
(Figure 7D), indicating that our nanoplatform showed no acute
toxicity in vivo. These results further highlighted the promis-
ing potential of our nanoplatform for spatiotemporally controlled
synergetic cancer therapy.
3. Conclusion
In summary, a TRPV1 targeted NO-releasing nanoplatform has
been fabricated and successfully applied for specific cancer ther-
apy. In this design, the biocompatible HCuS@PDA acted as not
only Fenton-like catalysts to evoke CDT but also nanocarriers for
encapsulation of NIR-responsive NO donors BNN6. Upon NIR
irradiation, the PDA layer can absorb the NIR light and trans-
form the photons to active electrons, inducing NO release from
BNN6 and then specific activation of TRPV1 channels. This NIR
controlled mode enabled the nanoplatform to exert its therapeu-
tic effects below the threshold temperature (43°C) to induce pho-
tothermal cell damage, minimizing the damage to normal tissue.
Together with its CDT action, the designed nanoplatform exhib-
ited an amplified anticancer activity with negligible systematic
toxicity both in vitro and in vivo. As an example of NO-releasing
material based TRPV1 agonist, this work provides a promising
strategy for site-specific cancer synergetic therapy in a spatiotem-
porally controlled manner.
4. Experimental Section
Synthesis of HCuS NPs: HCuS NPs were prepared using a reported
method with slight modifications.[32] Briefly, copper chloride solution
(0.5 m, 100 μL) was mixed with polyvinylpyrrolidone (PVP-K30) solution
(0.24 g, 25 mL) under continuous stirring. Next, the reaction system was
diluted with an aqueous NaOH solution (pH 9.0, 25 mL), followed by the
addition of hydrazine anhydrous solution (50%, 6.4 μL) within 5 min. After
mixed with the sodium sulphide aqueous solution (320 mg mL−1 , 200 μL),
the reaction was allowed to proceed at 60°C for 2 h. Then, the precipitate
was collected by centrifugation at 10,000 rpm for 10 min and washed with
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Figure 7. A) The mean tumor volumes of U373-tumor-bearing nude mice after different treatments within 16 days (n = 6, ** p ˂ 0.01, *** p ˂ 0.001). For the
phototherapeutic groups, mice at 4 h after intravenously injecting with different nanocomposites (2.8 mg kg−1 B.W.) were exposed to the 808 nm laser
(0.4 W cm−2 , 5 min). B) Representative digital photos of mice in day 0 and 16 after different treatments. C) The H&E-staining images of the dissected
tumor tissues after 16 days of treatment. Scale bar = 50 μm. D) The body weights of mice from different groups within 16 days (n = 6). Note that
1–6 represents different groups: 1) saline, 2) saline + 808 nm laser, 3) HCuS@PDA-TRPV1, 4) HCuS@PDA-TRPV1 + 808 nm laser, 5) HCuS@PDA-
TRPV1/BNN6, and 6) HCuS@PDA-TRPV1/BNN6 + 808 nm laser.

D.I. water and ethanol for three times, respectively. The product was finally
dried under vacuum.
Preparation of HCuS@PDA/BNN6: The HCuS@PDA/BNN6
nanocomposites were synthesized through in situ polymerization
method. HCuS NPs (1 mg) were first diluted to the tris(hydroxymethyl)
aminomethane solution (10 mm, pH 8.5, 10 mL) under magnetic stirring.
Then, a DMSO solution of BNN6 (0.48 mg, 1 mL) and different weight of
dopamine hydrochloride (2 mg, 1 mg and 0.5 mg) were added into the
suspension. Afterward, the reaction mixture was further stirred overnight
at 25°C. Finally, the HCuS@PDA/BNN6 nanocomposites were obtained
via centrifugation and washing several times with D.I. water. The loading
capacity of BNN6 was measured by assessing its UV–vis absorption at
252 nm. The BNN6 loading efficiency of nanocomposites was calculated
as follows:
Loading efficiency (%) = (m0 − m1 ) ∕m × 100% (1)
where m0 and m1 were the initial and equilibrium mass of BNN6, respec-
tively, and m was the mass of HCuS@PDA.
Preparation of HCuS@PDA-TRPV1/BNN6: The HCuS@PDA/BNN6
(10 mg) and 50% glutaraldehyde solution (1 mL) were added into phos-
phate buffer (pH 7.4, 20 mL), followed by stirring at 25°C for 2 h. After
addition of anti-TRPV1 antibody (60 μg), the reaction was continued for
24 h under 8°C with constant stirring. The product was centrifuged, and
washed with D.I. water for several times.
Detection of NO Release In Vitro: The release of NO from
HCuS@PDA/BNN6 was triggered by NIR laser irradiation. Specifi-
cally, the HCuS@PDA/BNN6 suspension (0.5 mg mL−1 ) was exposed to
an 808 nm laser (0.5 W cm−2 ). After irradiation for different times, the
supernatant was obtained via centrifugation at 12 000 rpm for 2 min. NO
assay kit was applied to monitor the amount of released NO. Moreover,
the NO release behavior of HCuS@PDA/BNN6 at different temperatures
(25, 37, and 60°C) was also detected with the same protocols.
Cell Toxicity Assays: U373 cells (5 × 103 cells per well) were cultured in
96-well plates for 24 h and treated by HCuS@PDA-TRPV1 or HCuS@PDA-
TRPV1/BNN6 at different concentrations. For phototherapeutic groups,
the cells were exposed to 808 nm laser irradiation (0.5 W cm−2 , 5 min) at
2 h after pretreated with nanocomposites. After further incubated for 24 h,
standard MTT assay was employed to evaluate the cell viability.
Intracellular Calcium Analysis: U373 cells or HeLa cells (1 × 105 cells
per well) were cultured in 24-well plates for 24 h. Then, fresh medium con-
taining CaCl2 (150 mg L−1 ) was added to replace the culture medium.
In the case of Cpz treatment groups, cells were pre-incubated with Cpz
(1 mm) for 30 min, followed by the addition of HCuS@PDA-TRPV1 or
HCuS@PDA-TRPV1/BNN6 (40 μg mL−1 ). After 2 h of treatment, the cells
were loaded with Fluo-3 AM for 1 h in the dark at 37°C following the stan-
dard protocol. Finally, the cells were irradiated by an 808 nm laser (0.5 W
cm−2 ) for 5 min, and the intracellular calcium images were collected by
fluorescence microscope.
Intracellular Mitochondrial Membrane Potential Test: Briefly, U373 cells
were cultured in 24-well plates (1 × 105 cells per well) for 24 h. After that,
the culture medium was changed by the fresh medium containing CaCl2
(150 mg L−1 ). Subsequently, the cells were treated with HCuS@PDA-
TRPV1 or HCuS@PDA-TRPV1/BNN6 (40 μg mL−1 ) for 2 h. Then, the cells
were irradiated by an 808 nm laser (0.5 W cm−2 ) for 5 min, followed by a
further incubation of 12 h. Finally, the membrane potential dye JC-1 was
used to stain with the cells following the standard protocol. The images
were recorded by fluorescence microscope.
Animals Experiment: All animal experimental and handling procedures
were in accordance with the guidelines of the Hebei committee for care
and use of laboratory animals, and approved by the Ethics Committee
for Animal Experimentation of Hebei Medical University (IACUC-Hebmu-
2022040). All the animals were housed in a controlled environment with
free access to food and water.
In Vivo Toxicity Test: Six-week-old female BALB/c mice were provided
by the Experimental Animal Center of the Chinese Academy of Medical Sci-
ences. To test the toxicity of the nanocomposites in vivo, the mice were ran-
domized into three groups (n = 6), and then were intravenously injected
with saline, saline containing HCuS NPs or HCuS@PDA (5 mg kg−1 B.W.),
respectively. The body weights of the mice were recorded every day. 21 days

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after treatment, the hearts, livers, spleens, lungs, kidneys and brains of the
mice were harvested and fixed with 4% paraformaldehyde for 24 h. After-
ward, the organs were embedded in paraffin and cut into 4 μm sections to
carry out the Hematoxylin-eosin (H&E) staining. Images were taken using
an optical microscope.
In Vivo Biodistribution: The nude mice bearing subcutaneous U373 tu-
mors were treated with HCuS@PDA-TRPV1 (2.8 mg kg−1 B.W.) via intra-
venous injection. After administration for 0, 2, 4, 8, and 24 h, the tumor
sites were irradiated by an 808 nm laser (0.5 W cm−2 ) for 5 min. And the
photothermal images were collected using FLIR infrared camera.
The U373 tumor-bearing mice were treated with saline or HCuS@PDA-
TRPV1 (25 mg kg−1 B.W.) via intravenous injection. After 4 h, mice were
sacrificed, hearts, livers, spleens, lungs, kidneys and tumors of the mice
were collected to determine the Cu content in the samples by ICP-AES.
In Vivo Tumor Therapy: The tumor-bearing models were established
by subcutaneous injection of 1 × 108 U373 cells suspended in PBS
(100 μL) into six weeks-old female BALB/c nude mice. Then, the mice
were randomized into six groups (n = 6) for different treatment when
the tumor volume reached ≈ 110–130 mm3 : 1) saline, 2) saline +
808 nm laser, 3) HCuS@PDA-TRPV1, 4) HCuS@PDA-TRPV1 + 808 nm
laser, 5) HCuS@PDA-TRPV1/BNN6, and 6) HCuS@PDA-TRPV1/BNN6 +
808 nm laser. On day 0, the mice were intravenously injected with saline,
HCuS@PDA-TRPV1 (2.8 mg kg−1 B.W.) or HCuS@PDA-TRPV1/BNN6
(2.8 mg kg−1 B.W.). For the phototherapeutic groups, the tumor sites of
the mice were exposed to 808 nm laser irradiation (0.4 W cm−2 , 5 min) at
4 h after administration. The tumor size and body weight of all mice were
recorded every other day. The equation V = a × b2 /2 was used to calculate
the tumor volume, where a and b represent the longest and shortest tu-
mor diameters, respectively. In addition, the tumor growth inhibition rate
(TGI%) was determined via using the following equation:
TGI (%) = (Vc − Vt ) ∕ (Vc − V0 ) × 100% (2)
where Vc and Vt refer to the mean tumor volumes of mice in the control
and treated groups on day 16, respectively, and V0 refers to the mean tu-
mor volume of control group on day 0.
Statistical Analysis: All of the data in this study were presented as mean
± standard deviation. One-way analysis of variance (ANOVA) was used to
evaluate the statistical significance between different groups. Significant
differences were denoted by * p < 0.05, ** p < 0.01 and *** p < 0.001, re-
spectively.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
S.W. and Y.W. contributed equally to this work. This work was supported by
the Central Guidance on Local Science and Technology Development Fund
of Hebei Province (226Z2601G), the Youth Top-notch Talents Support-
ing Plan of Hebei Province (QNBJ19004), the Scientific Research Foun-
dation of Hebei Province for the Returned Overseas Chinese Scholars
(C20220508), the Science and Technology Project of Hebei Education De-
partment (no. ZD2021072), and the Postdoctoral Fund of Hebei Medical
University.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
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Keywords
chemodynamic therapy, NO nanogenerators, remote manipulation, spe-
cific cancer therapy, TRPV1 channels
Received: October 17, 2023
Revised: December 19, 2023
Published online: January 2, 2024
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