Conducting polymer-stabilized nanozymes alleviate sepsis-induced myocardial injury by inhibiting iron accumulation and lipid peroxidation

Domain: chemistry
Published: 2026-03-12
Editorial outcome: major_revision

Abstract
Septic cardiomyopathy (SC) is a severe complication of sepsis that significantly increases patient
mortality. The onset of SC is associated with excessive reactive oxygen species (ROS), leading to
increased oxidative stress and iron accumulation. Herein, we develop a library of conductive
polypyrrole copolymerized polythiophene (PPy-co-PTh)-stabilized nanozymes by coordinating
different metal ions within the copolymer framework. From this library, we identify a ruthenium-
based nanozyme (PPy-co-PTh stabilized Ruzyme) that exhibits significant catalase- and
superoxide dismutase-like activity, as well as nitrogen radical scavenging capability. Modifying
the conductive interface significantly improves the catalytic activity and stability of the Ruzyme
by lowering the catalytic energy barrier, reducing excessive oxidation caused by ROS, and
facilitating the continuous decomposition of ROS. To achieve precise targeted delivery to
myocardial mitochondria, 5-carboxypentyl (triphenyl)phosphonium bromide (TPP-COOH) and a
cardiac-targeting peptide (CTP) are conjugated onto the surface of the PPy-co-PTh stabilized
Ruzyme. In vitro, the stabilized nanozymes effectively scavenge ROS, reduce iron accumulation,
and inhibit iron-dependent cell death, thereby lowering lipid peroxidation. In male SC mice, they
improve cardiac function, demonstrating therapeutic benefits. Given the growing interest in
nanozymes for cardiovascular diseases, this study provides a robust foundation for the
development of therapeutic agents for severe cardiomyopathy and related conditions.

Introduction
Sepsis is a syndrome characterized by a systemic inflammatory response and organ dysfunction
resulting from the body's uncontrolled reaction to infection1. This dysregulated response can lead
to damage to and failure of the heart, kidneys, lungs, and other organs2, 3. Sepsis is one of the
leading causes of death worldwide4. According to statistics, the number of people diagnosed with
sepsis in 2017 was close to 49 million, and the number of related deaths was approximately 11
million, accounting for about 20% of the deaths in that year5. Septic cardiomyopathy (SC) is a
common complication of sepsis2 and presents as an acute but reversible heart disease induced by
sepsis6, 7. SC can resolve within 7-10 days in the early stages, affecting approximately 40% of
sepsis patients8, 9, with an incidence rate ranging from 13.8% to 40%7. The main pathological
features are left and/or right ventricular systolic and diastolic dysfunction10, 11, reduced left
ventricular output12, and decreased ejection fraction (EF)1, 8. Without timely intervention, the
patient’s condition progressively worsens, leading to septic shock, which causes systemic organ
dysfunction and failure. This can result in mortality rates as high as 70%-90%13. Currently, early
clinical diagnosis of SC primarily involves assessing hemodynamics14, electrocardiogram,
echocardiography8, and monitoring afterload-related cardiac function (ACP)11. Certain serum
biomarkers, such as procalcitonin (PCT), C-reactive protein (CRP), and N-terminal forebrain
natriuretic peptide (NT-pro-BNP)11, 15, are also detected to aid in diagnosis. Furthermore, clinical
treatment primarily involves supportive therapies, such as fluid resuscitation and antibiotics, in the
early stages of the disease16; however, targeted interventions are still lacking3. Early diagnosis of
SC, along with a thorough understanding of its pathogenesis and the development of targeted
treatments, is essential for improving patient outcomes and achieving a favorable prognosis.

The pathophysiological mechanism of SC is complex and has not been fully elucidated6. The
contributing factors include systemic cytokine storms (such as IL-1β and TNF-α)17, imbalance of
nitric oxide and reactive oxygen species (R OS)18, endotoxin release8, 19, mitochondrial structural
damage and dysfunction20, high mobility group protein B1 (HMGB1) dysregulation21,
ferroptosis22, calcium overload in cardiomyocytes23, endoplasmic reticulum stress3 and
pyroptosis24. Mitochondria play a central role, as they are the primary site of adenosine
triphosphate (ATP) production. In the adult heart, the ATP needed for cardiac contraction is
produced mainly through mitochondrial oxidative phosphorylation and glycolysis, with oxidative
phosphorylation accounting for 95% of ATP generation25. Damage to mitochondria disrupts
oxidative phosphorylation, resulting in excessive free radical production and subsequent
cardiomyocyte apoptosis, as demonstrated in numerous animal studies6, 26. Several clinical studies
have shown that mitochondrial dysfunction can directly contribute to the negative outcomes of
sepsis20. Ferroptosis is a form of regulated cell death (RCD) that depends on iron and ROS22. It
differs from other forms of cell death, such as necrosis, apoptosis, pyroptosis, and autophagy27.
Intracellular iron overload and an abnormal redox system are the main features of ferroptosis28.
Fe2+ accumulates in cells, promotes the production of ROS, and exacerbates oxidative stress and
lipid peroxidation, thereby leading to cell damage29. Iron chelators or lipid peroxidation inhibitors
can prevent the occurrence of ferroptosis30. Iron-mediated cell death has been implicated in the
development of various cardiomyopathies, including SC, cardiac hypertrophy, diabetic
cardiomyopathy, and adriamycin-induced cardiotoxicity22, 27, 31. For example, Shen et al. were the
first to demonstrate the therapeutic potential of melanin nanoparticles as cardioprotective agents,
showing that they alleviate sepsis-induced myocardial injury by scavenging ROS and preventing
iron accumulation, thereby inhibiting ferroptosis32. Wang et al. confirmed that microRNA-130b-
3p (miR-130b-3p) effectively reduces sepsis-induced myocardial injury by inhibiting ferroptosis
in cardiomyocytes10. Thus, previous studies have confirmed that targeting mitochondria and
inhibiting ferroptosis may be effective approaches for alleviating SC.

Oxidative stress is the cornerstone of sepsis pathogenesis, mainly caused by ROS and reactive
nitrogen species (RNS). ROS typically include superoxide anion (O •–), hydrogen peroxide (H O ),
2 2 2
and hydroxyl radical (•OH). RNS mainly include nitric oxide (NO) in free radical form and
peroxynitrite (ONOO–) in nonfree radical form33, 34. Antioxidant therapy is a pharmacological
treatment in which antioxidants help reduce inflammation by scavenging free radicals in the
inflammatory microenvironment35. The body contains various antioxidants, including small
molecules such as reduced glutathione, vitamin A, vitamin E, and trace elements, as well as
enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), and glutathione
peroxidase (GPx)36. Under normal physio logical conditions, small molecule antioxidants and
enzymatic antioxidants work to scavenge reactive oxygen and nitrogen species (RONS),
maintaining redox homeostasis. However, during sepsis, the production of RONS exceeds the
scavenging capacity of these antioxidants, disrupting redox balance. This leads to oxidative stress,
which in turn produces even more RONS, creating a vicious cycle37. It is therefore necessary to
introduce exogenous antioxidants to scavenge excess RONS to mitigate oxidative damage in
tissues and cells.

Nanozymes are nanomaterials with inherent enzymatic properties38. Compared with natural
enzymes, they not only mimic the activity of natural enzymes but also offer advantages such as
lower production costs, greater stability, more diverse functions, and more easily regulated
catalytic activity39. Antioxidant nanozymes may exhibit activities similar to those of SOD, CAT,
or GPx33. Among these, SOD-like nanozymes can convert O •– into H O with relatively low
2 2 2
reactivity, CAT-like nanozymes can catalyze the decomposition of H O into O and H O, and
2 2 2 2
GPx-like nanozymes can decompose H O into H O33, 36. By clearing ROS in the inflammatory
2 2 2
microenvironment, antioxidant nanozymes can help alleviate oxidative stress. Currently, various
nanozymes have been extensively studied in the context of ROS-related diseases, such as the Fe-
Curcumin@Tannin acid (Fe-Cur@TA) nanozyme for targeted myocardial infarction therapy40, the
Prussian blue nanozyme for alleviating Parkinson’s disease41, the Pt-NC single-atom antioxidant
nanozyme for alleviating myocardial ischemia‒reperfusion injury42, and the multifunctional
selenium nanozyme for the treatment of acute lung injury43. However, some nanozymes present
several limitations, including overoxidation, low catalytic specificity, and inadequate stability in
complex physiological environments44. Interfacial modification can effectively enable the precise
regulation of the catalytic activity of nanozymes45. Therefore, a suitably modified interfacial
nanozyme has the potential to represent a clinically applicable category of antioxidants. These
nanozymes can mimic the structure and function of natural enzymes, effectively scavenging
reactive oxygen and nitrogen species (RONS) while restoring redox balance.

In this study, pyrrole and thiophene were used as the frameworks to coordinate various metal
ions, resulting in a conductive polypyrrole copolymerized polythiophene (PPy-co-PTh) stabilized
nanozyme library containing eleven polymers. Following a series of enzymatic activity screenings
and characterizations, a ruthenium-based nanozyme (Ruzyme) was selected for its improved CAT-
like, SOD-like, and nitrogen radical scavenging abilities. To enable precise mitochondrial targeting,
5-carboxypentyl (triphenyl)phosphonium bromide (TPP-COOH) was modified onto the surface of
the Ruzyme, creating the ICT-Ruzyme. Subsequently, ICT-Ruzyme was further modified with a
cardiac-targeting peptide (CTP) to achieve cascade targeting to the myocardial mitochondria,
resulting in CICT-Ruzyme (Fig. 1A). A series of studies revealed that, compared with
nonpolymeric nanozymes (NTCT-Ruzymes), PPy-co-PTh stabilized Ruzymes (Ruzyme, ICT-
Ruzyme, and CICT-Ruzyme) can better prevent themselves from overoxidation in the environment
of excessive H O and have better conductivity and antioxidant properties. Moreover, electrostatic
2 2
forces between conductive polymer nanoparticles can inhibit the aggregation of nanozymes,
thereby enhancing their solubility and dispersion in aqueous solutions. This property allows
nanozymes targeted at the mitochondria and myocardium to disperse uniformly within the heart,
forming an efficient conductive pathway. Additionally, through myocardial and mitochondrial
targeting, PPy-co-PTh stabilized Ruzymes effectively scavenge ROS and chelate Fe2+ in
cardiomyocytes, reducing lipid peroxidation and alleviating oxidative stress, thereby achieving a
precise therapeutic effect on SC (Fig. 1B).

Results
Synthesis and characterization of PPy-co-PTh stabilized nanozymes
Using polyvinyl alcohol, pyrrole, and thiophene as the basic framework, various metal ions were
coordinated to synthesize 11 types of PPy-co-PTh stabilized nanozymes (Fig. S1, Supplementary
Information). A series of structural characterizations were subsequently performed on the 11 types
of PPy-co-PTh stabilized nanozymes. Transmission electron microscopy (TEM) confirmed that
the nanozymes exhibited good dispersion (Fig. S2, Supplementary Information). UV‒vis
spectroscopy revealed a distinct absorption peak at 378 nm for all 11 types of PPy-co-PTh
stabilized nanozymes (Fig. S3, Supplementary Information). Dynamic light scattering (DLS) was
used to characterize the PPy-co-PTh stabilized nanozymes. The results showed that their sizes
were predominantly in the range of 50–400 nm, with particle size distribution indices (PDI) all
below 0.7, indicating good dispersion of the nanozyme solutions (Fig. S4, S5, S6, Supplementary
Information). All the PPy-co-PTh stabilized nanozymes showed positive potentials (Fig. S7,
Supplementary Information). Furthermore, as illustrated in Fig. S8, the PPy-co-PTh stabilized
nanozymes containing Fe, Co, Ni, Cu, Mn, Ce, Ru, Rh, Dy, and Pt were found to have an
amorphous structure. However, the PPy-co-PTh stabilized Au-based nanozyme exhibited a distinct
crystalline structure (Fig. S8, Supplementary Information). Overall, a library of 11 polypyrrole
nanobioc catalysts with different metal ion ligands was successfully constructed.

Characterization and screening of the enzyme-like activities of the PPy-co-PTh stabilized
nanozymes
The scavenging of RONS is a key property of nanozymes that enables their antioxidant
mechanisms. The enzyme-like activities of the PPy-co-PTh stabilized nanozymes were
systematically evaluated to identify one with improved antioxidant properties. Peroxidase (POD)
activity catalyzes the decomposition of H O into toxic •OH, and O can be converted to H O by
2 2 2 2 2
accepting electrons under the action of oxidase (OXD) (Fig. 2A). The POD-like and OXD-like
activities of the PPy-co-PTh stabilized nanozymes were evaluated by the 3,3’,5,5’-
tetramethylbenzidine (TMB) probe assay. Almost none of the PPy-co-PTh stabilized nanozymes
produced •OH or H O under the neutral pH conditions typically required for antioxidant effects
2 2
(Fig. 2B-C). Additionally, given the sensitivity of the TMB chromogenic substrate to pH variations,
we further validated the POD-like and OXD-like activities of eleven PPy-co-PTh stabilized
nanozymes at pH 7.4 using o-phenylenediamine (OPD) and 2,2’-azino-bis (3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS) as alternative substrates. The results demonstrated
that, regardless of the substrate (OPD or ABTS), all eleven PPy-co-PTh stabilized nanozymes
exhibited minimal, even negligible, POD-like and OXD-like activities (Fig. S9, Fig. S10,
Supplementary Information).

Nanozymes with CAT-like activity catalyze the decomposition of H O into O and H O (Fig.
2 2 2 2
2D). Since H O has a characteristic absorption peak at 240 nm, the CAT-like activity of the
2 2
nanozymes can be assessed by measuring the decrease in the absorbance of H O at this
2 2
wavelength during the reaction with the nanozymes. Among the 11 types of PPy-co-PTh stabilized
nanozymes, Ruzyme demonstrated the most significant reduction in H O content over time,
2 2
followed by Ptzyme (Fig. 2E). Moreover, O generation during the reaction was monitored to
2
assess the CAT-like activity of nanozymes. Compared with the other PPy-co-PTh stabilized
nanozymes, Ruzyme significantly increased the O concentration in the system and produced more
2
bubbles (Fig. S11, Supplementary Information). These findings indicate that Ruzyme has a strong
ability to catalyze the decomposition of H O into H O and O . Although Ptzyme and Rhzyme
2 2 2 2
simultaneously exhibited CAT-like activity , their catalytic performance was inferior to that of
Ruzyme (Fig. 2E-F; Fig. S11, Supplementary Information).

SOD is an important cellular antioxidant enzyme that can convert O •– to H O 46 (Fig. 2D). The
2 2 2
SOD-like activity of the PPy-co-PTh stabilized nanozymes was assessed using the cytochrome C
assay. In this reaction, O •– interacts with oxidized cytochrome C, generating reduced cytochrome
2
C, which has a specific absorption peak at 550 nm. In the presence of SOD, a portion of O •– is
2
consumed, reducing the absorption of reduced cytochrome C at 550 nm. Thus, the SOD-like
activity of the nanozymes was indirectly evaluated by measuring the inhibition rate. When the
PPy-co-PTh stabilized nanozymes were compared, Ruzyme effectively scavenged O •– even at
2
lower concentrations, with an IC of 0.009368 mg/mL (Fig. 2G; Fig. S12, Supplementary
50
Information).

Both ROS and RNS have the potential to damage DNA in tissue cells, which can subsequently
trigger the development of inflammation47. Therefore, the ability of PPy-co-PTh stabilized
nanozymes to scavenge nitrogen free radicals was examined next, using 2,2’-azino-bis-3-
ethylbenzothiazoline-6-sulfonic acid radical (ABTS•+) and 1,1-diphenyl-2-picrylhydrazine radical
(DPPH•) as substrates (Fig. 2D). The results showed that Ruzyme was relatively more effective at
clearing RNS in a concentration-dependent manner, with its ability to scavenge RNS nearly
matching that of the positive control (vitamin C) (Fig. 2H-I).

Based on a series of enzyme-like activity characterizations of all the PPy-co-PTh stabilized
nanozymes in the library, Ruzyme was selected for its greater ability to scavenge both ROS and
RNS (Fig. 2J-K). Therefore, we chose PPy-co-PTh stabilized Ruzyme for subsequent experimental
studies.

Synthesis and characterization of Ruzyme, ICT-Ruzyme and CICT-Ruzyme
Ruzyme was identified from eleven PPy-co-PTh stabilized nanozymes through high-throughput
screening of enzyme-like activities for further research. ICT-Ruzyme and CICT-Ruzyme were
subsequently synthesized by sequentially modifying the surface of Ruzyme with TPP-COOH and
CTP, as outlined in Fig. 1A. Moreover, nonpolymer nanozymes synthesized using BSA as a ligand
(NCT-Ruzyme) and Ru-nanozymes with the polyvinylpyrrolidone framework (PVP@Ru) were
selected as controls (Fig. S13, Supplementary Information). The EDS mapping diagram displayed
the distributions of Ru, C, N, O, and S elements within the Ruzyme, confirming the presence of
Ru signals in the polymer and providing direct evidence of the successful synthesis of the PPy-co-
PTh stabilized Ruzyme (Fig. S14, S15B, Supplementary Information). The distribution of each
element in the Ruzyme, along with the specific Ru content, was subsequently analyzed via
inductively coupled plasma‒mass spectrometry (ICP‒MS), revealing a Ru content of about 16.42%
(Fig. S15A, Supplementary Information). Additionally, the contents of phosphorus (P) in Ruzyme
and ICT-Ruzyme were detected by ICP‒MS, respectively. The content of P in ICT-Ruzyme was
approximately 0.0057%, but it was not detected in Ruzyme. The addition of P further indicated the
successful ligation of TPP-COOH on the surface of the Ruzyme, and the amount of grafted TPP-
COOH accounted for approximately 0.0844% of the total nanozyme. We subsequently measured
the Fourier transform infrared (FT-IR) spectra of the two nanozymes, ICT-Ruzyme and CICT-
Ruzyme. After CTP modification, distinct stretching vibrations were observed at 1644 and 1343
cm-1 in the spectrum of the CICT-Ruzyme, corresponding to the C=O bonds of CTP, confirming
the successful conjugation of CTP (Fig. S16, Supplementary Information). The Ru contents of the
Ruzyme, ICT-Ruzyme, and CICT-Ruzyme nanozymes detected by ICP-MS were 16.42%, 9.83%,
and 8.69%, respectively (Fig. S17, Supplementary Information). EDS elemental mapping also
revealed the distributions of Ru in the ICT-Ruzyme and CICT-Ruzyme, respectively (Fig. S18,
Supplementary Information). The TEM micrographs demonstrate that the nanoparticles exhibit a
relatively uniform distribution, as illustrated in Fig. 3A and Fig. S19A of the Supplementary
Information. Additionally, the amorphous nature of the nanozyme complex is consistent with the
findings from the high-angle annular dark-field (HAADF) image, which are presented in Fig. S19B
in the Supplementary Information (Fig. S19B, Supplementary Information). Atomic force
microscopy (AFM) measurements demonstrated that the thickness of the Ruzyme was
approximately 6–15 nm, the NCT-Ruzyme had a thickness of about 23–35 nm, and the ICT-
Ruzyme exhibited a thickness of around 20 nm (Fig. 3B-C; Fig. S20, Supplementary Information).
The hydrated particle sizes of the NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme ranged
from 50 to 100 nm (Fig. 3D). The smaller PDI values of Ruzyme, ICT-Ruzyme, and CICT-Ruzyme
indicated better dispersion of their aqueous solutions than that of the NCT-Ruzyme (Fig. 3E). NCT-
Ruzyme exhibited a negative zeta potential of -34.08 mV, whereas Ruzyme had a zeta potential of
5.272 mV. The presence of lipophilic cations (TPP-COOH) and myocardial targeting peptide (CTP)
in the ICT-Ruzyme and CICT-Ruzyme resulted in increased zeta potentials for both (Fig. 3F). Fig.
3G displays the stability of each nanozyme stored in different biological media (DI water, saline,
PBS, FBS, and DMEM) on days 1 and 7. After 1 mg/mL nanozyme aqueous solutions were
prepared, the NCT-Ruzyme rapidly precipitated in several biological media, whereas the PPy-co-
PTh stabilized Ruzymes maintained better dispersibility over seven days with negligible changes
in particle size (Fig. 3G; Fig. S21, Table S1, Supplementary Information). These results further
indicate that electrostatic forces between conductive polymer nanoparticles can inhibit the
aggregation of nanozymes and that the modification of TPP-COOH and CTP effectively increases
the dispersibility of Ruzyme. X-ray diffraction (XRD) analysis further demonstrated that both
Ruzyme and ICT-Ruzyme exhibit amorphous structures, a finding that aligns with the results
presented in Fig. S19B (Fig. 3H; Fig. S19B, Supplementary Information). Both polypyrrole and
polythiophene are commonly used conductive polymers. Among them, polypyrrole offers
advantages such as easy preparation, high conductivity, stability, and solubility. To assess the
electrical conductivity of the PPy-co-PTh stabilized Ruzyme, electrochemical impedance
spectroscopy (EIS) was conducted, with the nonpolymer NCT-Ruzyme used as a control. The size
of the semicircle diameter in the high-frequency region of the EIS data directly reflects the
electrical impedance of the material, providing insight into the electron transfer dynamics on the
electrode surface. A smaller semicircle diameter indicates lower electron transfer resistance and
better conductivity, whereas a larger diameter suggests higher impedance and poorer conductivity.
As shown in Fig. 3I, compared with the NCT-Ruzyme, the PPy-co-PTh stabilized Ruzyme
exhibited a smaller semicircle diameter in the high-frequency region (Fig. 3I). This indicates that
the PPy-co-PTh stabilized Ruzyme, with polypyrrole and polythiophene as the framework, has a
lower charge transfer resistance. This suggests that the nanozyme facilitates faster charge transfer
during the catalytic reaction, resulting in better electrical conductivity. In conclusion, the
incorporation of polypyrrole and polythiophene effectively enhanced the electrochemical
properties of the nanozyme, linking the electrochemical behavior to the catalytic activity. This
provides support for the subsequent validation of the catalytic performance of the PPy-co-PTh
stabilized Ruzyme.

Characterization of the CAT-like activity of nanozymes and investigation of the mechanism
of H O decomposition
2 2
Next, the CAT-like activity of Ruzyme was thoroughly characterized, with NCT-Ruzyme and/or
PVP@Ru used as controls. The generation of O bubbles, a typical catalytic phenomenon of CAT
2
enzymes, was observed. H O decomposition was effectively catalyzed by Ruzyme in the buffer
2 2
solution, leading to bubble formation, followed by ICT-Ruzyme and CICT-Ruzyme (Fig. S22,
Supplementary Information). This difference in activity was likely attributed to the presence of
TPP-COOH and CTP, which may have partially shielded the active site. The results show that the
PPy-co-PTh stabilized Ruzymes have significant CAT-like activity. Dynamic monitoring of H
2
O
2
decomposition confirmed that the PPy-co-PTh stabilized Ruzymes could efficiently catalyze H O
2 2
decomposition and O generation (Fig. 4A).
2
In addition, the ability of the nanozymes to decompose H O was evaluated using classical UV‒
2 2
Vis absorption spectroscopy with the H O indicator Ti(SO ) . The H O concentration in the
2 2 4 2 2 2
system decreased significantly over time when it was incubated with PPy-co-PTh stabilized
Ruzymes, accompanied by a noticeable color change. NCT-Ruzyme showed almost no ability to
decompose H O (Fig. 4B; Fig. S23A-D, Supplementary Information). Among them, CICT-
2 2
Ruzyme was able to deplete H O in a time- and dose-dependent manner, gradually changing the
2 2
color of the system from yellow to colorless (Fig. 4B-C; Fig. S23D, Supplementary Information).
To further examine the catalytic kinetics of the nanozymes, the time-dependent decomposition of
H O was monitored for each nanozyme. The results revealed that Ruzyme and the single-targeted
2 2
ICT-Ruzyme were able to decompose nearly 80% of H O within the first 10 min, reaching a
2 2
plateau by 20 min. In comparison, the dual-targeted CICT-Ruzyme depleted almost 40% of H O
2 2
within the first 10 min and reached a plateau at 60 min. The control group, however, showed little
to no change in H O concentration throughout the incubation period (Fig. 4D). Consistent results
2 2
were obtained based on the color change observed in each system during the detection process
(Fig. S23E, Supplementary Information). The catalytic properties of Ruzyme were further
evaluated using PVP@Ru, RuO @BSA and several other previously reported nanozymes as
2
controls. As illustrated in Fig. S24A, Ruzyme exhibited the ability to eliminate nearly 70% of H O
2 2
within the first five minutes, accompanied by a significant color change. This was followed by
Ru@MOF, PVP@Ru, and Ru@PAM. In contrast, RuO @BSA did not scavenge H O (Fig. S24A,
2 2 2
J; Supplementary Information). We analyzed the decomposition of H O at the same concentration
2 2
and different incubation times. As anticipated, Ruzyme demonstrated superior catalytic ability for
the decomposition of H O , resulting in a more pronounced color change in the system (Fig. S24B-
2 2
G, K, Supplementary Information). Subsequently, we assessed the sustained catalytic properties
and kinetics of this series of nanozymes. After four cycles, Ruzyme maintained strong catalytic
activity, whereas the performance of the other nanozymes progressively decreased. NCT-Ruzyme
and RuO @BSA displayed negligible sustained catalytic activity (Fig. S24H, Supplementary
2
Information). Additionally, we investigated the kinetics of their catalase-like activities and
summarized the Michaelis‒Menten constant (K ) and maximum initial velocity (V ) for several
M max
previously reported nanozymes. With respect to H O scavenging kinetics, Ru@MOF and
2 2
PVP@Ru demonstrated slightly higher H O affinities (1/K ) than Ruzyme; however, Ruzyme
2 2 M
achieved a greater catalytic rate (V ) compared with the other 11 nanozymes (Fig. 4E; Fig. S24I;
max
Table S2, Supplementary Information). Additionally, the calculated turnover number (TON) value
of Ruzyme for CAT-like activity is 6.053 s−1, which is significantly greater than that of Ru@CoSe
(2.004 s−1) (Table S2, Supplementary Information). Overall, Ruzyme exhibited a relatively high
catalytic rate and favorable affinity for H O , demonstrating optimal catalase-like kinetic
2 2
properties for H O scavenging. Furthermore, both the single-targeted ICT-Ruzyme and the dual-
2 2
targeted CICT-Ruzyme effectively decomposed H O repetitively (Fig. S25, Supplementary
2 2
Information).

To further investigate the role of Ruzyme in scavenging H O and its catalytic mechanism, in
2 2
situ Fourier transform infrared (in situ FTIR) spectroscopy was performed. Fig. 4F displays the in
situ FTIR spectra of the Ruzyme measured at voltages ranging from 0.4 V to 1.2 V. At voltages
between 0.4 V and 1.0 V, a distinct OOH bending vibration peak appeared near 1400 cm-1, which
was attributed to the adsorption of H O on the nanozyme surface. As the voltage increased, the
2 2
intensity of this peak decreased, and the characteristic peak gradually disappeared when the
voltage reached 1.1 V, indicating that H O was consumed. By 1.1 V, H O was almost entirely
2 2 2 2
depleted. The double peaks at 1160 cm-1 and 1102 cm-1, corresponding to the O-O antisymmetric
and symmetric stretching vibrations, respectively, also decreased in intensity with increasing
voltage. This suggests that the generation of O may be inhibited after H O is depleted as the
2 2 2
voltage increases (Fig. 4F). Under conditions with no H O , a constant voltage, and varying
2 2
treatment times, an O-O stretching vibration peak appeared at 1094 cm-1, and an OOH bending
vibration peak appeared at 1428 cm-1. As the treatment time increased, the intensity of these peaks
also increased, indicating that, in the absence of H O and at a constant voltage, the catalytic
2 2
activity of the nanozymes increased over time during the electrolysis process, suggesting that the
nanozymes became more effective at catalyzing H O decomposition as electrolysis continued
2 2
(Fig. 4G). As shown in Fig. 4H, the consumption of H O was indicated by the gradual
2 2
intensification of the OOH bending vibration peak at 1428 cm-1 as the voltage increased. The
emergence of the characteristic O-O stretching vibration peak at 1070 cm-1 (indicating O ), along
2
with the noticeable increase in the O-O antisymmetric stretching vibration and symmetric
stretching vibration peaks at 1100 cm-1 and 1166 cm-1, respectively, as well as the prominent OH
bending vibration peak at 3214 cm-1 (a characteristic peak of H O), demonstrate the production of
2
O and H O (Fig. 4H). Under the same voltage condition, as time increased, the O-O stretching
2 2
vibration peak at 1100 cm-1 appeared, and its intensity increased with time. Similarly, the OOH
bending vibration peak at 1426 cm-1 became more pronounced over time. These results suggested
that the catalytic ability of the Ruzyme in decomposing H O was enhanced with increasing
2 2
electrolysis time when H O was present and the voltage was kept constant (Fig. 4I). These results
2 2
not only demonstrated the excellent CAT-like activity of the Ruzyme but also highlighted its strong
catalytic stability, providing a solid foundation for potential biotherapeutic applications.

Investigating the mechanism of PPy-co-PTh stabilized Ruzyme to prevent excessive
oxidation and enhance catalytic stability
To investigate the specific mechanisms regulating the activity of the conductive PPy-co-PTh
stabilized Ruzyme, the valence changes of both the PPy-co-PTh stabilized Ruzyme and the BSA-
supported Ruzyme (NCT-Ruzyme) were analyzed before and after the catalytic reaction via X-ray
photoelectron spectroscopy (XPS). XPS analysis of the NCT-Ruzyme and PPy-co-PTh stabilized
Ruzyme before and after the reaction with H O demonstrated that the PPy-co-PTh stabilized
2 2
Ruzyme exhibited greater valence stability under identical catalytic conditions (Fig. 5A-B; Fig.
S26, Supplementary Information). In contrast, the NCT-Ruzyme shows larger shifts, suggesting
greater susceptibility to changes in the valence state. The conductive nature of PPy-co-PTh likely
stabilizes the electronic structure of the Ruzyme, preventing excessive oxidation or reduction. This
stability ensures consistent catalytic activity over multiple cycles, which is crucial for applications
such as RONS removal in sepsis-induced cardiac injury. Density functional theory (DFT)
calculations were performed to elucidate the catalytic mechanisms underlying the SOD- and CAT-
like activities of the NCT-Ruzyme and PPy-co-PTh stabilized Ruzyme. The dynamic equilibrium
structures of the NCT-Ruzyme and PPy-co-PTh stabilized Ruzyme, derived from ab initio
molecular dynamics (AIMD) simulations, were used as the initial models. These structures, which
involve nonpolymer nanozymes and PPy-co-PTh molecules adsorbed on their surfaces, provide
the foundation for investigating the catalytic mechanisms (Fig. 5C). Fig. 5D-G, Fig. S27A-B and
Fig. S28A-B illustrate the proposed mechanisms underlying the SOD- and CAT-like activities of
the NCT-Ruzyme and PPy-co-PTh stabilized Ruzyme. In the SOD pathway, the rate-determining
step for the NCT-Ruzyme is *H O-OOH → *OH + H O , with an energy barrier of 1.465 eV. For
2 2 2
the PPy-co-PTh stabilized Ruzyme, where an electron-conductive polymer is grafted onto the
surface, this energy barrier decreases to 0.702 eV. The rate-determining step then shifts to the final
O desorption step, with an energy barrier of 1.032 eV (Fig. 5D-E; Fig. S27A, S28A
2
Supplementary Information). The catalytic performance significantly improved after grafting with
PPy-co-PTh. In the CAT pathway, the rate-determining step for the NCT-Ruzyme is the final
oxygen desorption, which has an energy barrier of 1.907 eV. For the PPy-co-PTh stabilized
Ruzyme, where an electron-conductive polymer is grafted onto the surface, this energy barrier
decreases to 1.154 eV (Fig. 5F-G; Fig. S27B, S28B, Supplementary Information). This
enhancement is attributed to the PPy-co-PTh modification, which weakens O adsorption and
2
thereby promotes a more efficient catalytic effect. These findings offer important insights into the
molecular mechanisms driving the cascade of SOD- and CAT-like activities in nanozymes, as well
as the mechanisms that contribute to their catalytic stability.

Characterization of nitrogen radical scavenging ability and iron chelation activity of NCT-
Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme
As anticipated, ICT-Ruzyme effectively scavenged oxygen radicals such as O •⁻ and 1O
2 2,
while simultaneously demonstrating robust nitrogen radical scavenging capability, as evidenced by the
reduced amplitude in electron paramagnetic resonance (EPR) measurements (Fig. 6A-C). Given
that excessive production of RNS is closely associated with the onset and progression of
inflammation, ABTS•+ and DPPH• assays were conducted to further assess the RNS-scavenging
capabilities of the nanozymes. The results indicated that the nanozymes effectively scavenged
ABTS•+ and DPPH• in a dose-dependent manner (Fig. 6D, F; Fig. S29B-E, S30B-E,
Supplementary Information). The color of the reaction system gradually changed from dark blue
to colorless and from dark purple to yellow (Fig. S29A, S30A, Supplementary Information). The
PPy-co-PTh stabilized Ruzymes exhibited a clearance effect nearly equivalent to that of the
positive control, vitamin C (vit C), as shown more clearly in the full-wavelength scanning image
under identical concentration conditions. NCT-Ruzyme had a less effective clearance effect (Fig.
6E, G). The PPy-co-PTh stabilized Ruzymes demonstrated strong scavenging abilities for both
RNS and ROS.

Iron is a transition metal that readily alternates between Fe2+ and Fe3+ states under certain
conditions. This property gives it a strong pro-oxidative effect, which plays a significant role in its
biological activity29. Studies have shown that excess iron in the body can lead to the accumulation
of highly reactive nontransferrin-bound iron (NTBI) in cardiac tissues. This NTBI forms
intracellular iron pools, which promote the generation and accumulation of RONS, resulting in
mitochondrial dysfunction and lipid peroxidation. Over time, these processes contribute to cellular
damage48, 49. Testing the iron-chelating ability of nanozymes is therefore well justified. The
compound 1,10-phenanthroline reacts with Fe2+ to form an orange‒red complex that exhibits a
characteristic absorption peak at 510 nm. The absorbance of this complex at 510 nm correlates
linearly with Fe2+ concentrations ranging from 0–100 μM, accompanied by a noticeable color
change (Fig. S31A, B, Supplementary Information). This property makes 1,10-phenanthroline an
effective and sensitive indicator for detecting free Fe²⁺ in the system. The results demonstrated that
the PPy-co-PTh stabilized Ruzymes could chelate free Fe2+ in a concentration-dependent manner,
achieving a chelation efficiency of up to 95% at 500 mg/L. The nonpolymer NCT-Ruzyme,
however, exhibited a much lower chelation efficiency of only 30% (Fig. 6H; Fig. S31C-F,
Supplementary Information). This conclusion was more clearly illustrated in the scanning graph
of the system at the same concentration, which demonstrated the strong iron chelation ability of
the nanozymes (Fig. 6I).

Fe2+ can undergo the Fenton-like reaction with H O to generate highly toxic •OH50. Therefore,
2 2
we further investigated whether the iron-enriched nanozyme (PPy-co-PTh stabilized Fezyme,
referred to as Fezyme) itself could initiate the Fenton-like reaction under neutral conditions where
antioxidant activity is typically needed. The results showed that in a pH 7.4 buffer system,
regardless of whether TMB or ABTS was used as the substrate, Fezyme did not exhibit Fenton-
like reaction activity; that is, it failed to effectively catalyze the production of •OH (Fig. S32,
Supplementary Information). This finding indicates that although the nanozyme contains abundant
iron ions, it does not initiate a typical Fenton-like reaction at neutral pH, where antioxidant activity
is typically needed.

After confirming that Ruzyme could effectively chelate Fe2+, we evaluated the potential
leaching risk of metal ions in the relevant biological environment (including the presence of Fe2+).
We simulated the relevant biological environment (PBS buffer) and incubated Ruzyme in iron-
containing (with added Fe2+) and iron-free conditions for 72 hours to perform the ion leaching
experiments. We employed a dialysis method to separate the leached metal ions and used ICP‒MS
for highly sensitive quantitative detection of Ru ions in the dialysate. The results showed that the
relative leaching amount of Ru ions was very low (<0.0002%) in both groups. Although the
leaching rate in the iron-containing group showed a slight increasing trend compared to the iron-
free group (P = 0.0503), the absolute values remained negligible (Fig. S33, Supplementary
Information). This suggests that the risk of Ru ions leaching from the material is negligible,
demonstrating its good stability in biological environments. Moreover, this finding also indicates
from another perspective that the ruthenium sites remain highly stable.

In vitro antioxidant capacity of conducting polymer-stabilized nanozymes
The above results confirm the excellent scavenging properties of PPy-co-PTh stabilized
Ruzymes for ROS and RNS. The safety of the nanozymes was then evaluated using the rat
cardiomyocyte cell line (H9C2) (Fig. S34, Supplementary Information). The results demonstrated
that both polymeric and nonpolymeric nanozymes exhibited good biocompatibility in H9C2 cells,
with minimal cytotoxicity observed at concentrations up to 400 μg/mL (Fig. 7A).

Lipopolysaccharide (LPS)-induced H9C2 cells were used to establish an in vitro model of septic
cardiomyopathy (SC) to evaluate the potential application of PPy-co-PTh stabilized nanozymes
for cardiomyocyte protection in SC (Fig. 7B). The DCFH-DA probe was used to stain
cardiomyocytes treated with nanozymes and/or LPS, and confocal laser scanning microscopy
(CLSM) was employed to observe the intracellular ROS levels. As shown in Fig. 7C, compared
with that in the untreated group, the green fluorescence signal was significantly enhanced
following LPS treatment, indicating that LPS stimulated H9C2 cells to produce and accumulate
ROS. However, after treatment with Ruzyme and ICT-Ruzyme, the green fluorescence intensity
was markedly reduced, suggesting that the nanozymes effectively scavenged intracellular ROS.
However, the NCT-Ruzyme showed no significant effect on the ROS levels (Fig. 7C, Fig. S35,
Supplementary Information). Consistent results were obtained when O •– was used as an inducer
2
to examine the role of nanozymes in scavenging ROS (Fig. S36, Supplementary Information).

Previous results have demonstrated that PPy-co-PTh stabilized Ruzyme exhibits good
biocompatibility and effective ROS scavenging ability. TPP, a commonly used lipophilic cation,
can target mitochondria by enhancing the electrostatic interaction between the delivery vector and
the mitochondria, enabling efficient drug accumulation within the mitochondria51. Mitochondria
are crucial organelles involved in energy production within the cell52. Therefore, to further clarify
the targeting mechanism of the as-prepared nanozyme, we first determined its suborgan
localization in H9C2 cells. However, owing to the low electron density of the polymeric nanozyme,
we were unable to directly observe the accumulation of ICT-Ruzyme in the mitochondria or
lysosomes at the ultrastructural level (Fig. S37, Supplementary Information). Therefore, we further
verified the ability of ICT-Ruzyme to target mitochondria at the cellular level via fluorescence
confocal microscopy. As shown in Fig. 7D, the nuclei labeled with the dye DAPI displayed blue
fluorescence, whereas the nanozymes labeled with the dye Cy5.5 emitted red fluorescence.
Mitochondrial-targeting fluorescent probes, which can penetrate cell membranes, have been used
for specific fluorescence staining of mitochondria in cells53. After 1 hour of coincubation with the
nanozymes, a small amount began to enter the cells. As the coincubation time increased, the red
fluorescence signal gradually intensified, indicating progressive cellular uptake across all systems.
However, different nanozyme systems at the same concentration exhibited distinct uptake
behaviors. Specifically, throughout the incubation period, the fluorescence signal of ICT-Ruzyme
was significantly greater than that of Ruzyme (Fig. 7D). This finding indicates that the
incorporation of (5-carboxypentyl) (triphenyl)phosphonium bromide (TPP-COOH) enables the
nanozyme to target mitochondria, thereby increasing the cellular uptake. To further confirm that
TPP-COOH modification enhances the cellular uptake of ICT-Ruzyme by H9C2 cells, we
compared the ruthenium (Ru) content in cells treated with either Ruzyme or ICT-Ruzyme at
equivalent Ru contents. The ICP–MS results showed that the Ru content in the ICT-Ruzyme group
was approximately 1.2 times higher than that in the Ruzyme group (Fig. 7E). Moreover, TEM
revealed that all the nanozymes maintained mitochondrial structural integrity, demonstrating their
biosafety, which is consistent with the cytotoxicity results from the CCK-8 assay (Fig. S38,
Supplementary Information). Mitochondria are the primary organelles responsible for generating
O •⁻ and inducing cellular oxidative damage, whereas lysosomes serve as the main organelles for
2
phagocytosing exogenous materials54. Therefore, we next investigated the behavior of Ruzyme
and ICT-Ruzyme entry into lysosomes. After H9C2 cells were incubated with the nanozymes for
24 hours, the lysosomes were stained with LysoTracker. The results, as shown in Fig. S39, indicate
that both Ruzyme and ICT-Ruzyme accumulated in the lysosomal compartments, with Pearson’s
correlation coefficients of 0.69 and 0.65, respectively (Fig. S39, Supplementary Information).
In summary, ICT-Ruzyme significantly accumulated in both lysosomes and mitochondria.

Mitochondria are the main site of intracellular ROS production55. Mitochondrial ROS (mtROS)
were detected by staining with the mitochondrial superoxide indicator MitoSOX to assess
oxidative damage. After LPS-induced oxidative damage, a strong red fluorescence signal was
observed in the mitochondria, which decreased following treatment with Ruzyme and ICT-
Ruzyme (Fig. 7F). Quantitative analysis further revealed that both Ruzyme and ICT-Ruzyme
treatments significantly reduced mtROS levels compared to the model group, whereas the NCT-
Ruzyme had no significant effect (Fig. 7G). These results indicate that ICT-Ruzyme effectively
targets the mitochondria of cardiomyocytes, scavenges ROS in the mitochondrial region, and
prevents the generation of intracellular free radicals.

Evaluation of the in vivo biocompatibility of the PPy-co-PTh stabilized Ruzymes
Before studying the use of PPy-co-PTh stabilized Ruzymes for the treatment of SC in mice,
the biocompatibility and biosafety of the CICT-Ruzyme were systematically evaluated in vivo to
assess its clinical translational potential. A total of 20 C57BL/6J mice were randomly assigned to
4 groups: the control group and three CICT-Ruzyme groups (20, 40, and 60 mg/kg). The mice were
then continuously administered the treatments and monitored for 2 weeks (Fig. 8A). The daily
weight changes of each mouse were recorded during the experiment, and no significant behavioral
changes were observed (Fig. 8B). After 2 weeks of treatment, blood biochemical analysis and
complete blood cell analysis were performed. The results showed no significant differences in liver
and kidney function, as well as blood routine parameters, between the control group and the
different doses of CICT-Ruzyme (Fig. 8C-D). These findings indicated that CICT-Ruzyme
exhibited negligible toxicity to the liver, kidneys, and blood of the mice. Similarly, histological
analysis of the major organs of the mice was performed via H&E staining. The results revealed
that CTCT-Ruzyme had no significant histological toxicity, even at the high dose of 60 mg/kg (Fig.
8E). Moreover, the hemolysis test confirmed that the PPy-co-PTh stabilized Ruzymes exhibited
good blood compatibility and did not induce significant hemolysis (Fig. S40, Supplementary
Information). These results indicate that the PPy-co-PTh stabilized Ruzymes possess high
biosafety and biocompatibility.

To subsequently verify the ability of the PPy-co-PTh stabilized Ruzymes to alleviate LPS-
induced cardiac injury, this study first evaluated their cardiac targeting efficacy via in vivo imaging.
For this purpose, Cy5.5-labeled Ruzyme, ICT-Ruzyme, and CICT-Ruzyme nanoparticles were
prepared. Studies have shown that the cardiac-targeting peptide (CTP) with the amino acid
sequence CSTSMLKAC exhibits significant selectivity toward the pathological myocardium56.
Therefore, this experiment was performed using an LPS-induced SC mouse model. Specifically,
SC mice were intravenly injected with equal doses of three types of Cy5.5-labeled nanozyme
dispersions (Cy5.5-Ruzyme, ICT-Ruzyme and CICT-Ruzyme). The average fluorescence intensity
of each nanoenzyme in the organs at different time points (1 h, 3 h, 6 h, and 24 h) was detected via
an in vivo imaging system. Ex vivo fluorescence imaging revealed that the accumulation of
nanoparticles in myocardial tissue increased over time. Moreover, in the SC mouse model,
compared with that in the nontargeted group, the myocardial fluorescence signal in the group
injected with the CTP-targeted peptide-containing nanozyme was the strongest at all time points,
indicating that CTP modification significantly enhanced the targeted accumulation of nanozymes
in the pathological myocardium (Fig. 8F, and Fig. S41, Supplementary Information). To further
validate the targeting efficacy of CTP-modified nanozymes toward pathological myocardium, LPS-
induced SC mice were intravenously administered three types of nanozymes with equivalent Ru
contents. Heart tissues were collected 12 hours post-injection to compare the ruthenium (Ru)
content. The ICP‒MS results demonstrated that the Ru level in the cardiac tissue of the CTP-
targeted peptide nanozyme (CICT-Ruzyme) group was approximately 2.3 times greater than that
in the nontargeted group (ICT-Ruzyme) (Fig. 8G). This result further confirms the ability of CTP
to target pathological myocardium and facilitates subsequent in vivo animal experiments.
Moreover, the accumulation of the three nanozymes in the liver, spleen, lungs, and kidneys was
monitored over time to reflect their metabolic profiles. The results indicated that all three
nanozymes were distributed to varying degrees in each organ, with the most significant
accumulation observed in the liver and kidneys, which is consistent with conventional drug
metabolic pathways (Fig. S42, Supplementary Information).

In vivo therapeutic efficacy of PPy-co-PTh stabilized Ruzymes
Studies have reported that once nanoparticles enter the bloodstream, proteins and other
biomolecules in the blood may interact with them, forming a so-called “biomolecular corona” on
their surface, which alters the intrinsic bioactivity and therapeutic efficacy of the drugs57, 58.
Therefore, prior to conducting in vivo experiments, the PPy-co-PTh stabilized Ruzymes were
incubated with 10% fetal bovine serum (FBS) at 37°C for 0, 3, 6, and 18 hours, respectively, to
evaluate the effect of the formed biomolecular corona on the activity of the nanozyme. The
dissolved oxygen content produced by the catalytic decomposition of hydrogen peroxide by
nanozymes after incubation with 10% FBS for varying durations was measured via a dissolved
oxygen meter. The results indicated that as the incubation time with 10% FBS increased, the
biomolecular corona on the surface of the nanoparticles gradually formed, leading to a
corresponding decrease in their CAT-like activity to varying degrees (Fig. S43A-C, Supplementary
Information). This may be attributed to the partial obscuration of the nanozymes’ active sites by
the formed biomolecular corona. Subsequently, their capacity to scavenge reactive nitrogen species
(RNS) was evaluated using ABTS•+ and DPPH• as substrates. The results demonstrated that when
ABTS•+ was used as the detection substrate, the biomolecular corona formed on each nanozyme
after varying incubation periods with 10% FBS had a negligible effect on their ability to scavenge
ABTS•+ radicals. The fact that the color of the reaction system did not change also accurately
confirmed this conclusion (Fig. S44A-E, Supplementary Information). Meanwhile, when DPPH•
was used as the detection substrate, the RNS scavenging capacity of all the nanozymes slightly
decreased with prolonged incubation time, accompanied by varying degrees of color change.
Among them, CICT-Ruzyme exhibited the most significant reduction. Nevertheless, even after 18
hours of coincubation, CICT-Ruzyme retained 60% of its RNS scavenging activity (Fig. S45A-E,
Supplementary Information). We then further evaluated the uptake of ICT-Ruzyme by H9C2 cells
both before and after incubation with 10% FBS. As the coincubation time of the nanozymes with
the cells increased, the red fluorescence signal within the cells tended to increase. However, the
cellular uptake of the nanozyme with a “biomolecular corona” was slightly weaker than that of the
nanozyme without incubation (Fig. S46, Supplementary Information). This finding indicates,
from another perspective, that the “biomolecular corona” formed on the surface of the nanozyme does
indeed affect the efficiency of its cellular uptake. Therefore, PPy-co-PTh stabilized Ruzymes with
a surface biomolecular corona still exhibit considerable ROS and RNS scavenging capacity, as
well as cellular uptake efficiency, which lays a foundation for subsequent validation of the
therapeutic efficacy of these nanozymes in vivo.

To better assess the in vivo therapeutic efficacy of PPy-co-PTh stabilized Ruzymes, an SC
mouse model was established using LPS, and the effect of induction time on cardiac function was
first explored. After intravenous injection of LPS for 12 or 24 h, echocardiography, and histological
examination were performed to evaluate changes in cardiac function and histology, to determine
the optimal induction time (Fig. S47A, Supplementary Information). Cardiac echocardiography
evaluation showed that, compared with the untreated group, a 12-h induction time significantly
altered the cardiac function of the mice, as evidenced by a more pronounced reduction in the left
ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (Fig. S47B-
D, Supplementary Information). Furthermore, histological analysis revealed more inflammatory
cell infiltration, red blood cell accumulation, and ROS production when the induction time was 12
h (Fig. S47E-F, Supplementary Information). Since SC is reversible within 7–10 days of onset, the
cardiac function of the mice may have entered a compensatory phase after 24 h of induction,
leading to the restoration of cardiac function. Therefore, the induction time of 12 h was ultimately
selected for the subsequent animal experiments.

Next, the SC mouse model was established by intravenously injecting LPS for an induction time
of 12 h. We then used NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme for treatment (Fig.
9A). According to the results of echocardiography, compared with the control, LPS damaged the
cardiac function of the mice, which significantly reduced the LVEF and LVFS. After PPy-co-PTh
stabilized Ruzymes treatment, the LVEF and LVFS values significantly increased, indicating that
the cardiac function of the mice improved, but the effect of the nonpolymer nanozyme, NCT-
Ruzyme, was not obvious (Fig. 9B-D). In addition, there was no difference in the ratio of heart
weight to body weight among the groups (Fig. S48, Supplementary Information). Serological and
histological assays were then conducted at 12 h to assess the serum concentrations of CK-MB and
LDH, two markers of myocardial injury. The results revealed a significant increase in the serum
levels of these markers in the LPS group compared with those in the control group. However, in
the mice treated with Ruzyme, ICT-Ruzyme, or CICT-Ruzyme, the levels of both myocardial
injury markers were effectively reduced (Fig. 9E-F). As shown in Fig. 9G, histological analysis
demonstrated that, compared with those in the control group, the myocardial tissue of the mice
treated with LPS exhibited noticeable pathological changes. These included infiltration of
inflammatory cells, disordered arrangement of myocardial cells, cell rupture and dissolution,
significantly widened intercellular spaces, and accumulation of red blood cells, indicating
successful induction of myocardial injury by LPS. After treatment with the NCT-Ruzyme, the
intercellular space was restored; however, the infiltration of inflammatory cells and red blood cell
accumulation persisted. However, treatment with the PPy-co-PTh stabilized Ruzymes effectively
improved the tissue structure. The myocardial morphology became clearer, the cells were more
neatly arranged, and pathological damage was markedly reduced (Fig. 9G). As depicted in Fig. 9H,
Fig. S49 and S50, LPS-induced myocardial injury resulted in the generation of ROS, which
increased the level of oxidative stress in myocardial tissue and promoted the apoptosis of
myocardial cells. The CICT-Ruzyme with myocardial targeting ability effectively reduced
oxidative stress levels and the number of apoptotic myocardial cells (Fig. 9H; Fig. S49, S50,
Supplementary Information). Additionally, the RNS scavenging capacity of the PPy-co-PTh
stabilized Ruzymes was verified at the tissue level via the fluorescent probe O52. The results
showed that LPS significantly increased RNS levels in myocardial tissue, whereas treatment with
PPy-co-PTh stabilized Ruzymes effectively reduced RNS levels. In contrast, the NCT-Ruzyme
exhibited no alleviating effect (Fig. S51, Supplementary Information). Subsequently, PVP@Ru,
RuO @BSA, and three previously reported nanozymes were employed as controls to further
2
examine the mitigating effect of the PPy-co-PTh stabilized Ruzyme on SC (Fig. S52A,
Supplementary Information). The results indicated that, compared with the control, LPS
significantly decreased both LVEF and LVFS in mice, leading to a reduction in cardiac function.
Following treatment with nanozymes, cardiac function improved across all groups; however, CTP-
Ruzyme treatment resulted in the most pronounced effects (Fig. S52B-D, Supplementary
Information). PPy-co-PTh stabilized Ruzymes can thus effectively alleviate LPS-induced
myocardial injury and improve cardiac function.

Mechanisms underlying the therapeutic effects of CICT-Ruzyme in alleviating SC
To further investigate the mechanisms through which PPy-co-PTh stabilized Ruzymes alleviate
LPS-induced SC, high-throughput transcriptomics sequencing analysis was conducted on mouse
heart tissues. DESeq2 analysis revealed 2,919 differentially expressed mRNAs between the model
group and the control group, indicating that the SC model was successfully established (Fig. S53,
Supplementary Information). A total of 2,096 mRNAs were differentially expressed following
CICT-Ruzyme treatment compared with the model group, with 1,120 mRNAs upregulated and
1,076 mRNAs downregulated. These findings indicate significant transcriptomic differences
between the model group and the CICT-Ruzyme treatment group (Fig. 10A-C). Gene Ontology
(GO) analysis of the differentially expressed mRNAs indicated that CICT-Ruzyme treatment was
involved in signaling pathways related to the inflammatory response, receptor complex, and
receptor ligand activity (Fig. 10D; Table S3, Supplementary Information). To gain a deeper
understanding of the biological functions and pathways associated with the differentially expressed
genes (DEGs) induced by CICT-Ruzyme, KEGG enrichment analysis was conducted. The top 30
enriched pathways were visualized in a KEGG bubble map. Several pathways, including the
ferroptosis pathway, were identified as being involved in the effects of CICT-Ruzyme treatment
(Fig. 10E).

When the intracellular Fe2+ concentration becomes excessive, the Fenton reaction can generate
a substantial amount of ROS, leading to oxidative stress and lipid peroxidation, which in turn
triggers ferroptosis in cells59. In previous characterization studies, the Fe2+ chelating ability of PPy-
co-PTh stabilized Ruzymes was demonstrated. Building upon these findings, the role of the CICT-
Ruzyme in the ferroptosis pathway was further investigated in cardiomyocytes, guided by previous
characterization and transcriptome analysis results. An iron overload model was established in
vitro using LPS on cardiomyocytes. Iron accumulation in the cells was assessed using FerroOrange,
a fluorescent probe for Fe2+. Excess Fe2+ accumulated in LPS-treated cells, but preincubation with
Ruzyme and ICT-Ruzyme significantly inhibited this process. However, the effect of the
nonpolymer nanozyme, NCT-Ruzyme, was minimal (Fig. 10F; Fig. S54A, Supplementary
Information). Intracellular iron accumulation is a hallmark of ferroptosis, promoting the generation
of ROS and lipid ROS (LOS), which specifically increases oxidative stress within the
inflammatory microenvironment60. To further investigate this, intracellular lipid peroxide (LPO)
levels were measured after incubating the cells with nanozymes and/or LPS using the lipid
peroxidation fluorescent probe (Liperfluo). Coincubation with LPS significantly elevated LPO
accumulation. However, pretreatment with Ruzyme and ICT-Ruzyme led to a significant reduction
in LPO levels, whereas the effect of the NCT-Ruzyme was comparatively weaker (Fig. 10G). The
relative quantitative results were consistent with the above results (Fig. S54B, Supplementary
Information). Prussian blue staining of cardiac tissue showed that iron levels increased in the
myocardial tissue after LPS induction. Treatment with CICT-Ruzyme effectively reduced the
cardiac iron content (Fig. S55, Supplementary Information). These results indicated that the PPy-
co-PTh stabilized Ruzymes effectively reduced ferroptosis-related characteristics, including Fe2+
accumulation and lipid peroxidation, in LPS-induced cardiomyocytes.

RSL3, a GPX4 inhibitor, has been widely used to induce ferroptosis61. Therefore, we employed
RSL3 as an inducer to further validate the inhibitory effect of PPy-co-PTh stabilized Ruzymes on
ferroptosis. We first evaluated the cytotoxicity of RSL3 to H9C2 cells to determine the
concentration that induced approximately 50% cell death, which was identified as 12 μM (Fig.
S56A, Supplementary Information). Subsequently, H9C2 cells were treated with 12 μM RSL3 to
induce ferroptosis in cardiomyocytes, allowing observation of the rescue effects of various
nanozymes (Fig. 10H). The results demonstrated that treatment with PPy-co-PTh stabilized
Ruzymes restored cell viability to over 75%, whereas the NCT-Ruzyme showed no significant
protective effect (Fig. 10I). Liproxstatin-1 (Lip-1), a small molecule that protects cells from death
caused by inactivation of ferroptosis inhibitors62, was used as a positive control. Malondialdehyde
(MDA), an aldehyde produced by the decomposition of lipid peroxides, causes functional
impairment of proteins and DNA in cells and tissues, and serves as a biomarker of lipid oxidation
to evaluate ferroptosis levels63. After confirming the safety of Lip-1 (Fig. S56B, Supplementary
Information), MDA levels were subsequently measured in cells subjected to the various treatments.
As expected, MDA levels were significantly higher in the RSL3-induced cardiomyocytes than in
the control cardiomyocytes. Nanozyme treatment markedly reduced the elevated MDA levels
induced by RSL3, providing evidence that the nanozymes inhibited lipid peroxidation (Fig. 10J).
Ferrostatin-1 (Fer-1), a small-molecule inhibitor that specifically suppresses ferroptosis64. The
cytotoxicity of Fer-1 in H9C2 cardiomyocytes was evaluated, and 2 μM was selected as the
working concentration for subsequent experiments (Fig. S56C, Supplementary Information). The
inhibitory effect of the nanozyme on ferroptosis was subsequently validated by Western blotting.
Acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), a key promoter of ferroptosis,
facilitates the incorporation of free polyunsaturated fatty acids (PUFAs) into cell membranes,
thereby accelerating lipid peroxidation65. Following RSL3 induction, ACSL4 expression increased
in cardiomyocytes. Pre-protection with both ICT-Ruzyme and Fer-1 suppressed ferroptosis, with
Fer-1 exhibiting more pronounced effects than the ICT-Ruzyme (Fig. 10K, Fig. S57,
Supplementary Information). Conversely, glutathione peroxidase 4 (GPX4), a specific biomarker
associated with ferroptosis, protects cells against lipid peroxidation damage and maintains the
intracellular redox balance66. RSL3 directly inactivates GPX4 through binding, leading to
intracellular lipid peroxide accumulation and ferroptosis initiation61, 66. After treatment with ICT-
Ruzyme or Fer-1, GPX4 expression level was significantly increased (Fig. 10K, Fig. S57,
Supplementary Information). GPX4 enzymatic activity was then measured in drug-treated cells.
The results demonstrated that RSL3 induction significantly reduced GPX4 activity in
cardiomyocytes, whereas both ICT-Ruzyme and Fer-1 effectively ameliorated the RSL3-induced
reduction in GPX4 activity (Fig. 10L). These findings indicate that the ROS-scavenging and
antioxidant properties of nanozymes effectively mitigate lipid peroxidation in RSL3-induced
cardiomyocytes. At the tissue level, GPX4 activity was assessed in LPS-induced myocardial
tissues. Compared with that in the control group, GPX4 activity decreased in the model group but
was effectively restored by PPy-co-PTh stabilized Ruzymes (Fig. 10M). 4-Hydroxy-2-nonenal (4-
HNE), a lipid peroxidation metabolite and critical ferroptosis marker66, 67. The expression and
distribution of 4-HNE in different treatment groups were evaluated by performing microstructural
and quantitative analyses on four discontinuous sections from each cardiac tissue sample. The
results showed that LPS induction effectively elevated 4-HNE expression, whereas treatment with
the PPy‑co‑PTh‑stabilized nanozyme suppressed 4‑HNE levels in multiple cardiac sections,
although the extent of suppression varied among sections and treatment groups, with statistically
significant reductions observed in selected sections (Fig. S58, Supplementary Information).
Collectively, these findings demonstrate that PPy-co-PTh stabilized Ruzymes effectively alleviate
ferroptosis-induced cardiomyocyte damage by significantly inhibiting lipid peroxidation, restoring
GPX4 activity, and modulating key ferroptosis regulators.

Overall, this study further demonstrated the efficacy of conducting polymer-stabilized
nanozymes in alleviating LPS-induced cardiac ferroptosis and lipid peroxidation. The conducting
polymer–stabilized Ruzymes not only exhibit excellent electrical conductivity but also show
enhanced enzyme-like activity and a strong ability to scavenge nitrogen-free radicals, thereby
effectively preventing excessive oxidation and improving nanozyme stability. Their pronounced
antioxidant and iron-chelation properties contribute to the improvement of cardiac function,
highlighting their potential for the treatment of sepsis-related cardiac injury. Moreover, PPy-co-
PTh stabilized Ruzymes exhibit good biocompatibility and safety, providing a solid basis for
potential clinical applications. Beyond the present system, the modification of nanozymes with a
conductive polymer interface offers a general strategy to enhance the catalytic efficiency, stability,
and biocompatibility of conventional nanozymes. This approach may be particularly relevant for
therapeutic applications in diseases associated with electrical conductivity, especially those
affecting the heart and nervous system. Nevertheless, further studies are required to elucidate the
underlying molecular mechanisms and to evaluate the translational potential of these nanozymes
in clinical settings.

Methods
Ethics statement
All animal experiments were conducted in accordance with the guidelines for the care and use of
laboratory animals and were approved by the Institutional Animal Care and Use Committee of the
Central China Subcenter of National Center for Cardiovascular Diseases (Approval No.
CCSNCCD-IACUC-2024000).

Construction of the conductive PPy-co-PTh stabilized nanozymes library
Polyvinyl alcohol (PVA, grade 2099, MW 31,000–50,000, 87–89% hydrolyzed, Bidepharm, 100
mg) was dissolved in 10 mL of deionized (DI) water by heating at 90℃, and ammonium persulfate
(APS, ≥98%, Bidepharm) solutions (40 μmol) containing different metal ions were prepared. The
APS solutions containing different metal ions were slowly added to PVA solutions that had cooled
to room temperature (RT) and stirred vigorously for 1 h at RT. Pyrrole (≥98%, Macklin, 100 μL)
and thiophene (≥99%, J&K Scientific, 100 μL) were added dropwise, and the mixture was stirred
thoroughly for 4 h at 4°C. The molar ratio of the metal ions to thiophene was fixed at 1:4. The pH
of the system was subsequently adjusted to 5.5-6.0 with 0.1 M NaOH/NaBH (analytical grade,
4
Bidepharm), and the resulting mixture was dialyzed (MWCO 8-14 kDa) to remove unbound metal
ions and then lyophilized to obtain a black cotton-like solid. Among them, the selected metal ions
are FeCl ·6H O (≥99%, Macklin), CoCl ·6H O (≥99%, Macklin), NiCl ·6H O (≥99%, Adamas-
3 2 2 2 2 2
beta), CeCl ·7H O (≥99%, Bidepharm), MnCl ·4H O (≥98%, Macklin), CuSO ·5H O (≥99%,
3 2 2 2 4 2
Aladdin), HAuCl ·3H O (≥99.9%, Adamas-beta), H PtCl ·6H O (≥99.9%, Adamas-beta),
4 2 2 6 2
RuCl ·xH O (≥99%, Adamas-beta), RhCl ·3H O (≥99%, Macklin), and DyCl ·6H O (≥99.9%,
3 2 3 2 3 2
Macklin). All experimental procedures were conducted in a well-ventilated fume hood using
appropriate personal protective equipment due to the involvement of strong oxidizing agents,
reducing agents, corrosive alkaline reagents, and volatile organic monomers.

Synthesis of ICT-Ruzyme
5-carboxypentyl (triphenyl)phosphonium bromide (TPP-COOH, ≥98%, Adamas-beta, 3.7 mg), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCL, TCI, 2.4 mg) and N-
hydroxysuccinimide (NHS, ≥98%, Aladdin, 1.2 mg) were dissolved in DI water (10 mL) and
stirred at room temperature for 30 min to activate the carboxyl group. Subsequently, Ruzyme (50
mg) was added to the above mixture, and the reaction was allowed to proceed under continuous
stirring at RT for 1 h. After completion, the reaction mixture was dialyzed (MWCO 8-14 kDa) for
24 h to remove unreacted small molecules. The resulting product was collected by lyophilization
to afford ICT-Ruzyme as a black cotton-like solid. All experimental procedures were conducted
in a well-ventilated fume hood using appropriate personal protective equipment due to the use of
reactive coupling reagents.

Synthesis of CICT-Ruzyme
DSPE-PEG2000-MAL (≥95%, Ponsure, 5 mg) was dissolved in dichloromethane (CH Cl , 500
2 2
μL). ICT-Ruzyme (25 mg) was then added, and the biphasic mixture was stirred overnight at RT
to allow conjugation and removal of the organic solvent. Subsequently, cardiac-targeting peptide
(CTP, ≥99%, MCE, 2.5 mg) was added, and the reaction mixture was further stirred at RT for 2 h.
After completion of the reaction, the mixture was subjected to vacuum freeze-drying to afford
CICT-Ruzyme as a black cotton-like solid. All experimental procedures were conducted in a well-
ventilated fume hood using appropriate personal protective equipment due to the use of volatile
organic solvents and reactive functional groups.

Synthesis of the nonpolymer nanozyme NCT-Ruzyme
Ruthenium(III) chloride aqueous solution (RuCl ·xH O, ≥99%, Adamas-beta, 0.1 M, 5.0 mL, 0.50
3 2
mmol) was mixed with bovine serum albumin solution (BSA Albumin Fraction V, ≥98%,
Neofroxx; 25 mg/mL, 45 mL, 1.125 g) under vigorous stirring at room temperature for 5 min to
ensure complete homogenization. Separately, sodium hydroxide solution (NaOH, 2.0 M, 1.5 mL,
3.0 mmol) and sodium borohydride solution (NaBH , 2.0 M, 1.5 mL, 3.0 mmol) were prepared
4
and mixed at a volume ratio of 1:1 to obtain a NaOH/NaBH mixture (total volume: 3.0 mL).
4
Subsequently, 1.5 mL of this mixture (containing NaOH and NaBH ) was added to the system to
4
reduce Ru3+. The reaction mixture was then incubated at 37 °C for 24 h under continuous stirring.
After completion, the mixture was dialyzed (MWCO 8-14 kDa) for 24 h to remove unbound metal
ions and then lyophilized to afford NCT-Ruzyme as a cotton-like solid. All experimental
procedures were conducted in a well-ventilated fume hood using appropriate personal protective
equipment due to the involvement of strong reducing agents and corrosive alkaline reagents.

Synthesis of PVP@Ru
Polyvinylpyrrolidone (PVP, K30, MW ≈ 30 kDa, Bidepharm, 133 mg) was dissolved in
anhydrous methanol (MeOH, analytical grade, 180 mL) under continuous stirring until a
homogeneous solution was obtained. Separately, ruthenium (III) chloride hydrate (RuCl ·xH O,
3 2
19.2 mg) was dissolved in DI water (20 mL) to form an aqueous RuCl solution. The RuCl
3 3
aqueous solution was added dropwise to the PVP methanolic solution, followed by vortex mixing
to ensure thorough homogenization. The resulting mixture was then reacted at 70°C for 3 h under
continuous stirring. After completion of the reaction, most of the methanol was removed by rotary
evaporator. The remaining small amount of methanol was used to resuspend the solid, which was
transferred into a clean centrifuge tube. Subsequently, chloroform (CHCl , analytical grade, equal
3
volume to the residual methanol) and n-hexane (analytical grade; five times the volume of the
residual methanol) were added. The mixture was vortexed thoroughly and centrifuged at 7000 × g
for 10 min. The supernatant was discarded and the precipitate was retained. This washing process
was repeated three times. Finally, the precipitate was dried in a vacuum drying oven to afford
PVP@Ru as a solid product. All experimental procedures were conducted in a well-ventilated
fume hood using appropriate personal protective equipment due to the use of volatile and
flammable organic solvents and toxic halogenated solvents.

Statistical analysis
Statistical comparisons of all data were performed by GraphPad Prism 8.0.2. P values were
determined by unpaired two-tailed t tests between two groups. One-way ANOVA was performed
for data from three or more groups. Two-way ANOVA was performed for data with two factors.
When performing enrichment analysis on transcriptomic results, the Fisher’s exact test was used,
and the BH method was applied for P value adjustment.

Data availability statement
The RNA sequencing data generated in this study have been deposited in the NCBI Sequence Read
Archive (SRA) database under accession code PRJNA1400643. All the data necessary for
evaluating the conclusions in this paper are included within the paper itself, the Supplementary
Information, or the Source Data. All data underlying this study are available from the
corresponding author upon request. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents an innovative approach using conducting polymer-stabilized Ruzyme nanozymes to mitigate\nsepsis-induced myocardial injury, focusing on iron accumulation and lipid peroxidation. The study is well-structured and\nprovides compelling evidence that Ruzyme alleviates oxidative stress and iron overload, both of which are key contributors\nto ferroptosis. While the findings suggest a potential link to ferroptosis, the authors appropriately avoid making definitive\nclaims about ferroptosis inhibition. However, additional biochemical validation of ferroptosis markers and mechanistic\ninsights could further strengthen the study’s impact. Nevertheless, this paper is suitable for publication.\nBelow are some recommendations to enhance the manuscript’s clarity and scientific robustness.\nSuggested Enhancements:\n1. The study provides compelling evidence for reduced iron accumulation and lipid peroxidation but does not assess\nferroptosis-specific markers (e.g., GPX4, ACSL4). The authors could consider further analysing and graphing the\ntranscriptome data to highlight specific changes in key ferroptosis-related genes (e.g., GPX4). As an additional step, they\ncould validate protein levels using Western blot to determine which ferroptosis-related pathways or genes are influenced by\nRuzyme. These additions would further support the study’s conclusions without requiring major experimental changes.\n2. Ruzyme’s effect on iron homeostasis and oxidative stress is promising, but it remains unclear whether its cardioprotective\nbenefits stem from general antioxidant activity or specific ferroptosis modulation. The authors could consider comparing\nRuzyme’s effects with well-known ferroptosis inhibitors, such as ferrostatin-1 (Fer-1; radical-trapping agent), liproxstatin-1\n(lipid peroxidation inhibitor), and deferoxamine (iron chelator). This comparison would help clarify whether Ruzyme’s\nprotective mechanism involves ferroptosis regulation.\n3. The study effectively demonstrates Ruzyme’s therapeutic potential, but in vivo ferroptosis-specific validation would\nstrengthen the claims. If the authors have spare myocardial tissue sections, they could perform immunohistochemistry (IHC)\nstaining for oxidative damage markers (e.g., 4-HNE, MDA, or GPX4) to visualise oxidative stress in cardiac tissues.\nAdditionally, Prussian blue staining or ICP-MS analysis could be used to confirm iron accumulation in myocardial tissue.\nThese additional analyses would further support the in vivo findings.", "reviewer_2": "The manuscript titled “Conducting Polymer-Stabilized Nanozymes Alleviate Sepsis-\ninduced Myocardial Injury by Inhibiting Iron Accumulation and Lipid Peroxidation”\npresents a comprehensive and well-executed analysis of conducting polymer-stabilized\nnanozymes for the treatment of septic cardiomyopathy (SC). Through extensive\ncharacterization and rigorous testing, the authors successfully demonstrate that\nRuzyme, a ruthenium-based nanozyme, exhibits excellent catalytic efficiency, stability,\n, stability,\nand ROS-scavenging capabilities. The study provides strong evidence that targeted\ndelivery of these nanozymes to myocardial mitochondria effectively mitigates oxidative\nstress, iron accumulation, and lipid peroxidation, ultimately improving cardiac function\nin SC models. The thorough experimental validation and in-depth mechanistic insights\nmake this work highly valuable for the field of nanozyme-based therapeutics. I support the\npublication of this manuscript; however, the following comments should be\naddressed through revision:\n1. In the title: “Conducting Polymer-Stabilized Nanozymes Alleviate Sepsis-induced\nMyocardial Injury by Inhibiting Iron Accumulation and Lipid Peroxidation”. The word\n“induced” should be capitalized.\n2. The subscript and superscript formatting of radicals in the text is too small, making\nthem difficult to notice at times. It is recommended to adjust the formatting to make the\nsuperscripts and subscripts more distinct. For example: O •–, •OH, ONOO–.\n2\n3. Figure 2A appears to be essentially the same as Scheme 1A, raising the question of\nits necessity. Consider removing Figure 2A to avoid redundancy.\n4. It is difficult to clearly observe the described insolubility and solute aggregation in\nFigure 2H. The differences between the samples are not visually distinct, making it\nchallenging to validate the claims in the text. I suggest magnifying the relevant areas\nor providing zoomed-in insets to enhance clarity.\n5. In the legend for Figure 3B and 3C, please explicitly indicate the H\n2\ndose used in\nFigure 3B and the time points measured in Figure 3C.\n6. The numbering of supplementary tables should follow a sequential order in the main\ntext. Currently, Table S1 appears far later than Table S2. Please ensure that Table S1 is\ncited before Table S2 in the text.\n7. Could you provide a bit more explanations on how the XPS analysis supports the\nconclusion that PPy-co-PTh stabilized Ruzyme exhibits greater valence stability as\ndescribed in Section 2.6: “The XPS analysis of NCT-Ruzyme and PPy-co-PTh\nstabilized Ruzyme before and after the reaction with H O demonstrated that PPy-co-\n2 2\nPTh stabilized Ruzyme exhibited greater valence stability under identical catalytic\nconditions”?\n8. Could you please double-check the atomic labels in Figure 4C? Six distinct atom\ntypes are present, but only five are labeled. It seems that the silver atom might be\nnitrogen rather than hydrogen. If this is the case, correcting the labels.\n9. In Figures 4D and 4G, the top-view perspectives are really difficult to clearly observe\nthe adsorbed molecules. Consider improving the clarity by, for example, using a\ndifferent color scheme, or different atom style, or highlighting adsorption sites, or\nproviding additional in the supplementary information.\n10. In the legend of Figure 4, it states: “Surface configurations of different modeled\ninitial,\ntransition, and final states for the simulated …...” However, based on my understanding,\nthe results and the DFT method described in the supplementary indicate that you didn’t\nperform transition state calculations, such as NEB or dimer methods. Instead, you\ncalculated different. adsorbed configurations of the adsorbates and compared their\nenergies. So, the legend seems to be somewhat misleading, please consider revising it\nto something like: “Surface configurations of different adsorbed states for the\nsimulated …...”.\n11. A bit more details on the DFT calculation method are needed to ensure\nreproducibility. Can DFT accurately reproduce the lattice parameters and band structure of RuO ? A comparison\n2\nwith experimental or previous computational results would be helpful. Additionally, which\nRuO2 surface was used, what is the size of the supercell, and whether the bottom layers were\nfrozen during calculations (and if so, how many layers were fixed). Furthermore, specifying\nthe thermostat used for the AIMD simulations.\n12. I might be wrong but in Section 2.5, second paragraph, all the notations for Fe2+ and Fe3+\nappear incorrect. It looks like the superscripts are displayed in a smaller font\nstyle rather than being properly formatted as superscripts (which often happens after\ntext refinement by GPT). Please verify whether the correct superscript formatting has\nbeen applied.\n13. In Section 2.5, I noticed multiple formatting inconsistencies and errors in the text\nand figure legend. Please carefully review and refine the text to ensure consistency with\nthe previous sections.\n14. Additionally, Section 2.5 appears after Section 2.6, which seems out of order. Please\ndoublecheck the paper format to ensure the correct section numbering and overall\nstructure.\n15. Just out of curiosity, in Figure 7B, the daily weight changes of the mice in the low\nconcentration CICT-Ruzyme group (blue line: 20 mg/kg) seem to show slightly more\nvariation compared to the higher-concentration group. Is there a specific reason for\nthis?\n16. In Figure 7E, I do not see significant changes in most organs; however, the heart\nappears to show a relatively larger variation. Could you provide an explanation for\nthis?\n17. Some subtitles use sentence case (e.g., 2.1 Synthesis and characterization of PPy-\nco-PTh stabilized nanozymes), while some use title case (e.g., 2.10 Mechanisms\nUnderlying the Therapeutic Effects of CICT-Ruzyme in Alleviating SC). Please ensure\nconsistency in formatting across all subtitles.", "reviewer_3": "The topic is very interesting and the work presented showed some very promising data.\nHowever, the methodological/experimental description/execution, especially in the first\npart of the article (preparation and comparison among different nanozyme candidates)\ndoes not meet at the current status the standards of excellence required for a publication\nin Nature Communications. The main findings of the paper (Ruzymes showed a good\npotential in preventing LPS-induced damages in myocardial cells in vitro and in vivo\nthanks to their iron and ROS scavenging activity) get buried in a set of weaker data that\nlower the overall quality of the work. The authors might take into consideration\nremoving (or moving to SI) the initial comparative study with different nanozymes that\nactually need substantial improvements. Nevertheless, major revisions are necessary\nbefore re-evaluating the manuscript for publication in Nature Communications:", "reviewer_4": "This study presents a conductive polymer-stabilized ruthenium-based nanozyme (Ruzyme) designed to alleviate sepsis-\ninduced myocardial injury by targeting mitochondrial iron accumulation and lipid peroxidation. The authors synthesized a\nlibrary of polypyrrole-thiophene copolymer (PPy-co-PTh) nanozymes, identifying Ruzyme for its superior catalase-,\nsuperoxide dismutase-like, and nitrogen radical scavenging activities. Through interface modification with TPP-COOH and\ncardiac-targeting peptides, the nanozyme achieved myocardial and mitochondrial targeting, demonstrating enhanced\nROS/RNS scavenging, reduced ferroptosis, and improved cardiac function in LPS-induced septic mice. However, more in-\ndepth mechanistic studies are warranted for further exploration.\n1. FITR data should be provided to demonstrate the structure of the nanozymes.\n2. Figure S3 shows that all 11 types of PPy-co-PTh nanozymes exhibit absorption around ~240 nm. Could this absorption\npotentially interfere with the UV-Vis detection of the CAT activity of the nanozymes?\n3. Is there an error in the caption of Figure S9?\n4. In Figure 6, why is there an absence of validation regarding the effects of the CICT-Ruzyme on H9C2 cells?\n5. While cardiac targeting is claimed, quantitative biodistribution data (e.g., organ-specific accumulation) are absent. Include\nimaging or ICP-MS data to validate targeting efficiency in vivo.\n6. In in vivo experiments, the levels of ROS and RNS in myocardial tissue should be additionally detected to verify the\nscavenging effects of the nanozymes.\n7. Transcriptomic analysis suggests that the ferroptosis pathway is implicated in the effects of CICT-Ruzyme treatment.\nHowever, key markers (e.g., GPX4, ACSL4) are not experimentally validated. Western blotting or qPCR of these targets\nwould strengthen the mechanistic claims."}, "author_response": "Below, please find our responses to the comments and questions from the reviewers:\n------------------------------------------------------------------------------------------------------\nReview 1:\nThis manuscript presents an innovative approach using conducting polymer-stabilized\nRuzyme nanozymes to mitigat sepsis-induced myocardial injury, focusing on iron\naccumulation and lipid peroxidation. The study is well-structured and provides\ncompelling evidence that Ruzyme alleviates oxidative stress and iron overload, both of\nwhich are key contributors to ferroptosis. While the findings suggest a potential link to\nferroptosis, the authors appropriately avoid making definitive claims about ferroptosis\ninhibition. However, additional biochemical validation of ferroptosis markers and\nmechanistic insights could further strengthen the study’s impact. Nevertheless, this\npaper is suitable for publication.\nResponse: We appreciate the positive comments.\nBelow are some recommendations to enhance the manuscript’s clarity and scientific\nrobustness.\nSuggested Enhancements:\n1. The study provides compelling evidence for reduced iron accumulation and lipid\nperoxidation but does not assess ferroptosis-specific markers (e.g., GPX4, ACSL4). The\nauthors could consider further analysing and graphing the transcriptome data to\nhighlight specific changes in key ferroptosis-related genes (e.g., GPX4). As an\nadditional step, they could validate protein levels using Western blot to determine which\nferroptosis-related pathways or genes are influenced by Ruzyme. These additions would\nfurther support the study’s conclusions without requiring major experimental changes.\nResponse: Thanks for the helpful comments. In response to valuable suggestions to\nstrengthen evidence regarding ferroptosis-specific markers, we have expanded our\nexperimental validation.\nTo systematically evaluate the inhibitory effects of PPy-co-PTh-stabilized Ruzymes\n(hereafter termed “nanozymes”) on ferroptosis, we employed the well-established\nferroptosis inducer RSL-3, which is a specific GPX4 inhibitor widely used to trigger\nferroptosis pathways1. Before initiating the nanozyme treatment experiments, we\ndetermined the RSL-3 concentration (12 μM) required to induce approximately 50%\ndeath in H9C2 cardiomyocytes (Figure S54A, Supporting Information). To benchmark\nnanozyme performance, we included two classical ferroptosis inhibitors (ferrostatin-1,\nFer-1, and liproxstatin-1, Lip-1) as positive controls. Cytotoxicity assessments\nconfirmed that the experimental concentrations used for both inhibitors were safe in\nH9C2 cells (Figure S54B-C, Supporting Information).\nMalondialdehyde (MDA), an aldehyde compound generated during lipid peroxide\ndecomposition, causes damage to cellular proteins and DNA. As an established\nbiomarker of lipid peroxidation, MDA serves as a reliable indicator for assessing\nferroptosis levels2. Using Lip-1 as a positive control, we quantified the MDA levels in\nH9C2 cardiomyocytes treated with RSL-3 alone or with RSL-3 combined with\nnanozymes. As anticipated, RSL-3 induction significantly elevated MDA levels\ncompared with those in the control group (p < 0.01). Crucially, nanozyme treatment\neffectively reversed RSL-3-induced MDA accumulation, providing direct evidence of\ntheir ability to suppress lipid peroxidation (Figure 9J).\nTo address the reviewer’s key request regarding ferroptosis-specific markers, we\nperformed Western blot analysis of core regulators. RSL-3 induction robustly\nupregulated the expression of acyl-coenzyme A synthetase long-chain family member\n4 (ACSL4), which is a proferroptotic enzyme that integrates polyunsaturated fatty acids\n(PUFAs) into membrane lipids to potentiate peroxidation3. Both the nanozyme and Fer-\n1 pretreatment suppressed this ACSL4 upregulation, although Fer-1 exhibited greater\ninhibition. Conversely, glutathione peroxidase 4 (GPX4), which is the canonical\nferroptosis suppressor that prevents lipid peroxide accumulation 4, was significantly\ndownregulated following RSL-3-mediated inactivation. Critically, nanozyme treatment\neffectively restored GPX4 protein expression to levels comparable with those of Fer-1\n(Figure 9K), demonstrating its dual regulatory capacity against ferroptosis drivers.\nFurthermore, we functionally validated GPX4 reactivation by measuring its enzymatic\nactivity in dual experimental models.\nFurthermore, we functionally validated GPX4 reactivation through enzymatic assays.\nIn the cellular model, as anticipated, RSL-3 induced a significant decrease in GPX4\nactivity. Crucially, both the nanozyme and ferroptosis inhibitor treatments improved\nenzymatic function (Figure 9L). In the murine model of LPS challenge, decreased\ncardiac GPX4 activity was effectively improved by PPy-co-PTh-stabilized Ruzymes\n(Figure 9M).\nFigure S54. The viability of H9C2 cells was assessed after incubation with different\nconcentrations of (A) RSL-3 for 12 h, or with (B) Lip-1 or (C) Fer-1 for 24 h (n = 3).\nThe data are presented as the means ± SDs.\nFigure 9J. MDA levels in H9C2 cells incubated with 12 μM RSL-3 and different\nnanozymes (n = 3).\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were\ndetermined by western blot analysis. (a) Representative immunoblots of ferroptosis-\nrelated proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins\nin H9C2 cells (n = 3).\nFigure 9L. GPX4 activity in H9C2 cells incubated with 2 μM Fer-1 and different\nnanozymes (n = 3).\nFigure 9M. GPX4 activity in cardiac tissue (n = 5).\n2. Ruzyme’s effect on iron homeostasis and oxidative stress is promising, but it remains\nunclear whether its cardioprotective benefits stem from general antioxidant activity or\nspecific ferroptosis modulation. The authors could consider comparing Ruzyme’s\neffects with well-known ferroptosis inhibitors, such as ferrostatin-1 (Fer-1; radical-\ntrapping agent), liproxstatin-1 (lipid peroxidation inhibitor), and deferoxamine (iron\nchelator). This comparison would help clarify whether Ruzyme’s protective mechanism\ninvolves ferroptosis regulation.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding the\nmechanistic distinction between general antioxidant effects and specific ferroptosis\nmodulation.\nTo address this critical question, we conducted systematic comparisons of\nnanozymes (ICT-Ruzyme) with the classical ferroptosis inhibitors Fer-1 and Lip-1,\nleveraging a standardized ferroptosis model established by RSL-3 induction at a\nconcentration of 12 μM, which induced approximately 50% death in H9C2\ncardiomyocytes (Figure S54A, Supporting Information). Prior to comparative studies,\ncytotoxicity assessments confirmed safe experimental concentrations for Fer-1 and Lip-\n1 (Figure S54B-C, Supporting Information), confirming the validity of the positive\ncontrols.\nWestern blot analysis revealed that RSL-3 robustly upregulated the expression of the\nproferroptotic protein ACSL4, while both the nanozyme and the inhibitor Fer-1\nsignificantly suppressed this increase, with Fer-1 exhibiting the strongest inhibitory\neffect. Conversely, the RSL-3-mediated downregulation of the ferroptosis suppressor\nGPX4 was effectively reversed by nanozyme treatment, which outperformed Fer-1 in\nrestoring GPX4 expression (Figure 9K).\nThe GPX4 enzymatic activity assays conducted at the cellular level yielded\nconsistent findings, which functionally validated the protein-level observations (Figure\n9L). Further validation through the quantification of malondialdehyde (MDA), which\nis a terminal product of lipid peroxidation2, revealed that nanozyme treatment reduced\nRSL-3-induced MDA accumulation to levels comparable with those of Lip-1, providing\ndirect evidence of its ability to inhibit lipid peroxidation (Figure 9M). Crucially, the\nability of the nanozyme to concurrently antagonize ACSL4 upregulation, restore GPX4\nexpression, and resolve MDA accumulation, which achieves efficacy matching or\nexceeding that of established ferroptosis inhibitors, confirms that its cardioprotection\nstems from specific ferroptosis regulation rather than broad antioxidant activity.\nFigure S54. The viability of H9C2 cells was assessed after incubation with different\nconcentrations of (A) RSL-3 for 12 h, or with (B) Lip-1 or (C) Fer-1 for 24 h (n = 3).\nThe data are presented as the means ± SDs.\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were\ndetermined by western blot analysis. (a) Representative immunoblots of ferroptosis-\nrelated proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins\nin H9C2 cells (n = 3).\nFigure 9L. GPX4 activity in H9C2 cells incubated with 2 μM Fer-1 and different\nnanozymes (n = 3).\nFigure 9M. GPX4 activity in cardiac tissue (n = 5).\nFigure 9J. MDA levels in H9C2 cells incubated with 12 μM RSL-3 and different\nnanozymes (n = 3).\n3. The study effectively demonstrates Ruzyme’s therapeutic potential, but in vivo\nferroptosis-specific validation would strengthen the claims. If the authors have spare\nmyocardial tissue sections, they could perform immunohistochemistry (IHC) staining\nfor oxidative damage markers (e.g., 4-HNE, MDA, or GPX4) to visualise oxidative\nstress in cardiac tissues. Additionally, Prussian blue staining or ICP-MS analysis could\nbe used to confirm iron accumulation in myocardial tissue. These additional analyses\nwould further support the in vivo findings.\nResponse: We sincerely appreciate the reviewer’s constructive suggestions regarding\nferroptosis-specific validation.\nTo address this point, we performed multilayered in vivo analyses.\nFirst, we quantified GPX4 enzymatic activity across treatment groups at the tissue\nlevel and demonstrated that PPy-co-PTh-stabilized Ruzymes significantly restored\nGPX4 function, which serves as a core inhibitor of ferroptosis.\nPrussian blue staining subsequently revealed increased iron deposition in LPS-\nchallenged myocardial tissue, which was mitigated by treatment with PPy-co-PTh\nstabilized Ruzymes.\nFurther immunohistochemical staining for 4-hydroxynonenal (4-HNE), a terminal\nproduct of lipid peroxidation1, confirmed that the intervention reduced 4-HNE\naccumulation, thereby alleviating oxidative stress and inhibiting lipid peroxidation.\nCollectively, these integrated results unequivocally demonstrate the therapeutic\nefficacy of PPy-co-PTh-stabilized Ruzymes in suppressing ferroptosis and attenuating\noxidative damage in cardiac tissue, confirming our original conclusions. Relevant data\nare presented below.\nFigure 9M. GPX4 activity in cardiac tissue (n = 5).\nFigure S53. Changes in iron content in cardiac tissue were detected via Prussian blue\nstaining. Scale bar = 50 µm.\nFigure S55. Representative immunohistochemical staining of 4-HNE in cardiac tissue\nsamples from SC mice. Scale bar = 100 µm.\n--------------------------------------------------------------------------------\nReview 2:\nThe manuscript titled “Conducting Polymer-Stabilized Nanozymes Alleviate Sepsis-\ninduced Myocardial Injury by Inhibiting Iron Accumulation and Lipid Peroxidation”\npresents a comprehensive and well-executed analysis of conducting polymer-stabilized\nnanozymes for the treatment of septic cardiomyopathy (SC). Through extensive\ncharacterization and rigorous testing, the authors successfully demonstrate that\nRuzyme, a ruthenium-based nanozyme, exhibits excellent catalytic efficiency, stability,\nand ROS-scavenging capabilities. The study provides strong evidence that targeted\ndelivery of these nanozymes to myocardial mitochondria effectively mitigates oxidative\nstress, iron accumulation, and lipid peroxidation, ultimately improving cardiac function\nin SC models. The thorough experimental validation and in-depth mechanistic insights\nmake this work highly valuable for the field of nanozyme-based therapeutics. I support the\npublication of this manuscript; however, the following comments should be addressed\nthrough revision:\nResponse: We appreciate the positive comments.\n1. In the title: “Conducting Polymer-Stabilized Nanozymes Alleviate Sepsis-induced\nMyocardial Injury by Inhibiting Iron Accumulation and Lipid Peroxidation”. The word\n“induced” should be capitalized.\nResponse: We sincerely appreciate the reviewer’s careful reading and constructive\nsuggestion regarding the capitalization of “induced” in the manuscript title. We fully\nagree with this observation and have accordingly revised the title to:\n“Conducting Polymer-Stabilized Nanozymes Attenuate Sepsis-Induced Myocardial\nInjury by Suppressing Iron Accumulation and Lipid Peroxidation”\nThanks for the valuable input, which has helped improve the clarity and accuracy of\nour manuscript.\n2. The subscript and superscript formatting of radicals in the text is too small, making\nthem difficult to notice at times. It is recommended to adjust the formatting to make the\nsuperscripts and subscripts more distinct. For example: O •–, •OH, ONOO–.\nproofread the entire manuscript and adjusted the formatting of all superscripts and\nsubscripts to improve clarity.\n3. Figure 2A appears to be essentially the same as Scheme 1A, raising the question of\nits necessity. Consider removing Figure 2A to avoid redundancy.\nResponse: Thanks for the helpful suggestion. After careful evaluation, we concur with\nthe reviewer's observation regarding the similarity between Figure 2A and Scheme 1A.\nIn response, we have removed Figure 2A to eliminate redundancy and have enhanced\nScheme 1A to ensure that all critical information is clearly presented. These\nmodifications have improved both the conciseness and logical flow of the manuscript.\nThe revised Scheme 1 is as follows:\nScheme 1. Mechanistic diagram illustrating the role of conductive PPy-co-PTh-\nstabilized nanozymes in alleviating sepsis-induced cardiac injury through myocardial\nand mitochondrial targeting. (A) Schematic diagram of the construction of the PPy-co-\nPTh-stabilized nanozyme library and the synthesis of PPy-co-PTh-stabilized Ruzymes\n(Ruzyme, ICT-Ruzyme, CICT-Ruzyme). (B) CICT-Ruzyme was directed to the\nmitochondria of myocardial cells, effectively removing reactive oxygen and nitrogen\nspecies (RONS) in the inflammatory microenvironment, inhibiting oxidative stress, and\nmitigating myocardial injury associated with sepsis.\n4. It is difficult to clearly observe the described insolubility and solute aggregation in\nFigure 2H. The differences between the samples are not visually distinct, making it\nchallenging to validate the claims in the text. I suggest magnifying the relevant areas\nor providing zoomed-in insets to enhance clarity.\nResponse: We sincerely appreciate the reviewers’ valuable comments.\nIn response to the reviewer’s third point, Figure 2 has been reorganized, with Figure\n2H now designated Figure 2G. In response, we have revised Figure 2G and verified the\nchanges in the hydrated particle size of the PPy-co-PTh-stabilized Ruzymes when\nstored for 1, 3, 5, and 7 days in different biological media (DI water, saline, PBS, FBS,\nand DMEM).\nFurthermore, we have provided expanded explanations of these results in the revised\nmanuscript to offer a more comprehensive interpretation.\nThe details are as follows:\n“Figure 2G displays the stability of each nanozyme stored in different biological media\n(DI water, saline, PBS, FBS, and DMEM) on days 1 and 7. After 1 mg/mL nanozyme\naqueous solutions were prepared, the NCT-Ruzyme rapidly precipitated in several\nbiological media, whereas the PPy-co-PTh-stabilized Ruzymes maintained better\ndispersibility over seven days with negligible changes in particle size (Figure 2G;\nFigure S21, Supporting Information).\nFigure 2G. Liquid images of NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme\nstored in various biological media on days 1 and 7.\nFigure S21. Changes in the sizes of (A) Ruzyme, (B) ICT-Ruzyme and (C) CICT-\nRuzyme in various biological media incubated for different durations. The data are\npresented as the means ± SDs.\n5. In the legend for Figure 3B and 3C, please explicitly indicate the H\n2\ndose used in\nFigure 3B and the time points measured in Figure 3C.\nResponse: We sincerely thank the reviewer for this valuable suggestion. We agree that\nexplicitly stating critical experimental parameters in the figure legends enhances clarity\nfor readers. Accordingly, we have revised the manuscript to explicitly indicate the H₂O₂\nconcentration (30 mM) used in Figure 3B and the incubation time point (30 min)\nmeasured in Figure 3C within their respective figure legends.\nAdditionally, we have updated the corresponding experimental methods in the\nSupplementary Information (Assays of the CAT-like activity and H O decomposition\n2 2\nability of nanozymes) to provide comprehensive methodological support.\nThese modifications ensure the immediate visibility of these key experimental\nconditions within the figure presentations. We greatly appreciate this constructive\nfeedback, which has significantly improved the manuscript's clarity.\n6. The numbering of supplementary tables should follow a sequential order in the main\ntext. Currently, Table S1 appears far later than Table S2. Please ensure that Table S1 is\ncited before Table S2 in the text.\nResponse: We thank the reviewer for identifying the requirement for sequential\ncitations of supplementary tables relative to their appearance in the main text. We\nconfirm that the reference order of Tables S1 and S2 has been corrected in the revised\nmanuscript. This adjustment ensures consistent numbering alignment with textual\ncitations throughout the manuscript. We appreciate the reviewer's meticulous\nassessment, which has refined the logical progression of the supplementary information.\n7. Could you provide a bit more explanations on how the XPS analysis supports the\nconclusion that PPy-co-PTh stabilized Ruzyme exhibits greater valence stability as\ndescribed in Section 2.6: “The XPS analysis of NCT-Ruzyme and PPy-co-PTh\nstabilized Ruzyme before and after the reaction with H O demonstrated that PPy-co-\n2 2\nPTh stabilized Ruzyme exhibited greater valence stability under identical catalytic\nconditions”?\nResponse: We sincerely appreciate the reviewers’ valuable comments. In the revised\nmanuscript, we have expanded the explanation of the XPS analysis, which was used to\nexamine the valence stability of the PPy-co-PTh-stabilized Ruzyme compared with the\nNCT-Ruzyme before and after the reaction with H O .\n2 2\n“In contrast, the NCT-Ruzyme shows larger shifts, suggesting greater susceptibility\nto changes in the valence state. The conductive nature of PPy-co-PTh likely stabilizes\nthe electronic structure of the Ruzyme, preventing excessive oxidation or reduction.\nThis stability ensures consistent catalytic activity over multiple cycles, which is crucial\nfor applications such as RONS removal in sepsis-induced cardiac injury”\n8. Could you please double-check the atomic labels in Figure 4C? Six distinct atom\ntypes are present, but only five are labeled. It seems that the silver atom might be\nnitrogen rather than hydrogen. If this is the case, correcting the labels.\nResponse: We sincerely thank the reviewer for their keen attention to detail in\nidentifying the labeling error in Figure 4C.\nUpon careful re-examination, we confirmed that the silver atom in the original figure\nwas incorrectly labeled as hydrogen. This error has been corrected in the revised\nmanuscript, where the silver atom is now accurately identified as nitrogen, and the\nmissing hydrogen atom has been appropriately labeled.\nThe revised high-resolution image has been incorporated into the manuscript, and\nthe figure legend has been verified to accurately reflect these corrections. We greatly\nappreciate the reviewer’s valuable feedback, which has played a critical role in ensuring\nthe accuracy of our structural representation.\nFigure 4C. Molecular models of NCT-Ruzyme and PPy-co-PTh-stabilized Ruzyme.\n9. In Figures 4D and 4G, the top-view perspectives are really difficult to clearly observe\nthe adsorbed molecules. Consider improving the clarity by, for example, using a\ndifferent color scheme, or different atom style, or highlighting adsorption sites, or\nproviding additional in the supplementary information.\nResponse: We thank the reviewer for their valuable suggestion regarding the\nobservation clarity of the adsorbed molecules in Figures 4D and 4G.\nIn response to the reviewer's comment, we have supplemented Figures 4D and 4G in\nthe Supplementary Information by adding multiangle visualizations of the SOD- and\nCAT-like activities of the PPy-co-PTh-stabilized Ruzyme to improve clarity.\nCorrespondingly, rotational views of the catalytic pathways of the NCT-Ruzyme for\nboth SOD-like and CAT-like mechanisms have been provided.\nFigure S27. Catalytic mechanisms of the NCT-Ruzyme. (A) SOD- and (B) CAT-like\ncatalytic pathways of the NCT-Ruzyme. (a), (b) and (c) represent different side view\nimages.\nFigure S28. Catalytic mechanisms of the PPy-co-PTh-stabilized Ruzyme. (A) SOD-\nand (B) CAT-like catalytic pathways of the PPy-co-PTh-stabilized Ruzyme. (a) and (b)\nrepresent different side-view images.\n10. In the legend of Figure 4, it states: “Surface configurations of different modeled\ninitial,\ntransition, and final states for the simulated …...” However, based on my understanding,\nthe results and the DFT method described in the supplementary indicate that you didn’t\nperform transition state calculations, such as NEB or dimer methods. Instead, you\ncalculated different. adsorbed configurations of the adsorbates and compared their\nenergies. So, the legend seems to be somewhat misleading, please consider revising it\nto something like: “Surface configurations of different adsorbed states for the\nsimulated …...”.\nResponse: We sincerely thank the reviewer for this meticulous observation regarding\nthe description of Figure 4. We acknowledge that the original legend inaccurately\nimplied the calculation of transition states, which was not performed in our study.\nAs correctly noted, our DFT simulations focused solely on comparing the energies\nof different adsorbed configurations of adsorbates rather than conducting formal\ntransition state calculations.\nWe have revised the legend to “Surface configurations of different adsorbed states\nfor the simulated (E) SOD- and (F) CAT-like catalytic processes of the NCT-Ruzyme-\nand PPy-co-PTh-stabilized Ruzyme”.\nThis amendment more accurately reflects the scope of our computational work. The\nupdated figure has been incorporated into the manuscript. Additionally, we have\ncarefully reviewed all the computational figure legends to ensure terminology\nconsistency and prevent similar ambiguities.\nWe greatly appreciate the reviewer’s expertise in identifying this oversight, which\nhas strengthened the precision of our presentation.\n11. A bit more details on the DFT calculation method are needed to ensure\nreproducibility. Can DFT accurately reproduce the lattice parameters and band structure of RuO ? A comparison\n2\nwith experimental or previous computational results would be helpful. Additionally, which\nRuO2 surface was used, what is the size of the supercell, and whether the bottom layers were\nfrozen during calculations (and if so, how many layers were fixed). Furthermore, specifying\nthe thermostat used for the AIMD simulations.\nResponse: We sincerely thank the reviewer for these valuable suggestions.\nRutile-RuO with a number of mp-825 from the Materials Project database was used,\n2\nand the crystal parameters of a, b, c, α, β, and γ were 4.480 Å, 4.480 Å, 3.110 Å, 90°,\n90° and 90°, respectively.\nTwo layers of the (110) surface were cleaved with a vacuum of 20 Å, and the surface\nwas expanded to the (4×2) supersurface to construct the slab model. The optimized\nRuO crystal was heated from 0 K to 3000 K by velocity scaling over 3 ps every MD\n2\nstep and equilibrated at 3000 K for 0.5 ps via the Nose‒Hoover thermostat with a time\nstep of 1 fs to obtain the amorphous RuO structure. These methodological details have\n2\nbeen incorporated into the Supporting Information to ensure full transparency and\nreproducibility.\n12. I might be wrong but in Section 2.5, second paragraph, all the notations for Fe2+ and Fe3+ appear incorrect. It looks like the superscripts are displayed in a smaller font style rather than being properly formatted as superscripts (which often happens after text refinement by GPT). Please verify whether the correct superscript formatting has been applied.\nResponse: We sincerely appreciate the reviewer’s meticulous attention to detail\nregarding the manuscript. Following their comment, we have conducted a\ncomprehensive review and uniformly corrected all superscript/subscript notations\nthroughout the manuscript.\n13. In Section 2.5, I noticed multiple formatting inconsistencies and errors in the text\nand figure legend. Please carefully review and refine the text to ensure consistency with\nthe previous sections.\nResponse: We thank the reviewer for this helpful comment. We have carefully\nreviewed the text and figure legends in Section 2.5 and have revised them to ensure full\nconsistency with the formatting used in the previous sections. These changes have been\nincorporated into the manuscript.\n14. Additionally, Section 2.5 appears after Section 2.6, which seems out of order. Please\ndoublecheck the paper format to ensure the correct section numbering and overall\nstructure.\nResponse: We sincerely appreciate the reviewer’s meticulous attention to the structural\nintegrity of our manuscript. Regarding the sequencing of section numbers highlighted\nin the comment, we have thoroughly reexamined the paper’s hierarchical organization\nand confirmed an inadvertent error in section numbering. This has now been rectified\nto ensure strict adherence to sequential logic, with all subsections following the\nstandardized format.\n15. Just out of curiosity, in Figure 7B, the daily weight changes of the mice in the low\nconcentration CICT-Ruzyme group (blue line: 20 mg/kg) seem to show slightly more\nvariation compared to the higher-concentration group. Is there a specific reason for\nthis?\nResponse: Thanks for the thorough observation of the body weight changes in the low-\ndose CICT-Ruzyme group (20 mg/kg). We greatly appreciate your attention to this\ndetail.\nStatistical analysis revealed that these fluctuations remained within the range of\nnormal biological variation and were not significantly different from those of the\ncontrol group when analyzed over the entire study period (all interday comparisons of\nweight variation within the 20 mg/kg group yielded p values > 0.05). We thank you\nagain for raising this valuable point.\n16. In Figure 7E, I do not see significant changes in most organs; however, the heart\nappears to show a relatively larger variation. Could you provide an explanation for\nthis?\nResponse: Thanks for the insightful observation regarding the organ-specific changes\nshown in Figure 7E. We agree that the heart exhibits more pronounced morphological\nalterations than other organs do. As a metabolically active organ with continuous\ncontractile function and high mitochondrial density, the heart is particularly sensitive\nto systemic interventions. The observed changes (such as mild blurring of myocardial\nstriations and slight widening of the interstitium at higher doses) likely reflect adaptive\nphysiological responses to the treatment rather than pathological toxicity. These\nalterations were dose dependent and reversible, as no severe structural damage or cell\ndeath was detected in any group.\nWe further corroborated these histological findings with functional assays, including\nin vivo measurements of cardiac enzyme levels. No significant elevation in LDH or\nCK-MB was observed, confirming the absence of irreversible injury. We acknowledge\nthat the responsiveness of the heart to the compound warrants further investigation into\nlong-term adaptive mechanisms. Future studies will focus on the temporal dynamics of\ncardiac-specific responses to treatment.\n17. Some subtitles use sentence case (e.g., 2.1 Synthesis and characterization of PPy-\nco-PTh stabilized nanozymes), while some use title case (e.g., 2.10 Mechanisms\nUnderlying the Therapeutic Effects of CICT-Ruzyme in Alleviating SC). Please ensure\nconsistency in formatting across all subtitles.\nResponse: We sincerely appreciate the reviewer’s meticulous attention to the\nconsistency of subtitle formatting throughout the manuscript. In response to this\ncomment, we have conducted a comprehensive review of all the subtitles and\nimplemented necessary revisions to ensure uniform formatting.\n--------------------------------------------------------------------------------\nReview 3:\nThe topic is very interesting and the work presented showed some very promising data.\nHowever, the methodological/experimental description/execution, especially in the first\npart of the article (preparation and comparison among different nanozyme candidates)\ndoes not meet at the current status the standards of excellence required for a publication\nin Nature Communications. The main findings of the paper (Ruzymes showed a good\npotential in preventing LPS-induced damages in myocardial cells in vitro and in vivo\nthanks to their iron and ROS scavenging activity) get buried in a set of weaker data that\nlower the overall quality of the work. The authors might take into consideration\nremoving (or moving to SI) the initial comparative study with different nanozymes that\nactually need substantial improvements. Nevertheless, major revisions are necessary\nbefore re-evaluating the manuscript for publication in Nature Communications:\nResponse: We appreciate the positive comments.\n1. In Abstract and Introduction define all acronyms/short names at the first use. PPy?\nPTh? Tpp? MiR130B-3P? Fe-Cur@TA?\nResponse: We sincerely appreciate the reviewer’s meticulous attention to the\nstandardization of acronym usage. We have thoroughly reviewed all acronyms in the\nAbstract and Introduction sections, ensuring that each is expanded to its full form upon\nfirst occurrence and implemented necessary revisions to the manuscript accordingly.\n2. “the nanozymes exhibits overoxidation and inadequate stability”…maybe some of\nthem, but several actually have been proven to present good stability. I think the authors\nmade an overstatement in this context.\nResponse: We thank the reviewer for this valuable comment. We agree that the original\nstatement may have been overly absolute. Indeed, this issue is highly material specific,\nand not all nanozymes necessarily face such challenges. We have revised the relevant\ndescription in the manuscript to more accurately reflect the current understanding in the\nfield. We greatly appreciate the reviewer’s input, which has helped us improve the rigor\nof our manuscript.\n3. “Ruzymes (PPy-co-PTh stabilized Ruzyme [Ruzyme], ICT-Ruzyme, and CICT-\nRuzyme) can better prevent themselves from overoxidation in the environment of\nexcessive H O , and has more excellent conductivity and antioxidant properties.”\n2 2\nCompared to what?\nResponse: We sincerely thank the reviewer for this insightful comment. Regarding the\nclaim of superior conductivity, we conducted electrochemical impedance spectroscopy\n(EIS) measurements with the nonpolymer NCT-Ruzyme used as a control.\nThe size of the semicircle diameter in the high-frequency region of the EIS\ninstrument directly reflects the electrical impedance of the material, providing insight\ninto the electron transfer dynamics on the electrode surface. A smaller semicircle\ndiameter indicates lower electron transfer resistance and better conductivity, whereas a\nlarger diameter suggests higher impedance and poorer conductivity.\nAs shown in Figure 2I, compared with the NCT-Ruzyme, the PPy-co-PTh-stabilized\nRuzyme exhibited a smaller semicircle diameter in the high-frequency region (Figure\n2I). This indicates that the PPy-co-PTh-stabilized Ruzyme, with polypyrrole and\npolythiophene as the framework, has a lower charge transfer resistance. This suggests\nthat the nanozyme facilitates faster charge transfer during the catalytic reaction,\nresulting in better electrical conductivity.\nConcerning the antioxidant properties, we used the NCT-Ruzyme as a control and\ncomprehensively evaluated the ability of the Ruzyme to catalyze the decomposition of\nhydrogen peroxide. Furthermore, comparisons with PVP@Ru and several reported\nnanozymes (including Ru@PAM, Ru@MOF, and RuO₂@BSA) confirmed the superior\ncatalytic stability of Ruzyme. Additionally, the antioxidant efficacy was assessed at\nboth the cellular and animal levels, where treatment with Ruzymes resulted in a marked\ndownregulation of ROS levels and lipid peroxidation in H9C2 cells as well as in heart\ntissues, indicating alleviation of oxidative stress.\nWe have therefore amended the statement in the manuscript to “A series of studies\nhave shown that, compared with nonpolymeric nanozymes (NCT-Ruzymes), PPy-co-\nPTh-stabilized Ruzymes can better prevent themselves from overoxidation in\nenvironments with excessive H O and have better conductivity and antioxidant\n2 2\nproperties.”\nThe relevant section has been updated accordingly. We greatly appreciate the\nreviewer’s careful review, which has helped us improve the clarity and accuracy of our\nwork.\n4. The author claims stability of the prepared nanozymes and that the PDI below 0.7 by\nDLS indicates good dispersion. However, all the PDI reported are above 0.2 and\nseveral above 0.4 indicating highly polydisperse samples for all the PPy-co-PTh\nnanozymes containing the different metal ions including the different Ruzymes. Actually,\nwith such a high PDI there is some risk even in extrapolating the peak maxima by\nnumber distribution and z potential when using a Malvern instrument…those numbers\nmight not be so solid. DLS spectra expressed in intensity should also be reported in SI.\nResponse: We thank the reviewers for their valuable comments. We agree that PDI\nvalues alone are insufficient to define nanozyme quality. We have provided DLS spectra\nexpressed in terms of intensity for the eleven types of nanozymes in the Supporting\nInformation. We are grateful for the reviewer's expertise in prompting these essential\nimprovements.\nFigure S5. Particle size profiles (intensity (percent)) of eleven PPy-co-PTh-supported\nnanozymes.\n5. Further TEM measurements should also be provided (more micrographs and higher\nquality).\nResponse: We thank the reviewer for this suggestion. Additional TEM micrographs of\nthe Ruzyme, including high-angle annular dark-field (HAADF) and select area electron\ndiffraction (SAED) images, are provided in the Supplementary Information.\nFigure S19. Morphological and structural characterization of the Ruzyme. (A)\nRepresentative TEM image of a Ruzyme. Scale bar (left) = 500 nm. Scale bar (right) =\n500 nm. (B) Atomic HAADF image of a Ruzyme with the SAED pattern inset in the\ntop right corner. Scale bar = 100 nm.\n6. Figure 2B do not really show “relatively uniform spherical morphology”.\nResponse: We thank the reviewer for their valuable comments. We have revised the\ndescription in the manuscript and replaced it with “The TEM micrographs demonstrate\nthat the nanoparticles exhibit a relatively uniform distribution, as illustrated in Figure\n2A and Figure S19A of the Supporting Information. Additionally, the amorphous nature\nof the nanozyme complex is consistent with the findings from the high-angle annular\ndark-field (HAADF) image, which are presented in Figure S19B in the Supporting\nInformation (Figure S19B, Supporting Information).”. We thank the reviewer for the\ncareful review, which has improved the accuracy of our work.\n7. Potential leaching of metal ions in the relevant biological environments (including\nin the presence of iron) should be evaluated.\nResponse: We sincerely thank the reviewer for raising this important point. We fully\nagree with the reviewer’s comment regarding the necessity of evaluating the potential\nleaching of metal ions in relevant biological environments, particularly in the presence\nof iron, as this is crucial for assessing the safety and stability of the material in\nbiological applications. In response, we simulated the relevant biological environment\n(PBS buffer), conducted ion leaching experiments under both iron-containing (with\nadded Fe2+) and iron-free conditions, and incubated for 72 hours.\nWe employed a dialysis method to separate any leached metal ions and used\ninductively coupled plasma‒mass spectrometry (ICP-MS) for highly sensitive\nquantitative detection of ruthenium (Ru) ions in the dialysate. The results indicated that\nthe relative leaching amount of Ru ions was extremely low (< 0.0002%), with no\nsignificant difference observed between the Fe2+-containing and iron-free environments.\nThis suggests that, under our experimental conditions, the risk of Ru ions leaching from\nthe material is negligible, demonstrating excellent stability in biological environments.\nWe agree that this supplementary experiment significantly strengthens the\ncompleteness and persuasiveness of our study. We have included these experimental\nresults and corresponding discussion in the revised manuscript, clearly addressing the\npotential risk of metal ion leaching and the material’s chemical stability in biological\ncontexts. Once again, we sincerely appreciate your constructive feedback, which has\ngreatly enhanced the quality of our paper. We look forward to your further review.\nFigure S33. Relative leaching rates of Ru ions under different conditions as determined\nby ICP‒MS (n = 3). The data are presented as the means ± SDs.\n8. ICP-MS and EDS should be performed on all samples and not only on the Ruzyme\nto allow for ions-dependent meaningful comparisons of the enzyme-like activities.\nResponse: We appreciate the reviewer’s suggestion to perform ICP‒MS and EDS on\nall samples to enable meaningful comparisons of enzyme-like activities. In response, we\nconducted ICP‒MS analysis on the NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and\nCICT-Ruzyme, quantified the elemental composition and presented the results in Figure\nS17.\nAdditionally, EDS analysis was performed to map the elemental distribution within\nthe nanostructures, with maps included in Supplementary Figures S14 and S18. These\nanalyses provide crucial insights into the concentration and spatial distribution of key\nelements, facilitating more accurate comparisons of catalytic activities. These additions\nsignificantly enhance the robustness and interpretability of our study, and the\nmanuscript has been updated accordingly.\nFigure S17. Measurement of Ru content in Ruzyme, ICT-Ruzyme, and CICT-Ruzyme\nby ICP‒MS.\nFigure S14. EDS elemental mapping of Ruzyme. Scale bar = 200 nm.\nFigure S18. EDS elemental mapping of ICT-Ruzyme and CICT-Ruzyme. The scale bar\nin the upper image is 700 nm, and the scale bar in the lower image is 100 nm.\n9. If the activity is supposed to be metal ion type-dependent, considering the\npolydispersity index of the samples and the different sizes of nanozymes, a\nnormalization by mass is not an adequate approach for a meaningful comparison.\nEspecially since it is not clear from the characterization how many atoms of the\ndifferent metals are present per nanoparticles. The current literature suggest that you\nshould normalize the available active sites and measure the turnover frequency (TOF)\nto ensure the activity is catalytic (cycling) (https://doi.org/10.1021/acsnano.1c07520).\nResponse: We thank the reviewer for their valuable comments. We have carefully read\nthe suggested reference (https://doi.org/10.1021/acsnano.1c07520) provided by the\nreviewer, which states that calculating the catalytic turnover rate (K ) requires\ncat\ninformation on the nanozyme concentration.\nThe key issue lies in how to quantify the active sites. The reference explicitly\nmentions: “First, on the one hand, we do not recommend calculating all the metal ions\nin the whole NP, since the interior ones are not involved in a catalytic reaction (Figure\n6B). On the other hand, considering the entire particle as one enzymatic unit also does\nnot reflect the true chemical picture (Figure 6A). A more accurate estimation is to\ncalculate the number of surface sites (Figure 6C).” However, quantifying the exact\nnumber of active sites on nanoparticles remains challenging, but calculating the number\nof surface metal atoms can provide a good reference or a reasonable approximation.\nThis is because “it is unlikely that the number of active sites is more than the number\nof surface atoms. Whether each surface metal atom is active or not would require more\ndetailed surface science and mechanistic studies. But if a catalytic turnover can be\nachieved by counting all surface atoms, one can safely conclude the catalysis.” This\nnecessarily involves morphological parameters.\nSpecifically, the referenced literature highlights three key morphological parameters:\nparticle size, crystal facet orientation, and geometric shape. First, the size and shape of\nthe nanoparticles need to be obtained through electron microscopy. Additionally,\ndifferent exposed crystal facets possess different atomic densities. Second, surface area\nserves as the foundation for quantifying the number of atoms, and neglecting the\nspecific surface area can lead to severe deviations in the TON calculation (such as\n“Since only 0.32% of the total Fe atoms in the 300 nm NPs are the surface atoms,\nconsidering all the Fe O units as active sites could lead to a drastic underestimation of\n3 4\nthe catalytic turnover”).\nThe referenced literature indicates that the surface area of nanoparticles can be\ncalculated via morphological parameters (such as diameter and lattice constant) or via\ntechniques such as Brunauer‒Emmett–Teller (BET) adsorption. By integrating both the\nmorphological parameters and specific surface area, this study calculated the TON\nvalues for Fe O NPs, AuNPs (GOx), CeO NPs, and single-atom nanozymes. In\n3 4 2\nsummary, the morphology of nanoparticles (size, crystal facets, porosity) serves as the\nfundamental basis for TON calculations, and the specific surface area (determined\nthrough geometric modeling or experimental measurement) is an essential parameter\nfor quantifying the number of surface atoms. Neglecting morphological factors will\nlead to deviations in the TON.\nHowever, in our manuscript, on the basis of TEM observations, our Ruzyme\nexhibited an irregular spherical structure. Furthermore, XRD analysis indicates that\nRuzyme is an amorphous polymer without a specific and defined crystal lattice\nmorphology. Consequently, with the currently available information on Ruzyme, it is\nnot feasible to calculate the surface active sites of the nanoparticles as described in the\nreferenced literature.\nAdditionally, for the nanozymes cited from other literature in our manuscript, we\nintended only to perform a longitudinal comparison to demonstrate that the Ruzyme\nhas a relatively high catalytic rate and superior affinity for H O , revealing its optimal\n2 2\nCAT-like kinetic characteristics for H O scavenging. Moreover, many of the\n2 2\nnanomaterials in these references lack the necessary data for such analysis (Table S1),\nmaking it impossible to perform the corresponding calculations.\nAccordingly, we will continue to explore more scientific and accurate calculation\nmethods suitable for determining the active sites of such amorphous nanoparticles. We\nonce again thank the reviewer for their valuable comments, which provide important\nguidance for our future research.\n10. I am afraid that the authors fall into a common analytical mistake. The TMB is not\nsuitable to be used for pH dependent or temperature dependent studies as performed in\nthis manuscript (monitoring the absorbance at 652 nm), because the TMB chromogenic\nsubstrate itself is known to be sensitive to the pH variation, leading to remarkable\nspectral changes and in addition the TMBox dimer is unstable at neutral pH\n(https://doi.org/10.1002/adfm.202315587).\nResponse: We sincerely thank the reviewer for pointing out this critical issue regarding\nthe use of TMB as a chromogenic substrate in pH-dependent studies. We appreciate the\nprovided reference (https://doi.org/10.1002/adfm.202315587), which helped us better\nunderstand the limitations of TMB under neutral conditions. In response to your\ncomment, we have conducted additional experiments to further validate our findings.\nWhile we noted that some previous studies have used TMB to evaluate the enzyme-like\nactivity of nanozymes under near-neutral conditions (https://doi.org/10.1038/s41467-\n024-53047-1, https://doi.org/10.1016/j.cej.2024.152265), we fully acknowledge its\nlimitations for quantitative pH-dependent analyses, as rightly emphasized by the\nreviewer. Therefore, to ensure the robustness of our conclusions, we performed\nadditional catalytic assays using two alternative substrates, o-phenylenediamine (OPD)\nand 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), at pH 7.4. These\nexperiments confirmed that all eleven types of PPy-co-PTh-stabilized nanozymes\nexhibit negligible peroxidase-like (POD) and oxidase-like (OXD) activities at neutral\npH, which is consistent with our initial observations using TMB. The supplementary\ndata from these additional experiments have been included in the revised manuscript\n(Figure S9, Figure S10, Supporting Information). We believe these results further\nreinforce the main conclusions of our study. Once again, we are grateful for the\nreviewer’s insightful suggestion, which has significantly improved the quality and\nreliability of our work. The experimental results are presented below.\nFigure S9. Evaluation of the POD-like activity of the PPy-co-PTh-stabilized\nnanozymes with OPD and ABTS as substrates. The data are presented as the means ±\nSDs.\nFigure S10. Evaluation of the OXD-like activity of the PPy-co-PTh-stabilized\nnanozymes with OPD and ABTS as substrates. The data are presented as the means ±\nSDs.\n11. As a general comment the methodologies reported are not properly detailed and\nshould be implemented.\nResponse: We appreciate the reviewer’s constructive comment. In response, we have\nrevised and expanded the “Methods” section both in the Supplementary Information\nand the main text regarding the experimental procedures. Furthermore, all the newly\nadded experimental results in the revised manuscript are now accompanied by clearly\ndescribed methodologies. These modifications enhance the reproducibility and\ntransparency of our study. If further clarification is needed, we are pleased to provide\nadditional details.\n12. Some of the tests are performed in buffers, including in PBS. The authors should\nshow that the nanozymes are colloidally stable in these conditions.\nResponse: We sincerely thank the reviewer for raising this important point regarding\nthe colloidal stability of nanozymes in buffer conditions. In response to this comment,\nwe have conducted additional experiments to evaluate the colloidal stability of\nnanozymes (NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme) across various\nbiological media, including deionized water, saline, phosphate-buffered saline (PBS),\nfetal bovine serum (FBS), and DMEM. After 1 mg/mL nanozyme aqueous solutions\nwere prepared, the NCT-Ruzyme rapidly precipitated in several biological media,\nwhereas the PPy-co-PTh-stabilized Ruzymes maintained better dispersibility over\nseven days with negligible changes in particle size (Figure 2G; Figure S21, Supporting\nInformation). The experimental results are presented below.\nFigure 2G. Liquid images of NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme\nstored in various biological media on days 1 and 7.\nFigure S21. Changes in the sizes of (A) Ruzyme, (B) ICT-Ruzyme and (C) CICT-\nRuzyme in various biological media incubated for different durations. The data are\npresented as the means ± SDs.\n13. The authors state: “It was observed that Ruzyme significantly increased the O\n2\nconcentration in the system and produced more bubbles compared to the other PPy-co-\nPTh stabilized nanozymes” It is not clear how the oxygen production was measured.\nDid you use an oxygen sensor or this is only based on a naked-eye qualitative\nassessment?\nResponse: We thank the reviewer for this valuable comment. In response to the\nquestion regarding oxygen measurement, we confirmed that the oxygen concentration\ngenerated by the nanozymes was quantitatively monitored via a dissolved oxygen meter.\nSimultaneously, bubble formation was observed qualitatively as a supplementary\nindicator. Additionally, we have provided further details on this experimental method\nin the Supporting Information. The corresponding results are shown in the figure below.\nFigure S11. Characterization of the CAT-like activities of the PPy-co-PTh-supported\nnanozymes. (A) Detection of the dissolved oxygen content produced by the\ndecomposition of H O catalyzed by nanozymes or reference samples (n = 3). (B)\n2 2\nPicture of eleven nanozymes breaking down H O to produce bubbles (O ). The data\n2 2 2\nare presented as the means ± SDs.\n14. The authors should report a full characterization (including TEM and size\ndistribution) of PVP@Ru and RuO @BSA and all the other nanozymes involved in this\n2\nstudy. In the NCTRuzyme preparation the ruthenium precursor is prepared in the\npresence of BSA by NaBH reduction. Considering the strong reducing agent involved,\n4\nhow the protein structure will appear after synthesis? Are you sure the NPs structure\nwill resemble to the one reported in Figure S10A, Ruthenium clusters embedded in\nunfolded BSA? What kind of interactions keep them together? I am concerned that the\nchosen controls are not appropriate, as we know very little about them.\nResponse: Thanks for the insightful comments regarding the structural characterization\nof the nanozyme and the stability of the NCT-Ruzyme during the synthesis process.\nWe understand the reviewer’s concern about the potential impact of strong reducing agents\non protein conformation and nanoparticle assembly. We have supplemented the size\nand potential of the nanozymes mentioned in the manuscript, as shown in Table S1.\nWith respect to the impact of NaBH reduction on the structure of BSA during the\n4\nsynthesis of the NCT-Ruzyme, we would like to clarify that even under strongly\nreducing conditions, the protein can retain a functional conformation that facilitates the\nformation and stabilization of metal nanoclusters. The evidence in the literature\nsuggests that proteins such as BSA can maintain structural integrity through specific\nmetal-coordinating interactions, despite partial unfolding under alkaline or reducing\nconditions.\nFor example, in the study by Xinyue Wang et al.2, during the design and development\nof a single-atom nanozyme (BSA-Cu SAN) that highly mimics the natural enzyme\nstructure based on bovine serum albumin (BSA) with copper (Cu) as the active metal\ncenter, a strong reducing agent, NaOH, was similarly used to adjust the pH to 12. The\nselected area electron diffraction (SAED) pattern confirmed the amorphous nature of\nthe BSA matrix (Fig. 1c), and high-angle annular dark-field (HAADF) imaging further\nrevealed uniformly dispersed isolated copper atoms in the BSA matrix (Fig. 1c). Fourier\ntransform infrared (FT-IR) spectroscopy (Supplementary Fig. 5) revealed characteristic\nbands at 1656 and 1538 cm-1, whereas Raman spectroscopy (Supplementary Fig. 6)\nconfirmed the presence of BSA and amide bond. Additionally, molecular docking\nsimulations (Fig. 1i–j) demonstrated that Cu+ forms stable coordination bonds with\nHis246/His67 (via N-coordination) and Asp248 (via O-coordination) in BSA.\nIn summary, amino acid residues in BSA (such as histidine and aspartic acid) can\ncoordinate with metal ions (e.g., Cu+ or Ru) through N/O atoms. Even under highly\nreductive conditions, this coordination effectively stabilizes the metal clusters and\nmaintains the “protein-encapsulated nanocluster” structure\n(https://doi.org/10.1038/s41392-023-01491-8,\nhttps://doi.org/10.1002/anbr.202200144).\nFurthermore, the study by Shengyang Fu et al.3 on CeO @BSA demonstrated that\n2\nalthough the biomineralization process under alkaline conditions (pH 12) may induce\npartial unfolding of BSA, its abundant functional groups (such as carboxyl and amino\ngroups) can still effectively anchor metal ions, leading to the formation of uniformly\ndispersed nanoclusters (as shown in HRTEM, Fig. 1B).\nTable S1. CAT-mimicking reaction kinetic analysis of the PPy-co-PTh-stabilized\nRuzyme and other nanozymes reported previously.\nV : the maximal reaction velocity; K : the Michaelis constant; [E]: the nanozyme\nmax M\nconcentration; TON: the maximum number of substrate molecules converted per active\ncatalytic center per unit of enzyme activity.\n15. If the metal ions are supposed to be the active sites during the iron ions scavenging,\nis the nanozyme itself, enriched of iron ions, able to perform a Fenton-like reaction?\nIs the Iron replacing Ruthenium ions during the scavenging process?\nResponse: We thank the reviewer for raising this important question. In response, we\nconducted experimental validation focusing on the following two aspects.\nFirst, to evaluate whether the iron-enriched nanozyme (PPy-co-PTh-stabilized\nFezyme, referred to as the Fezyme) would undergo a Fenton-like reaction under\nphysiologically relevant conditions, we conducted catalytic experiments in a buffer\nsystem at pH 7.4 using TMB and ABTS as characteristic substrates. The results\ndemonstrated that Fezyme did not exhibit significant Fenton-like activity under these\nconditions, indicating that it was unable to effectively catalyze the production of\nhydroxyl radicals (•OH). This finding indicates that although the nanozyme contains\nabundant iron ions, it does not initiate a typical Fenton-like reaction at neutral pH,\nwhere antioxidant activity is typically needed.\nSecond, to determine whether iron ions displace ruthenium (Ru) sites during the iron\nremoval process, we conducted an in-depth investigation through ion leaching\nexperiments. We simulated the relevant biological environment (PBS buffer) and\nincubated Ruzyme in iron-containing (with added Fe2+) and iron-free conditions for 72\nhours to perform the ion leaching experiments. We employed a dialysis method to\nseparate any leached metal ions and used ICP‒MS for highly sensitive quantitative\ndetection of Ru ions in the dialysate. The results revealed that the relative leaching\namount of Ru ions was extremely low (<0.0002%), with no significant difference\nbetween iron-containing and iron-free conditions, indicating the absence of exchange\nor displacement behavior between iron ions and ruthenium ions.\nIn conclusion, our experiments demonstrate that the iron-rich nanozyme (Fezyme)\nitself does not initiate the Fenton-like reaction and that the ruthenium site in the Ruzyme\nstructure is highly stable and has not been replaced by iron ions.\nFigure S32. Time-dependent measurement of •OH generation via TMB and ABTS for\nFezyme + H O .\n2 2\nFigure S33. Relative leaching rates of Ru ions under different conditions as determined\nby ICP‒MS (n = 3). The data are presented as the means ± SDs.\n16. Structural, compositional, and colloidal stability of the presented nanozymes before\nand after exposure to the relevant biological media (cell culture media and mice plasma)\nshould be assessed.\nResponse: We sincerely thank the reviewer for their important comments regarding the\nstability evaluation of nanozymes in biological media. We fully concur on the necessity\nof systematically assessing the structural stability, compositional stability, and colloidal\nstability of nanozymes in complex biological environments, which is crucial for their\nbiomedical applications.\nWith respect to the structural and compositional stability of the nanozyme, we\nevaluated the potential leaching risk of metal ions (including the presence of Fe2+) from\nthe nanozyme in the relevant biological environment. We simulated the relevant\nbiological environment (PBS buffer) and incubated Ruzyme in iron-containing (with\nadded Fe2+) and iron-free conditions for 72 hours to perform the ion leaching\nexperiments. We employed a dialysis method to separate any leached metal ions and\nused ICP‒MS for highly sensitive quantitative detection of Ru ions in the dialysate. The\nresults revealed that the relative leaching amount of Ru ions was extremely low\n(<0.0002%), with no significant difference between iron-containing and iron-free\nconditions (Figure S33, Supporting Information). This indicates that the risk of Ru ion\nleaching from the material is negligible, and the ruthenium sites remain highly stable\nwithout being replaced by iron ions, which effectively demonstrates its excellent\nstructural and compositional stability in the biological environment.\nWe also evaluated the changes in the enzymatic activity of the nanozymes after\nincubation in biological medium (FBS) for 0, 3, 6, and 18 hours. Although the catalytic\nactivity decreased to varying degrees with prolonged incubation time, they still\nexhibited considerable ROS and RNS scavenging capacity, which functionally\ndemonstrates the well-preserved structural and compositional integrity.\nFurthermore, on the basis of the demonstrated stability of similar polymer-based\nnanozymes in biological media, as reported in the literature\n(https://doi.org/10.1002/advs.202308251), the polymer nanozymes developed in this\nstudy also exhibit excellent structural and compositional stability. To assess colloidal\nstability, we evaluated the stability of the nanozymes stored in different biological\nmedia (DI water, saline, PBS, FBS, and DMEM) for 1, 3, 5, and 7 days. After 1 mg/mL\nnanozyme aqueous solutions were prepared, the NCT-Ruzyme rapidly precipitated in\nseveral biological media, whereas the PPy-co-PTh-stabilized Ruzymes maintained\nbetter dispersibility over seven days with negligible changes in particle size (Figure 2G;\nFigure S21, Supporting Information).\nWe once again thank the reviewers for their insightful comments, which have greatly\ncontributed to the refinement of this research.\nFigure S33. Relative leaching rates of Ru ions under different conditions as determined\nby ICP‒MS (n = 3). The data are presented as the means ± SDs.\nFigure S43. Changes in the CAT-like activities of (A) Ruzyme, (B) ICT-Ruzyme and\n(C) CICT-Ruzyme after coincubation with 10% fetal bovine serum (FBS) for 0 h, 3 h,\n6 h, and 18 h.\nFigure S44. Changes in the nitrogen free radical (ABTS•+) scavenging activity of the\nPPy-co-PTh-stabilized Ruzymes after coincubation with 10% FBS for 0 h, 3 h, 6 h, and\n18 h. (A) Scavenging rates of the PPy-co-PTh-stabilized Ruzymes. ABTS•+ scavenging\nability of (B) Ruzyme, (C) ICT-Ruzyme and (D) CICT-Ruzyme as assessed by UV‒vis.\n(E) Representative optical photograph of ABTS•+ cleared by nanozymes.\nFigure S45. Changes in the nitrogen free radical (DPPH•) scavenging activity of the\nPPy-co-PTh-stabilized Ruzymes after coincubation with 10% FBS for 0 h, 3 h, 6 h, and\n18 h. (A) Scavenging rates of the PPy-co-PTh-stabilized Ruzymes. DPPH• scavenging\nability of (B) Ruzyme, (C) ICT-Ruzyme and (D) CICT-Ruzyme as assessed by UV‒vis.\n(E) Representative optical photograph of DPPH• cleared by nanozymes.\nFigure 2G. Liquid images of NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme\nstored in various biological media on days 1 and 7.\nFigure S21. Changes in the sizes of (A) Ruzyme, (B) ICT-Ruzyme and (C) CICT-\nRuzyme in various biological media incubated for different durations. The data are\npresented as the means ± SDs.\n17. In this study the potential role of biomolecular corona on the colloidal stability,\nenzymatic-like activity, targeting and internalization/cellular distribution\n(https://doi.org/10.1038/s41565-021-00860-0,\nhttps://doi.org/10.1021/acsnano.0c00629,\nhttps://doi.org/10.1021/acs.nanolett.3c01479) should be taken into account. The\namount of proteins present in cell culture medium supplemented with 10% FBS is\nsufficient to cover the Ruzyme surface at the concentration used in in vitro experiments?\nIdeally, a large excess of protein per NZ’s surface area available should be guaranteed\nto mimic the in vivo scenario.\nResponse: We sincerely thank the reviewer for this insightful comment regarding the\ncrucial role of the biomolecular corona.\nWe agree that understanding corona formation is essential for evaluating nanozyme\nbehavior in biological environments. First, Giulia Tarricone et al.\n(https://doi.org/10.1021/acs.nanolett.3c01479) compared the catalytic activity of\nPtNPs-BSA under 10% FBS (in vitro-like) and 50% FBS (in vivo-like) conditions. The\nresults in Fig. 2B-E show that all enzymatic activities (POD/OX/CAT/SOD) were\nsignificantly suppressed in 10% FBS (activity decreased to approximately 50%),\nwhereas 50% FBS only provided an additional 25% inhibition, indicating that the\ninhibition mechanism was already complete at the 10% concentration. Xianzhi Zhang\net al. (https://doi.org/10.1021/acsnano.0c00629) suggested that even as low as 1%\nserum can form a protein corona that inhibits activity, and 10% FBS far exceeds this\nconcentration, which is sufficient to form a more stable inhibitory state.\nFurthermore, endogenous proteases within endosomes and lysosomes can restore\nnanozyme activity through proteolysis, independent of the serum concentration.\nTherefore, 10% FBS is sufficient to simulate physiological inhibitory effects\neffectively.\nStudies have reported that once nanoparticles enter the bloodstream, proteins and\nother biomolecules in the blood may interact with them, forming a so-called\n“biomolecular corona” on their surface, which alters the intrinsic bioactivity and\ntherapeutic efficacy of the drugs4, 5. Therefore, we coincubated the nanozymes with\n10% FBS in vitro to promote the formation of a \"biomolecular corona\" on their surface.\nWe first tested the stability of the nanozymes in FBS. After 1 mg/mL nanozyme aqueous\nsolutions were prepared, the NCT-Ruzyme rapidly precipitated in several biological\nmedia, whereas the PPy-co-PTh-stabilized Ruzymes maintained better dispersibility\nover seven days with negligible changes in particle size (Figure 2G; Figure S21,\nSupporting Information). Subsequently, the PPy-co-PTh-stabilized Ruzymes were\nincubated with 10% fetal bovine serum (FBS) at 37 °C for 0, 3, 6, and 18 hours to\nevaluate the effect of the formed biomolecular corona on the activity of the nanozyme.\nThe dissolved oxygen content produced by the catalytic decomposition of hydrogen\nperoxide by nanozymes after incubation with 10% FBS for various durations was\nmeasured via a dissolved oxygen meter. The results indicated that as the incubation time\nwith 10% FBS increased, the biomolecular corona on the surface of the nanoparticles\ngradually formed, leading to a corresponding decrease in their CAT-like activity to\nvarying degrees (Figure S43A-C, Supporting Information). This may be attributed to\nthe partial obscuration of the nanozymes' active sites by the formed biomolecular\ncorona.\nThe capacity of these compounds to scavenge reactive nitrogen species (RNS) was\nsubsequently evaluated via the use of ABTS•+ and DPPH• as substrates. The results\ndemonstrated that when ABTS•+ was used as the detection substrate, the biomolecular\ncorona formed on each nanozyme after varying incubation periods with 10% FBS had\na negligible effect on their ability to scavenge ABTS•+ radicals. The fact that the color\nof the reaction system did not change accurately confirmed this conclusion (Figure\nS44A-D, Supporting Information). Moreover, when DPPH• was used as the detection\nsubstrate, the RNS scavenging capacity of all the nanozymes slightly decreased with\nprolonged incubation time, accompanied by varying degrees of color change. Among\nthem, CICT-Ruzyme exhibited the most significant reduction. Nevertheless, even after\n18 hours of coincubation, CICT-Ruzyme retained 60% of its RNS scavenging activity\n(Figure S45A–D, Supporting Information).\nWe then further evaluated the uptake of ICT-Ruzyme by H9C2 cells both before and\nafter incubation with 10% FBS. As the coincubation time of the nanozymes with the\ncells increased, the red fluorescence signal within the cells tended to increase. However,\nthe cellular uptake of the nanozyme with a “biomolecular corona” was slightly weaker\nthan that of the nanozyme without incubation (Figure S46, Supporting Information).\nThis finding indicates, from another perspective, that the “biomolecular corona” formed\non the surface of the nanozyme does indeed affect the efficiency of its cellular uptake.\nTherefore, PPy-co-PTh-stabilized Ruzymes with a surface biomolecular corona still\nexhibit considerable ROS and RNS scavenging capacity, as well as cellular uptake\nefficiency, which lays a foundation for subsequent validation of the therapeutic efficacy\nof these nanozymes in vivo.\nGiven the impact of the “biomolecular corona” on the targeting capability of\nnanozymes, we believe that the cardiac targeting study of CICT-Ruzyme is sufficient\nto address this issue. This is because the experiment was conducted in an LPS-induced\nSC mouse model, which is more persuasive than simulating the in vivo\nmicroenvironment by coincubating nanozymes with FBS. Specifically, SC mice were\nintravenly injected with equal doses of three types of Cy5.5-labeled nanozyme\ndispersions (Cy5.5-Ruzyme, ICT-Ruzyme and CICT-Ruzyme). The average\nfluorescence intensity of each nanoenzyme in the organs at different time points (1 h, 3\nh, 6 h, and 24 h) was detected via an in vivo imaging system. Ex vivo fluorescence\nimaging revealed that the accumulation of nanoparticles in myocardial tissue increased\nover time. Moreover, in the SC mouse model, compared with that in the nontargeted\ngroup, the myocardial fluorescence signal in the group injected with the CTP-targeted\npeptide-containing nanozyme was the strongest at all time points, indicating that CTP\nmodification significantly enhanced the targeted accumulation of nanozymes in the\npathological myocardium (Figure 7F; Figure S41, Supporting Information).\nTo further validate the targeting efficacy of CTP-modified nanozymes toward\npathological myocardium, LPS-induced SC mice were intravenously administered\nthree types of nanozymes at equal concentrations. Heart tissues were collected 12 hours\npost-injection to compare the ruthenium (Ru) content. The ICP‒MS results\ndemonstrated that the Ru level in the cardiac tissue of the CTP-targeted peptide\nnanozyme (CICT-Ruzyme) group was approximately 2.3 times greater than that in the\nnontargeted group (ICT-Ruzyme) (Figure 7G). This result further confirms the ability\nof CTP to target pathological myocardium and illustrates the influence of the in vivo\nmicroenvironment on the targeting ability of nanozymes.\nFigure 2G. Liquid images of NCT-Ruzyme, Ruzyme, ICT-Ruzyme, and CICT-Ruzyme\nstored in various biological media on days 1 and 7.\nFigure S21. Changes in the sizes of (A) Ruzyme, (B) ICT-Ruzyme and (C) CICT-\nRuzyme in various biological media incubated for different durations. The data are\npresented as the means ± SDs.\nFigure S43. Changes in the CAT-like activities of (A) Ruzyme, (B) ICT-Ruzyme and\n(C) CICT-Ruzyme after coincubation with 10% fetal bovine serum (FBS) for 0 h, 3 h,\n6 h, and 18 h.\nFigure S44. Changes in the nitrogen free radical (ABTS•+) scavenging activity of the\nPPy-co-PTh-stabilized Ruzymes after coincubation with 10% FBS for 0 h, 3 h, 6 h, and\n18 h. (A) Scavenging rates of the PPy-co-PTh-stabilized Ruzymes. ABTS•+ scavenging\nability of (B) Ruzyme, (C) ICT-Ruzyme and (D) CICT-Ruzyme as assessed by UV‒vis.\n(E) Representative optical photograph of ABTS•+ cleared by nanozymes.\nFigure S45. Changes in the nitrogen free radical (DPPH•) scavenging activity of the\nPPy-co-PTh-stabilized Ruzymes after coincubation with 10% FBS for 0 h, 3 h, 6 h, and\n18 h. (A) Scavenging rates of the PPy-co-PTh-stabilized Ruzymes. DPPH• scavenging\nability of (B) Ruzyme, (C) ICT-Ruzyme and (D) CICT-Ruzyme as assessed by UV‒vis.\n(E) Representative optical photograph of DPPH• cleared by nanozymes.\nFigure S46. Fluorescence images of Cy5.5-ICT-Ruzyme before and after incubation\nwith 10% FBS and coincubation with H9C2 cells for 1, 4 and 12 hours. Scale bar = 50\nµm.\nFigure 7F. Cy5.5-labeled Ruzyme, ICT-Ruzyme or CICT-Ruzyme (100 μL) was\nadministered via I.V. injection for ex vivo imaging to evaluate the distribution of\ndifferent nanoparticles in SC and normal mice at different time points (1, 3, 6, and 24\nh).\nFigure S41. Time-course quantification of fluorescence signals from the heart (n = 3).\nThe data are presented as the means ± SDs.\nFigure 7G. ICP‒MS analysis of the ruthenium (Ru) distribution in the heart across the\nexperimental groups. The data are presented as the means ± SDs.\n18. The author should perform ICP-MS to confirm the uptake of the different Ruzymes\nand controls (assessing the metal content per cell) and negative staining TEM to\nconfirm the mitochondria targeting/lysosomal accumulation (and demonstrating no\nmetal ion leaching).\nResponse: We sincerely thank the reviewer for this insightful suggestion, which has\ngreatly improved the quality of our manuscript. After co-incubating the cells with the\nnanozymes (Ruzyme and ICT-Ruzyme) for 24 hours, the cells were collected, and the\nmetal content per cell was assessed via ICP‒MS. The ICP‒MS results revealed that the\nRu content in the ICT-Ruzyme group was approximately 1.2 times greater than that in\nthe Ruzyme group (Figure 6E). Additionally, we observed the mitochondrial targeting\nof ICT-Ruzyme in H9C2 cells via TEM. However, owing to the low electron density of\nthe polymeric nanozyme, we were unable to directly observe the accumulation of the\nICT-Ruzyme in the mitochondria or lysosomes at the ultrastructural level. Therefore,\nwe further verified the ability of ICT-Ruzyme to target mitochondria at the cellular level\nvia fluorescence confocal microscopy. Moreover, TEM revealed that all the nanozymes\nmaintained mitochondrial structural integrity, demonstrating their biosafety, which is\nconsistent with the cytotoxicity results from the CCK-8 assay.\nFigure 6E. In vitro uptake of Ruzyme and ICT-Ruzyme by H9C2 cells by ICP‒MS\nmeasurements (n = 3). The coincubation time was 24 hours.\nFigure S37. H9C2 cells treated with ICT-Ruzyme for 24 h were subjected to ultrathin\nsectioning, and the process of the cellular uptake of ICT-Ruzyme was observed via\ntransmission electron microscopy. Scale bars are 5 μm, 2 μm (enlarged), and 500 nm\n(enlarged).\nFigure S38. H9C2 cells treated with ICT-Ruzyme for 24 h were subjected to ultrathin\nsectioning, and the mitochondrial structure in each group of cells was observed via\ntransmission electron microscopy. Scale bar = 2 µm.\n19. As a general comment the confocal representative images are very dim and the\nfluorescent signal is difficult to see on all channels.\nResponse: We thank the reviewer for this constructive feedback regarding the clarity\nof the confocal microscopy images. We acknowledge that the fluorescence signals\nappear dim in the representative images provided in the original submission. To\nimprove image clarity and visibility across all channels, we reprocessed the figures by\noptimizing the contrast and brightness settings while ensuring that all adjustments were\napplied uniformly and did not alter the interpretation of the results. The updated images\nnow offer a better visual representation of the fluorescence distribution and intracellular\nlocalization. These revised figures have been included in the manuscript.\n20. The authors state that the nanozymes are endocytosed. However, the appearence of\nICT-Ruzymes distribution in cells is very different from the one of most of nanomaterials\naccumulating in organelles. Why only the uptake of ICT-Ruzyme is shown and not\ncompared with Ruzymes?\nResponse: We thank the reviewer for this insightful comment regarding the\nendocytosis and comparative cellular uptake of the nanozymes. In response, we have\nconducted additional experiments to compare the internalization of Ruzyme and ICT-Ruzyme in\nH9C2 cells after coincubation for 1 h, 4 h, and 12 h. The results indicate that both\nnanozymes were internalized within 1 h of treatment, and the fluorescence signals\ngradually increased over time with prolonged incubation. However, different nanozyme\nsystems with the same concentration exhibited distinct cellular uptake behaviors.\nSpecifically, throughout the incubation period, the fluorescence signal of ICT-Ruzyme\nwas significantly greater than that of Ruzyme. This finding indicates that the\nincorporation of (5-carboxypentyl)(triphenyl)phosphonium bromide (TPP-COOH)\nenables the nanozyme to target mitochondria, thereby increasing their cellular uptake.\nRepresentative fluorescence images illustrating the uptake of both nanozymes in H9C2\ncells are provided below.\nFigure 6D. Fluorescence images and fluorescence quantification of Ruzyme and ICT-\nRuzyme coincubated H9C2 cells for 1, 4, and 12 hours. Scale bar = 50 µm.\n21. In general, a comparison between Ruzyme and ICT-Ruzyme is missing: how can the\nauthors state that the overlap with mitochondria is due to the ICT without this\ncomparison? In Figures 6F and 6G the results for Ruzyme and ICT-Ruzymes in terms\nof ROS and mtROS scavenging are comparable.\nResponse: We sincerely thank the reviewer for your valuable comments regarding the\nvalidation of mitochondrial targeting. The lack of direct comparisons between Ruzyme\nand ICT-Ruzyme prompted us to perform additional experiments to further verify the\nTPP-COOH–mediated targeting mechanism.\nIn response, we carried out mitochondrial colocalization assays to systematically\ncompare the mitochondrial localization efficiency of both nanozymes in\ncardiomyocytes. As shown in Figure 6D, the nuclei labeled with the dye DAPI\ndisplayed blue fluorescence, whereas the nanozymes labeled with the dye Cy5.5\nemitted red fluorescence. Mitochondrial-targeting fluorescent probes, which can\npenetrate cell membranes, were used for specific fluorescence staining of mitochondria\nin the cells. After 1 hour of coincubation with the nanozymes, a small amount began to\nenter the cells. As the coincubation time increased, the red fluorescence signal gradually\nintensified, indicating progressive cellular uptake across all systems. However,\ndifferent nanozyme systems at the same concentration exhibited distinct uptake\nbehaviors.\nSpecifically, throughout the incubation period, the fluorescence signal of ICT-\nRuzyme was significantly greater than that of Ruzyme (Figure 6D). This finding\nindicates that the incorporation of (5-carboxypentyl)(triphenyl)phosphonium bromide\n(TPP-COOH) enables the nanozyme to target mitochondria, thereby increasing their\ncellular uptake.\nTo further validate the targeting of the TPP-COOH-modified ICT-Ruzyme to H9C2\ncells, we compared the ruthenium (Ru) content in cells treated with the same\nconcentration of either Ruzyme or ICT-Ruzyme. The ICP‒MS results revealed that the\nRu content in the ICT-Ruzyme group was approximately 1.2 times greater than that in\nthe Ruzyme group (Figure 6E). To compare the mitochondrial ROS scavenging effects,\nwe re-evaluated the scavenging capacity of Ruzyme and ICT-Ruzyme toward\nmitochondrial ROS (mtROS) and performed a statistical analysis. The results\ndemonstrated that, compared with Ruzyme, ICT-Ruzyme significantly enhanced\nmtROS clearance by 28% (p < 0.05) (Figure 6G). This finding is consistent with the\nmitochondrial colocalization results, indicating that the mitochondrion-targeted\nnanozyme (ICT-Ruzyme) more effectively facilitates mtROS elimination. We once\nagain thank the reviewer for their insightful comments, which prompted us to refine the\nin-depth characterization of the targeted nanozymes.\nFigure 6D. Fluorescence images and fluorescence quantification of Ruzyme and ICT-\nRuzyme coincubated H9C2 cells for 1, 4, and 12 hours. Scale bar = 50 µm.\nFigure 6E. In vitro uptake of Ruzyme and ICT-Ruzyme by H9C2 cells by ICP‒MS\nmeasurements (n = 3). The coincubation time was 24 hours.\nFigure 6F‒G. Confocal fluorescence images and corresponding quantitative analysis\nof the effects of the NCT-Ruzyme, Ruzyme, and ICT-Ruzyme on mitochondrial\noxidative injury via the use of mitoSOX as an O •– indicator (n=3). Scale bar = 50 µm.\n2\n22. Do the Ruzymes co-localize with lysosomes after 24h incubation as commonly\nhappens to nanoparticles?\nResponse: We appreciate the reviewer’s attention to the subcellular localization of the\nnanozymes.\nNanomaterials primarily enter cells through endocytosis and are initially\nencapsulated within endosomes, which subsequently fuse with lysosomes. This is the\ninitial destination for the vast majority of internalized nanomaterials6.\nOur lysosome colocalization results also indicate that both Ruzyme and ICT-Ruzyme\naccumulate in the lysosome compartment, with Pearson’s correlation coefficients of\n0.69 and 0.65, respectively. It is important to recognize that the lysosome, characterized\nby its slightly acidic environment, presents a unique context for enzymatic activity. Our\nnanozyme demonstrates peroxidase-like (POD-like) activity at pH 4.5 (Figure 1B).\nHowever, a critical consideration must be made regarding the availability of its essential\nsubstrate, hydrogen peroxide (H O ). In this context, the nanozyme examined in this\n2 2\nstudy exhibits robust antioxidant enzyme-like activity, effectively scavenging the low\nlevels of ROS produced by cells. In summary, the cellular antioxidant system ensures\nthat ROS, including H O , are consistently maintained at a low concentration in the\n2 2\nnormal cellular environment. Under normal physiological conditions, cells maintain\nlow levels of hydrogen peroxide (H O ), through a series of defense mechanisms7.\n2 2\nMoreover, the antioxidant function of the nanozyme in this study renders the peroxidase\n(POD)-like catalytic reaction, which is dependent on H O in lysosomes, extremely\n2 2\nweak. Additionally, the nanozyme does not exhibit oxidase-like (OXD-like) activity\nunder acidic conditions (Figure 1C).\nConsequently, even within the acidic milieu of the lysosome, the nanozyme is unable\nto leverage its enzyme-mimicking capabilities to produce the toxic hydroxyl radical\n(•OH). This underscores the nuanced interplay between the nanozyme’s activity profile\nand the specific biochemical environment of the lysosome, highlighting the importance\nof substrate availability and pH-dependent activity in determining its functional\noutcomes.\nIn addition, the intracellular trafficking of nanomaterials is typically a highly\ndynamic process: extracellular nanoparticles continuously enter through endocytic\npathways and accumulate in lysosomes, while at the same time, nanoparticles within\nlysosomes continually escape into the cytoplasm. In addition to this, the crosstalk\nbetween damaged mitochondria and the endoplasmic reticulum can lead to changes in\nthe acidity of lysosomes, which in turn alters the microenvironment in which the\nnanozyme can exert its POD-like activity8. Furthermore, our mitochondrial\ncolocalization images showed that the TPP-COOH modified nanozyme (ICT-Ruzyme)\nexhibited significant mitochondrial colocalization at 1 h, 4 h, and 12 h (stronger than\nRuzyme). This suggests that ICT-Ruzyme has a propensity to target mitochondria over\ntime, which may have implications for its intracellular distribution and potential\ninteractions with mitochondrial components.\nIt is noteworthy that mitochondria are the primary organelles responsible for\ngenerating O •– and inducing oxidative cellular damage. The mitochondrial targeting\n2\ncapability of ICT-Ruzyme provides a strong assurance for efficiently eliminating\nintracellular ROS.\nWe once again thank the reviewer for their thorough critique, which has prompted us\nto explore the intracellular behavior of the nanozymes in greater depth.\nFigure S39. Confocal images showing the colocalization of Ruzyme and ICT-Ruzyme\nwith lysosomes. Pearson’s correlation coefficient was obtained via Fiji ImageJ. Scale\nbar = 50 µm.\nFigure 6D. Fluorescence images and fluorescence quantification of Ruzyme and ICT-\nRuzyme coincubated H9C2 cells for 1, 4, and 12 hours. Scale bar = 50 µm.\n23. The efficacy of CICT-Ruzyme in targeting cardiomyocytes in virtue of the CT peptide\nhas never been demonstrated in the manuscript, making these data useless, as the effect\nof CICT-Ruzyme is very similar to ICT-Ruzyme and Ruzyme (Figure 8 C,D,E,F). A\ntrend is visible, but is it significant? Eventually, a biodistribution study (via ICP-MS)\nshould confirm the effectiveness of the specific in vivo targeting.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\nvalidation of cardiac-targeting efficiency. To address this point, we performed\nquantitative biodistribution analyses through two complementary approaches. First, we\nperformed ex vivo fluorescence imaging and relative quantification on hearts isolated\nfrom LPS-induced SC model mice to verify that CTP modification significantly\nenhanced targeted accumulation in the pathological myocardium. After intravenous\nadministration of the three nanozymes (Ruzyme, ICT-Ruzyme, and CICT-Ruzyme) to\nSC mice for 12 h, cardiac tissues were subsequently excised, and ruthenium (Ru)\naccumulation was quantified via ICP‒MS, with comparative analysis across\nexperimental groups. We have supplemented the imaging data, and the results are as\nfollows:\nFigure 7F. Cy5.5-labeled Ruzyme, ICT-Ruzyme or CICT-Ruzyme (100 μL) was\nadministered via I.V. injection for ex vivo imaging to evaluate the distribution of\ndifferent nanoparticles in SC and normal mice at different time points (1, 3, 6, and 24\nh).\nFigure S41. Time-course quantification of fluorescence signals from the heart (n = 3).\nThe data are presented as the means ± SDs.\nFigure 7G. ICP‒MS analysis of the ruthenium (Ru) distribution in the heart across the\nexperimental groups. The data are presented as the means ± SDs.\n24. Page 19, line 463: “Consistent results were obtained when using O •–….” Figure\ncytometry spectra. In addition, the data are not presented as an average of n=3, as\nstated in the caption.\nResponse: We sincerely thank the reviewer for this insightful comment. As noted above,\nthe original data presented for the O •– scavenging assay were insufficient. In response,\n2\nwe repeated the flow cytometry experiments to assess the ability of our nanozymes to\nscavenge O •– more accurately in H9C2 cells. The new results, which are based on three\n2\nindependent replicates (n = 3), are presented in the figure below. These data clearly and\nconsistently demonstrate the ability of our nanozymes to scavenge ROS (O •–).\nupdated the manuscript and supplementary materials with these new results and\napologize for any confusion caused by the initial presentation.\nFigure S36. Effects of O •– on H9C2 cell viability and intracellular ROS levels. (A)\n2\nand (C) corresponding quantitative results of intracellular ROS in H9C2 cells (DCFH-\nDA staining) (n = 3). (D) Gating strategy used to determine the level of cellular ROS\npresented in Figure S36B. The data are presented as the means ± SDs. The data were\nanalyzed via one-way ANOVA with Tukey’s post hoc test.\n25. Not clear how you exploited here the electrical conductivity of the Ruzyme.\nResponse: We thank the reviewer for raising this important point. We used the\nnonconductive nanozyme NCT-Ruzyme as a control to measure the conductivity of the\nRuzyme. The size of the semicircle diameter in the high-frequency region of the EIS\ninstrument directly reflects the electrical impedance of the material, providing insight\ninto the electron transfer dynamics on the electrode surface. A smaller semicircle\ndiameter indicates lower electron transfer resistance and better conductivity, whereas a\nlarger diameter suggests higher impedance and poorer conductivity. As shown in Figure\n2J, compared with the NCT-Ruzyme, the PPy-co-PTh-stabilized Ruzyme exhibited a\nsmaller semicircle diameter in the high-frequency region (Figure 2I). This indicates that\nthe PPy-co-PTh-stabilized Ruzyme, with polypyrrole and polythiophene as the\nframework, has a lower charge transfer resistance. This suggests that the nanozyme\nfacilitates faster charge transfer during the catalytic reaction, resulting in better\nelectrical conductivity. Specifically, the conductive framework formed by the\npolypyrrole‒polythiophene copolymer facilitates rapid electron transfer, effectively\nenhancing the electrochemical properties of the nanozyme. This enhancement was\nachieved by lowering the catalytic energy barrier, reducing excessive oxidation caused\nby ROS (Figure 4), and facilitating the continuous catalysis of ROS decomposition\n(Figure 3; Figure S20).\n--------------------------------------------------------------------------------\nReview 4:\nThe authors have addressed the raised issues appropriately. I would like to recommend\nthis work to be published.\nResponse: We appreciate the positive comments.\n1. FITR data should be provided to demonstrate the structure of the nanozymes.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\nstructural validation of the nanozymes. As recommended, we have supplemented the\nFourier transform infrared (FT-IR) spectra of both ICT-Ruzyme and CICT-Ruzyme for\ncomparative analysis. After CTP modification, distinct stretching vibrations were\nobserved at 1644 and 1343 cm-1 in the spectrum of the CICT-Ruzyme, corresponding\nto the C=O bonds of CTP, confirming the successful conjugation of CTP (Figure S16,\nSupporting Information).\nAdditionally, the contents of phosphorus (P) in Ruzyme and ICT-Ruzyme were\ndetected via ICP‒MS. The content of P in ICT-Ruzyme was approximately 0.0057%,\nbut it was not detected in Ruzyme. Therefore, the addition of P further indicated the\nsuccessful ligation of TPP-COOH on the surface of the Ruzyme, and the amount of\ngrafted TPP-COOH accounted for approximately 0.0844% of the total nanozyme.\nFigure S16. FT-IR spectra of ICT-Ruzyme and CICT-Ruzyme.\n2. Figure S3 shows that all 11 types of PPy-co-PTh nanozymes exhibit absorption\naround ~240 nm. Could this absorption potentially interfere with the UV-Vis detection\nof the CAT activity of the nanozymes?\nResponse: We sincerely thank the reviewer for raising this important concern regarding\npotential interference from the UV absorption of the PPy-co-PTh nanozymes at ~240\nnm. We acknowledge that all eleven types of PPy-co-PTh nanozymes exhibit absorption\nat 240 nm, whereas H O displays a characteristic absorption peak at this wavelength.\n2 2\nWe monitored the decrease in the absorbance of H O at 240 nm in the reaction system\n2 2\nof 11 types of PPy-co-PTh nanozymes with H O to determine the CAT-like activity of\n2 2\nthe nanozymes. The monitoring lasted for a total of 10 minutes. Crucially, the reported\ndata represent ΔA values (absorbance difference at each time point minus the initial\nabsorbance). Since the initial measurement already accounts for the background\nabsorption of both the nanozyme and H O , the subsequent changes in ΔA exclusively\n2 2\nreflect H O decomposition. This differential measurement approach effectively\n2 2\neliminates interference from the intrinsic absorption of nanozymes, ensuring the\nreliability of our CAT-like activity assessment. We have clarified this methodology in\nthe revised Supporting Information.\n3. Is there an error in the caption of Figure S9?\nResponse: We sincerely thank the reviewer for identifying this significant oversight. In\nthe original manuscript, an error was present in the caption of Figure S9, which has\nnow been thoroughly corrected in the revised version of the manuscript, and the figure\nhas been renumbered as Figure S12. We apologize for any confusion that this may have\ncaused and greatly appreciate the reviewer's meticulous attention to detail, which has\nundoubtedly enhanced the accuracy and clarity of our work.\n4. In Figure 6, why is there an absence of validation regarding the effects of the CICT-\nRuzyme on H9C2 cells?\nResponse: Thanks for raising this insightful question regarding the lack of CICT-\nRuzyme validation in cell-based experiments. We acknowledge that H9C2 cells are\nwidely used for cardiac drug screening (https://doi.org/10.1038/s41467-024-45927-3,\nhttps://doi.org/10.1002/adma.202006570). In this study, the core of the CICT-Ruzyme\ndesign lies in the targeted delivery system (cardiac-targeting peptide CTP), whose\nfunction can be activated only in the presence of a specific pathological environment in\nvivo. CTP and other targeted components need to be combined with proteins exposed\nin the damaged myocardium (such as myosin light chain), but H9C2 cells cultured in\nvitro in a single layer lack such a damaged microenvironment, resulting in the inability\nof in vitro experiments to reflect the true targeting efficiency. We understand the\nreviewers’ concern about the in vitro validation. However, the targeting mechanism of\nCICT-Ruzyme and its pathogenic dependence make it difficult to replicate in a\nsimplified model. Therefore, we prefer to conduct more validations of CICT-Ruzyme\nat the animal level. We once again thank the reviewer for raising this important issue.\n5. While cardiac targeting is claimed, quantitative biodistribution data (e.g., organ-\nspecific accumulation) are absent. Include imaging or ICP-MS data to validate\ntargeting efficiency in vivo.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding the\nvalidation of cardiac-targeting efficiency. To address this point, we performed\nquantitative biodistribution analyses through two complementary approaches. First, we\nperformed ex vivo fluorescence imaging and relative quantification on hearts isolated\nfrom LPS-induced SC model mice to verify that CTP modification significantly\nenhanced targeted accumulation in the pathological myocardium. After intravenous\nadministration of the three nanozymes (Ruzyme, ICT-Ruzyme, and CICT-Ruzyme) to\nSC mice for 12 h, cardiac tissues were subsequently excised, and ruthenium (Ru)\naccumulation was quantified via ICP‒MS, with comparative analysis across\nexperimental groups. We have supplemented the imaging data, and the results are as\nfollows:\nFigure 7F. Cy5.5-labeled Ruzyme, ICT-Ruzyme or CICT-Ruzyme (100 μL) was\nadministered via I.V. injection for ex vivo imaging to evaluate the distribution of\ndifferent nanoparticles in SC and normal mice at different time points (1, 3, 6, and 24\nh).\nFigure S41. Time-course quantification of fluorescence signals from the heart (n = 3).\nThe data are presented as the means ± SDs.\nFigure 7G. ICP‒MS analysis of the ruthenium (Ru) distribution in the heart across the\nexperimental groups. The data are presented as the means ± SDs.\n6. In in vivo experiments, the levels of ROS and RNS in myocardial tissue should be\nadditionally detected to verify the scavenging effects of the nanozymes.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding the\nvalidation of the scavenging effects of ROS/RNS in myocardial tissue. To evaluate the\nROS levels in the myocardial tissues across the experimental groups, we performed\ndihydroethidium (DHE) staining (Fig. 8H). Additionally, we validated the ability of the\nnanozymes to scavenge reactive nitrogen species (RNS) in cardiac tissues. The specific\nexperimental results are as follows:\nFigure 8H. DHE staining of the cardiac tissues of the mice in each group following\nPBS or nanozyme treatment. Scale bar = 200 μm.\nFigure S49. RNS levels in the heart tissue of each mouse group (n = 5).\n7. Transcriptomic analysis suggests that the ferroptosis pathway is implicated in the\neffects of CICT-Ruzyme treatment. However, key markers (e.g., GPX4, ACSL4) are not\nexperimentally validated. Western blotting or qPCR of these targets would strengthen\nthe mechanistic claims.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding the\nexperimental validation of key ferroptosis markers. To better verify the inhibitory effect\nof nanoenzymes on ferroptosis, we used RSL-3, which has been widely reported to\ninduce ferroptosis9, as an inducer. We determined that the concentration of RSL-3 that\ncaused approximately 50% H9C2 cell death was 12 μΜ. Additionally, we used Fer-1\nas a positive control and evaluated the safety of the usage concentration of Fer-1 on\nH9C2 cardiomyocytes. Acyl-coenzyme A synthetase long-chain family member 4\n(ACSL4) is an important promoter of ferroptosis that is capable of integrating free\npolyunsaturated fatty acids (PUFAs) into the cell membrane, thereby promoting the\nlipid peroxidation process10. Glutathione peroxidase 4 (GPX4) is another specific\nmarker related to ferroptosis that can protect cells from lipid peroxidation damage and\nmaintain redox balance within cells11. RSL-3 can directly bind to (GPX4) and inactivate\nit, leading to the accumulation of lipid peroxides within cells and triggering ferroptosis.\nTherefore, we focused on ACSL4 and GPX4 and used the ferroptosis inhibitor Fer-1 as\na positive control. We verified the inhibitory effect of the nanozyme on ferroptosis\nthrough WB. In addition, we also examined the changes in GPX4 enzyme activity in\neach group after treatment with various drugs at the cellular and tissue levels. These\nresults indicated that after induction by RSL-3, GPX4 enzyme activity in\ncardiomyocytes significantly decreased. After the effects of the nanozyme and\nferroptosis inhibitor, the GPX4 enzyme activity in each treatment group improved, and\nthe effect of nanozyme was better. Moreover, we also evaluated the changes in GPX4\nenzyme activity in each drug-treated group after induction with LPS at the tissue level.\nIn summary, by verifying the key markers of ferroptosis, the PPy-co-PTh-stabilized\nRuzymes effectively inhibited the process of lipid peroxidation and prevented the\noccurrence of ferroptosis. The specific results are as follows:\nFigure S54. The viability of H9C2 cells was assessed after incubation with different\nconcentrations of (A) RSL-3 for 12 h, or with (B) Lip-1 or (C) Fer-1 for 24 h (n = 3).\nThe data are presented as the means ± SDs.\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were\ndetermined by western blot analysis. (a) Representative immunoblots of ferroptosis-\nrelated proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins\nin H9C2 cells (n = 3).\nFigure 9L. GPX4 activity in H9C2 cells incubated with 2 μM Fer-1 and different\nnanozymes (n = 3).\nFigure 9M. GPX4 activity in cardiac tissue (n = 5).\nReference\n1. Liu L, et al. Deubiquitinase OTUD5 as a Novel Protector against 4‐HNE‐Triggered\nFerroptosis in Myocardial Ischemia/Reperfusion Injury. Advanced Science 10, (2023).\n2. Wang X, et al. Destroying pathogen-tumor symbionts synergizing with catalytic therapy of\ncolorectal cancer by biomimetic protein-supported single-atom nanozyme. Signal Transduction\nand Targeted Therapy 8, (2023).\n3. Fu S, et al. ROS-Targeted Depression Therapy via BSA-Incubated Ceria Nanoclusters. Nano\nLetters 22, 4519-4527 (2022).\n4. Tarricone G, et al. Catalytic Bioswitch of Platinum Nanozymes: Mechanistic Insights of\nReactive Oxygen Species Scavenging in the Neurovascular Unit. Nano Letters 23, 4660-4668\n(2023).\n5. Dawson KA, Yan Y. Current understanding of biological identity at the nanoscale and future\nprospects. Nature Nanotechnology 16, 229-242 (2021).\n6. Liu C, et al. Red Emissive Carbon Dot Superoxide Dismutase Nanozyme for Bioimaging and\nAmeliorating Acute Lung Injury. Advanced Functional Materials 33, (2023).\n7. Halliwell B. Understanding mechanisms of antioxidant action in health and disease. Nature\nReviews Molecular Cell Biology 25, 13-33 (2023).\n8. Li S, et al. Impaired mitochondria-initiated crosstalk with lysosomes reciprocally aggravates\nmitochondrial defect through LManVI. Nature Communications 16, (2025).\n9. Xu S, Min J, Wang F. Ferroptosis: an emerging player in immune cells. Science Bulletin 66,\n2257-2260 (2021).\n10. Wang X, et al. Adipocyte-derived ferroptotic signaling mitigates obesity. Cell Metabolism 37,\n673-691.e677 (2025).\n11. Feng Y, et al. A ferroptosis-targeting ceria anchored halloysite as orally drug delivery system\nfor radiation colitis therapy. Nature Communications 14, (2023).\nBelow please find our responses to the comments and questions from the reviewers:\n-----------------------------------------------------------------------------------------------------\nReviewer #1 (Remarks to the Author):\nThank you for addressing my comments in your rebuttal and for the additional data\nprovided. I appreciate the effort to clarify the results. However, several important issues\nremain, particularly regarding the western blotting and in vivo IHC datasets. To ensure\ntransparency and reproducibility, I ask that the following points be addressed:\nResponse: Thank you for acknowledging our supplementary data and responses, and\nfor highlighting the remaining concerns regarding the Western blot and in vivo IHC\ndatasets. To address these important issues thoroughly, we have now conducted\nrepeated experiments and additional analyses as detailed below.\n1. Western blot data integrity (ACSL4, GPX4, loading controls)\nThe ACSL4 blots show multiple bands, raising concerns about antibody specificity. A\nclear, single band at ~80–88 kDa is expected. Please provide uncropped, full-length\nblots with visible molecular weight markers for all WB data, including loading controls,\nalong with the original raw files. Validation of ACSL4 is required (e.g., knockdown/KO,\npeptide competition, or a second antibody). I recommend repeating the experiment with\na more specific antibody; several validated options are commercially available.\nResponse: We thank the reviewer for this critical comment. In accordance with the\nsuggestion, we have now repeated the experiment for the key results involving ACSL4.\nFurthermore, as requested, we have provided uncropped, full-length blots for all\nWestern blot data (including ACSL4, GPX4, and GAPDH) in the Supplementary\nInformation, with all molecular weight markers clearly visible. The raw data images\nhave also been updated in the Source Data. We believe these additional data adequately\naddress the concern and hope the reviewer finds the updated figures and source data\nsatisfactory.\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were\ndetermined by western blot analysis. (a) Representative immunoblots of ferroptosis-\nrelated proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins\nin H9C2 cells (n = 3).\nFigure S57. Raw data of Western blot image in Figure 9K. (A) Uncropped, full-length\nmembranes for all Western blot data. (B) Full membrane index for the Western blot\nimages of ACSL4, GPX4, and GAPDH in Figure 9K (a).\n2. Replication and quantification of western blots\nProvide quantitative densitometry from ≥3 independent biological replicates,\nnormalized to loading control, with mean ± SEM and appropriate statistical testing.\nClarify whether ACSL4/GPX4 panels and loading controls derive from the same\nmembranes (stripped/re-probed) or from separate gels.\nResponse: We thank the reviewer for this suggestion to enhance the quantitative rigor\nof our data. As recommended, we have repeated the Western blot experiments to\nvalidate the inhibitory effect of the nanozyme on ferroptosis with three independent\nbiological replicates (n=3). The quantitative densitometry analysis for ACSL4 and\nGPX4, normalized to loading control (GAPDH), is now presented in the revised figures\nas mean ± SEM, and appropriate statistical analyses have been performed to assess the\nsignificance between groups. Furthermore, we confirm that the blotting images for\nACSL4, GPX4, and GAPDH shown in the figure panels were indeed derived from the\nsame PVDF membrane (stripped/re-probed). We appreciate the reviewer's insightful\ncomment, which has helped us improve the data presentation and statistical robustness\nof our study.\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were\ndetermined by western blot analysis. (a) Representative immunoblots of ferroptosis-\nrelated proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins\nin H9C2 cells (n = 3).\nFigure S57. Raw data of Western blot image in Figure 9K. (A) Uncropped, full-length\nmembranes for all Western blot data. (B) Full membrane index for the Western blot\nimages of ACSL4, GPX4, and GAPDH in Figure 9K (a).\n3. In vivo IHC (heart sections)\nOnly one representative image per group is provided in the rebuttal, which is\ninsufficient. Please provide quantified IHC data from >6 animals per group, analyzed\nacross multiple sections per heart, using blinded or automated methods. Provide mean\n± SEM with statistical comparisons.\nResponse: We thank the reviewer for the constructive feedback. In response to the\ncomment regarding the need for quantified IHC data, we have conducted\ncomprehensive quantitative analyses. The study now includes IHC data from more than\n6 animals per group. For each animal, quantification was performed on four non-\nconsecutive cardiac sections, analyzed in a blinded manner using automated image\nanalysis software. The results, expressed as mean ± SEM, have been incorporated into\nthe revised manuscript (Supplementary Figure S58), along with statistical comparisons\nbetween groups using one-way ANOVA. We believe these additional data address the\nreviewer's concern and strengthen the manuscript.\nFigure S58. Immunohistochemical analysis of 4‑HNE expression in cardiac tissue\nsamples. (A) Representative immunohistochemical staining images of 4-HNE in\nmultiple sections per heart. Scale bar = 1000 µm. (B) Relative quantitative analysis of\n4-HNE staining across experimental groups in multiple cardiac sections. The data are\npresented as the means ± SEM, and p values were provided in the figures using one-\nway analysis of variance (ANOVA) followed by Tukey’s post hoc test.\n4. Supplementary data and transparency\nAll uncropped western blot images (with markers, ladders, and all replicates) should\nbe uploaded as supplementary source data. Include a “raw blot index” mapping each\ncropped panel to its corresponding full-length blot.\nResponse: We thank the reviewer for this recommendation to ensure full transparency\nof our Western blot data. In accordance with the suggestion, we have now uploaded all\nuncropped Western blot images, which include molecular weight markers, ladders, and\nall experimental replicates, as part of the Supplementary Source Data. Additionally, a\n“raw blot index” that clearly maps each cropped panel featured in the main figures to\nits corresponding full-length, uncropped blot has been provided in both the Source Data\nfile and the Supplementary Information. We believe these materials will provide a\ncomplete and clear overview of the original data.\nFigure S57. (B) Full membrane index for the Western blot images of ACSL4, GPX4,\nand GAPDH in Figure 9K (a).\nProvide raw IHC quantification spreadsheets to support the representative figure\nconclusion.\nResponse: We sincerely thank the reviewer for the valuable comments on this\nmanuscript. In response to the suggestions, we have updated the manuscript,\nsupplementary materials, and source data accordingly. We hope these revisions have\nenhanced the quality and completeness of the manuscript. Once again, we greatly\nappreciate the reviewer’s insightful feedback.\nReviewer #2 (Remarks to the Author):\nThe authors have satisfactorily addressed my previous comments. I support the\npublication of this manuscript after minor revision:\nResponse: Thank you very much for your positive assessment and for supporting the\npublication of our manuscript. We have carefully checked the text and figures to\nincorporate all remaining minor adjustments, as detailed in the point-by-point response.\nAll changes are highlighted in the revised manuscript and supplementary materials.\n1. In the Method section, the phrase “every MD step” is ambiguous and potentially\nmisleading. The heating procedure from 0 K to 3000 K within 3 ps using velocity scaling\ntypically refers to a continuous temperature ramp during the molecular dynamics\nsimulation, rather than an action performed at each integration step. For clarity, this\nsentence could be revised as: “The optimized RuO crystal was heated from 0 K to 3000\nHoover thermostat with a time step of 1 fs to obtain the amorphous RuO structure.”\nmanuscript and their valuable comments regarding the potential ambiguity of the phrase\n“every MD step” in the Methods section. The reviewer’s observation is highly pertinent,\nand we fully agree that the original wording could lead to misunderstanding about the\nspecific implementation of the heating process. Following the reviewer’s suggestion,\nwe have revised the relevant sentence to: “The optimized RuO crystal was heated from\n2\n0.5 ps via the Nose-Hoover thermostat with a time step of 1 fs to obtain the amorphous\nRuO structure.” This revision clarifies that the heating represents a continuous process\n2\ncarried out over 3 ps, rather than an operation performed at each integration step,\nthereby making the methodological description clearer and more accurate. Once again,\nwe greatly appreciate the reviewer's input, which has been instrumental in improving\nthe quality of our manuscript.\n2. In the Method section, although many published articles do not explicitly cite\nresources such as the Materials Project database or the Nosé-Hoover thermostat, I\nbelieve it is good scholarly practice to include the appropriate references as a sign of\nrespect to the original sources and to enhance transparency and reproducibility. In this\ncontext, the Materials Project can be cited as https://doi.org/10.1063/1.4812323, while\nthe Nosé-Hoover thermostat should be attributed to both\nhttps://doi.org/10.1080/00268970110089108 and\nhttps://doi.org/10.1103/PhysRevA.31.1695.\nResponse: We fully agree that explicitly citing key resources such as the Materials\nProject database and the Nosé-Hoover thermostat in the Methods section represents\ngood scholarly practice, as it both acknowledges the original contributors and enhances\nthe transparency and reproducibility of our study. Following the reviewer’s\nrecommendation, we have incorporated the suggested references at the appropriate\nlocations in the manuscript. These revisions are reflected in the latest version of the\npaper. We believe these additions have made our methodological description completer\nand more rigorous. Once again, we appreciate the reviewer’s insightful comments,\nwhich have been essential in improving the quality of our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have undertaken significant additional work, leading to a clear\nimprovement in the scientific level of the manuscript, which is now suitable for\npublication.\nThe manuscript show the therapeutic potential of polymer-coated ruthenium based\nnanozymes engineered to perform protective RONS scavenging to counteract septic\ncardiomyopathy, showing promising results in vitro and in vivo. Some methodological\nlittle detail might still be improved, understandably given the complexity and the size\nof the work done, nevertheless, the general scientific outcome appear to be solid and\nimpactful, and especially relevant for the Nanomedicine community.\nResponse: Thank you very much for your positive assessment and recognition. We are\nalso pleased to observe that, following the additional experiments and textual revisions\nin this round, the scientific rigor and completeness of the manuscript have been further\nenhanced. In response to your comment that “some methodological little detail might\nstill be improved,” we have carefully reviewed each point and provided supplementary\ninformation and clarifications accordingly.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed the raised issues appropriately. I would like to recommend\nthis work to be published.\nResponse: Thank you very much for your positive feedback and for recommending our\nwork for publication. We truly appreciate your constructive comments throughout the\nreview process, which have helped us improve the quality of our manuscript.\nROUND 1 REVIEWER 2 ATTACHMENT:\nThe manuscript titled “Conducting Polymer-Stabilized Nanozymes Alleviate Sepsis-induced\nMyocardial Injury by Inhibiting Iron Accumulation and Lipid Peroxidation” presents a\ncomprehensive and well-executed analysis of conducting polymer-stabilized nanozymes for the\ntreatment of septic cardiomyopathy (SC). Through extensive characterization and rigorous testing,\nthe authors successfully demonstrate that Ruzyme, a ruthenium-based nanozyme, exhibits\nexcellent catalytic efficiency, stability, and ROS-scavenging capabilities. The study provides\nstrong evidence that targeted d elivery of these nanozymes to myocardial mitochondria effectively\nmitigates oxidative stress, iron accumulation, and lipid peroxidation, ultimately improving cardiac\nfunction in SC models. The thorough experimental validation and in-depth mechanistic insights\nmake this work highly valuable for the field of nanozyme-based therapeutics. I support the\npublicatio n of this manuscript; however, the following comments should be addressed through\nr evision:\n1. In the title: “Conducting Polymer-Stabilized Nanozymes Alleviate Sepsis-induced Myocardial\nInjury by Inhibiting Iron Accumulation and Lipid Peroxidation”. The word “induced” should\nbe capitalized.\n2. The subscript and superscript formatting of radicals in the text is too small, making them\ndifficult to notice at times. It is recommended to adjust the formatting to make the superscripts\nand subscripts more distinct. For example: O•–, •OH, ONOO–.\n2\n3. Figure 2A appears to be essentially the same as Scheme 1A, raising the question of its necessity.\nConsider removing Figure 2A to avoid redundancy.\n4. It is difficult to clearly observe the described insolubility and solute aggregation in Figure 2H.\nThe differences between the samples are not visually distinct, making it challenging to validate\nthe claims in the text. I suggest magnifying the relevant areas or providing zoomed-in insets to\nenhance clarity.\n5. In the legend for Figure 3B and 3C, please explicitly indicate the H O dose used in Figure 3B\n2 2\nand the time points measured in Figure 3C.\n6. The numbering of supplementary tables should follow a sequential order in the main text.\nCurrently, Table S1 appears far later than Table S2. Please ensure that Table S1 is cited before\nTable S2 in the text.\n7. Could you provide a bit more explanations on how the XPS analysis supports the conclusion\nthat PPy-co-PTh stabilized Ruzyme exhibits greater valence stability as described in section\n2.6: “The XPS analysis of NCT-Ruzyme and PPy-co-PTh stabilized Ruzyme before and after\nthe reaction with H O demonstrated that PPy-co-PTh stabilized Ruzyme exhibited greater\n2 2\nvalence stability under identical catalytic conditions”?\n8. Could you please double-check the atomic labels in Figure 4C? Six distinct atom types are\npresent, but only five are labeled. It seems that the silver atom might be nitrogen rather than\nhydrogen. If this is the case, correcting the labels.\n9. In Figures 4D and 4G, the top-view perspectives are really difficult to clearly observe the\nadsorbed molecules. Consider improving the clarity by, for example, using a different color\nscheme, or different atom style, or highlighting adsorption sites, or providing additional side-\nview images in the supplementary information.\n10. In the legend of Figure 4, it states: “Surface configurations of different modeled initial,\ntransition, and final states for the simulated …...” However, based on my understanding, the\nresults and the DFT method described in the supplementary indicate that you didn’t perform\ntransition state calculations, such as NEB or dimer methods. Instead, you calculated different\nadsorbed configurations of the adsorbates and compared their energies. So, the legend seems\nto be somewhat misleading, please consider revising it to something like: “Surface\nconfigurations of different adsorbed states for the simulated …...”.\n11. A bit more details on the DFT calculation method are needed to ensure reproducibility. Can\nDFT accurately reproduce the lattice parameters and band structure of RuO ? A comparison\n2\nwith experimental or previous computational results would be helpful. Additionally, which\nRuO surface was used, what is the size of the supercell, and whether the bottom layers were\n2\nfrozen during calculations (and if so, how many layers were fixed). Furthermore, specifying\nthe thermostat used for the AIMD simulations.\n12. I might be wrong but in Section 2.5, second paragraph, all the notations for Fe2+ and Fe3+\nappear incorrect. It looks like the superscripts are displayed in a smaller font style rather than\nbeing properly formatted as superscripts (which often happens after text refinement by GPT).\nPlease verify whether the correct superscript formatting has been applied.\n13. In Section 2.5, I noticed multiple formatting inconsistencies and errors in the text and figure\nlegend. Please carefully review and refine the text to ensure consistency with the previous\nsections.\n14. Additionally, Section 2.5 appears after Section 2.6, which seems out of order. Please double-\ncheck the paper format to ensure the correct section numbering and overall structure.\n15. Just out of curiosity, in Figure 7B, the daily weight changes of the mice in the low-\nconcentration CICT-Ruzyme group (blue line: 20 mg/kg) seem to show slightly more\nvariation compared to the higher-concentration group. Is there a specific reason for this?\n16. In Figure 7E, I do not see significant changes in most organs; however, the heart appears to\nshow a relatively larger variation. Could you provide an explanation for this?\n17. Some subtitles use sentence case (e.g., 2.1 Synthesis and characterization of PPy-co-PTh\nstabilized nanozymes), while some use title case (e.g., 2.10 Mechanisms Underlying the\nTherapeutic Effects of CICT-Ruzyme in Alleviating SC). Please ensure consistency in\nformatting across all subtitles.\nOnce these revisions are made, the manuscript should meet the standards for publication.\nROUND 1 REVIEWER 3 ATTACHME NT:\nThe topic is very interesting and the work presented showed some very promising data.\nHowever, the methodological/experimental description/execution, especially in the first part\nof the article (preparation and comparison among different nanozyme candidates) does not\nmeet at the current status the standards of excellence required for a publication in Nature\nCommunications. The main findings of the paper (Ruzymes showed a good potential in\npreventing LPS-induced damages in myocardial cells in vitro and in vivo thanks to their iron\nand ROS scavenging activity) get buried in a set of weaker data that lower the overall quality\nof the work.\nThe authors might take into consideration removing (or moving to SI) the initial comparative\nstudy with different nanozymes that actually need substantial improvements.\nNevertheless, major revisions are necessary before re-evaluating the manuscript for\npublication in Nature Communications:\n- In Abstract and Introduction define all acronyms/short names at the first use. PPy?\nPTh? Tpp? MiR130B-3P? Fe-Cur@TA?\n“ the nanozymes exhibits overoxidation and inadequate stability”…maybe some of\nthem, but several actually have been proven to present good stability. I think the\nauthors made an overstatement in this context.\n- “Ruzymes (PPy-co-PTh stabilized Ruzyme [Ruzyme], ICT-Ruzyme, and CICT-Ruzyme) can\nbetter prevent themselves from overoxidation in the environment of excessive H2O2, and\nhas more excellent conductivity and antioxidant properties.” Compared to what?\n- The author claims stability of the prepared nanozymes and that the PDI below 0.7 by DLS\nindicates good dispersion. However, all the PDI reported are above 0.2 and several above\n0.4 indicating highly polydisperse samples for all the PPy-co-PTh nanozymes containing\nthe different metal ions including the different Ruzymes. Actually, with such a high PDI\nthere is some risk even in extrapolating the peak maxima by number distribution and z-\npotential when using a Malvern instrument…those numbers might not be so solid.\nDLS spectra expressed in intensity should also be reported in SI.\n- Further TEM measurements should also be provided (more micrographs and higher\nquality).\n- Figure 2B do not really show “relatively uniform spherical morphology”.\n- Potential leaching of metal ions in the relevant biological environments (including in the\npresence of iron) should be evaluated.\n- ICP-MS and EDS should be performed on all samples and not only on the Ruzyme to allow\nfor ions-dependent meaningful comparisons of the enzyme-like activities.\n- If the activity is supposed to be metal ion type-dependent, considering the polydispersity\nindex of the samples and the different sizes of nanozymes, a normalization by mass is\nnot an adequate approach for a meaningful comparison. Especially since it is not clear\nfrom the characterization how many atoms of the different metals are present per\nnanoparticles. The current literature suggest that you should normalize the available\nactive sites and measure the turnover frequency (TOF) to ensure the activity is catalytic\n(cycling) (https://doi.org/10.1021/acsnano.1c07520).\n- I am afraid that the authors fall into a common analytical mistake. The TMB is not suitable\nto be used for pH dependent or temperature dependent studies as performed in this\nmanuscript (monitoring the absorbance at 652 nm), because the TMB chromogenic\nsubstrate itself is known to be sensitive to the pH variation, leading to remarkable\nspectral changes and in addition the TMBox dimer is unstable at neutral pH\n(https://doi.org/10.1002/adfm.202315587).\n- As a general comment the methodologies reported are not properly detailed and\nshould be implemented.\n- Some of the tests are performed in buffers, including in PBS. The authors should show\nthat the nanozymes are colloidally stable in these conditions.\n- The authors state: “It was observed that Ruzyme significantly increased the O2\nconcentration in the system and produced more bubbles compared to the other PPy-co-\nPTh stabilized nanozymes” It is not clear how the oxygen production was measured. Did\nyou use an oxygen sensor or this is only based on a naked-eye qualitative assessment?\n- The authors should report a full characterization (including TEM and size distribution) of\nPVP@Ru and RuO @BSA and all the other nanozymes involved in this study. In the NCT-\nNaBH reduction. Considering the strong reducing agent involved how the protein\n4\nstructure will appear after synthesis? Are you sure the NPs structure will resemble to the\none reported in Figure S10A, Ruthenium clusters embedded in unfolded BSA? What kind\nof interactions keep them together? I am concerned that the chosen controls are not\nappropriate as we know very little about them.\n- If the metal ions are supposed to be the active sites during the iron ions scavenging,\nis the nanozyme itself, enriched of iron ions, able to perform a Fenton-like reaction? Is\nthe Iron replacing Ruthenium ions during the scavenging process?\n- Structural, compositional, and colloidal stability of the presented nanozymes before\nand after exposure to the relevant biological media (cell culture media and mice plasma)\nshould be assessed.\n- In this study the potential role of biomolecular corona on the colloidal stability,\nenzymatic-like activity, targeting and internalization/cellular distribution\n(https://doi.org/10.1038/s41565-021-00860-0,\nhttps://doi.org/10.1021/acsnano.0c00629,\nhttps://doi.org/10.1021/acs.nanolett.3c01479) should be taken into account. The\namount of proteins present in cell culture medium supplemented with 10% FBS is\nsufficient to cover the Ruzyme surface at the concentration used in in vitro experiments?\nIdeally, a large excess of protein per NZ’s surface area available should be guaranteed to\nmimic the in vivo scenario.\n- The author should perform ICP-MS to confirm the uptake of the different Ruzymes and\ncontrols (assessing the metal content per cell) and negative staining TEM to confirm the\nmitochondria targeting/lysosomal accumulation (and demonstrating no metal ion\nleaching).\n- As a general comment the confocal representative images are very dim and the\nfluorescent signal is difficult to see on all channels.\n- The authors state that the nanozymes are endocytosed. However, the appearence of\nICT-Ruzymes distribution in cells is very different from the one of most of\nnanomaterials accumulating in organelles. Why only the uptake of ICT-Ruzyme is\nshown and not compared with Ruzymes?\n- In general, a comparison between Ruzyme and ICT-Ruzyme is missing: how can the\nauthors state that the overlap with mitochondria is due to the ICT without this\ncomparison? In Figures 6F and 6G the results for Ruzyme and ICT-Ruzymes in terms\nof ROS and mtROS scavenging are comparable.\n- Do the Ruzymes co-localize with lysosomes after 24h incubation as commonly\nhappens to nanoparticles?\n- The efficacy of CICT-Ruzyme in targeting cardiomyocytes in virtue of the CTpeptide\nhas never been demonstrated in the manuscript, making these data useless, as the\neffect of CICT-Ruzyme is very similar to ICT-Ruzyme and Ruzyme (Figure 8 C,D,E,F). A\ntrend is visible, but is it significant? Eventually, a biodistribution study (via ICP-MS)\nshould confirm the effectiveness of the specific in vivo targeting.\n- Page 19, line 463: “Consistent results were obtained when using O2- ….” Figure S24B\ndoes not show consistent results, and the ROS recovery is not clear from the flow\ncytometry spectra. In addition, the data are not presented as an average of n=3, as\nstated in the caption.\n- Not clear how you exploited here the electrical conductivity of the Ruzyme."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for addressing my comments in your rebuttal and for the additional data provided. I appreciate the effort to clarify the results. However, several important issues remain, particularly regarding the western blotting and in vivo IHC datasets. To ensure transparency and reproducibility, I ask that the following points be addressed:\n1. Western blot data integrity (ACSL4, GPX4, loading controls)\nThe ACSL4 blots show multiple bands, raising concerns about antibody specificity. A clear, single band at ~80–88 kDa is expected. Please provide uncropped, full-length blots with visible molecular weight markers for all WB data, including loading controls, along with the original raw files. Validation of ACSL4 is required (e.g., knockdown/KO, peptide competition, or a second antibody). I recommend repeating the experiment with a more specific antibody; several validated options are commercially available.\n2. Replication and quantification of western blots\nProvide quantitative densitometry from ≥3 independent biological replicates, normalized to loading control, with mean ± SEM and appropriate statistical testing. Clarify whether ACSL4/GPX4 panels and loading controls derive from the same membranes (stripped/re-probed) or from separate gels.\n3. In vivo IHC (heart sections)\nOnly one representative image per group is provided in the rebuttal, which is insufficient. Please provide quantified IHC data from >6 animals per group, analyzed across multiple sections per heart, using blinded or automated methods.Provide mean ± SEM with statistical comparisons.\n4. Supplementary data and transparency\nAll uncropped western blot images (with markers, ladders, and all replicates) should be uploaded as supplementary source data. Include a “raw blot index” mapping each cropped panel to its corresponding full-length blot.\nProvide raw IHC quantification spreadsheets to support the representative figure conclusion.", "reviewer_2": "The authors have satisfactorily addressed my previous comments. I support the publication of this manuscript after minor revision:\n1. In the Method section, the phrase \"every MD step\" is ambiguous and potentially misleading. The heating procedure from 0 K to 3000 K within 3 ps using velocity scaling typically refers to a continuous temperature ramp during the molecular dynamics simulation, rather than an action performed at each integration step. For clarity, this sentence could be revised as:\n\"The optimized RuO crystal was heated from 0 K to 3000 K over 3 ps using velocity scaling and equilibrated at 3000 K for\n2\n0.5 ps via the Nose-Hoover thermostat with a time step of 1 fs to obtain the amorphous RuO structure.\"\n2\n2. In the Method section, although many published articles do not explicitly cite resources such as the Materials Project database or the Nosé-Hoover thermostat, I believe it is good scholarly practice to include the appropriate references as a sign of respect to the original sources and to enhance transparency and reproducibility. In this context, the Materials Project can be cited as https://doi.org/10.1063/1.4812323, while the Nosé-Hoover thermostat should be attributed to both\nhttps://doi.org/10.1080/00268970110089108 and https://doi.org/10.1103/PhysRevA.31.1695.", "reviewer_3": "The authors have undertaken significant additional work, leading to a clear improvement in the scientific level of the manuscript, which is now suitable for publication.\nThe manuscript show the therapeutic potential of polymer-coated ruthenium based nanozymes engineered to perform protective RONS scavenging to counteract septic cardiomyopathy, showing promising results in vitro and in vivo. Some methodological little detail might still be improved, understandably given the complexity and the size of the work done, nevertheless, the general scientific outcome appear to be solid and impactful, and especially relevant for the Nanomedicine community.", "reviewer_4": "The authors have addressed the raised issues appropriately. I would like to recommend this work to be published."}, "author_response": "Response: Thank you for acknowledging our supplementary data and responses, and for highlighting the remaining concerns regarding the Western blot and in vivo IHC datasets. To address these important issues thoroughly, we have now conducted repeated experiments and additional analyses as detailed below.\n1. Western blot data integrity (ACSL4, GPX4, loading controls)\nThe ACSL4 blots show multiple bands, raising concerns about antibody specificity. A clear, single band at ~80–88 kDa is expected. Please provide uncropped, full-length blots with visible molecular weight markers for all WB data, including loading controls, along with the original raw files. Validation of ACSL4 is required (e.g., knockdown/KO, peptide competition, or a second antibody). I recommend repeating the experiment with a more specific antibody; several validated options are commercially available.\nResponse: We thank the reviewer for this critical comment. In accordance with the suggestion, we have now repeated the experiment for the key results involving ACSL4. Furthermore, as requested, we have provided uncropped, full-length blots for all Western blot data (including ACSL4, GPX4, and GAPDH) in the Supplementary Information, with all molecular weight markers clearly visible. The raw data images have also been updated in the Source Data. We believe these additional data adequately address the concern and hope the reviewer finds the updated figures and source data satisfactory.\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were determined by western blot analysis. (a) Representative immunoblots of ferroptosis-related proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins in H9C2 cells (n = 3).\nFigure S57. Raw data of Western blot image in Figure 9K. (A) Uncropped, full-length membranes for all Western blot data. (B) Full membrane index for the Western blot images of ACSL4, GPX4, and GAPDH in Figure 9K (a).\n2. Replication and quantification of western blots\nProvide quantitative densitometry from ≥3 independent biological replicates, normalized to loading control, with mean ± SEM and appropriate statistical testing. Clarify whether ACSL4/GPX4 panels and loading controls derive from the same membranes (stripped/re-probed) or from separate gels.\nResponse: We thank the reviewer for this suggestion to enhance the quantitative rigor of our data. As recommended, we have repeated the Western blot experiments to validate the inhibitory effect of the nanozyme on ferroptosis with three independent biological replicates (n=3). The quantitative densitometry analysis for ACSL4 and GPX4, normalized to loading control (GAPDH), is now presented in the revised figures as mean ± SEM, and appropriate statistical analyses have been performed to assess the significance between groups. Furthermore, we confirm that the blotting images for ACSL4, GPX4, and GAPDH shown in the figure panels were indeed derived from the same PVDF membrane (stripped/re-probed). We appreciate the reviewer's insightful comment, which has helped us improve the data presentation and statistical robustness of our study.\nFigure 9K. The protein expression levels of ACSL4 and GPX4 in H9C2 cells were determined by western blot analysis. (a) Representative immunoblots of ferroptosis-related proteins in H9C2 cells. (b) Quantitative analysis of ferroptosis-related proteins in H9C2 cells (n = 3).\nFigure S57. Raw data of Western blot image in Figure 9K. (A) Uncropped, full-length membranes for all Western blot data. (B) Full membrane index for the Western blot images of ACSL4, GPX4, and GAPDH in Figure 9K (a).\n3. In vivo IHC (heart sections)\nOnly one representative image per group is provided in the rebuttal, which is insufficient. Please provide quantified IHC data from >6 animals per group, analyzed across multiple sections per heart, using blinded or automated methods. Provide mean ± SEM with statistical comparisons.\nResponse: We thank the reviewer for the constructive feedback. In response to the comment regarding the need for quantified IHC data, we have conducted comprehensive quantitative analyses. The study now includes IHC data from more than 6 animals per group. For each animal, quantification was performed on four non-consecutive cardiac sections, analyzed in a blinded manner using automated image analysis software. The results, expressed as mean ± SEM, have been incorporated into the revised manuscript (Supplementary Figure S58), along with statistical comparisons between groups using one-way ANOVA. We believe these additional data address the reviewer's concern and strengthen the manuscript.\nFigure S58. Immunohistochemical analysis of 4‑HNE expression in cardiac tissue samples. (A) Representative immunohistochemical staining images of 4-HNE in multiple sections per heart. Scale bar = 1000 µm. (B) Relative quantitative analysis of 4-HNE staining across experimental groups in multiple cardiac sections. The data are presented as the means ± SEM, and p values were provided in the figures using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.\n4. Supplementary data and transparency\nAll uncropped western blot images (with markers, ladders, and all replicates) should be uploaded as supplementary source data. Include a “raw blot index” mapping each cropped panel to its corresponding full-length blot.\nResponse: We thank the reviewer for this recommendation to ensure full transparency of our Western blot data. In accordance with the suggestion, we have now uploaded all uncropped Western blot images, which include molecular weight markers, ladders, and all experimental replicates, as part of the Supplementary Source Data. Additionally, a “raw blot index” that clearly maps each cropped panel featured in the main figures to its corresponding full-length, uncropped blot has been provided in both the Source Data file and the Supplementary Information. We believe these materials will provide a complete and clear overview of the original data.\nFigure S57. (B) Full membrane index for the Western blot images of ACSL4, GPX4, and GAPDH in Figure 9K (a).\nProvide raw IHC quantification spreadsheets to support the representative figure conclusion.\nResponse: We sincerely thank the reviewer for the valuable comments on this manuscript. In response to the suggestions, we have updated the manuscript, supplementary materials, and source data accordingly. We hope these revisions have enhanced the quality and completeness of the manuscript. Once again, we greatly appreciate the reviewer’s insightful feedback.\nReviewer #2 (Remarks to the Author):\nThe authors have satisfactorily addressed my previous comments. I support the publication of this manuscript after minor revision:\nResponse: Thank you very much for your positive assessment and for supporting the publication of our manuscript. We have carefully checked the text and figures to incorporate all remaining minor adjustments, as detailed in the point-by-point response. All changes are highlighted in the revised manuscript and supplementary materials.\n1. In the Method section, the phrase “every MD step” is ambiguous and potentially misleading. The heating procedure from 0 K to 3000 K within 3 ps using velocity scaling typically refers to a continuous temperature ramp during the molecular dynamics simulation, rather than an action performed at each integration step. For clarity, this sentence could be revised as: “The optimized RuO crystal was heated from 0 K to 3000 K over 3 ps using velocity scaling and equilibrated at 3000 K for 0.5 ps via the Nose-Hoover thermostat with a time step of 1 fs to obtain the amorphous RuO structure.”\nResponse: We sincerely thank the reviewer for their thorough evaluation of our manuscript and their valuable comments regarding the potential ambiguity of the phrase “every MD step” in the Methods section. The reviewer’s observation is highly pertinent, and we fully agree that the original wording could lead to misunderstanding about the specific implementation of the heating process. Following the reviewer’s suggestion, we have revised the relevant sentence to: “The optimized RuO crystal was heated from 0 K to 3000 K over 3 ps using velocity scaling method and equilibrated at 3000 K for 0.5 ps via the Nose-Hoover thermostat with a time step of 1 fs to obtain the amorphous RuO structure.” This revision clarifies that the heating represents a continuous process carried out over 3 ps, rather than an operation performed at each integration step, thereby making the methodological description clearer and more accurate. Once again, we greatly appreciate the reviewer's input, which has been instrumental in improving the quality of our manuscript.\n2. In the Method section, although many published articles do not explicitly cite resources such as the Materials Project database or the Nosé-Hoover thermostat, I believe it is good scholarly practice to include the appropriate references as a sign of respect to the original sources and to enhance transparency and reproducibility. In this context, the Materials Project can be cited as https://doi.org/10.1063/1.4812323, while the Nosé-Hoover thermostat should be attributed to both https://doi.org/10.1080/00268970110089108 and https://doi.org/10.1103/PhysRevA.31.1695.\nResponse: We fully agree that explicitly citing key resources such as the Materials Project database and the Nosé-Hoover thermostat in the Methods section represents good scholarly practice, as it both acknowledges the original contributors and enhances the transparency and reproducibility of our study. Following the reviewer’s recommendation, we have incorporated the suggested references at the appropriate locations in the manuscript. These revisions are reflected in the latest version of the paper. We believe these additions have made our methodological description completer and more rigorous. Once again, we appreciate the reviewer’s insightful comments, which have been essential in improving the quality of our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have undertaken significant additional work, leading to a clear improvement in the scientific level of the manuscript, which is now suitable for publication.\nThe manuscript show the therapeutic potential of polymer-coated ruthenium based nanozymes engineered to perform protective RONS scavenging to counteract septic cardiomyopathy, showing promising results in vitro and in vivo. Some methodological little detail might still be improved, understandably given the complexity and the size of the work done, nevertheless, the general scientific outcome appear to be solid and impactful, and especially relevant for the Nanomedicine community.\nResponse: Thank you very much for your positive assessment and recognition. We are also pleased to observe that, following the additional experiments and textual revisions in this round, the scientific rigor and completeness of the manuscript have been further enhanced. In response to your comment that “some methodological little detail might still be improved,” we have carefully reviewed each point and provided supplementary information and clarifications accordingly.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed the raised issues appropriately. I would like to recommend this work to be published.\nResponse: Thank you very much for your positive feedback and for recommending our work for publication. We truly appreciate your constructive comments throughout the review process, which have helped us improve the quality of our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have satisfied my comments."}, "author_response": ""}]

Decision letter