Abstract

   Pyroptosis inducer, a powerful anti‐tumor agent that causes obvious
   programmed cell death and immune stimulation, has been challenged to
   trigger specific pyroptotic tumor cell death while keeping pyroptosis
   silence in normal cells. Here, an intelligent inducer is reported that
   acts as a reactive oxygen species (ROS) scavenger in normal cells to
   keep pyroptosis silence, while serving as ROS generator to induce
   obvious pyroptotic tumor cells death dependent on high hydrogen
   peroxide levels and near‐infrared laser irradiation. This switchable
   activity ensures this inducer to precisely kill the tumor cells with
   augmented immunogenicity while causing minimal damage to normal cells.
   Moreover, the catalase‐like activity endows this inducer to overcome
   limitation of tumor hypoxia on ROS generation and show significant
   pyroptosis activation, further initiating the immune response to
   inhibit the tumor metastases in vivo. This study provides valuable
   insights into design new pyroptosis inducer with controllable
   pyroptosis activity to specifically induce programmed tumor cell
   pyroptosis for precise and augmented tumor therapy with minimal side
   effects.

   Keywords: H[2]O[2] responsive, precise tumor therapy, ROS
   scavenger/generator, specific pyroptosis
     __________________________________________________________________

   The hydrogen peroxide responsive specific pyroptosis inducer triggers
   specific pyroptotic tumor cell death and maintains pyroptosis silence
   through differentially controlling reactive oxygen species levels in
   tumor and normal cells, precisely inhibiting the growth of primary and
   metastatic tumors with negligible side effect.

   graphic file with name ADVS-12-2407713-g008.jpg

1. Introduction

   Tumor immunotherapy, which enhances the natural immune system to
   defense the tumor cells, has shown a prospective future in tumor
   inhibition.^[ [44]^1 ^] However, the therapeutic efficiency of
   immunotherapy in solid tumors has been severely challenged by the low
   immunogenicity of tumor microenvironment (TME).^[ [45]^2 ^] This
   limitation emphasizes the urgent need to ameliorate TME and improve the
   immunogenicity for effective tumor therapy. Pyroptosis, as a mode of
   programmed cell death, is distinguished from apoptosis and necrosis by
   the distinctive features of cell swelling, membrane rupture,
   bubble‐like protrusions appear, pro‐inflammatory cytokines, and
   cellular contents release.^[ [46]^3 ^] It is an effective mode to
   enhance tumor immunogenicity and initiate immune response. For the
   typical pathway of pyroptosis, it is primarily induced by the
   stimulation of bacteria, viruses, toxins, or chemotherapeutic drugs
   from external or intracellular, and is associated with the recruitment
   and activation of caspase‐1. Specifically, the gasdermin D (GSDMD) is
   cleaved by activated caspase‐1 to produce GSDMD‐N domain to generate
   pore in the plasma membrane with size of about 1 µm, allowing the
   release of inflammatory cytokines like IL‐1β and IL‐18.^[ [47]^4 ^]
   These phenomena result in the strong inflammation and massive exposure
   of antigen epitopes to augment immunogenicity. Hence, pyroptosis
   inducers provide innovative study guideline to activate pyroptotic
   tumor cell death, synergistically increasing tumor immunogenicity and
   enhancing tumor therapy efficiency.^[ [48]^5 ^]

   Growing evidence has revealed that reactive oxygen species (ROS), as a
   pyroptosis inducer, initiates pyroptosis for effective tumor therapy
   via the csapase‐3/GSDME pathway.^[ [49]^6 ^] ROS accumulation induces
   Bax recruitment in the mitochondrial membrane and release cytochrome c
   (cyt c).^[ [50]^3 , [51]^7 ^] The released cyt c activates caspase‐3 to
   cleave GSDME and leads to the release of massive inflammatory cytokines
   and damage‐associated molecular patterns (DAMPs), triggering an immune
   response and suppressing tumor growth.^[ [52]^8 ^] To minimize the
   damage in normal tissues caused by the pyroptosis, the activatable
   photosensitizers with the remarkable capacity to specifically increase
   the ROS levels in tumor and induce pyroptotic tumor cell death have
   been extensively studied.^[ [53]^9 ^] These intelligent pyroptosis
   inducers show assembled state in normal tissue, resulting in the “off”
   state on triggering pyroptosis.^[ [54]^10 ^]Alternatively, these
   inducers respond to TME, e.g., acidic environment or high glutathione
   (GSH) level, and disassemble to achieve “on” state on triggering
   pyroptosis.^[ [55]^11 ^] These novel studies provide insights into the
   design of specific pyroptosis inducers with minimal side effects.
   Unfortunately, pragmatic application of these activatable strategies is
   critically limited by the ineluctable inflammation response of normal
   tissue, which is characterized by oxidative stress evoked by elevated
   ROS level caused by the released inflammatory factor from dead tumor
   cells.^[ [56]^12 ^] Besides, TME with hypoxia nature, which imparts
   resistance to ROS generation, resulting the inefficient activation of
   pyroptosis and tumor therapeutic efficacy with the low
   immunogenicity.^[ [57]^13 ^] Thus, it is desirable to develop an
   intelligent and highly efficient pyroptosis inducer that acts as ROS
   scavenger in normal tissue to keep pyroptosis silence for normal cells
   protection, while serving as an efficient ROS generator with TME
   regulation capacity to trigger significant pyroptosis and
   synergistically increase tumor immunogenicity for robust tumor therapy.

   Herein, we develop a TME responsive specific iron ruthenium pyroptosis
   inducer (SIRPI) that acts as a highly specific ROS generator to
   effectively initiate the tumor cell pyroptosis while serving as a ROS
   scavenger to maintain the pyroptosis related protein in low levels.
   Specifically, the SIRPI exhibits reducibility under physiological
   conditions to effectively scavenge the excess ROS in normal tissues,
   resulting in negligible pyroptosis and toxicity. Whereas, the SIRPI
   switches from a ROS scavenger to a ROS generator in tumor site owning
   to the H[2]O[2] responsive oxidation of Ru and Fe elements. This
   transformation enables SIRPI to generate amounts of hydroxyl radical
   (•OH) through oxidated Fe and Ru ions‐based Fenton reactions and
   singlet oxygen (^1O[2]) through the free electrons transferring to
   oxygen under near‐infrared (NIR) laser simulation, ensuring SIRPI
   induces significant pyroptosis in tumor cells. Additionally, SIRPI
   relieves the hypoxia of tumor based on its excellent catalase‐like
   (CAT‐like) enzyme activity, enabling efficient ROS generation to
   trigger specific pyroptotic tumor cell death via the caspase‐3/GSDME
   pathway in tumor tissue. The specific tumor pyroptosis leads to the
   release of inflammatory cytokines and DAMPs into the extracellular,
   exposing antigenic recognition sites, promoting dendritic cells (DCs)
   maturation, and amplifying systemic immune responses to inhibit the
   primary and metastasis lesions. Moreover, the SIRPI with T [2] imaging
   performance will facilitate real‐time monitoring of tumor progression
   and therapeutic efficiency (Scheme [58]1 ). This specific pyroptosis
   mediated by SIRPI, which works as vibrant/invalid pyroptosis inducer in
   tumor/normal tissue, offers a new possibility for the efficient and
   safety tumor therapy.

Scheme 1.

   Scheme 1
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   Schematic diagram of SIRPI act as ROS scavenger/generator to induce
   specific pyroptosis for enhancing immune response. The mechanism of
   SIRPI, which is responds with H[2]O[2] to act as ROS
   scavenger/generator, to augment the immune response and accurately
   suppress the tumors by inducing specific pyroptotic tumor cell death
   via the caspase‐3/GSDME pathway.

2. Results and Discussion

2.1. Synthesis and Characterization of Specific Iron Ruthenium Pyroptosis
Inducer (SIRPI)

   We synthesized the specific iron ruthenium pyroptosis inducer (SIRPI)
   by thermo‐decomposition of the mixture containing ruthenium
   acetylacetonate and iron acetylacetonate in 1‐Octadecene. Transmission
   electron microscopy (TEM) images indicate that the as‐prepared SIRPI
   exhibit uniform morphology with the diameter of approximately 2.4 ± 0.3
   nm (Figure [60]S1, Supporting Information). The uniformly lattice
   fringes across the entire nanoparticles are clearly observed in
   high‐resolution TEM (HRTEM) image, affirming the excellent
   crystallinity of SIRPI (Figure [61]1A). To further verify the
   composition of SIRPI, the energy dispersive X‐ray (EDX) elemental
   mapping was performed. It appears that the iron (Fe) and ruthenium (Ru)
   signals are homogeneously disperse in the whole nanoparticles,
   suggesting the simultaneously presence of Fe and Ru element in a SIRPI
   particle (Figure [62]1B). Additionally, the X‐ray diffraction (XRD)
   analysis exhibits the characteristic diffraction peaks of SIRPI match
   well with iron ruthenium alloy (JCPDS No.03‐065‐6545), which
   coordinately revealing the basic crystal structure of as‐prepared SIRPI
   (Figure [63]1C). The magnetic property of SIRPI was investigated using
   superconducting quantum interference device (SQUID) magnetometer at
   room temperature (300 K). The field‐dependent magnetization (M‐H)
   curves show that SIRPI exhibits superparamagnetic behavior without any
   hysteresis at room temperature, which is expected to be used as a T [2]
   contrast agent for magnetic resonance imaging (Figure [64]1D).
   Subsequently, we transferred SIRPI into aqueous by hydrophilic
   1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐ [amino (polyethylene
   glycol)‐2000] (DSPE‐PEG2000 amine). The hydrodynamic size and zeta
   potential of water‐soluble SIRPI were measured by dynamic light
   scattering (DLS). It displays that the size and potential of SIRPI are
   about 10.8 nm and + 3.4 mV, promising the SIRPI with the well potential
   to remain stable in the circulation (Figure [65]S2, Supporting
   Information).

Figure 1.

   Figure 1
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   Characterization and reducibility of SIRPI. A) TEM and High‐resolution
   TEM images of SIRPI, the scale bar is 20 nm and 5 nm, respectively. B)
   HAADF‐STEM image and elemental mappings of Fe and Ru elements in SIRPI,
   the scale bar is 2 nm. C) XRD patterns and D) smooth M–H curves of
   SIRPI, indicating the ferromagnetic properties of SIRPI have been
   successfully synthesized. XPS high‐resolution spectra of E) Ru 3p and
   F) Fe 2p in SIRPI. G) Elimination of O[2] ^·− with different
   concentrations of SIRPI (4.6–36.7 µg mL^−1). H) ESR spectra of ^1O[2]
   scavenging and I) UV‐vis spectra of •OH with different concentrations
   of SIRPI (0, 25, 50 µg mL^−1).

2.2. Reducibility‐Mediated ROS Scavenging Activity of SIRPI in Physiological
Condition

   To assess the oxidation‐reduction property of SIRPI, we performed the
   X‐ray photoelectron spectroscopy (XPS) to analyze Fe and Ru valence
   states in mimic physiological condition. The XPS peak‐fitting analyses
   reveal that the ratios of Ru^0 (at 461.6 eV) to Ru^3+/4+ (at 464.5 eV)
   and Fe^0 (at 709.5 eV) to Fe^2+ (at 711.1 eV) is about 3.3:1 and 0.6:1
   (Figure [67]1E, F). The large proportion of Ru^0 imparts SIRPI with the
   reducibility, which is expected to act as ROS scavenger in the
   physiological condition^34. With this hypothesis, we assessed the
   potential of SIRPI to eliminate superoxide anion (O[2] ^· ^−), singlet
   oxygen (^1O[2]), and hydroxyl radical (·OH) in physiological condition,
   respectively. We first measured the scavenging activity of O[2] ^· ^−
   through adding different concentration of SIRPI (4.6‐36.7 µg mL^−1)
   into a mixture of L‐methionine and riboflavin after 15 min ultraviolet
   light irradiation and used nitrotetrazolium blue chloride (NBT) as
   probe. It appears that SIRPI shows high O[2] ^· ^– scavenging activity
   in concentration‐dependent manner. The O[2] ^· ^– elimination ratio is
   as high as ≈92.7% when SIRPI concentration reaches to 36.7 µg mL^−1
   (Figure [68]1G). Subsequently, electron spin resonance (ESR) spectra
   were applied to detect ^1O[2] levels with or without SIRPI using
   2,2,6,6‐tetramethylpiperidin‐1‐oxygen (TEMP) as spin trap.
   Interestingly, the signal intensity of TEMP/^1O[2] at the
   characteristic peaks are effectively reduced by SIRPI in concentration
   dependence manner. It should note that complete scavenging is observed
   when the concentration of SIRPI is as low as 50 µg mL^−1, implying the
   effective elimination of ^1O[2] by SIRPI at physiological environment
   (Figure [69]1H). The •OH scavenging activity was investigated by adding
   SIRPI into the solution containing •OH using methylene blue (MB) as
   specific probe. Remarkably, we observed gradual recovery of the
   characteristic absorbance peak of MB with the increase of SIRPI
   concentration, indicating the effective scavenging of •OH by SIRPI
   (Figure [70]1I). These results prove that SIRPI with the reducibility
   exhibits the superior ROS elimination performance under physiological
   conditions, which is expected to minimize the ROS induced pyroptosis in
   normal tissues.

2.3. ^1O[2] Generation Activity of SIRPI under NIR Laser Irradiation

   Ru‐based complexes have been widely used as photosensitizers to
   generate ^1O[2] and work as potential photodynamic therapy agent.^[
   [71]^14 ^] To investigate the ^1O[2] generation activity of SIRPI, we
   exposed SIRPI to an 808 nm NIR laser for 10 min and monitored ^1O[2]
   levels using 1,3‐diphenylisobenzofuran (DPBF) as probe. We observed a
   9.2% decrease in the DPBF characteristic absorbance peak at 412 nm
   after NIR laser irradiation (Figure [72]2A). Consistently, the signal
   intensity of TEMP/^1O[2] in ESR spectra increased with the adding of
   SIRPI (Figure [73]2B). These results clearly indicate that SIRPI
   successfully generates ^1O[2] under NIR laser irradiation. However, the
   signal changes in both DPBF and TEMP/^1O[2] are limited compared to the
   control group, indicating relatively low ^1O[2] generation efficiency
   of SIRPI under NIR laser irradiation in physiological conditions. These
   phenomena could be ascribed to the partially elimination of generated
   ^1O[2] based on the ROS scavenging activity of SIRPI in physiological
   condition, which is beneficial to diminish ^1O[2] generation in normal
   tissue in further therapy. To understand the detailed physical and
   chemical mechanisms of ^1O[2] generation by SIRPI under NIR laser
   irradiation, we performed density functional theory (DFT) analyses.^[
   [74]^15 ^] The surface energies of low index facets of Fe─Ru
   nanostructure were first calculated. It appears that (110) facet of
   iron ruthenium nanostructure shows the lowest surface energy,
   indicating that (110) facet is more likely to form stable structures
   and preferentially exposed on the surface of SIRPI. We noticed that the
   Ru‐d orbital is upshifted after O[2] adsorption compared to the clean
   slab in the partial density of state (PDOS) spectra, indicating the
   interaction between Ru d and O[2] and suggesting that the ^1O[2] would
   form based on the activated electron transfer to O[2] under extra
   energy (Figure [75]2C). According to the previous research, the shared
   pairs of electrons of the broken Fe−Ru bond may dominate to the O[2]
   activation. We employed the PDOS and crystal orbital Hamilton
   population (COHP) to analyze the Fe−Ru bond strength as function of
   O[2]activation. We noticed obvious hybridization that occurred between
   the Fe─d and Ru‐d orbitals in PDOS (Figure [76]2E). These results
   suggest that the main component in the Fe─Ru bond are Fe─d and Ru‐d.
   Besides, electron deposition between Fe─Ru bond could be observed,
   implying the adequate free electron supplying during the breaking of
   Fe─Ru bond (Figure [77]2D). For COHP, the Fe─Ru bond interactions are
   mainly between −9.0 eV and 4.0 eV with the antibonding state above −1.0
   eV. These results are close to the Fermi energy level, which finally
   causes the activation of O atoms. The integral COHP (ICOHP) was
   employed to estimate the bond strength. In theory, the stronger bond
   is, the more negative ICOHP would be observed. The Fe─Ru bond strength
   is calculated to be about –0.75. The low bond strength between Fe and
   Ru ensures that Fe─Ru bond could be easily broken by the extra energy
   and provide sufficient free electrons to the adsorbed O[2] and generate
   ^1O[2] (Figure [78]2F). Additionally, we noticed that the ESR spectra
   of SIRPI exhibited a pair of inverted pinnacles (Figure [79]S3,
   Supporting Information). This result implies the existence of
   high‐density defects in SIRPI, which endow SIRPI with the efficient
   exposure of active sites and optimized electronic structures to enhance
   the ^1O[2] yield.^[ [80]^16 ^] Overall, the prepared SIRPI effectively
   transfers free electrons from the broken of Fe─Ru bond to O[2] and
   generate ^1O[2] under NIR laser irradiation (Figure [81]2G–I). On the
   other hand, the ROS scavenging activity of SIRPI in physiological
   condition make SIRPI eliminate partially generated ^1O[2]. This
   double‐edged performance results in the relatively low ^1O[2]
   generation in physiological environment and limited ^1O[2] generation
   in normal tissue, diminishing the potential side effects during
   treatment.

Figure 2.

   Figure 2
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   ^1O[2] generation activity of SIRPI. A) UV‐vis spectra of DPBF and B)
   ESR spectra of TEMP)/ ^1O[2] to detect ^1O[2] by SIRPI under NIR laser
   irradiation. C) The pre‐ and post‐ O[2] adsorption PDOS for the Ru d
   orbital. D) Electron deposition between Fe─Ru bond. The public electron
   pair in the Fe─Ru bond is represented by the red circle, while the
   yellow color represents the charge increase and the cyan color
   represents the charge decrease. E) Fe─d and Ru‐d hybridization during
   O[2] adsorption. F) The Fe−Ru bond strength analysis using the COHP. G)
   The O[2] adsorption structure of SIRPI. H) Simulated structure for
   ^1O[2] generation. I) Schematic illustration of the ^1O[2] generation
   by SIRPI under 808 nm NIR laser stimulation.

2.4. ROS Generation of SIRPI in Mimic Tumor Microenvironment (TME)

   TME is characterized by mild acidity, high H[2]O[2] concentration, and
   massive enrichment of GSH, distinguishing it from the normal tissues.^[
   [83]^17 ^] To explore the specific responsiveness of SIRPI to the TME,
   we investigated the structural changes of SIRPI in mimic TME. The XPS
   analyses show a notable increase in the valence states of Ru element in
   SIRPI in the mimic TME with high H[2]O[2] level compared to the
   physiological environment. The main valence state forms of Ru changed
   from reductive Ru^0 to oxidative Ru^3+/4+ (Figure [84]3A). This
   H[2]O[2]responsive structural and valence change of SIRPI is promising
   to specifically generate ROS based the oxidation state in tumor.^[
   [85]^18 ^] Meanwhile, we noted that SIRPI exhibited strong
   catalase‐like (CAT‐like) activity to accelerate the decomposition of
   H[2]O[2] to generate O[2] within a very short time (Figure [86]3B).
   This efficient CAT‐like activity is highly promising to alleviate the
   hypoxic TME and provides sufficient O[2] to increase the ^1O[2]
   generation in tumor for augmenting the efficiency of tumor therapy.^[
   [87]^19 ^] With the above hypothesis we further investigated the ^1O[2]
   generation of SIRPI in the presence of H[2]O[2]. Interestingly, we
   observed significantly absorbance decrease of DPBF at 412 nm after
   introducing H[2]O[2] (Figure [88]3C; Figures [89]S4–[90]S6, Supporting
   Information). This phenomenon could be attributed to the synergy
   between the elevated valence of Ru and enhanced O[2] supplying based on
   CAT‐like activity[,] which result in the decrease of ROS elimination
   and provide sufficient O[2] as the free electron receptor to improve
   the ^1O[2] generation efficiency. Consistent with the UV–vis analyses,
   the signal intensity of TEMP/^1O[2] in ESR spectra increased from the
   group without H[2]O[2] to the group with H[2]O[2], proving the
   efficient H[2]O[2] responsive ^1O[2] generation of SIRPI
   (Figure [91]3D). Moreover, the ^1O[2] generation of SIRPI shows a
   significant time and concentration dependence in the presence of
   H[2]O[2] under NIR laser irradiation. SIRPI enables to generate
   abundant ^1O[2] in a short time (less than 10 min) with low
   concentration (7–11 µg mL^−1), confirming that SIRPI endorses an
   efficient ^1O[2] generation ability in the mimic TME with high H[2]O[2]
   level (Figure [92]3E).

Figure 3.

   Figure 3
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   Multiple ROS generation of SIRPI in mimic tumor environment (TME). A)
   XPS high‐resolution spectra of Ru 3p in SIRPI without and with H[2]O[2]
   incubation (Top: without H[2]O[2]; bottom: with H[2]O[2]). B) CAT‐like
   activity of SIRPI to accelerate the decomposition of H[2]O[2] to
   generate O[2]. C) The absorbance of DPBF at 665 nm and D) ESR spectra
   of TEMP/ ^1O[2] to analyze the ^1O[2] generation efficiency by SIRPI
   under different treatments. E)The absorbance of DPBF at 665 nm with
   different times of irradiation and different concentration of SIRPI to
   explore the ^1O[2] generation rate. F) The absorbance of MB at 665 nm
   and G) ESR spectra of DMPO/•OH to analyze the •OH generation efficiency
   by SIRPI under different treatments. H) The ability of SIRPI to
   generate •OH and ^1O[2] under normoxia and hypoxia conditions,
   respectively, which is used to assess the CAT‐like activity of SIRPI
   overcome the limitation for SIRPI on ROS generation. I) Schematic
   illustration of the switchable ROS generation and scavenging in
   response with H[2]O[2].

   Since Fe^2+ and Ru^3+ have been proved to undergo Fenton and
   Fenton‐like reactions to generate •OH in tumor environments, we
   employed methylene blue (MB), a •OH probe, to test the •OH generation
   by SIRPI. The UV‐vis spectra show a significant decrease in the
   absorbance of MB at 665 nm after incubation with SIRPI and H[2]O[2] for
   30 minutes, suggesting that SIRPI performs the excellent •OH generation
   by Fe^2+ and Ru^3+. Notably, the •OH generation rate is further
   elevated by NIR laser irradiation (Figure [94]3F; Figure [95]S7,
   Supporting Information). This phenomenon suggests that NIR laser
   irradiation would not merely as the stimulator to generate ^1O[2], but
   also accelerates the ROS production rate by increasing the temperature
   based on the mild photothermal conversion ability of SIRPI (Figure
   [96]S8, Supporting Information). Additionally, the ESR spectra show
   obvious DMPO/•OH signals in the group treated by SIRPI and H[2]O[2],
   especially with NIR laser irradiation, confirming the excellent
   performance of SIRPI to generate •OH synergistically (Figure [97]3G).
   These results strongly suggest that SIRPI is expected to be diverse and
   large amounts of ROS producer in mimic TME. Moreover, the ^1O[2] and
   •OH generation efficiency of SIRPI is not dramatically weakened by
   exposing to the hypoxic condition, which is derived from the
   cooperative effect of CAT‐like activity and indicate the potential of
   SIRPI to overcome hypoxia environment to achieve high tumor therapeutic
   efficacy (Figure [98]3H; Figures [99]S9 and [100]S10, Supporting
   Information). Therefore, SIRPI is prospective for stable, specific, and
   highly efficient ROS generation in TME (Figure [101]3I).

2.5. Differentially Control the ROS Levels and Specific Pyroptosis by SIRPI
In Vitro

   ROS accumulation is enables to induce pyroptosis via the
   caspase‐3/gasdermin E (GSDME) pathway, resulting in cell membrane
   swelling and the releases of damage‐associated molecular patterns
   (DAMPs) and inflammatory cytokines.^[ [102]^4 , [103]^10 ^] Given the
   specific ROS generation feature of SIRPI in the mimic TME, a systematic
   mechanism of specific pyroptosis in normal and tumor cells by SIRPI is
   proposed (Figure [104]4A). Initially, we studied the intracellular
   uptake behavior of SIRPI. To visualize the intracellular uptake, we
   prepared SIRPI labeled with Rhodamine B. As shown in Figure [105]S11
   (Supporting Information), a strong red fluorescent signal is observed
   after treatment in 4T1, MCF‐7, and Hs578bst cells treated by Rhodamine
   B‐labeled SIRPI, indicating that SIRPI is successfully internalized by
   the cells. As the first organelle after the SIRPI entry, it is crucial
   to understand whether SIRPI escapes from the lysosome.^[ [106]^20 ^] To
   verify the lysosomal escape ability of SIRPI, we performed confocal
   imaging using Rhodamine B‐labeled SIRPI and the lysosomal marker
   (Lyso‐Tracker). Fluorescence images show that the red fluorescent
   signal (Rhodamine‐labeled SIRPI) overlap heavily with the green
   fluorescent signal (Lyso‐Tracker) at 2 h of SIRPI incubation,
   suggesting the successful internalization of SIRPI by the lysosomes.
   Additionally, as the incubation time of SIRPI increased to 6 h, the
   co‐localization of the red fluorescence with the green fluorescence
   decreased, indicating the SIRPI successfully escapes from the
   lysosomal, which is expected to be effective in inhibiting tumor cells
   growth (Figure [107]S12, Supporting Information). Based on these
   findings, we assessed the cytotoxicity of SIRPI on tumor cells (4T1 and
   MCF‐7) and normal cells (Hs578bst) under different conditions by
   cell‐counting‐kit‐8 (CCK‐8) assay. The viability of tumor cells
   gradually decreased with the increase of SIRPI concentration in all
   groups, especially in the SIRPI + Laser + H[2]O[2] group
   (Figure [108]4B; Figure [109]S13, Supporting Information). Notably, the
   viability of 4T1 cells reduced to 40% with the treatment of H[2]O[2]
   and 200 µM SIRPI under NIR laser irradiation, indicating the potent
   anti‐tumor efficacy of SIRPI. Conversely, there is negligible
   inhibition observed in Hs578bst cells with the same treatment, even in
   the group treated with both H[2]O[2] and Laser (Figure [110]4C). These
   exciting results reveal the minimal side effect of SIRPI in normal
   cells and imply its high biocompatibility in vitro. Consistent with the
   CCK‐8 analyses, the fluorescence images staining by Calcein
   acetoxymethyl ester (Calcein‐AM) and propidium iodide (PI) display a
   strong red and weak green fluorescence in 4T1 cells treated by SIRPI
   and H[2]O[2] under irradiation. In contrast, the Hs578bst cells treated
   with the same strategy exhibit a strong green fluorescence
   (Figure [111]4D). These findings indicate that SIRPI displays
   negligible cytotoxicity to normal cell under the same treatment
   conditions, suggesting its promising potential safety in normal cells
   while showing potential act as an effective anti‐tumor agent in tumor
   cells.

Figure 4.

   Figure 4
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   The anti‐tumor effect and ROS generation‐guided specific pyroptosis by
   SIRPI in vitro. A) Schematic illustration of the multiple ROS
   generation guided specific pyroptosis in normal and tumor cells by
   SIRPI. The relative viability of B) 4T1 cells and C) Hs578bst cells
   with different treatments (n = 4). D) The Calcein‐AM (live cells) and
   PI (dead cells) staining after different treatments, the scale bar is
   100 µm. E) Fluorescence images of DCFH‐DA staining in 4T1 cells and
   Hs578bst cells treated differently to detect ROS generation, the scale
   bar is 100 µm. F) Fluorescence images of 4T1 cells and Hs578bst cells
   incubated with Rosup, and Rosup + SIRPI using DCFH‐DA as indicator, the
   scale bar is 100 µm. G) Fluorescence images to detect mitochondria
   membrane potential measurement by JC‐1 probe, the scale bar is 100 µm.
   H) The morphology observation of 4T1 cells and Hs578bst cells with
   different treatment, the scale bar is 100 µm. Western blot analysis of
   the expressions of cleaved caspase‐3 and GSDME‐N in I) 4T1 cells and J)
   Hs578bst cells under different treatment regimens. Quantitative
   analysis of cleaved caspase‐3 and GSDME‐N expression in K) 4T1 cells
   and L) Hs578bst cells based on the western blot results (n = 3). The
   release of LDH and IL‐1β from M) 4T1 cells and N) Hs578bst cells after
   different treatments (n = 5). ATP release from O) 4T1 cells and P)
   Hs578bst cells after different treatments (n = 3). HMGB1 release from
   Q) 4T1 cells and R) Hs578bst cells after different treatments (n = 3).
   From G1 to G6, they are Control, Laser, SIRPI, SIRPI + Laser, SIRPI +
   H[2]O[2], SIRPI + Laser + H[2]O[2], respectively. Data are present as
   mean ± SD, *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001,
   compared to the Control group.

   To verify the specific anti‐tumor effect of SIRPI resulting from the
   differentially control of ROS levels in tumor and normal cells, we
   monitored the ROS levels in both 4T1 and Hs578bst cells using the
   2,7‐dichlorofluorescein diacetate (DCFH‐DA) as probe. The fluorescence
   images of 4T1 cells treated by SIRPI show visible green fluorescence.
   The most significant green fluorescence is detected in the SIRPI +
   Laser + H[2]O[2] group. Conversely, there is no apparent green
   fluorescence could be seen in the Hs578bst cells with the same
   treatment (Figure [113]4E). These results indicate that the ROS
   generation of SIRPI is specifically dependent on TME. To demonstrate
   the simultaneous generation of •OH and ^1O[2] by SIRPI in 4T1 cells, we
   chose 2',7'‐dichloro‐3'‐hydroxy‐6'‐(4‐hydroxy‐3,5‐diiodophenoxy) spiro
   [2‐benzofuran‐3,9'‐xanthene]‐1‐one (HKOH‐1r) and singlet oxygen sensor
   green (SOSG) as probe to detect the intracellular •OH and ^1O[2]
   accordingly. Consistent with the DCFH‐DA staining, we observed a
   remarkable green fluorescence in tumor cells treated with SIRPI by both
   HKOH‐1r and SOSG staining, indicating the diverse ROS generated by
   SIRPI in tumor cells (Figure [114]S14, Supporting Information). To
   confirm the precisely control of ROS levels in tumor and normal cells
   of SIRPI, we studied the ROS elimination efficiency in 4T1 and Hs578bst
   cells using Rosup to increase the ROS levels. Notably, the fluorescent
   intensity generated by the Rosup stimulation remarkably decreased in
   the presence of SIRPI in Hs578bst cells, directly showing the ROS
   scavenging activity of SIRPI in normal cells. Conversely, we noticed a
   slight increase on fluorescent intensity in 4T1 cells treated with
   SIRPI, indicating that SIRPI enables a sensitive response to the TME
   with structural alterations, presenting efficient ROS‐generating
   activity (Figure [115]4F). Subsequently, we evaluated the H[2]O[2]
   content in both Hs578bst cells and 4T1 cells. As expected, the H[2]O[2]
   content in 4T1 cells is about 2.9 times higher than that in Hs578bst
   cells (Figure [116]S15, Supporting Information). This obvious different
   H[2]O[2] levels in normal and tumor cells enables SIRPI to work as a
   ROS scavenger in normal cells for ROS elimination to minimize the side
   effect, while acting as a ROS generator in tumor cells for varying ROS
   generation to efficiently damage tumor cells. Mitochondria, as the
   energy supplier in cells, is sensitive to ROS offense and presents a
   decreased membrane potential in response to ROS damage.^[ [117]^21 ^]
   We monitored the mitochondrial membrane potential changes in different
   groups using 5,5,6,6‐tetrachloro‐1,1,3,3‐tetraethyl‐imidacarbocyanine
   iodide (JC‐1) as probe. The fluorescence images show significantly
   decrease in red fluorescent aggregates while increase in green
   fluorescent monomer in 4T1 cells treated with SIRPI + Laser and SIRPI +
   Laser + H[2]O[2]. Whereas no significant changes are observed in
   Hs578bst cells with similar treatments (Figure [118]4G). These results
   suggest that SIRPI induces a large amount of ROS and effectively
   reduces the mitochondria membrane potential in tumor cells, while
   protecting the mitochondria from ROS damage in normal cells.

   Since the SIRPI differentially control the ROS levels in tumor or
   normal cells, we explore its capacity to induced specific pyroptosis in
   tumor cells. Notably, a typical membrane swelling is observed in 4T1
   cells, which tend to be more marked following stimulation with H[2]O[2]
   and Laser irradiation. In contrast, the Hs578bst cells maintained their
   typical morphology without exhibiting any pyroptosis features with the
   same treatment (Figure [119]4H). To systematically evaluate the
   specific pyroptosis, we assessed the expression levels of cleaved
   caspase‐3 and GSDME‐N in tumor cells (4T1 and MCF‐7) and normal cells
   (Hs578bst) cells with different treatment by western blot assay. It
   appears that cleaved caspase‐3 and GSDME‐N expression levels are
   significantly upregulated in the 4T1 and MCF‐7 cells treated by SIRPI,
   especially in the group of SIRPI and H[2]O[2] under NIR laser
   irradiation (Figure [120]4I, K; Figures [121]S16‐[122]S18, Supporting
   Information). Conversely, no apparent changes are detected in Hs578bst
   cells treated by SIRPI (Figure [123]4J, L; Figure [124]S19, Supporting
   Information). Since lactate dehydrogenase (LDH) and IL‐1β are typical
   markers of pyroptosis, we detected the release of LDH and IL‐1β from
   4T1 and Hs578bst cells after different treatments. Remarkably, 4T1
   cells treated by SIRPI shows significant increase of LDH and IL‐1β
   release. The release ratio in the group of SIRPI + Laser + H[2]O[2] is
   as high as 48.1% for LDH and 38.6 (pg mL^−1) for IL‐1β, which is
   significantly higher than control group and suggest the notable
   pyroptosis in 4T1 cells treated by SIRPI (Figure [125]4M). Whereas the
   released LDH and IL‐1β in Hs578bst cells with same treatments are below
   5% (Figure [126]4N). These results clearly demonstrate that SIRPI
   successfully induce pyroptosis in tumor cells, while maintain
   pyroptosis silence in normal cells. Additionally, pyroptosis enabled
   the release of DAMPs, including adenosine triphosphate (ATP) and high
   mobility group protein B1 (HMGB1), which exposing the massive antigen
   epitope and enhancing the immunogenicity.^[ [127]^22 ^] We examined the
   release of ATP and HMGB1 in 4T1 and Hs578bst cells with different
   treatments as well. SIRPI results in an obvious increase in the release
   of ATP and HMGB1 in 4T1 cells, especially the group SIRPI + Laser +
   H[2]O[2] (Figure [128]4O, Q). Nevertheless, the released of ATP and
   HMGB1 in Hs578bst cells treated by SIRPI remained within normal ranges
   (Figure [129]4P, R). There results directly indicate that SIRPI enables
   specifically enhance the immunogenicity of tumor cells and is expected
   to augment the anti‐tumor immune response. Besides, the cell death is
   typically triggered by multiple mechanisms acting synergistically.
   Therefore, we explored its potential to induce cell death through the
   apoptotic pathway in addition to its ability to selectively induce
   tumor cell pyroptosis. Flow cytometry analyses reveal that SIRPI
   significantly activate the apoptotic pathway in 4T1 cells (Figure
   [130]S20, Supporting Information). These findings suggest that SIRPI
   acts through multiple mechanisms to inhibit tumor cells, offering
   stronger support for its role in precise tumor therapy.

2.6. Pyroptosis Induced by SIRPI in Hypoxia Condition

   Hypoxia, a specificity of the tumor microenvironment, significantly
   limit the ROS induced tumor pyroptotic death. Based on the CAT‐like
   activity of SIRPI, we assessed the hypoxia ameliorate ability and
   pyroptotic tumor cell death induced by SIRPI under hypoxic conditions.
   The CCK‐8 assay indicates that the viability of 4T1 cells is remarkably
   inhibited by SIRPI, especially the group treated with both H[2]O[2] and
   Laser (Figure [131]5A). The fluorescence images of Calcein‐AM and PI
   staining images show that the red fluorescence almost completely
   replaced the green fluorescence in the group of SIRPI + Laser +
   H[2]O[2](Figure [132]5B). These results suggest that SIRPI with
   CAT‐like activity catalyzes H[2]O[2] into O[2], providing sufficient
   O[2] to alleviate hypoxic and generate ^1O[2] to synergistically
   inhibit tumor cells progression. To verify this hypothesis, we
   investigated the tumor hypoxia regulation of SIRPI. The SIRPI treatment
   cause a decreased red fluorescence in the O[2] level assessment using
   [Ru(dpp)[3]] Cl[2] as indicator, showing the increase of O[2]
   concentration in 4T1 cells (Figure [133]5C). Besides, the 4T1 cells
   treated by SIRPI show downregulated expression of HIF‐1α compared to
   the 4T1 cells treated by phosphate buffer solution (PBS) or NIR laser
   (Figure [134]5D, E; Figure [135]S21, Supporting Information). These
   results verify the effectiveness of SIRPI to increase the O[2] level
   and regulate the hypoxia of tumor. Subsequently, we tested the ROS
   generation in 4T1 cells by SIRPI. It appears that significant green
   fluorescence could be observed in the cells treated by SIRPI in
   DCFH‐DA, HKOH‐1r, and SOSG staining, revealing the effective •OH and
   ^1O[2] generation (Figure [136]5F). Meanwhile, the fluorescence images
   of JC‐1 staining exhibit a remarkable green fluorescence in the cells
   treated with SIRPI, demonstrating the generation of ROS effectively
   reduced the mitochondrial membrane potential of tumor cells in the
   hypoxia condition (Figure [137]5G). To investigate the pyroptosis
   induced by SIRPI under hypoxia, we assessed the expressions of cleaved
   caspase‐3 and GSDME‐N in 4T1 cells using western blot. The expression
   levels of cleaved caspase‐3 and GSDME‐N in cells treated by SIRPI are
   significantly higher than the cells treated by PBS and laser
   (Figure [138]5H, I; Figure [139]S22, Supporting Information). Besides,
   the cells treated by SIRPI show typical membrane swelling and increased
   LDH and IL‐1β release compared to the cells treated by PBS and NIR
   laser irradiation. These results clearly show the noticeable pyroptosis
   induced by SIRPI under hypoxia, particularly following stimulation with
   H[2]O[2] and laser (Figure [140]5J, K; Figure [141]S23, Supporting
   Information). Meanwhile, the cells treated with SIRPI in hypoxia
   condition exhibit substantial ATP and HMGB1 release. These findings
   imply the effective performance of SIRPI to ameliorated tumor hypoxic
   environment and induce tumor pyroptosis to release DAMPs, enhancing the
   immunogenicity of tumor cells and potentially inhibiting the
   development of metastases (Figure [142]5L, M).

Figure 5.

   Figure 5
   [143]Open in a new tab

   TME amelioration of SIRPI in vitro. A) Relative cell viability of 4T1
   cells with different treatments incubated with 1% O[2] (n = 4).
   Fluorescence images of B) Calcein‐AM/PI staining and C)
   [Ru(dpp)[3]]^2+Cl staining of 4T1 cells with different treatments under
   hypoxia condition, the scale bar is 100 µm. D) Western blot result to
   analysis the expression levels of HIF‐1α under different treatment for
   hypoxia amelioration detection. E) Quantitative analysis of HIF‐1α
   expression in 4T1 cells incubated with 1% O[2] based on the western
   blot results (n = 3). Fluorescence images of F) DCFH‐Da, HKOH‐1r, and
   SOSG staining (to detect the generations of ROS, •OH, and ^1O[2],
   respectively), G) JC‐1 staining in hypoxia incubation, the scale bar is
   100 µm. H) Expressions of cleaved caspase‐3 and GSDME‐N in 4T1 cells
   under hypoxia condition using western blot analysis. I) Quantitative
   analysis of cleaved caspase‐3 and GSDME‐N expression of 4T1 cells in
   hypoxia condition based on the western blot results (n = 3). The
   release of J) IL‐1β, K) LDH, L) ATP, and M) HMGB1 from 4T1 cells after
   different treatments in 1% O[2] condition. (n = 3). From G1 to G6, they
   are Control, Laser, SIRPI, SIRPI + Laser, SIRPI + H[2]O[2], SIRPI +
   Laser + H[2]O[2], respectively. Data are present as mean ± SD, *p <
   0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001, compared to the
   Control group.

2.7. RNA Sequence Analyses of SIRPI

   To investigate the mechanism of SIRPI triggered pyroptosis, we
   performed transcriptome sequencing analyses of 4T1 cells from Control
   and SIRPI + Laser + H[2]O[2] groups. In comparison with the Control
   group, there are 1030 (53.4%) upregulated genes and 899 (46.6%)
   downregulated genes in the SIRPI + Laser + H[2]O[2] group (Figure
   [144]6A, B; Figure [145]S24A, Supporting Information). Subsequent Kyoto
   Encyclopedia of Genes and Genomes (KEGG) analyses show that the
   differentially expressed genes (DEGs) associate with SIRPI are mainly
   enriched in inflammatory signaling pathways, such as the oxidative
   phosphorylation, p53 signaling pathway, NOD‐like receptor signaling
   pathway, MAPK signaling pathway, and IL‐17 signaling pathway
   (Figure [146]6C). These results suggest the close association among the
   tumor cells inhibitory effect of SIRPI and the inflammatory response
   induced by oxidative stress.^[ [147]^4a ^] Subsequently, we selected
   the top 20 DEGs among the above pathways to perform gene ontology (GO)
   enrichment analyses. As expected, the results confirm the association
   between the DEGs and the oxidative stress and inflammatory pathways,
   verifying the critical effect of SIRPI as ROS‐generator in tumor
   suppression (Figure [148]S24B, Supporting Information). For systematic
   investigation of 4T1 cells inhibition by SIRPI triggered pyroptosis and
   inflammatory response, we extracted a subset of the DEGs to perform
   circus heatmap. Notably, the levels of Irf7, Hmox1, Trp53, Fos Nfkbib,
   Cxcl1, Il6, Ccl5, etc., are markedly upregulated, indicating
   significant enhancement of oxidative stress and inflammation caused by
   SIRPI. Additionally, the downregulation of Atp5j, Atp5k, Atp5l, Jun,
   etc., suggest that mitochondrial oxidative stress induced by SIRPI
   effectively reduces the mitochondrial membrane potential and inhibit
   tumor cell growth (Figure [149]6D). Based on the protein‐protein
   interaction (PPI) network analyses on relevant DEGs, we identified the
   correlation between oxidative stress, inflammatory response, and tumor
   suppression induced by SIRPI (Figure [150]6E). However, the
   transcriptomic sequencing only provides valuable insights into gene
   expression, which is not fully reflective of protein levels and other
   downstream effects. Therefore, we confirmed the ability of SIRPI as a
   ROS‐generator to specifically increase oxidative stress in tumor cells,
   boosting the inflammatory response in the tumor region, and effectively
   inducing specific pyroptotic tumor cell death by combining the
   transcriptome sequencing with western blot analysis of
   pyroptosis‐related proteins.^[ [151]^23 ^]

Figure 6.

   Figure 6
   [152]Open in a new tab

   RNA Sequence Analysis of SIRPI. A) Volcano plots and B) numbers of the
   differentially expressed genes between Control and SIRPI + Laser +
   H[2]O[2] group. C) Diagram of KEGG pathway enrichment analysis. D)
   Circus heatmap of related DEGs associate with oxidative stress and
   inflammation E) Protein‐protein interaction network. Treat is the SIRPI
   + Laser + H[2]O[2]‐treated group and Ctl is the PBS‐treated group.

2.8. Specific Pyroptosis by SIRPI In Vivo

   Before investigating the antitumor effect of SIRPI, we first analyzed
   biosafety of SIRPI in vivo. The hemolysis analyses show that the
   hemolysis rate of erythrocytes incubated with SIRPI remained within the
   safe range (less than 5.0%) even at a concentration of 200 µM (Figure
   [153]S25, Supporting Information). Consequently, intravenous
   administration of SIRPI in mice were followed by monitoring the
   biochemical indexes after 24 h. It appears that the main biochemical
   indexes, including aminotransferase (AST), alanine aminotransferase
   (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN),
   creatinine (CR), and uric acid (UA), in the SIRPI and Saline treated
   mice are within the normal range (Figure [154]S26, Supporting
   Information). Moreover, the hematoxylin‐eosin (H&E) staining of the
   major organs (heart, liver, spleen, lung, and kidney) in mice treated
   by SIRPI and laser display no significant histological damage,
   suggesting the superior biosafety and biocompatibility of SIRPI (Figure
   [155]S27, Supporting Information). To further verify the accumulation
   of SIRPI in tumor through enhanced permeability and retention effect,
   we quantified the uptake of SIRPI in tumors by inductively coupled
   plasma‐mass spectrometry (ICP‐MS) and Prussian blue staining. It
   appears that sufficient SIRPI is detected in tumor of mice treated by
   SIRPI, revealing the enrichment of SIRPI in tumor regions (Figures
   [156]S28 and [157]S29, Supporting Information). Based on the excellent
   T [2] contrast imaging performance of SIRPI in vitro, we proceeded to
   assess its imaging ability in solid tumors (Figure [158]S30, Supporting
   Information). T [2]‐weighted MR images acquired before and after
   injection of SIRPI reveal a noticeably darkened signal in the tumor
   region post‐injection (Figure [159] 7B). The signal‐to‐noise ratio
   (SNR) values following injection are about 69.8 and 80.9% in transverse
   and coronal planes (Figure [160]S31, Supporting Information). This
   significant contrast between tumor and normal tissue endows us to
   easily differentiate the tumor from surrounding normal tissue, which is
   promising for real‐time monitoring of the tumor therapy.

Figure 7.

   Figure 7
   [161]Open in a new tab

   Antitumor effect and specific pyroptosis of SIRPI in vivo. A)
   Experimental scheme of SIRPI for the antitumor effect and specific
   pyroptosis. B) T [2]‐weighted MR images of tumor bearing mice before
   after 1 h intravenous injection of SIRPI. C) Relative tumor volume, D)
   tumor weight of 4T1 tumor‐bearing mice with different treatments. E)
   Western blot analysis and F) immunohistochemistry images to evaluate
   the expression of HIF‐1α in the tumor tissues after differently
   treated, the scale bar is 50 µm. Protein expressions of cleaved
   caspase‐3 and GSDME‐N with different treatment in G) tumor and H)
   normal tissue. Quantitative analysis of I) cleaved caspase‐3 and J)
   GSDME‐N expression in tumors (n = 3). K) Quantitative analysis of
   GSDME‐N expression in normal tissue (n = 3). L) H&E, M) TUNEL, and N)
   Ki67 staining of tumors section from each group, the scale bar is 50
   µm. From G1 to G4, they are Saline, Saline + Laser, SIRPI, SIRPI +
   Laser, respectively. Data are present as mean ± SD, *p < 0.05, **p <
   0.01, and ***p < 0.001, ****p < 0.0001, compared to the Saline group.

   Subsequently, we assessed the tumor therapeutic efficacy of SIRPI in
   vivo (Figure [162]7A). The female 4T1 tumor‐bearing Balb/c mice were
   randomly assigned into 4 groups (n = 5): Saline, Saline + Laser, SIRPI,
   and SIRPI + Laser group. During the treatment, the mice weight
   fluctuates within the normal range with negligible difference among all
   groups (Figure [163]S32A, Supporting Information). We noticed that the
   tumor growth is significantly inhibited in SPIRI treated group,
   especially the mice subjected to SPIRI and NIR laser combination
   therapy (Figure [164]7C, D; Figure [165]S32B–D, Supporting
   Information). To evaluate the aggressive effect of SIRPI on ameliorate
   the tumor hypoxia environment, we evaluated the hypoxia levels in
   tumors of mice with different treatments by western blot and
   immunohistochemistry staining for HIF‐1α. It appears that the HIF‐1α
   expression in the SIRPI‐treated group is obviously lower than that in
   the Saline group, indicating the excellent CAT‐like activity of SIRPI
   in tumor tissues to alleviate the hypoxic condition and enhancing the
   therapeutic efficiency (Figure [166]7E, F; Figures [167]S33 and
   [168]S34, Supporting Information). Additionally, we analyzed the ROS
   levels in both tumor and normal tissues under different treatments.
   Dihydroethidium (DHE) staining results show a significantly increased
   ROS level in tumor tissues treated with SIRPI + Laser, while no
   significant changes were observed in normal tissues. The substantial
   ROS generation in tumor tissues is expected to mediate selective
   pyroptosis in tumor cells (Figure [169]S35, Supporting Information). To
   verify the significant pyroptosis in tumor induced by SIRPI in vivo, we
   detected the expression of cleaved caspase‐3 and GSDME‐N in tumor
   tissues by western blot analyses following intravenous administration
   of SIRPI. The expression of cleaved caspase‐3 and GSDME‐N in tumor
   tissue are significantly upregulated in response to the SIRPI as
   compared to the Saline group. In particularly, there is an elevation of
   nearly 1.7 and 3.8 times of cleaved caspase‐3 and GSDME‐N
   (Figure [170]7G, I, J; Figure [171]S36, Supporting Information).
   Meanwhile, we also analyzed the expression of cleaved caspase‐3 and
   GSDME‐N in normal tissue. Interestingly, we do not notice apparent
   change of cleaved caspase‐3 and GSDME‐N levels in mice treated with
   SIRPI compared to the mice treated with saline, indicating that SIRPI
   specifically induces the upregulation of pyroptosis associated proteins
   in tumor tissues while maintaining normal levels in normal tissues
   (Figures [172]S37–[173]S39, Supporting Information). These results
   demonstrate that SIRPI enables to precisely inhibit the growth of tumor
   through the pyroptosis pathway in vivo without pyroptosis‐inducing
   effect in normal tissues. However, the precise pyroptosis in tumor may
   be caused by the different accumulation amount of SIRPI in tumor and
   normal tissue. To investigate whether SIRPI induces pyroptosis in tumor
   tissues and maintaining pyroptosis silence in normal tissues by the
   switchable activity in response with different tissues, we
   subcutaneously injected an equal amount of SIRPI into normal tissues
   with laser irradiation and analyses the expression levels of cleaved
   caspase‐3 and GSDME‐N domain. The western blot analyses indicate that
   the SIRPI result in the negligible expression of cleaved caspase‐3 and
   GSDME‐N increase compared to the Saline group, even in the SIRPI +
   Laser group (Figure [174]7H, K; Figure [175]S40, Supporting
   Information). These results strongly prove that SIRPI shows pyroptosis
   silencing in normal tissues, demonstrating SIRPI enables the excellent
   property to induce selective cellular pyrolysis in the tumor region
   while negligible damage to the normal tissue. Additionally, we
   performed the H&E, TUNEL, and Ki67 staining to systematically evaluate
   the therapeutic effects of SIRPI. The H&E and TUNEL staining images
   show that SIRPI + Laser treatment severely damage tumor tissue
   structure (Figure [176]7L, M). The Ki67 expression in the SIRPI + Laser
   group is significantly lower than that in the Saline group, indicating
   the tumor cells proliferation is inhibited (Figure [177]7N). These
   results synergistically demonstrate the effective anti‐tumor effect of
   SIRPI.

2.9. Augmented Immune Effects Mediated by SIRPI in Inhibiting Lung Metastasis

   Building on the successful anti‐tumor effects of SIRPI, we explored the
   potential of SIRPI to enhance immune responses within the TME.
   Initially, we analyze the dendritic cells (DCs) maturation by assessing
   the expression of DC‐related markers CD80 and CD86 in both the spleen
   and tumor tissues. Flow cytometry results display that expression of
   CD80^+ and CD86^+ in the SIRPI‐treated group is significantly higher
   than that treated by Saline, implying an increase in DCs maturation
   (Figure [178]8A–C; Figure [179]S41A, Supporting Information).
   Specifically, the DCs maturation ratio in SIRPI + Laser group is about
   33.0% compared to the 18.8% in the Saline group in spleen, suggesting
   that the SIRPI + Laser synergistic therapy effectively promotes the
   maturation of DCs. Next, to systematically detect the immune cell
   infiltration, we conducted flow cytometry to quantify the various
   immune cells infiltration in the tumors.^[ [180]^9 , [181]^24 ^] These
   results reveal a significant increase in the proportion of CD4^+ and
   CD8^+ T cells in the SIRPI + Laser group, whereas the proportion of
   immunosuppressive regulatory T cells (Tregs) characterized by CD25 and
   Foxp3 expression was significantly reduced (Figure [182]8D–F;
   Figure [183]S41B–D, Supporting Information). Moreover, the
   immunofluorescence staining images of CD4^+ and CD8^+ T cells
   infiltration in the tumor tissues corroborated this finding, shows a
   significant increase in the intensity of red (CD4^+) and green (CD8^+)
   fluorescence in the SIRPI‐treated group compared to the Saline group
   (Figure [184]S42, Supporting Information). These findings demonstrate
   that SIRPI‐induced pyroptosis significantly promotes DCs maturation and
   reversing the immunosuppressive in TEM. However, while increased
   infiltration of CD4+ and CD8+ T cells is evident, we acknowledge that
   their specific activation or function in the tumor region remains to be
   further explored. Additionally, the levels of TNF‐α, IFN‐γ, and
   IL‐1β in the SIRPI‐treated group, especially in the synergistic treated
   by SIRPI and laser irradiation, are significantly higher than those in
   the Saline group (Figure [185]8G–I). These results confirm that SIRPI
   successfully promotes the expression of inflammatory factors and
   improving the systemic immune response, which is expected to inhibit
   the development of micro‐metastases. To further investigate the
   potential of SIRPI in inhibiting metastasis, we injected 4T1 cells into
   mice with multiple interventions of SIRPI to establish a metastatic
   tumor model. After 2 weeks, a large number of micro‐metastatic foci are
   visible in the lung tissues of the Saline‐treated group. The number of
   micro‐metastatic foci in Saline‐treated group is significantly higher
   than that in SIRPI‐treated group. Notably, it hard to observe typical
   metastases in SIRPI + Laser group, indicating the high efficiency of
   SIPRI on reduce the risk of metastases during tumor therapy
   (Figure [186]8J; Figure [187]S43, Supporting Information). Consistent
   with the optical images analyses, the H&E staining images of the lungs
   and the calculated ratio of metastatic tumors areas in the total lung
   tissue area indicate that the SIRPI treatment could effectively inhibit
   the metastases (Figure [188]8K; Figure [189]S44, Supporting
   Information). These findings confirm that SIRPI enhances cellular
   immunogenicity and increases the inflammatory factor levels through the
   specific pyroptosis, re‐modeling the immunosuppressive microenvironment
   by promoting DCs maturation and tumor T cell infiltration, thereby
   effectively inhibiting primary tumor growth and metastatic nodes.

Figure 8.

   Figure 8
   [190]Open in a new tab

   Augmented immune effects by SIRPI. A) Quantification of the proportions
   of CD80^+ CD86^+ DCs in the tumor (n = 3). B) Flow cytometry analyses
   of CD80^+ and CD86^+ on CD11c^+ cells in spleen after different
   treatments. C) Quantification of the proportions of CD80^+ CD86^+ DCs
   in the spleen (n = 3). D) Quantification of the proportions of CD4^+ T
   cells, E) CD8^+ T cells, and F) CD25^+ Foxp3^+ Tregs in the tumor (n =
   3). ELISA analysis of G) TNF‐α, H) IFN‐γ, and I) IL‐1β concentration in
   tumor‐bearing Balb/c mice serum after differently treated for 24 h (n =
   5). J) Statistics on the metastases number in lung at 30 days after
   different treatments (n = 3). K) H&E staining images of metastatic
   lesions of different treatment group. The metastatic regions were
   marked by the black arrows, red circles, and red arrows, the scale bar
   is 200 µm and 2 mm, respectively. From G1 to G4, they are Saline,
   Saline + Laser, SIRPI, SIRPI + Laser, respectively. Data are present as
   the mean ± SD, *p < 0.05, and ***p < 0.001, ****p < 0.0001, compared to
   the Saline group.

3. Conclusion

   In this work, we have developed a switchable reactive oxygen species
   (ROS) generator/ scavenger in response with hydrogen peroxide
   (H[2]O[2]) to trigger the specific pyroptosis in tumors, effectively
   promoting therapeutic efficiency. This intelligent inducer performs
   reductive properties to eliminate excessive ROS and minimize the side
   effects with the excellent biosafety in normal tissues. Notably, in
   tumor microenvironment (TME) with H[2]O[2] accumulation, the structure
   of specific iron ruthenium pyroptosis inducer (SIRPI) is altered and
   switched to an ROS generator, which specifically generate large amounts
   of singlet oxygen (^1O[2]) and hydroxyl radical (·OH) and trigger the
   specific pyroptotic tumor cell death via the caspase‐3/ gasdermin E
   (GSDME) pathway. This mode of programmed cell death effectively boosts
   inflammatory factors and damage‐associated molecular patterns (DAMPs)
   release, alleviates the immunosuppressive TME, enhances the immune
   response, and suppresses primary and metastatic tumors. Therefore, the
   selective pyroptosis in tumor cells mediated by structural alterations
   in the specific response of SIRPI to H[2]O[2] is anticipated to offer a
   novel strategy for the precisive tumor therapy.

4. Experimental Section

   Experimental details are provided in the Supporting Information.

Statistical Analysis

   Statistical analysis was attained by Graphpad Prism 8.0 and the
   statistical mode was analysis of one‐/two‐way analysis of variance
   (ANOVA) and Student's t‐test, the data are presented as mean + SD. p
   values of less than 0.05 was considered significant (*p < 0.05, **p <
   0.01, ***p <0.001, and ****p < 0.0001).

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   L.H., C.T., and Z.Z. conceived and designed the experiments. Z.Z.,
   L.H., S.Z., M.L., M.T., M.F., J.Z., J.Z., M.L., K.L., and C.T.
   performed the experiments. L.H., K.L., C.T., and Z.Z. analyzed the
   data, and L.H. and Z.Z. co‐wrote the paper. All authors discussed the
   results and commented on the manuscript.

Supporting information

   Supporting Information
   [191]ADVS-12-2407713-s001.docx^ (5.9MB, docx)

Acknowledgements