Tumor Microenvironment-Responsive CA@ZIF-8/MnO 2 Nanoreactor for Self-Reinforcing Cascade Chemodynamic Therapy and Immunomodulation

Tumor Microenvironment-Responsive CA@ZIF-8/MnO 2 Nanoreactor for Self-Reinforcing Cascade Chemodynamic Therapy and Immunomodulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tumor Microenvironment-Responsive CA@ZIF-8/MnO 2 Nanoreactor for Self-Reinforcing Cascade Chemodynamic Therapy and Immunomodulation Yawei Li, Xialin Sun, Yilin Huang, Shuang Mu, Zhengwei Zhu, Tingwen Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6676160/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chemodynamic therapy (CDT), which utilizes endogenous hydrogen peroxide (H 2 O 2 ) to generate hydroxyl radicals ( • OH) via Fenton-like reactions, faces critical limitations in clinical translation, including insufficient intratumoral H 2 O 2 levels and glutathione (GSH)-mediated ROS scavenging. To address these challenges, we developed a tumor microenvironment (TME)-responsive nanoreactor, CA@ZIF-8/MnO 2 (CZM), integrating dual functionalities of GSH-depleting and H 2 O 2 self-supplying for cascade-amplified CDT. The ZIF-8 framework serves as a biodegradable carrier for chlorogenic acid (CA), which converts superoxide (O 2 •− ) into H 2 O 2 , while the MnO 2 shell depletes GSH to yield Mn 2+ , a Fenton-like catalyst. Upon internalization by tumor cells, the MnO 2 shell reacts with GSH to produce Mn 2+ , which catalyzes the conversion of H 2 O 2 to • OH, while simultaneously depleting GSH to enhance CDT efficacy. Additionally, the acidic TME triggers the release of CA, which elevates H 2 O 2 levels through its self-oxidation property, creating a self-reinforcing cycle. In vitro and in vivo studies demonstrated that CZM NPs not only enhance • OH generation but also trigger immunogenic cell death (ICD), promoting antitumor immune responses. Furthermore, CZM NPs promote the polarization of tumor-associated macrophages towards the M1 antitumor phenotype, reshaping the immunosuppressive TME. RNA-seq and pathway analysis further revealed that CZM NPs modulate key signaling pathways, including NF-κB, to induce apoptosis and enhance antitumor immunity. Overall, these findings highlight the potential of CZM NPs as a multifunctional nanoplatform for cascade-amplified CDT and immunotherapy. Nanoreactor Cascade-amplified chemodynamic therapy GSH depletion H2O2 self-generation Immune regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction According to WHO statistics, cancer continues to be the leading cause of mortality worldwide, with its treatment remaining a central focus in medical research [ 1 – 3 ]. Chemodynamic therapy (CDT), a novel cancer treatment paradigm [ 4 , 5 ], leverages endogenous hydrogen peroxide (H 2 O 2 ) to produce hydroxyl radicals ( • OH), the most harmful reactive oxygen species (ROS) [ 6 – 8 ]. This process is facilitated through transition metal ion-mediated (e.g., Fe 2+ , Cu + , Mn 2+ ) Fenton or Fenton-like reactions, thereby achieving the effect of tumor cell destruction [ 9 – 11 ]. In contrast to conventional therapeutic approaches, CDT offers enhanced tumor selectivity, spatial and temporal controllability, minimal invasiveness, and reduced systemic toxicity [ 12 – 15 ]. Despite its promising potential, the advancement of CDT is confronted with several challenges [ 16 , 17 ]. Notably, the overexpression of glutathione (GSH) poses a significant obstacle [ 18 ]. As a critical component of the robust ROS scavenging system, GSH is upregulated (approximately 10 mM) in cancer cells to counteract the elevated ROS levels resulting from abnormal proliferation and heightened cellular respiration [ 19 ]. This upregulation assists in maintaining redox homeostasis; however, it concurrently leads to the rapid depletion of generated • OH before it can exert its cytotoxic effects, thereby augmenting cancer cell resistance to oxidative stress and diminishing the therapeutic efficacy of CDT [ 20 , 21 ]. Furthermore, despite the H 2 O 2 concentration (approximately 100 µM) in the tumor microenvironment (TME) being significantly higher than that in normal tissues [ 22 ], the endogenous H 2 O 2 is still insufficient to produce a sufficient amount of • OH to achieve satisfactory therapeutic outcomes, presenting another obstacle for CDT [ 23 – 25 ]. Consequently, it is urgent yet challenging to develop a nanoplatform with both self-supplying H 2 O 2 and self-consuming GSH functionalities to enhance the efficacy of CDT. In recent years, manganese dioxide (MnO 2 ) has emerged as a promising candidate for CDT nanoplatforms, attracting considerable research interest [ 26 ]. Primarily, the MnO 2 nanostructure exhibits unique redox reactivity with GSH, producing Mn 2+ ions that display exceptional Fenton-like catalytic activity [ 27 ]. This enables efficient conversion of endogenous H 2 O 2 into highly cytotoxic • OH [ 28 , 29 ]. Notably, this GSH-triggered degradation mechanism simultaneously reduces intracellular antioxidant defenses by depleting overexpressed tumor GSH, thereby minimizing • OH scavenging and establishing MnO 2 as an effective GSH-depleting agent [ 30 , 31 ]. Moreover, the GSH-responsive decomposition of MnO 2 nanostructures facilitates controlled drug release through gatekeeper-mediated payload delivery systems [ 32 ]. Concurrently, MnO 2 demonstrates catalase-like activity by decomposing excess H 2 O 2 into water and oxygen molecules, effectively ameliorating tumor hypoxia, a critical barrier in conventional cancer therapies [ 33 – 36 ]. Emerging evidence suggests that MnO 2 -induced oxidative stress elevation not only triggers immunogenic cell death (ICD) [ 37 , 38 ] but also promotes phenotypic reprogramming of tumor-associated macrophages (TAMs) [ 39 , 40 ]. Specifically, the substantial ROS generation facilitates polarization shift from pro-tumoral M2 macrophages to antitumor M1 variants [ 41 – 44 ]. Complementarily, the liberated Mn 2+ ions have been shown to independently induce macrophage M1 polarization through distinct signaling pathways [ 45 – 48 ]. This dual mechanism synergistically modulates the immunosuppressive TME, enhancing antitumor immunity through coordinated oxidative stress amplification and immune cell reprogramming. As previously mentioned, the efficacy of CDT is significantly influenced by the limited endogenous H 2 O 2 levels, making it crucial to incorporate the H 2 O 2 self-supplying capability into the nanosystem. Chlorogenic acid (CA), a prominent bioactive compound abundantly found in coffee, has been demonstrated to exhibit a wide range of pharmacological activities, including antibacterial, anti-inflammatory, and antitumor effects [ 49 – 51 ]. Notably, CA possesses a unique self-oxidation property, enabling it to efficiently convert superoxide anions (O 2 •− ) into H 2 O 2 [ 52 , 53 ]. This intrinsic H 2 O 2 -generating capability holds great potential for enhancing the therapeutic outcomes of CDT. Additionally, recent studies have highlighted that CA can also act as a promising immunomodulator by promoting M1-type macrophages and inhibiting the M2-phenotype [ 54 – 56 ]. Despite these advantages, the clinical application of CA is hindered by its inherent limitations, such as poor water solubility, short half-life, and low stability [ 57 , 58 ]. Therefore, maintaining high levels of intracellular catalysts (Mn 2+ ) and reactants (H 2 O 2 ), while reducing the content of ROS scavengers (GSH), can be accomplished by incorporating MnO 2 nanostructures and encapsulating CA into the nanocomplex. In light of the aforementioned considerations, this study presents the rational design and successful fabrication of a TME-responsive multifunctional nanoreactor designated as CA@ZIF-8/MnO 2 (CZM), which integrates self-consuming GSH and self-supplying H 2 O 2 capabilities for dual-enhanced CDT (Scheme 1 ). Central to this design is the implementation of zeolitic imidazolate framework-8 (ZIF-8), a representative material from the metal-organic framework (MOF) family, which serves dual functions: functioning as a nanocarrier for CA encapsulation while enabling direct surface deposition of MnO 2 nanoshells. Following cellular internalization of CZM NPs by tumor cells, the MnO 2 outer layer undergoes tumor-specific conversion into Mn 2+ ions through catalytic interaction with endogenous H 2 O 2 , and simultaneously depletes intracellular antioxidant GSH reserves to mitigate • OH scavenging, thereby potentiating CDT efficacy. Notably, this transformation concurrently induces ICD and promotes antitumor immune responses. Complementarily, the inner ZIF-8 framework undergoes rapid biodegradation under weakly acidic tumor conditions, enabling controlled release of encapsulated therapeutic agents. The released CA, exhibiting SOD-like activity, catalyzes O 2 •− conversion to H 2 O 2 , thereby establishing a self-amplifying cycle that elevates intratumoral H 2 O 2 concentrations and augments subsequent CDT performance. Significantly, CZM NPs demonstrated the capacity to promote macrophage polarization toward antitumor M1 phenotype. Comprehensive in vitro and in vivo evaluations confirmed the synergistic cooperation of all functional components within CZM NPs, with the “MnO 2 + CA loading” combination demonstrating superior CDT enhancement through a “1 + 1 > 2” therapeutic synergy. To elucidate the underlying molecular mechanisms, we employed RNA-seq coupled with bioinformatics analysis and western blot validation, providing systematic investigation of tumor cell gene expression profiles under CZM NPs treatment. Results and discussion Preparation and characterization of CZM NPs CZM NPs were prepared using a facile and environmentally friendly one-pot method. Briefly, two aqueous solutions, one containing Zn(NO 3 ) 2 ·6H 2 O and CA (solution A) and the other containing 2-MIM (solution B), were prepared in advance and then mixed under vigorous stirring. After several minutes of stirring, a light yellow emulsion formed, indicating the successful synthesis of CZ (CA@ZIF-8) NPs. Subsequently, MnO 2 nanosheets were easily decorated onto the surface of the CZ NPs by mixing the prepared NPs with an excess amount of permanganate, yielding CZM NPs (Fig. S1). The structure and surface morphology of the two samples were characterized using transmission electron microscopy (TEM) (Fig. 1A and B) and scanning electron microscopy (SEM) (Fig. 1C and D). Both CZ and CZM NPs exhibited three-dimensional dodecahedral structures, with a distinct MnO 2 layer visible on the surface of CZM NPs. The energy-dispersive spectroscopy (EDS) spectrum of CZM indicated the simultaneous presence of Zn and Mn elements (Fig. 1F), and the corresponding elemental mapping images further validated the uniform distribution of these elements (Fig. 1E). The hydrated particle sizes of these NPs were determined using dynamic light scattering (DLS). The CZM NPs exhibited a narrow size distribution (116.6 ± 2.7 nm), slightly larger than that of CZ NPs (100.7 ± 5.2 nm), which can be attributed to the MnO 2 coating (Fig. 1G). Additionally, DLS analysis demonstrated the favorable physiological stability of both CZ and CZM NPs, suggesting their potential for biomedical applications (Fig. 1H and S2). To further analyze the chemical composition and crystal phase of CZM NPs, X-ray photoelectron spectroscopy (XPS) and X-ray diffractometry (XRD) were employed. The XPS survey spectrum revealed the presence of Zn, Mn, O, N, and C elements in the nanocatalyst (Fig. 1I). The high-resolution Mn 2p spectrum (Fig. S3) displayed two peaks at 653.7 eV and 642.2 eV, corresponding to Mn 2p 1/2 and Mn 2p 3/2 , respectively, and these binding energies are consistent with those of manganese in MnO 2 , confirming the successful coating of MnO 2 on the NPs. Furthermore, the powder X-ray diffraction (PXRD) patterns of the as-synthesized ZIF-8, CZ, and CZM NPs (Fig. 1J) matched well with the simulated spectrum of ZIF-8, indicating that the drug loading and MnO 2 coating had negligible effects on the structural integrity of the NPs. Thermogravimetric analysis (TGA) was used to quantify the CA loading content. As shown in Fig. 1K, a significant weight loss difference was observed between the ZIF-8 and CZ NPs, attributed to the removal of encapsulated CA molecules. The drug loading capacity and encapsulation efficiency of CA were calculated to be approximately 11.8% and 69.6%, respectively, consistent with the results obtained from UV-vis spectroscopy (Fig. S4). Finally, UV-vis spectroscopy further confirmed the successful synthesis of CZM NPs (Fig. S5). In vitro evaluation of ROS generation, GSH depletion, and H 2 O 2 self-supplying The successful fabrication of the nanoreactor prompted us to explore its multifunctional capabilities (Fig. S6). As is well-established, iron-triggered Fenton chemistry is widely utilized to induce apoptosis in tumor cells by catalyzing endogenous H 2 O 2 into • OH, and Mn 2+ can achieve CDT by generating • OH through Fenton-like reaction [48,49]. To investigate the • OH-generating activity and the scavenging effect of GSH on • OH, we initially utilized methylene blue (MB), a dye susceptible to degradation by • OH, as an indicator. As depicted in Fig. 2A, a significant decrease in absorbance was observed when MB was incubated with H 2 O 2 and Mn 2+ in the presence of NaHCO 3 /CO 2 , whereas negligible change in MB absorbance was detected in other solutions lacking HCO 3 − , underscoring the pivotal role of bicarbonate (HCO 3 − ) in the Mn 2+ -driven Fenton-like reaction. Concurrently, Fig. 2B illustrated the H 2 O 2 concentration-dependent degradation profile of MB. As the H 2 O 2 concentration increased, the absorbance of MB diminished, accompanied by a gradual transition in solution color from blue to transparent, indicative of enhanced • OH generation. Notably, GSH, a ROS scavenger, can neutralize • OH and thereby compromise the efficacy of ROS-based therapies. The overexpression of GSH in cancer cells directly impedes CDT efficiency. Depleting intracellular GSH levels can augment the effectiveness of ROS-based therapies. In contrast to Mn 2+ , MnO 2 can initially consume GSH to yield Mn 2+ , thereby exhibiting a greater potential for • OH production. Fig. 2C corroborated that increasing GSH concentrations attenuated the efficiency of the Mn 2+ -mediated Fenton-like reaction. Conversely, in the context of CZM NPs-mediated Fenton-like catalysis, the inhibitory influence of GSH was markedly mitigated, with enhanced MB degradation observed across varying GSH concentrations (Fig. 2D). This phenomenon arises because, in physiological environments, GSH preferentially reacts with MnO 2 to generate Mn 2+ , enabling CZM NPs with augmented GSH-depleting capacity to effectively counteract this process and promote MB degradation. Subsequently, we evaluated the GSH depletion capability of CZM NPs using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as an indicator. As shown in Fig. 2E, pure DTNB exhibited a characteristic absorption peak at 325 nm, while all GSH + DTNB groups displayed a new absorption peak at 412 nm, primarily attributed to the reduction reaction between DTNB and the thiol groups of GSH (Fig. S7). Importantly, we observed a reduction in GSH levels in the CZM + GSH + DTNB group (Fig. 2F), as the outer MnO 2 layer of CZM NPs demonstrated exceptional responsiveness to GSH. And the characteristic absorbance peak at 412 nm decreased significantly with increasing CZM concentrations and prolonged reaction times. These findings suggest that CZM NPs can function as potent GSH-depleting agents, modulating intracellular redox homeostasis. Given the unique structural configuration of the MnO 2 shell and ZIF-8 core, we hypothesized that the nanoreactor would exhibit pH- and GSH-responsive characteristics. To validate this hypothesis, we systematically investigated the drug release profiles of CZM NPs under various simulated TME conditions, including different pH values (7.4 and 5.5) with or without GSH. As demonstrated in Fig. 2G, cumulative CA release reached 35.9% over 48 hours under acidic conditions (pH 5.5), while minimal release (5.6%) was observed at physiological pH (7.4), confirming the pH-responsive nature of the ZIF-8 framework. Notably, the release kinetics accelerated significantly (52.7% cumulative release) in acidic medium containing GSH compared to the equivalent pH condition without GSH (35.9%), highlighting the dual-gating mechanism mediated by the GSH-responsive properties of the MnO 2 shell and the pH sensitivity inherent to ZIF-8. These results collectively demonstrate that CZM NPs possess TME-specific responsiveness, enabling intelligent drug release while maintaining intrinsic H 2 O 2 self-supplying capabilities. Subsequently, we confirmed the ability of CA molecules to catalyze the production of H 2 O 2 from O 2 • − and O 2 through their self-oxidation pathway. Using a TiOSO 4 -based colorimetric assay, which forms a light-yellow peroxo-titanium complex (λmax = 415 nm) upon H 2 O 2 interaction, we quantitatively measured the production of H 2 O 2 . As demonstrated in Fig. 2H, substantial H 2 O 2 production was observed following CA administration, indicating that CA compounds exhibit intrinsic H 2 O 2 self-supplying capacity and can efficiently convert excessive O 2 • − into H 2 O 2 within neoplastic cellular environments. This conversion is particularly advantageous given that malignant cells typically exhibit 3-5 fold higher O 2 • − levels compared to normal cells, owing to their hypermetabolic state and dysfunctional mitochondrial respiration. Complementarily, it is widely recognized that MnO 2 can serve as an effective catalyst for the decomposition of H 2 O 2 into O 2 . As shown in Fig. 2I, dissolved oxygen levels remained negligible in control groups lacking MnO 2 , whereas CZM-containing solutions exhibited rapid oxygen generation proportional to CZM concentration. This oxygen generation not only confirms the catalytic proficiency of MnO 2 but also establishes a positive feedback loop that enhances the subsequent self-oxidation process of CA. Collectively, these sequential catalytic events bestow upon CZM NPs multifunctional capabilities, including ROS amplification, GSH depletion, and self-supplying H 2 O 2 , significantly enhancing the efficacy of CDT. Enhanced CDT efficacy of CZM NPs in vitro Effective intracellular delivery of nanocomposites is a prerequisite for achieving optimal antitumor efficacy, thus 4T1 murine breast cancer cells were employed to evaluate the phagocytic capacity of CZM NPs. For fluorescence tracking, CA molecules and Nile Red (NR) were co-encapsulated within the ZIF-8 framework. Confocal laser scanning microscopy (CLSM) images (Fig. 3A and S8) revealed distinct red fluorescence signals localized in the cytoplasm, with fluorescence intensity gradually enhancing as both incubation time and NPs concentration increased. This demonstrates time- and dose-dependent cellular internalization of CZM NPs. Following cellular uptake, the MnO 2 shell undergoes GSH-responsive dissociation, combined with the slightly acidic TME, effectively depletes the overexpressed intratumoral GSH to generate Mn 2+ ions and trigger CA release. This cascade establishes a self-amplifying system characterized by GSH depletion, H 2 O 2 self-supplying, and dual-enhanced CDT. Subsequent investigations validated the GSH-depleting capacity of MnO 2 and H 2 O 2 -generating property of CA at the cellular level. As anticipated, ZM NPs treatment significantly reduced intracellular GSH levels (Fig. 3B), while CA incubation induced dose-dependent H 2 O 2 accumulation (Fig. 3C). Intracellular ROS generation was quantitatively assessed using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Fig. 3D and S9 demonstrated negligible green fluorescence in control and CA-only groups, whereas the ZM group exhibited characteristic fluorescence attributed to the Mn 2+ -mediated Fenton-like reactions. Notably, the CZM group displayed the most intense fluorescence signal, attributable to CA-driven H 2 O 2 self-supplying coupled with MnO 2 -mediated GSH depletion, demonstrating the self-reinforcing CDT characteristics of CZM NPs. Given the exceptional • OH generation capability, the in vitro cytotoxicity assessment was subsequently conducted utilizing the conventional methyl thiazolyl tetrazolium (MTT) assay. In comparison to the control group, ZIF-8 exhibited high cell viability, even at relatively elevated concentrations (Fig. 3E), thereby indicating the favorable cytocompatibility of the synthesized nanocarrier. In contrast, both CA-loaded CZ NPs and MnO 2 -coated ZM NPs exhibited dose-dependent cytotoxicity through standalone chemotherapy or CDT, respectively. Remarkably, CZM NPs demonstrated superior cytotoxicity compared to monofunctional counterparts at equivalent CA concentrations, attributable to synergistic catalytic cascade reactions. Importantly, CZM NPs showed negligible toxicity towards 293T normal cells (Fig. 3F), indicating tumor-specific therapeutic potential. To elucidate apoptosis mechanisms, Annexin V-FITC/PI dual staining flow cytometry was performed. Quantitative analysis (Fig. 3G) revealed significantly higher apoptotic rates in the CZM group (21.36%) versus CA (7.79%) and ZM (11.43%) groups, further substantiating the therapeutic superiority of the self-amplifying CDT system. ICD induction and macrophage polarization by CZM NPs in vitro CDT has been widely recognized for its ability to trigger ICD in tumor cells, leading to the generation of endogenous damage-associated molecular patterns (DAMPs) [38]. These DAMPs subsequently stimulate antitumor immune responses by acting as “eat me” and “find me” signals, which are critical for antigen presentation [37,39]. To investigate the ICD-inducing potential of CZM NPs in tumor cells, we examined the associated DAMPs, as evidenced by the translocation of CRT, the nuclear export of HMGB1, and the extracellular release of ATP [41]. Immunofluorescence analysis was performed to evaluate HMGB1 localization after various treatments. As presented in Fig. 4A, the CZM NPs group exhibited the least green fluorescence intensity, indicating a rapid and efficient release of HMGB1 from 4T1 cells. Similarly, CRT translocation has also been revealed by CLSM. The CZM NPs group displayed intense green fluorescence at the cell membrane, while the CA and ZM NPs groups showed weaker signals (Fig. 4B). This suggests that CZM NPs treatment can significantly enhance CRT exposure on the cell surface, thereby promoting immune activation. Additionally, extracellular ATP levels were quantified using an ATP detection kit. The CZM NPs group demonstrated a remarkable increase in ATP release, with concentrations 2.9-fold and 2-fold higher than those in the CA and ZM NPs groups, respectively (Fig. 4C). This elevated ATP secretion is crucial for recruiting immature dendritic cells and enhancing immune surveillance. Collectively, these findings underscore the robust ICD-inducing capability of CZM NPs. The polarization of TAMs plays a pivotal role in modulating their antitumor functions. M1 macrophages are known for their antimicrobial and immunostimulatory properties, whereas M2 macrophages suppress T-cell activity and facilitate tumor progression, invasion, and metastasis. In this study, RAW264.7 macrophages were used to evaluate phenotypic polarization. Flow cytometry analysis revealed a significant upregulation of CD86, a marker for M1 macrophages, in the CZM NPs group compared to other treatments (Fig. 4D). Furthermore, cytokine profiling via quantitative PCR confirmed this polarization, showing that macrophages treated with CZM NPs secreted the highest levels of TNF-α and IL-12, both hallmark M1 cytokines, compared to the CA and ZM NPs groups (Fig. 4E and F). Together, these results demonstrate that CZM NPs effectively induce ICD in tumor cells and promote M1 macrophage polarization, thereby reshaping the immunosuppressive tumor microenvironment. Antitumor molecular mechanism of CZM NPs To further investigate the antitumor mechanism of CZM NPs, we employed RNA sequencing (RNA-seq) to analyze gene expression profiles in tumor cells following treatment. After ensuring the quality and integrity of RNA extracted from both CZM NPs-treated (NPs) and untreated (Con) groups, principal component analysis (PCA) was performed on the complete dataset to assess clustering patterns. As shown in Fig. S10, PCA results revealed clear separation between the two groups, indicating distinct transcriptional profiles and confirming the significant impact of CZM NPs on gene expression. Subsequently, differentially expressed genes (DEGs) were identified by comparing RNA-seq data from treated and untreated cells, with results visualized using volcano plots and heatmaps (Fig. 5). A total of 2,496 DEGs were identified, comprising 1,247 up-regulated and 1,249 down-regulated genes, highlighting the profound regulatory influence of CZM NPs on tumor cell transcription. Based on the RNA-seq findings, Gene Ontology (GO) and pathway enrichment analyses were conducted to explore the functional roles of the identified DEGs. GO analysis categorized the DEGs into three main domains: cellular component (CC), biological process (BP), and molecular function (MF). Within the CC domain, significant enrichment was observed for terms related to cytoplasm, cytosol, cytosolic ribosome, and nucleoplasm (Fig. 6A). In the BP domain, key processes such as translation, positive regulation of cell migration, cellular response to lipopolysaccharide and response to drug were prominently affected (Fig. 6B). For the MF domain, protein binding, structural constituents of ribosomes, ubiquitin protein ligase binding and translation regulator activity were the most significantly altered functions (Fig. 6C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that CZM NPs modulated a variety of signaling pathways. Notably, the IL-17 signaling pathway, TNF signaling pathway, Ribosome and Oxidative phosphorylation were among the top 20 enriched pathways (Fig. 6D). Further KEGG pathway classification (Fig. S11) revealed notable associations between DEGs and critical signaling cascades including MAPK, Nuclear factor-κB (NF-κB), and p53-mediated pathways. This multi-pathway regulation pattern suggests comprehensive immunomodulatory and pro-apoptotic effects of CZM NPs on tumor cells. Mechanistically, the NF-κB transcription factor complex, which consists of p50, p65, and IκBα subunits, functions as a pivotal regulator of inflammatory responses and immune homeostasis. Its canonical activation pathway involves the phosphorylation-dependent degradation of IκBα, mediated by the multimeric IκB kinase (IKK) complex. Importantly, both IL-17 and TNF-α signaling pathways converge on NF-κB activation, thereby orchestrating transcriptional programs that govern cell survival and anti-apoptotic processes. Based on our RNA-seq findings and existing literature, we hypothesized that CZM NPs may mediate antitumor immunity and apoptosis by inhibiting the NF-κB signaling pathway. Western blot validation confirmed this hypothesis (Fig. 7), demonstrating CZM NPs-induced upregulation of pro-apoptotic markers (Bax, cleaved caspase-3) concurrent with downregulation of anti-apoptotic Bcl-2 and phosphorylation events in both p65 and IκBα. Collectively, these findings established the suppression of NF-κB signaling as a critical mechanism underlying the dual action of CZM NPs in remodeling the tumor microenvironment and inducing apoptosis. In vivo antitumor efficacy evaluation of CZM NPs Building upon the promising in vitro antitumor characteristics of CZM NPs, we subsequently investigated their therapeutic potential in 4T1 tumor-bearing murine models. Mice were stratified into four cohorts (n=5): 1) PBS control, 2) free CA, 3) ZM NPs, and 4) CZM NPs, with groups 2-4 receiving equivalent CA or ZM doses via intravenous administration every 48 hours. To assess intracellular ROS levels within tumor tissues post-treatment, DCFH-DA staining was performed on tumor sections. Fig. 8A demonstrated that mice administered with CA exhibited weak green fluorescence signal compared to control group at 24 h post-injection, suggesting limited ROS production. In contrast, both ZM and CZM NPs groups displayed significantly enhanced DCFH fluorescence intensity, with the latter showing the most pronounced signal amplification. The therapeutic outcomes were subsequently monitored using identical treatment protocols. Fig. 8B illustrated that tumor volumes in the CA group showed modest reduction relative to controls, attributable to the intrinsic cytotoxicity of CA. Notably, ZM NPs administration resulted in substantial tumor growth inhibition, primarily mediated by MnO 2 shell-mediated Fenton-like reactions generating ROS. The CZM NPs cohort achieved superior tumor suppression, confirming the synergistic interplay between GSH depletion and H 2 O 2 self-supplying for enhanced CDT. The similar conclusion can be drawn from the average weight of resected tumors in each group following the final measurement. As illustrated in Fig. 8C and D, both the weight and size of tumors in the CZM NPs group were significantly reduced, as evidenced by the macroscopic examination of the dissected tumor tissues across all cohorts. Histopathological validation through H&E and TUNEL staining revealed marked nuclear condensation, cytoplasmic leakage, and apoptotic cell accumulation in the CZM NPs group (Fig. 8F), substantiating its therapeutic efficacy. Importantly, all treatment groups maintained stable body weights throughout the 12-day regimen (Fig. 8E), indicating the negligible systemic toxicity of these treatments to mice. Furthermore, H&E-stained major organ sections (Fig. S12) showed no evidence of inflammatory infiltration or histopathological abnormalities, confirming the biocompatibility of all formulations. To validate this CDT triggering antitumor immunity via ICD in vivo, we conducted immunocytochemistry staining assays to assess CRT exposure and HMGB1 release. Similar to the results in vitro, the CZM NPs-treated group exhibited markedly elevated CRT expression and robust HMGB1 release in tumor tissues compared to other groups (Fig. 9A), suggesting that CZM NPs treatment effectively triggered extensive ICD in vivo. Following this, we employed immunofluorescence to analyze the alterations in M1 and M2 phenotypes within the tumor, aiming to confirm the polarization capability of CZM NPs. Fig. 9B demonstrated a substantial increase in the CD86 fluorescence signal, indicative of M1 phenotype, in the CZM NPs-treated group relative to other groups. Conversely, the CD163 fluorescence signal, representing M2 phenotype, markedly reduced following CZM NPs treatment. Given that hallmark cytokines such as IFN-γ and TNF-α are associated with the M1 phenotype, and IL-10 with the M2 phenotype, we further evaluated the TAMs polarization by detection of the cytokines collected from tumor. As depicted in Fig. 9C-E, the secretion of IL-10 in the tumor lysates from the CZM NPs-treated group was significantly reduced by 2.64-fold compared to the control group, while the levels of TNF-α and IFN-γ were notably elevated, collectively indicating significant M2 to M1 polarization of TAMs in TME post CDT with CZM NPs. Conclusion In summary, we engineered a TME-responsive nanoreactor (CZM NPs) to overcome the intrinsic limitations of CDT through GSH-depleting, H 2 O 2 self-supplying, and immunomodulation. The MnO 2 shell effectively scavenges GSH to generate Mn 2+ , amplifying Fenton-like reactions, while the released CA enables persistent H 2 O 2 supply through O 2 •− -mediated conversion. This synergistic dual amplification strategy significantly enhances CDT efficacy both in vitro and in vivo. Importantly, CZM NPs could trigger ICD in tumor cells, promoting antitumor immune responses and polarizing TAMs from M2 to M1 phenotype, thus reshaping the immunosuppressive tumor microenvironment. Mechanistically, RNA-seq and bioinformatics analyses reveal NF-κB pathway inhibition and pro-apoptotic signaling activation as critical drivers of therapeutic efficacy. This study not only underscores the potential of CZM NPs as a promising nanoplatform for cascade-amplified CDT and immune regulation, but also provides a blueprint for designing multifunctional nanotherapeutics. Declarations All the experimental procedures to mouse described herein have gained approval from the Ethics Committee of Jilin Medical University and carried out corresponding to the regulation, principles, and guidelines of Chinese law concerning the protection of animal life. Supplementary information accompanies this paper at https://doi.org/10. 1186/s129 51 -025-#####-#. Authors’ contributions The manuscript was written through contributions of all authors. All authors read and approved the final manuscript Funding This work was supported by the Science and Technology Development Project of Jilin Province (No.20240101202JC and No.20220204038YY), the National Natural Sciences Foundation of China (No.32101154). Availability of data and materials All data generated or analysed during this study are included in this article and its additional file. Ethics approval and consent to participate Not applicable. Consent for publication All authors gave their consent for publication. Competing interests The authors declare that they have no competing interests. References Siegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. Ca- Cancer J. Clin. 2025;75(1):10-45. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. 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Ashraf K, Liu ZB, Zaman QU, Arshad M, Zaman WQ, Shan A, Yu J, Rehman TU, Zhuang Y, Guo M, Mohsin A. De Novo synthesis of selenium-doped CeO 2 @Fe 3 O 4 nanoparticles for improving secondary metabolite biosynthesis in Carthamus tinctorius cell suspension culture. Chem. Eng. J. 2025;505:159705. Metodiewa D, Jaiswal AK, Cenas N, Dickancaité E, Segura-Aguilar J. Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radical Biol. Med. 1999;26(1-2):107-16 Xing HZ, Bai XW, Pei XY, Zhang YQ, Zhang XY, Chen SJ, Li D, Lv B, Wang XY, Wu XL. Synergistic anti-oxidative/anti-inflammatory treatment for acute lung injury with selenium based chlorogenic acid nanoparticles through modulating Mapk8ip1/MAPK and Itga2b/PI3k-AKT axis. J. Nanobiotechnol. 2025;23(1):37. Sun Q, Chen W, Zhang Q, Han Y, Gao Y, Ye J, Wang H, Zhang Y, Gao L, Liu Y, Yang Y. A pH transformable nanocarrier for gradual and precise delivery of a natural immunomodulator and chemotherapy agent to trigger tumor apoptosis. Chem. Eng. J. 2024;495:153209. Ye J, Yang Y, Jin J, Ji M, Gao Y, Feng Y, Wang H, Chen X, Liu Y. Targeted delivery of chlorogenic acid by mannosylated liposomes to effectively promote the polarization of TAMs for the treatment of Glioblastoma. Bioact. Mater. 2020;5(3):694-708. Xue N, Zhou Q, Ji M, Jin J, Lai F, Chen J, Zhang M, Jia J, Yang H, Zhang J, Li W, Jiang J, Chen X. Chlorogenic acid inhibits glioblastoma growth through repolarizating macrophage from M2 to M1 phenotype. Sci. Rep. 2017;7:39011. Li Q, Sun L, Xu L, Jia Y, Wang Z, Shen Z, Bi K. Determination and pharmacokinetic study of syringin and chlorogenic acid in rat plasma after administration of Aidi lyophilizer. Biomed. Chromatogr. 2006;20(12):1315-20. Zhang J, Chen M, Ju W, Liu S, Xu M, Chu J, Wu T. Liquid chromatograph/tandem mass spectrometry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study. J. Pharm. Biomed. Anal. 2010;51(3):685-90. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx floatimage1.jpeg Scheme 1Schematic illustration of a) the preparation of CZM NPs and b) their application for cascade-amplified CDT and immunotherapy. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6676160","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459030792,"identity":"435a4463-fb40-49bc-aa9e-5c1d73eb1169","order_by":0,"name":"Yawei Li","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yawei","middleName":"","lastName":"Li","suffix":""},{"id":459030793,"identity":"7b6aa08d-5aff-45cc-8caf-471f38c6953d","order_by":1,"name":"Xialin Sun","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xialin","middleName":"","lastName":"Sun","suffix":""},{"id":459030794,"identity":"4a7a23ac-5831-430a-a30c-c19260266017","order_by":2,"name":"Yilin Huang","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yilin","middleName":"","lastName":"Huang","suffix":""},{"id":459030795,"identity":"544580ac-1b59-4043-b566-785e3a367e01","order_by":3,"name":"Shuang Mu","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Mu","suffix":""},{"id":459030796,"identity":"9ace64ed-96d7-410b-a5a5-b1e5ea9395a6","order_by":4,"name":"Zhengwei Zhu","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengwei","middleName":"","lastName":"Zhu","suffix":""},{"id":459030797,"identity":"bd398f38-9c5c-4913-8074-41f0e1b12d4b","order_by":5,"name":"Tingwen Zhang","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingwen","middleName":"","lastName":"Zhang","suffix":""},{"id":459030798,"identity":"a773cf70-e0c2-4fb1-8c54-45531741439c","order_by":6,"name":"Xianmin Feng","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianmin","middleName":"","lastName":"Feng","suffix":""},{"id":459030800,"identity":"1a72adb7-3911-444f-aad9-e5de7a62a982","order_by":7,"name":"Wenhe Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACAwYGxgMJDAwJ/MzMhx8Qq4UBrEWynS3NgHgtQJxgcJ5HQYIoLeYSyQcOPPhzOM/4MA9Qf41NNEEtljPSEg4k8BwuNjvMe+ABw7G03AaCDruRY3AgQeJ24rbDfAkGjA2HidVicDtxczOPgQQJWhJuJ25gJlrLmWdAvxz4nzjjMDCQE4jyy/Hkgw9//ElL7O8/fPjBhxobwlpQQQJpykfBKBgFo2AU4AIAhylHKmngFTIAAAAASUVORK5CYII=","orcid":"","institution":"Jilin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Wenhe","middleName":"","lastName":"Zhu","suffix":""},{"id":459030803,"identity":"4ade92ac-b92c-493b-a4da-259fba601f82","order_by":8,"name":"Zhigang Xie","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-05-16 01:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6676160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6676160/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83274840,"identity":"9c72bdbc-0dea-434b-8a8f-e728a2f80ed8","added_by":"auto","created_at":"2025-05-22 08:48:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214633,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of (\u003cstrong\u003eA\u003c/strong\u003e) CZ and (\u003cstrong\u003eB\u003c/strong\u003e) CZM NPs. SEM images of (\u003cstrong\u003eC\u003c/strong\u003e) CZ and (\u003cstrong\u003eD\u003c/strong\u003e) CZM NPs. (\u003cstrong\u003eE\u003c/strong\u003e) HAADF-STEM image and elemental mapping for CZM NPs. (\u003cstrong\u003eF\u003c/strong\u003e) EDS spectrum of CZM NPs. (\u003cstrong\u003eG\u003c/strong\u003e) Size distribution of prepared NPs. (\u003cstrong\u003eH\u003c/strong\u003e) Changes of hydrodynamic diameter of CZM NPs in water and in PBS with FBS (10%). (\u003cstrong\u003eI\u003c/strong\u003e) XPS spectrum of CZM NPs. (\u003cstrong\u003eJ\u003c/strong\u003e) PXRD of ZIF-8, CZ, CZM and simulated ZIF-8. (\u003cstrong\u003eK\u003c/strong\u003e) TGA curves of ZIF-8 and CZ NPs.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/f25b238f7a0240c0d59aeed7.jpeg"},{"id":83274844,"identity":"9b223658-3ab9-4df8-85bf-92a21e39a9b2","added_by":"auto","created_at":"2025-05-22 08:48:29","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":211725,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) UV-vis absorption spectra and photo (inset) of MB degradation by Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reaction under varying conditions. (\u003cstrong\u003eB\u003c/strong\u003e) MB degradation by Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reaction at varying concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (\u003cstrong\u003eC\u003c/strong\u003e) MB degradation by Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reaction at varying concentrations of GSH. (\u003cstrong\u003eD\u003c/strong\u003e) MB degradation by CZM NPs-induced Fenton-like reaction at varying concentrations of GSH. In (A)-(D): [HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e] = 25 mM, [MB] = 25 μg mL\u003csup\u003e−1\u003c/sup\u003e, [Mn\u003csup\u003e2+\u003c/sup\u003e] = 0.5 mM, [H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e] = 8 mM. (\u003cstrong\u003eE\u003c/strong\u003e) UV-vis absorbance spectra of DTNB with varying concentrations of GSH. (\u003cstrong\u003eF\u003c/strong\u003e) Detection of GSH consumption by varying concentrations of CZM NPs at specific time points. In (E)-(F): [HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e] = 25 mM, [DTNB] = 50 μM, [GSH] = 50 μM. (\u003cstrong\u003eG\u003c/strong\u003e) CA release profile of CZM NPs under different conditions. (\u003cstrong\u003eH\u003c/strong\u003e) Generation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e under different conditions detected by TiOSO\u003csub\u003e4\u003c/sub\u003e (1.TiOSO\u003csub\u003e4\u003c/sub\u003e, 2.O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•−\u003c/sup\u003e+TiOSO\u003csub\u003e4\u003c/sub\u003e, 3-5.O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•−\u003c/sup\u003e+TiOSO\u003csub\u003e4\u003c/sub\u003e+CA (125, 250, 500 μM CA)). (\u003cstrong\u003eI\u003c/strong\u003e) Dissolved O\u003csub\u003e2\u003c/sub\u003e concentrations with varying concentrations of CZM NPs.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/d1aded11b53826ae5135f3aa.jpeg"},{"id":83275519,"identity":"450d7817-8876-42b8-a1ea-853fa00895f5","added_by":"auto","created_at":"2025-05-22 08:56:28","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231873,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) CLSM images of 4T1 cells incubated with CZM NPs ([CA] = 50 μg mL\u003csup\u003e−1\u003c/sup\u003e) at 37\u003csup\u003eo\u003c/sup\u003eC for different time. Scale bars: 20 μm. (\u003cstrong\u003eB\u003c/strong\u003e) Intercellular concentration of GSH after treatment with various concentrations of ZM NPs for 24 h. (\u003cstrong\u003eC\u003c/strong\u003e) Intercellular concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e after treatment with various concentrations of CA for 24 h. (\u003cstrong\u003eD\u003c/strong\u003e) Detection of intracellular ROS generation by DCFH-DA ([CA] = 50 μg mL\u003csup\u003e−1\u003c/sup\u003e, [Mn] = 75 μg mL\u003csup\u003e−1\u003c/sup\u003e). Scale bars: 20 μm. (\u003cstrong\u003eE\u003c/strong\u003e) Cell viabilities of 4T1 cells after treatment with different samples for 24 h. (\u003cstrong\u003eF\u003c/strong\u003e) Cell viabilities of 293T cells after treatment with different concentrations of CZM NPs. (\u003cstrong\u003eG\u003c/strong\u003e) Flow cytometry analysis of 4T1 cells after treatment with different samples ([CA] = 50 μg mL\u003csup\u003e−1\u003c/sup\u003e, [Mn] = 75 μg mL\u003csup\u003e−1\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/aec887b2dec65decb27fad0f.jpeg"},{"id":83275522,"identity":"65744a08-c8f6-49d8-b616-3d46cbdd739b","added_by":"auto","created_at":"2025-05-22 08:56:29","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144380,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence images of (\u003cstrong\u003eA\u003c/strong\u003e) HMGB1 and (\u003cstrong\u003eB\u003c/strong\u003e) CRT in 4T1 cells with different treatments. (\u003cstrong\u003eC\u003c/strong\u003e) Detection of extracellular ATP content by ATP assay kit. (\u003cstrong\u003eD\u003c/strong\u003e) Expression levels of CD86 on macrophages after cultured with different treatments. The levels of cytokines of (\u003cstrong\u003eE\u003c/strong\u003e) TNF-α and (\u003cstrong\u003eF\u003c/strong\u003e) IL-12 released after different stimulations.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/ff770deca01d71b886a01415.jpeg"},{"id":83274843,"identity":"f377bb18-5ddc-4f32-9503-7cadb162ccd2","added_by":"auto","created_at":"2025-05-22 08:48:28","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54618,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Volcano plots to determine DEGs of CZM NPs treatment group and control group. The x-axis represents the log 2.0-fold changes (FCs) of genes and the y-axis represents the -log10 of the p-values for the various condition pairs. Each dot represents a gene. The gray points represent a non-statistically significant difference in gene expression. The red field represents the upregulated genes and the green field represents the downregulated genes. (\u003cstrong\u003eB\u003c/strong\u003e) Heat map displaying the overview of the differentially expressed genes induced by CZM NPs treatment.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/e0f42363f5db8fb0caa535fb.jpeg"},{"id":83274853,"identity":"0bb8e594-6c2e-409c-9ab9-10a489bf22fe","added_by":"auto","created_at":"2025-05-22 08:48:29","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112955,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG pathway functional enrichment analysis of DEGs in CZM NPs treated 4T1 cells. (\u003cstrong\u003eA\u003c/strong\u003e) Cellular components. (\u003cstrong\u003eB\u003c/strong\u003e) Biochemical processes. (\u003cstrong\u003eC\u003c/strong\u003e) Molecular function. (\u003cstrong\u003eD\u003c/strong\u003e) KEGG pathway functional enrichment of DEGs.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/30251f148b675f7eaa8ca00e.jpeg"},{"id":83275749,"identity":"76d19f2c-8516-4636-b63e-132418ef6090","added_by":"auto","created_at":"2025-05-22 09:04:29","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":69582,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Apoptosis-related protein expressions of Bcl-2, Bax, and cleaved caspase-3 were analyzed using western blotting and (\u003cstrong\u003eB\u003c/strong\u003e) quantified in relation to β-actin. (\u003cstrong\u003eC\u003c/strong\u003e) NF-κB signaling pathway relative protein expressions of p65 and IκBα were analyzed and (\u003cstrong\u003eD\u003c/strong\u003e) quantified in relation to β-actin. Densitometric values were normalized by β-actin and expressed as mean ± SD, n = 3. Statistical significance: *p \u0026lt; 0.05, and **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/cedbc99f8c26ffbd570cd6ec.jpeg"},{"id":83275754,"identity":"a155b753-4692-4bc1-8bb3-21907667f470","added_by":"auto","created_at":"2025-05-22 09:04:29","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":184329,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo therapeutic efficacy evaluation. (\u003cstrong\u003eA\u003c/strong\u003e) DCFH-DA staining images of tumor tissue sections obtained from tumor-bearing mice with various treatments. (\u003cstrong\u003eB\u003c/strong\u003e) Tumor growth curves and (\u003cstrong\u003eC\u003c/strong\u003e) tumor weights of 4T1 breast tumor-bearing mice after different treatments. (\u003cstrong\u003eD\u003c/strong\u003e) Photographs of excised tumors after the last treatment. (\u003cstrong\u003eE\u003c/strong\u003e) Body weight changes of tumor-bearing mice in each group during treatments. (\u003cstrong\u003eF\u003c/strong\u003e) Representative images of tumor tissue sections after H\u0026amp;E and TUNEL staining. Scale bars: 50 μm. Data are expressed as mean ± SD (n\u003cem\u003e \u003c/em\u003e= 3). Statistical significance: **p\u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/469327fdcebf14f3e2de06aa.jpeg"},{"id":83274856,"identity":"cdced999-17fd-4875-a321-1e9e1eaf7544","added_by":"auto","created_at":"2025-05-22 08:48:29","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":123894,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunohistochemical staining of HMGB1 and CRT in tumor tissues. (\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence staining of M1 macrophage marker CD86 and M2 macrophage marker CD163 in tumor tissues. Cytokine levels of (\u003cstrong\u003eC\u003c/strong\u003e) TNF-α, (\u003cstrong\u003eD\u003c/strong\u003e) IFN-γ, and (\u003cstrong\u003eE\u003c/strong\u003e) IL-10 in tumor tissues.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/d9cd1ae9bc55a275923b3116.jpeg"},{"id":83276893,"identity":"491d373a-0ab5-4abb-b92b-52252c5e3cde","added_by":"auto","created_at":"2025-05-22 09:20:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2154421,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/f0e20b7c-6032-4826-9ddf-6f5b156acd90.pdf"},{"id":83274851,"identity":"b4249c05-d8bc-4966-9b64-d66d4c170cb1","added_by":"auto","created_at":"2025-05-22 08:48:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":668564,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/b502c4752266a9a0bd6ef6be.docx"},{"id":83276603,"identity":"7a3aa623-9dee-489c-bc49-7579fd565ef6","added_by":"auto","created_at":"2025-05-22 09:12:29","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":113053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003eSchematic illustration of a) the preparation of CZM NPs and b) their application for cascade-amplified CDT and immunotherapy.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6676160/v1/f028b11e3b3d042e5318ce75.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor Microenvironment-Responsive CA@ZIF-8/MnO 2 Nanoreactor for Self-Reinforcing Cascade Chemodynamic Therapy and Immunomodulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to WHO statistics, cancer continues to be the leading cause of mortality worldwide, with its treatment remaining a central focus in medical research [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Chemodynamic therapy (CDT), a novel cancer treatment paradigm [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], leverages endogenous hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to produce hydroxyl radicals (\u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH), the most harmful reactive oxygen species (ROS) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This process is facilitated through transition metal ion-mediated (e.g., Fe\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e) Fenton or Fenton-like reactions, thereby achieving the effect of tumor cell destruction [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast to conventional therapeutic approaches, CDT offers enhanced tumor selectivity, spatial and temporal controllability, minimal invasiveness, and reduced systemic toxicity [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Despite its promising potential, the advancement of CDT is confronted with several challenges [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Notably, the overexpression of glutathione (GSH) poses a significant obstacle [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As a critical component of the robust ROS scavenging system, GSH is upregulated (approximately 10 mM) in cancer cells to counteract the elevated ROS levels resulting from abnormal proliferation and heightened cellular respiration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This upregulation assists in maintaining redox homeostasis; however, it concurrently leads to the rapid depletion of generated \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH before it can exert its cytotoxic effects, thereby augmenting cancer cell resistance to oxidative stress and diminishing the therapeutic efficacy of CDT [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, despite the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration (approximately 100 \u0026micro;M) in the tumor microenvironment (TME) being significantly higher than that in normal tissues [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is still insufficient to produce a sufficient amount of \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH to achieve satisfactory therapeutic outcomes, presenting another obstacle for CDT [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Consequently, it is urgent yet challenging to develop a nanoplatform with both self-supplying H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and self-consuming GSH functionalities to enhance the efficacy of CDT.\u003c/p\u003e \u003cp\u003eIn recent years, manganese dioxide (MnO\u003csub\u003e2\u003c/sub\u003e) has emerged as a promising candidate for CDT nanoplatforms, attracting considerable research interest [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Primarily, the MnO\u003csub\u003e2\u003c/sub\u003e nanostructure exhibits unique redox reactivity with GSH, producing Mn\u003csup\u003e2+\u003c/sup\u003e ions that display exceptional Fenton-like catalytic activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This enables efficient conversion of endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into highly cytotoxic \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Notably, this GSH-triggered degradation mechanism simultaneously reduces intracellular antioxidant defenses by depleting overexpressed tumor GSH, thereby minimizing \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH scavenging and establishing MnO\u003csub\u003e2\u003c/sub\u003e as an effective GSH-depleting agent [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, the GSH-responsive decomposition of MnO\u003csub\u003e2\u003c/sub\u003e nanostructures facilitates controlled drug release through gatekeeper-mediated payload delivery systems [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Concurrently, MnO\u003csub\u003e2\u003c/sub\u003e demonstrates catalase-like activity by decomposing excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into water and oxygen molecules, effectively ameliorating tumor hypoxia, a critical barrier in conventional cancer therapies [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Emerging evidence suggests that MnO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress elevation not only triggers immunogenic cell death (ICD) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] but also promotes phenotypic reprogramming of tumor-associated macrophages (TAMs) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Specifically, the substantial ROS generation facilitates polarization shift from pro-tumoral M2 macrophages to antitumor M1 variants [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Complementarily, the liberated Mn\u003csup\u003e2+\u003c/sup\u003e ions have been shown to independently induce macrophage M1 polarization through distinct signaling pathways [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This dual mechanism synergistically modulates the immunosuppressive TME, enhancing antitumor immunity through coordinated oxidative stress amplification and immune cell reprogramming.\u003c/p\u003e \u003cp\u003eAs previously mentioned, the efficacy of CDT is significantly influenced by the limited endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels, making it crucial to incorporate the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying capability into the nanosystem. Chlorogenic acid (CA), a prominent bioactive compound abundantly found in coffee, has been demonstrated to exhibit a wide range of pharmacological activities, including antibacterial, anti-inflammatory, and antitumor effects [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Notably, CA possesses a unique self-oxidation property, enabling it to efficiently convert superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This intrinsic H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-generating capability holds great potential for enhancing the therapeutic outcomes of CDT. Additionally, recent studies have highlighted that CA can also act as a promising immunomodulator by promoting M1-type macrophages and inhibiting the M2-phenotype [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Despite these advantages, the clinical application of CA is hindered by its inherent limitations, such as poor water solubility, short half-life, and low stability [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Therefore, maintaining high levels of intracellular catalysts (Mn\u003csup\u003e2+\u003c/sup\u003e) and reactants (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), while reducing the content of ROS scavengers (GSH), can be accomplished by incorporating MnO\u003csub\u003e2\u003c/sub\u003e nanostructures and encapsulating CA into the nanocomplex.\u003c/p\u003e \u003cp\u003eIn light of the aforementioned considerations, this study presents the rational design and successful fabrication of a TME-responsive multifunctional nanoreactor designated as CA@ZIF-8/MnO\u003csub\u003e2\u003c/sub\u003e (CZM), which integrates self-consuming GSH and self-supplying H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e capabilities for dual-enhanced CDT (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Central to this design is the implementation of zeolitic imidazolate framework-8 (ZIF-8), a representative material from the metal-organic framework (MOF) family, which serves dual functions: functioning as a nanocarrier for CA encapsulation while enabling direct surface deposition of MnO\u003csub\u003e2\u003c/sub\u003e nanoshells. Following cellular internalization of CZM NPs by tumor cells, the MnO\u003csub\u003e2\u003c/sub\u003e outer layer undergoes tumor-specific conversion into Mn\u003csup\u003e2+\u003c/sup\u003e ions through catalytic interaction with endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and simultaneously depletes intracellular antioxidant GSH reserves to mitigate \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH scavenging, thereby potentiating CDT efficacy. Notably, this transformation concurrently induces ICD and promotes antitumor immune responses. Complementarily, the inner ZIF-8 framework undergoes rapid biodegradation under weakly acidic tumor conditions, enabling controlled release of encapsulated therapeutic agents. The released CA, exhibiting SOD-like activity, catalyzes O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e conversion to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, thereby establishing a self-amplifying cycle that elevates intratumoral H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations and augments subsequent CDT performance. Significantly, CZM NPs demonstrated the capacity to promote macrophage polarization toward antitumor M1 phenotype. Comprehensive in vitro and in vivo evaluations confirmed the synergistic cooperation of all functional components within CZM NPs, with the \u0026ldquo;MnO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;CA loading\u0026rdquo; combination demonstrating superior CDT enhancement through a \u0026ldquo;1\u0026thinsp;+\u0026thinsp;1\u0026thinsp;\u0026gt;\u0026thinsp;2\u0026rdquo; therapeutic synergy. To elucidate the underlying molecular mechanisms, we employed RNA-seq coupled with bioinformatics analysis and western blot validation, providing systematic investigation of tumor cell gene expression profiles under CZM NPs treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003ePreparation and\u0026nbsp;characterization of\u0026nbsp;CZM NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCZM NPs were prepared using a facile and environmentally friendly one-pot method. Briefly, two aqueous solutions, one containing Zn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO and CA (solution A) and the other containing 2-MIM (solution B), were prepared in advance and then mixed under vigorous stirring. After several minutes of stirring, a light yellow emulsion formed, indicating the successful synthesis of CZ (CA@ZIF-8) NPs. Subsequently, MnO\u003csub\u003e2\u003c/sub\u003e nanosheets were easily decorated onto the surface of the CZ NPs by mixing the prepared NPs with an excess amount of permanganate, yielding CZM NPs (Fig. S1). The structure and surface morphology of the two samples were characterized using transmission electron microscopy (TEM) (Fig. 1A and B) and scanning electron microscopy (SEM) (Fig. 1C and D). Both CZ and CZM NPs exhibited three-dimensional dodecahedral structures, with a distinct MnO\u003csub\u003e2\u003c/sub\u003e layer visible on the surface of CZM NPs. The energy-dispersive spectroscopy (EDS) spectrum of CZM indicated the simultaneous presence of Zn and Mn elements (Fig. 1F), and the corresponding elemental mapping images further validated the uniform distribution of these elements (Fig. 1E). The hydrated particle sizes of these NPs were determined using dynamic light scattering (DLS). The CZM NPs exhibited a narrow size distribution (116.6 \u0026plusmn; 2.7 nm), slightly larger than that of CZ NPs (100.7 \u0026plusmn; 5.2 nm), which can be attributed to the MnO\u003csub\u003e2\u003c/sub\u003e coating (Fig. 1G). Additionally, DLS analysis demonstrated the favorable physiological stability of both CZ and CZM NPs, suggesting their potential for biomedical applications (Fig. 1H and S2). To further analyze the chemical composition and crystal phase of CZM NPs, X-ray photoelectron spectroscopy (XPS) and X-ray diffractometry (XRD) were employed. The XPS survey spectrum revealed the presence of Zn, Mn, O, N, and C elements in the nanocatalyst (Fig. 1I). The high-resolution Mn 2p spectrum (Fig. S3) displayed two peaks at 653.7 eV and 642.2 eV, corresponding to Mn 2p\u003csub\u003e1/2\u003c/sub\u003e and Mn 2p\u003csub\u003e3/2\u003c/sub\u003e, respectively, and these binding energies are consistent with those of manganese in MnO\u003csub\u003e2\u003c/sub\u003e, confirming the successful coating of MnO\u003csub\u003e2\u003c/sub\u003e on the NPs. Furthermore, the powder X-ray diffraction (PXRD) patterns of the as-synthesized ZIF-8, CZ, and CZM NPs (Fig. 1J) matched well with the simulated spectrum of ZIF-8, indicating that the drug loading and MnO\u003csub\u003e2\u003c/sub\u003e coating had negligible effects on the structural integrity of the NPs. Thermogravimetric analysis (TGA) was used to quantify the CA loading content. As shown in Fig. 1K, a significant weight loss difference was observed between the ZIF-8 and CZ NPs, attributed to the removal of encapsulated CA molecules. The drug loading capacity and encapsulation efficiency of CA were calculated to be approximately 11.8% and 69.6%, respectively, consistent with the results obtained from UV-vis spectroscopy (Fig. S4). Finally, UV-vis spectroscopy further confirmed the successful synthesis of CZM NPs (Fig. S5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro evaluation of ROS generation, GSH depletion, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe successful fabrication of the nanoreactor prompted us to explore its multifunctional capabilities (Fig. S6). As is well-established, iron-triggered Fenton chemistry is widely utilized to induce apoptosis in tumor cells by catalyzing endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH, and Mn\u003csup\u003e2+\u003c/sup\u003e can achieve CDT by generating \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH through Fenton-like reaction [48,49]. To investigate the \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH-generating activity and the scavenging effect of GSH on \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH, we initially utilized methylene blue (MB), a dye susceptible to degradation by \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH, as an indicator. As depicted in Fig. 2A, a significant decrease in absorbance was observed when MB was incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e in the presence of NaHCO\u003csub\u003e3\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e, whereas negligible change in MB absorbance was detected in other solutions lacking HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, underscoring the pivotal role of bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) in the Mn\u003csup\u003e2+\u003c/sup\u003e-driven Fenton-like reaction. Concurrently, Fig. 2B illustrated the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration-dependent degradation profile of MB. As the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration increased, the absorbance of MB diminished, accompanied by a gradual transition in solution color from blue to transparent, indicative of enhanced \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH generation. Notably, GSH, a ROS scavenger, can neutralize \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH and thereby compromise the efficacy of ROS-based therapies. The overexpression of GSH in cancer cells directly impedes CDT efficiency. Depleting intracellular GSH levels can augment the effectiveness of ROS-based therapies. In contrast to Mn\u003csup\u003e2+\u003c/sup\u003e, MnO\u003csub\u003e2\u003c/sub\u003e can initially consume GSH to yield Mn\u003csup\u003e2+\u003c/sup\u003e, thereby exhibiting a greater potential for \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH production. Fig. 2C corroborated that increasing GSH concentrations attenuated the efficiency of the Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reaction. Conversely, in the context of CZM NPs-mediated Fenton-like catalysis, the inhibitory influence of GSH was markedly mitigated, with enhanced MB degradation observed across varying GSH concentrations (Fig. 2D). This phenomenon arises because, in physiological environments, GSH preferentially reacts with MnO\u003csub\u003e2\u003c/sub\u003e to generate Mn\u003csup\u003e2+\u003c/sup\u003e, enabling CZM NPs with augmented GSH-depleting capacity to effectively counteract this process and promote MB degradation. Subsequently, we evaluated the GSH depletion capability of CZM NPs using 5,5\u0026prime;-dithiobis(2-nitrobenzoic acid) (DTNB) as an indicator. As shown in Fig. 2E, pure DTNB exhibited a characteristic absorption peak at 325 nm, while all GSH + DTNB groups displayed a new absorption peak at 412 nm, primarily attributed to the reduction reaction between DTNB and the thiol groups of GSH (Fig. S7). Importantly, we observed a reduction in GSH levels in the CZM + GSH + DTNB group (Fig. 2F), as the outer MnO\u003csub\u003e2\u003c/sub\u003e layer of CZM NPs demonstrated exceptional responsiveness to GSH. And the characteristic absorbance peak at 412 nm decreased significantly with increasing CZM concentrations and prolonged reaction times. These findings suggest that CZM NPs can function as potent GSH-depleting agents, modulating intracellular redox homeostasis.\u003c/p\u003e\n\u003cp\u003eGiven the unique structural configuration of the MnO\u003csub\u003e2\u003c/sub\u003e shell and ZIF-8 core, we hypothesized that the nanoreactor would exhibit pH- and GSH-responsive characteristics. To validate this hypothesis, we systematically investigated the drug release profiles of CZM NPs under various simulated TME conditions, including different pH values (7.4 and 5.5) with or without GSH. As demonstrated in Fig. 2G, cumulative CA release reached 35.9% over 48 hours under acidic conditions (pH 5.5), while minimal release (5.6%) was observed at physiological pH (7.4), confirming the pH-responsive nature of the ZIF-8 framework. Notably, the release kinetics accelerated significantly (52.7% cumulative release) in acidic medium containing GSH compared to the equivalent pH condition without GSH (35.9%), highlighting the dual-gating mechanism mediated by the GSH-responsive properties of the MnO\u003csub\u003e2\u003c/sub\u003e shell and the pH sensitivity inherent to ZIF-8. These results collectively demonstrate that CZM NPs possess TME-specific responsiveness, enabling intelligent drug release while maintaining intrinsic H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying capabilities. Subsequently, we confirmed the ability of CA molecules to catalyze the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and O\u003csub\u003e2\u003c/sub\u003e through their self-oxidation pathway. Using a TiOSO\u003csub\u003e4\u003c/sub\u003e-based colorimetric assay, which forms a light-yellow peroxo-titanium complex (\u0026lambda;max = 415 nm) upon H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e interaction, we quantitatively measured the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. As demonstrated in Fig. 2H, substantial H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production was observed following CA administration, indicating that CA compounds exhibit intrinsic H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying capacity and can efficiently convert excessive O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e within neoplastic cellular environments. This conversion is particularly advantageous given that malignant cells typically exhibit 3-5 fold higher O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e levels compared to normal cells, owing to their hypermetabolic state and dysfunctional mitochondrial respiration. Complementarily, it is widely recognized that MnO\u003csub\u003e2\u003c/sub\u003e can serve as an effective catalyst for the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into O\u003csub\u003e2\u003c/sub\u003e. As shown in Fig. 2I, dissolved oxygen levels remained negligible in control groups lacking MnO\u003csub\u003e2\u003c/sub\u003e, whereas CZM-containing solutions exhibited rapid oxygen generation proportional to CZM concentration. This oxygen generation not only confirms the catalytic proficiency of MnO\u003csub\u003e2\u003c/sub\u003e but also establishes a positive feedback loop that enhances the subsequent self-oxidation process of CA. Collectively, these sequential catalytic events bestow upon CZM NPs multifunctional capabilities, including ROS amplification, GSH depletion, and self-supplying H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, significantly enhancing the efficacy of CDT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnhanced CDT efficacy of CZM NPs in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEffective intracellular delivery of nanocomposites is a prerequisite for achieving optimal antitumor efficacy, thus 4T1 murine breast cancer cells were employed to evaluate the phagocytic capacity of CZM NPs. For fluorescence tracking, CA molecules and Nile Red (NR) were co-encapsulated within the ZIF-8 framework. Confocal laser scanning microscopy (CLSM) images (Fig. 3A and S8) revealed distinct red fluorescence signals localized in the cytoplasm, with fluorescence intensity gradually enhancing as both incubation time and NPs concentration increased. This demonstrates time- and dose-dependent cellular internalization of CZM NPs. Following cellular uptake, the MnO\u003csub\u003e2\u003c/sub\u003e shell undergoes GSH-responsive dissociation, combined with the slightly acidic TME, effectively depletes the overexpressed intratumoral GSH to generate Mn\u003csup\u003e2+\u003c/sup\u003e ions and trigger CA release. This cascade establishes a self-amplifying system characterized by GSH depletion, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying, and dual-enhanced CDT. Subsequent investigations validated the GSH-depleting capacity of MnO\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-generating property of CA at the cellular level. As anticipated, ZM NPs treatment significantly reduced intracellular GSH levels (Fig. 3B), while CA incubation induced dose-dependent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation (Fig. 3C). Intracellular ROS generation was quantitatively assessed using 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Fig. 3D and S9 demonstrated negligible green fluorescence in control and CA-only groups, whereas the ZM group exhibited characteristic fluorescence attributed to the Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reactions. Notably, the CZM group displayed the most intense fluorescence signal, attributable to CA-driven H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying coupled with MnO\u003csub\u003e2\u003c/sub\u003e-mediated GSH depletion, demonstrating the self-reinforcing CDT characteristics of CZM NPs.\u003c/p\u003e\n\u003cp\u003eGiven the exceptional \u003cstrong\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e\u003c/strong\u003eOH generation capability, the in vitro cytotoxicity assessment was subsequently conducted utilizing the conventional methyl thiazolyl tetrazolium (MTT) assay. In comparison to the control group, ZIF-8 exhibited high cell viability, even at relatively elevated concentrations (Fig. 3E), thereby indicating the favorable cytocompatibility of the synthesized nanocarrier. In contrast, both CA-loaded CZ NPs and MnO\u003csub\u003e2\u003c/sub\u003e-coated ZM NPs exhibited dose-dependent cytotoxicity through standalone chemotherapy or CDT, respectively. Remarkably, CZM NPs demonstrated superior cytotoxicity compared to monofunctional counterparts at equivalent CA concentrations, attributable to synergistic catalytic cascade reactions. Importantly, CZM NPs showed negligible toxicity towards 293T normal cells (Fig. 3F), indicating tumor-specific therapeutic potential. To elucidate apoptosis mechanisms, Annexin V-FITC/PI dual staining flow cytometry was performed. Quantitative analysis (Fig. 3G) revealed significantly higher apoptotic rates in the CZM group (21.36%) versus CA (7.79%) and ZM (11.43%) groups, further substantiating the therapeutic superiority of the self-amplifying CDT system. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICD induction and macrophage polarization by CZM NPs in vitro\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCDT has been widely recognized for its ability to trigger ICD in tumor cells, leading to the generation of endogenous damage-associated molecular patterns (DAMPs) [38]. These DAMPs subsequently stimulate antitumor immune responses by acting as \u0026ldquo;eat me\u0026rdquo; and \u0026ldquo;find me\u0026rdquo; signals, which are critical for antigen presentation [37,39]. To investigate the ICD-inducing potential of CZM NPs in tumor cells, we examined the associated DAMPs, as evidenced by the translocation of CRT, the nuclear export of HMGB1, and the extracellular release of ATP [41]. Immunofluorescence analysis was performed to evaluate HMGB1 localization after various treatments. As presented in Fig. 4A, the CZM NPs group exhibited the least green fluorescence intensity, indicating a rapid and efficient release of HMGB1 from 4T1 cells. Similarly, CRT translocation has also been revealed by CLSM. The CZM NPs group displayed intense green fluorescence at the cell membrane, while the CA and ZM NPs groups showed weaker signals (Fig. 4B). This suggests that CZM NPs treatment can significantly enhance CRT exposure on the cell surface, thereby promoting immune activation. Additionally, extracellular ATP levels were quantified using an ATP detection kit. The CZM NPs group demonstrated a remarkable increase in ATP release, with concentrations 2.9-fold and 2-fold higher than those in the CA and ZM NPs groups, respectively (Fig. 4C). This elevated ATP secretion is crucial for recruiting immature dendritic cells and enhancing immune surveillance. Collectively, these findings underscore the robust ICD-inducing capability of CZM NPs. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe polarization of TAMs plays a pivotal role in modulating their antitumor functions. M1 macrophages are known for their antimicrobial and immunostimulatory properties, whereas M2 macrophages suppress T-cell activity and facilitate tumor progression, invasion, and metastasis. In this study, RAW264.7 macrophages were used to evaluate phenotypic polarization. Flow cytometry analysis revealed a significant upregulation of CD86, a marker for M1 macrophages, in the CZM NPs group compared to other treatments (Fig. 4D). Furthermore, cytokine profiling via quantitative PCR confirmed this polarization, showing that macrophages treated with CZM NPs secreted the highest levels of TNF-\u0026alpha; and IL-12, both hallmark M1 cytokines, compared to the CA and ZM NPs groups (Fig. 4E and F). Together, these results demonstrate that CZM NPs effectively induce ICD in tumor cells and promote M1 macrophage polarization, thereby reshaping the immunosuppressive tumor microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntitumor molecular mechanism of CZM NPs\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the antitumor mechanism of CZM NPs, we employed RNA sequencing (RNA-seq) to analyze gene expression profiles in tumor cells following treatment. After ensuring the quality and integrity of RNA extracted from both CZM NPs-treated (NPs) and untreated (Con) groups, principal component analysis (PCA) was performed on the complete dataset to assess clustering patterns. As shown in Fig. S10, PCA results revealed clear separation between the two groups, indicating distinct transcriptional profiles and confirming the significant impact of CZM NPs on gene expression. Subsequently, differentially expressed genes (DEGs) were identified by comparing RNA-seq data from treated and untreated cells, with results visualized using volcano plots and heatmaps (Fig. 5). A total of 2,496 DEGs were identified, comprising 1,247 up-regulated and 1,249 down-regulated genes, highlighting the profound regulatory influence of CZM NPs on tumor cell transcription.\u003c/p\u003e\n\u003cp\u003eBased on the RNA-seq findings, Gene Ontology (GO) and pathway enrichment analyses were conducted to explore the functional roles of the identified DEGs. GO analysis categorized the DEGs into three main domains: cellular component (CC), biological process (BP), and molecular function (MF). Within the CC domain, significant enrichment was observed for terms related to cytoplasm, cytosol, cytosolic ribosome, and nucleoplasm (Fig. 6A). In the BP domain, key processes such as translation, positive regulation of cell migration, cellular response to lipopolysaccharide and response to drug were prominently affected (Fig. 6B). For the MF domain, protein binding, structural constituents of ribosomes, ubiquitin protein ligase binding and translation regulator activity were the most significantly altered functions (Fig. 6C).\u003c/p\u003e\n\u003cp\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that CZM NPs modulated a variety of signaling pathways. Notably, the IL-17 signaling pathway, TNF signaling pathway, Ribosome and Oxidative phosphorylation were among the top 20 enriched pathways (Fig. 6D). Further KEGG pathway classification (Fig. S11) revealed notable associations between DEGs and critical signaling cascades including MAPK, Nuclear factor-\u0026kappa;B (NF-\u0026kappa;B), and p53-mediated pathways. This multi-pathway regulation pattern suggests comprehensive immunomodulatory and pro-apoptotic effects of CZM NPs on tumor cells.\u003c/p\u003e\n\u003cp\u003eMechanistically, the NF-\u0026kappa;B transcription factor complex, which consists of p50, p65, and I\u0026kappa;B\u0026alpha; subunits, functions as a pivotal regulator of inflammatory responses and immune homeostasis. Its canonical activation pathway involves the phosphorylation-dependent degradation of I\u0026kappa;B\u0026alpha;, mediated by the multimeric I\u0026kappa;B kinase (IKK) complex. Importantly, both IL-17 and TNF-\u0026alpha; signaling pathways converge on NF-\u0026kappa;B activation, thereby orchestrating transcriptional programs that govern cell survival and anti-apoptotic processes. Based on our RNA-seq findings and existing literature, we hypothesized that CZM NPs may mediate antitumor immunity and apoptosis by inhibiting the NF-\u0026kappa;B signaling pathway. Western blot validation confirmed this hypothesis (Fig. 7), demonstrating CZM NPs-induced upregulation of pro-apoptotic markers (Bax, cleaved caspase-3) concurrent with downregulation of anti-apoptotic Bcl-2 and phosphorylation events in both p65 and I\u0026kappa;B\u0026alpha;. Collectively, these findings established the suppression of NF-\u0026kappa;B signaling as a critical mechanism underlying the dual action of CZM NPs in remodeling the tumor microenvironment and inducing apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo antitumor efficacy evaluation of CZM NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuilding upon the promising in vitro antitumor characteristics of CZM NPs, we subsequently investigated their therapeutic potential in 4T1 tumor-bearing murine models. Mice were stratified into four cohorts (n=5): 1) PBS control, 2) free CA, 3) ZM NPs, and 4) CZM NPs, with groups 2-4 receiving equivalent CA or ZM doses via intravenous administration every 48 hours. To assess intracellular ROS levels within tumor tissues post-treatment, DCFH-DA staining was performed on tumor sections. Fig. 8A demonstrated that mice administered with CA exhibited weak green fluorescence signal compared to control group at 24 h post-injection, suggesting limited ROS production. In contrast, both ZM and CZM NPs groups displayed significantly enhanced DCFH fluorescence intensity, with the latter showing the most pronounced signal amplification. The therapeutic outcomes were subsequently monitored using identical treatment protocols. Fig. 8B illustrated that tumor volumes in the CA group showed modest reduction relative to controls, attributable to the intrinsic cytotoxicity of CA. Notably, ZM NPs administration resulted in substantial tumor growth inhibition, primarily mediated by MnO\u003csub\u003e2\u003c/sub\u003e shell-mediated Fenton-like reactions generating ROS. The CZM NPs cohort achieved superior tumor suppression, confirming the synergistic interplay between GSH depletion and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying for enhanced CDT. The similar conclusion can be drawn from the average weight of resected tumors in each group following the final measurement. As illustrated in Fig. 8C and D, both the weight and size of tumors in the CZM NPs group were significantly reduced, as evidenced by the macroscopic examination of the dissected tumor tissues across all cohorts. Histopathological validation through H\u0026amp;E and TUNEL staining revealed marked nuclear condensation, cytoplasmic leakage, and apoptotic cell accumulation in the CZM NPs group (Fig. 8F), substantiating its therapeutic efficacy. Importantly, all treatment groups maintained stable body weights throughout the 12-day regimen (Fig. 8E), indicating the negligible systemic toxicity of these treatments to mice. Furthermore, H\u0026amp;E-stained major organ sections (Fig. S12) showed no evidence of inflammatory infiltration or histopathological abnormalities, confirming the biocompatibility of all formulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate this CDT triggering antitumor immunity via ICD in vivo, we conducted immunocytochemistry staining assays to assess CRT exposure and HMGB1 release. Similar to the results in vitro, the CZM NPs-treated group exhibited markedly elevated CRT expression and robust HMGB1 release in tumor tissues compared to other groups (Fig. 9A), suggesting that CZM NPs treatment effectively triggered extensive ICD in vivo. Following this, we employed immunofluorescence to analyze the alterations in M1 and M2 phenotypes within the tumor, aiming to confirm the polarization capability of CZM NPs. Fig. 9B demonstrated a substantial increase in the CD86 fluorescence signal, indicative of M1 phenotype, in the CZM NPs-treated group relative to other groups. Conversely, the CD163 fluorescence signal, representing M2 phenotype, markedly reduced following CZM NPs treatment. Given that hallmark cytokines such as IFN-\u0026gamma; and TNF-\u0026alpha; are associated with the M1 phenotype, and IL-10 with the M2 phenotype, we further evaluated the TAMs polarization by detection of the cytokines collected from tumor. As depicted in Fig. 9C-E, the secretion of IL-10 in the tumor lysates from the CZM NPs-treated group was significantly reduced by 2.64-fold compared to the control group, while the levels of TNF-\u0026alpha; and IFN-\u0026gamma; were notably elevated, collectively indicating significant M2 to M1 polarization of TAMs in TME post CDT with CZM NPs.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we engineered a TME-responsive nanoreactor (CZM NPs) to overcome the intrinsic limitations of CDT through GSH-depleting, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying, and immunomodulation. The MnO\u003csub\u003e2\u003c/sub\u003e shell effectively scavenges GSH to generate Mn\u003csup\u003e2+\u003c/sup\u003e, amplifying Fenton-like reactions, while the released CA enables persistent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e supply through O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e-mediated conversion. This synergistic dual amplification strategy significantly enhances CDT efficacy both in vitro and in vivo. Importantly, CZM NPs could trigger ICD in tumor cells, promoting antitumor immune responses and polarizing TAMs from M2 to M1 phenotype, thus reshaping the immunosuppressive tumor microenvironment. Mechanistically, RNA-seq and bioinformatics analyses reveal NF-κB pathway inhibition and pro-apoptotic signaling activation as critical drivers of therapeutic efficacy. This study not only underscores the potential of CZM NPs as a promising nanoplatform for cascade-amplified CDT and immune regulation, but also provides a blueprint for designing multifunctional nanotherapeutics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAll the experimental procedures to mouse described herein have gained approval from the Ethics Committee of Jilin Medical University and carried out corresponding to the regulation, principles, and guidelines of Chinese law concerning the protection of animal life.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e accompanies this paper at https://doi.org/10. 1186/s129 51 -025-#####-#. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Development Project of Jilin Province (No.20240101202JC and No.20220204038YY), the National Natural Sciences Foundation of China (No.32101154).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article and its additional file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave their consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. 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Anal. 2010;51(3):685-90.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanoreactor, Cascade-amplified chemodynamic therapy, GSH depletion, H2O2 self-generation, Immune regulation","lastPublishedDoi":"10.21203/rs.3.rs-6676160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6676160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemodynamic therapy (CDT), which utilizes endogenous hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to generate hydroxyl radicals (\u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH) via Fenton-like reactions, faces critical limitations in clinical translation, including insufficient intratumoral H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels and glutathione (GSH)-mediated ROS scavenging. To address these challenges, we developed a tumor microenvironment (TME)-responsive nanoreactor, CA@ZIF-8/MnO\u003csub\u003e2\u003c/sub\u003e (CZM), integrating dual functionalities of GSH-depleting and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e self-supplying for cascade-amplified CDT. The ZIF-8 framework serves as a biodegradable carrier for chlorogenic acid (CA), which converts superoxide (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, while the MnO\u003csub\u003e2\u003c/sub\u003e shell depletes GSH to yield Mn\u003csup\u003e2+\u003c/sup\u003e, a Fenton-like catalyst. Upon internalization by tumor cells, the MnO\u003csub\u003e2\u003c/sub\u003e shell reacts with GSH to produce Mn\u003csup\u003e2+\u003c/sup\u003e, which catalyzes the conversion of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH, while simultaneously depleting GSH to enhance CDT efficacy. Additionally, the acidic TME triggers the release of CA, which elevates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels through its self-oxidation property, creating a self-reinforcing cycle. In vitro and in vivo studies demonstrated that CZM NPs not only enhance \u003csup\u003e\u003cb\u003e\u0026bull;\u003c/b\u003e\u003c/sup\u003eOH generation but also trigger immunogenic cell death (ICD), promoting antitumor immune responses. Furthermore, CZM NPs promote the polarization of tumor-associated macrophages towards the M1 antitumor phenotype, reshaping the immunosuppressive TME. RNA-seq and pathway analysis further revealed that CZM NPs modulate key signaling pathways, including NF-κB, to induce apoptosis and enhance antitumor immunity. Overall, these findings highlight the potential of CZM NPs as a multifunctional nanoplatform for cascade-amplified CDT and immunotherapy.\u003c/p\u003e","manuscriptTitle":"Tumor Microenvironment-Responsive CA@ZIF-8/MnO 2 Nanoreactor for Self-Reinforcing Cascade Chemodynamic Therapy and Immunomodulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 08:48:24","doi":"10.21203/rs.3.rs-6676160/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"210f3594-7f1a-4234-8c15-b7b5f60a62cb","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-22T08:48:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 08:48:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6676160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6676160","identity":"rs-6676160","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}