Ferroptosis Process Research Articles

CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1

Abstract Objectives CDGSH iron sulfur domain 2 (CISD2) is essential to maintain iron (Fe) and reactive oxygen species (ROS) homeostasis, and ferroptosis suppressor protein 1 (FSP1) can protect cells from ferroptosis by inhibiting lipid peroxidation. Here, we investigate the role and potential mechanism of CISD2 and FSP1 in ferroptosis of hepatocellular carcinoma (HCC). Methods Human HCC cells were exposed to ferroptosis inducer Erastin, and the expression changes of CISD2 and FSP1 during ferroptosis were detected. Subsequently, we investigated the effect of overexpression of CISD2 on ferroptosis and FSP1 expression in HCC cells. Finally, we also investigated the effect of overexpression of FSP1 on ferroptosis in HCC cells. Results Erastin induced ferroptosis in hepatoma cells, and HepG2 cells were sensitive to Erastin. In addition, it was found that the expression of CISD2 was significantly upregulated and the expression of FSP1 was significantly downregulated in Erastin treated HepG2 cells. Subsequently, CISD2 was found to be highly expressed in HCC tissues, and overexpression of CISD2 reversed ferroptosis induced by Erastin in HepG2 cells and upregulated the expression of FSP1. Meanwhile, FSP1 showed a low expression level in HCC tissues and cells, and overexpression of FSP1 could reverse the ferroptosis induced by Erastin in HepG2 cells. Conclusion CISD2 and FSP1 are involved in the ferroptosis process of HCC induced by Erastin. CISD2 protects against the ferroptosis of HCC induced by Erastin by upregulating the expression of FSP1.

GSH and H2O2 dynamic correlation in the ferroptosis pathways revealed by engineered probe in tumor and kidney injury

Ferroptosis is a cell death mode of lipid peroxidation caused by iron metabolism disorder and redox imbalance, which is closely associated with the evolution of tumour and ischemia reperfusion injury and other diseases, and it involves the excessive accumulation of reactive oxygen species promoting ferroptosis pathway and thiols-associated anti-ferroptosis pathway. To study the dynamic correlation between reactive oxygen species and active sulfur in the two pathways is of great significance for revealing the precise regulation mechanism of ferroptosis. Hydrogen peroxide (H2O2) and glutathione (GSH), as very active redox species in organisms, participate dynamically in the ferroptosis process. In this study, hemicyanine dye conjugated H2O2 specific response site were coupled with coumarin derivative containing GSH specific response site, realizing distinguishing simultaneous detection of H2O2 and GSH through near-infrared and cyan fluorescence emission after response. At the cellular level, the dynamic correlation between H2O2 and GSH in the ferroptosis pathway was explored, and the synergistic ferroptosis inducer was screened for further application at the tumor level, then monitoring the process of ferroptosis inducer used for tumor ablation and ferroptosis inhibitor used to alleviate kidney ischemia–reperfusion injury by fluorescence imaging.

Comprehensive monitoring of mitochondrial viscosity variation during different cell death processes by a NIR mitochondria-targeting fluorescence probe

Cell death is a fundamental feature of multicellular organisms, in which mitochondria play crucial roles. Therefore, revealing and monitoring the microenvironment of mitochondria are significant to investigate cell death process. Herein, the mitochondrial viscosity variation behaviors of a series of different cell death processes were monitored with a NIR mitochondria-targeting fluorescence probe FLV. FLV was designed based on a rotatable flavylocyanine fluorophore that presented selective and sensitive NIR fluorescence enhancement response with the increase of environmental viscosity. Fluorescence imaging experiments of living cells incubated with nystatin or under different temperature indicated that FLV was capable of imaging the change of mitochondrial viscosity. Finally, FLV was applied for monitoring the mitochondrial viscosity variation during different cell death processes. It was found that there were obvious mitochondrial viscosity increases during apoptosis, necrosis and autophagy; however, no detectable mitochondrial viscosity variation was observed in ferroptosis process incubated with ferroptosis inducer erastin or RSL3 for 6 h. These results demonstrated that FLV is a viable tool for monitoring the mitochondrial viscosity variation and is likely to be used in the diagnosis of the mitochondrial viscosity-associated cell processes and diseases.

Inhalable Biomineralized Liposomes for Cyclic Ca2+-Burst-Centered Endoplasmic Reticulum Stress Enhanced Lung Cancer Ferroptosis Therapy

Lung cancer with the highest mortality poses a great threat to human health. Ferroptosis therapy has recently been raised as a promising strategy for lung cancer treatment by boosting the reactive species (ROS) production and lipid peroxidation (LPO) accumulation intracellularly. However, the insufficient intracellular ROS level and the unsatisfactory drug accumulation in lung cancer lesions hamper the efficacy of ferroptosis therapy. Here, an inhalable biomineralized liposome LDM co-loaded with dihydroartemisinin (DHA) and pH-responsive calcium phosphate (CaP) was constructed as a ferroptosis nanoinducer for achieving Ca2+-burst-centered endoplasmic reticulum (ER) stress enhanced lung cancer ferroptosis therapy. Equipped with excellent nebulization properties, about 6.80-fold higher lung lesions drug accumulation than intravenous injection made the proposed inhalable LDM an ideal nanoplatform for lung cancer treatment. The Fenton-like reaction mediated by DHA with peroxide bridge structure could contribute to intracellular ROS production and induce ferroptosis. Assisted by DHA-mediated sarco-/endoplasmic reticulum calcium ATPase (SERCA) inhibition, the initial Ca2+ burst caused by CaP shell degradation triggered the Ca2+-mediated intense ER stress and subsequently induced mitochondria dysfunction to further boost ROS accumulation, which strengthens ferroptosis. The second Ca2+ burst occurred as a result of Ca2+ influx through ferroptotic pores on cell membranes, thus sequentially constructing the lethal "Ca2+ burst-ER stress-ferroptosis" cycle. Consequently, the Ca2+-burst-centered ER stress enhanced ferroptosis process was confirmed as a cell swelling and cell membrane disruption process driven by notable intracellular ROS and LPO accumulation. The proposed LDM showed an encouraging lung retention property and extraordinary antitumor ability in an orthotropic lung tumor murine model. In conclusion, the constructed ferroptosis nanoinducer could be a potential tailored nanoplatform for nebulization-based pulmonary delivery and underscore the application of Ca2+-burst-centered ER stress enhanced lung cancer ferroptosis therapy.

Characterization and role exploration of ferroptosis-related genes in osteoarthritis.

Osteoarthritis (OA), viewing as a degenerative aseptic inflammatory disease, is characterized by joint pain and inflammation that significantly affects the quality of patients' life, especially for the elder. Although rapid progress has been achieved in elucidating the underlying mechanisms of OA occurrence and progression, there is still a lack of effective clinical therapeutics for OA patients. Currently the most common treatments including drug therapy and surgical operations are not very satisfactory in majority of cases, so it is worthy to explore new remedies. During the past few decades, a number of novel forms of regulated cell death have been reported widely, typified by ferroptosis, with its prominent features including reactive oxygen species (ROS) elevation, lipid peroxidation, iron accumulation and glutathione deprivation. Our study was designed to identify the functional roles of differentially expressed ferroptosis-related genes in OA, which were screened out by referring to GEO database via bioinformatics analyses. Human chondrocytes were applied to validate the above findings in the scenario of ferroptosis inhibitors administration. Results partially proved the consistency with bioinformatics analyses that ATF3 and TFRC were highly expressed in interleukin-1β (IL-1β)-stimulated chondrocytes whereas CXCL2 and JUN were downregulated. Besides, TFRC was firstly validated to be upregulated in IL-1β-stimulated chondrocytes, which could be reversed by ferroptosis inhibitors. In conclusion, our study reported two prominent ferroptosis-related genes, ATF3 and TFRC are upregulated in IL-1β-stimulated chondrocytes while CXCL2 and JUN are downregulated. And preliminary results demonstrated that TFRC might serve as an accomplice of ferroptosis process in IL-1β-stimulated chondrocytes and ferroptosis inhibitors have the potential to inhibit ROS in IL-1β-stimulated chondrocytes.

Evaluation of viscosity in inflammation and ferroptosis process based on a bithiophene-containing hemicyanine dye with diminished solvatochromic effect

To decrease solvatochromic effect and keep a strong selective fluorescence response to the change of viscosity, we have designed and synthesized a novel mitochondria-targetable hemicyanine fluorescence probe BT-In for viscosity detection during inflammation and ferroptosis process. By using high electron-rich bithiophene as a low polar electron donor, and the electron-withdrawing indolium group as electron acceptor as well as mitochondrial targeting group, BT-In shows high selectivity and rapid response for viscosity in vitro and high fluorescence-on signal enhancement at 614 nm with a large Stokes shift of 112 nm. Bioimaging experiments display that the probe has good mitochondrial-targeting ability (Pearson's correlation coefficient 0.99) and high-contrast imaging ability for detecting viscosity in living systems.

A Multifunctional Nanoplatform for Tumor‐Targeted Imaging and Enhanced Ferroptosis Therapy

AbstractWith the advantages of ferroptosis therapy (FT) in solving the apoptotic resistance of tumors, a smart strategy based on the combination of glucose oxidase (GOx) and ferroptosis‐inducing nanoagents is proposed to boost the FT. However, most of the nanoagents remain limited in complex synthesis, poor targeting specificity, and unclarified biological mechanisms. Herein, a multifunctional nanoplatform based on fluorescent apoferritin‐gold nanoclusters combined with GOx (AFt‐AuNCs@GOx) is designed for tumor‐targeted imaging and FT. The results show that AFt‐AuNCs@GOx catalyze the generation of toxic ·OH from H2O2 supplied by GOx. Consequently, AFt‐AuNCs@GOx induce the enhanced ferroptosis effect in 4T1 cells. Additionally, the transcriptome assay of 4T1 cells identifies the differential expression genes related to ferroptosis process after AFt‐AuNCs@GOx treatment. Moreover, with the merit of intrinsic fluorescence, the biodistribution both in vitro and in vivo of AFt‐AuNCs@GOx can be effectively visualized. In vivo evidences reveal that AFt‐AuNCs@GOx have the desired efficacy of FT and excellent biosafety. Overall, the AFt‐AuNCs@GOx nanoplatform offer remarkable advantages in simple architecture, fluorescent imaging, precise targeting, and tumor inhibition. The exploration of the ferroptosis effect and biological mechanisms of AFt‐AuNCs@GOx, as well as its application in tumor therapy, can introduce a new insight into the field of ferroptotic nanomedicine.

Emerging role of miRNAs in the regulation of ferroptosis.

Ferroptosis is a kind of cell death which has distinctive features differentiating it from autophagy, necrosis and apoptosis. This iron-dependent form of cell death is described by an increase in lipid reactive oxygen species, shrinkage of mitochondria and decrease in mitochondrial cristae. Ferroptosis is involved in the initiation and progression of many diseases and is regarded as a hotspot of investigations on treatment of disorders. Recent studies have shown that microRNAs partake in the regulation of ferroptosis. The impact of microRNAs on this process has been verified in different cancers as well as intervertebral disc degeneration, acute myocardial infarction, vascular disease, intracerebral hemorrhage, preeclampsia, hemorrhagic stroke, atrial fibrillation, pulmonary fibrosis and atherosclerosis. miR-675, miR-93, miR-27a, miR-34a and miR-141 have been shown to affect iron metabolism, antioxidant metabolism and lipid metabolism, thus influencing all pivotal mechanisms in the ferroptosis process. In the current review, we summarize the role of microRNAs in ferroptosis and their involvement in the pathetiology of malignant and non-malignant disorders.

Simultaneous two-photon intravital imaging of viscosity and superoxide radical anion by a styrylpyridinium-based fluorescent probe

Simultaneous sensing of cellular microenvironment parameters and redox signaling molecules by a single fluorescent probe allows an efficient access to numerous applications ranging from convenient diagnostic assays to investigation on their pathophysiological roles and cross-link. Focusing on a lack of efficient molecule tools for simultaneous intravital imaging of mitochondrial viscosity and superoxide radical anion (O2•-), we bridged diethylaminobenzene with diphenylphosphinate-modified pyridinium moiety by an olefinic bond to construct a styrylpyridinium-based two-photon fluorescent probe V-OS. V-OS triggered a turn-on response either at 625 nm to viscosity which blocks intramolecular rotation, or at 530 nm to O2•-via nucleophilic attack of O2•- followed by 1,6-elimination. With the aid of the two-photon probe, we not only identified the cross-talk between viscosity and O2•- but successfully mapped the burst of O2•- and the increased viscosity during either cellular ferroptosis process or cerebral ischemia reperfusion injury of living mice.

Long-term in-situ real-time fluorescence imaging of lipid droplets during cell ferroptosis process enabled by an epindolidione-based fluorescent probe

Ferroptosis, as a new form regulation of cell death discovered recently, is triggered by an iron-dependent accumulation of lipid reactive oxygen species and thus closely associated with cellular lipid droplets (LDs). However, directly limited by the un-sufficient photostability and low LDs specificity of common fluorescent probes, visualization of LDs during ferroptosis process particular in a long-term, in-situ, and real-time manner is highly preferred but extremely challenge. To this end, here a new LDs fluorescent probe Lipi-EP featuring with outstanding photostability and high LDs specificity have been rationally developed based on the four-ring-fused epindolidione. Experimentally, the photostability and LDs specificity of this new probe are much higher than the representative LDs probe BODIPY and Nile Red. Consequently, time-lapse multicolor imaging and time-lapse three-dimensional imaging employed this new probe have been successfully conducted to in-situ real-time monitor the LDs change during ferroptosis process up to 6 h. The morphology changes of cellular organelles (LDs, mitochondria and nucleus) and the variations of space distribution of LDs have been impressively visualized. Moreover, the sizes and numbers of LDs have been also determined based on the imaging. This work providing abundant information of LDs would significantly promote the in-depth study of LDs during ferroptosis.

Study of Ferroptosis Transmission by Small Extracellular Vesicles in Epithelial Ovarian Cancer Cells.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. The current treatment for EOC involves surgical debulking of the tumors followed by a combination of chemotherapy. While most patients achieve complete remission, many EOCs will recur and develop chemo-resistance. The cancer cells can adapt to several stress stimuli, becoming resistant. Because of this, new ways to fight resistant cells during the disease are being studied. However, the clinical outcomes remain unsatisfactory. Recently, ferroptosis, a novel form of regulated cell death trigged by the accumulation of iron and toxic species of lipid metabolism in cells, has emerged as a promising anti-tumor strategy for EOC treatment. This process has a high potential to become a complementary treatment to the current anti-tumor strategies to eliminate resistant cells and to avoid relapse. Cancer cells, like other cells in the body, release small extracellular vesicles (sEV) that allow the transport of substances from the cells themselves to communicate with their environment. To achieve this, we analyzed the capacity of epithelial ovarian cancer cells (OVCA), treated with ferroptosis inducers, to generate sEV, assessing their size and number, and study the transmission of ferroptosis by sEV. Our results reveal that OVCA cells treated with ferroptotic inducers can modify intercellular communication by sEV, inducing cell death in recipient cells. Furthermore, these receptor cells are able to generate a greater amount of sEV, contributing to a much higher ferroptosis paracrine transmission. Thus, we discovered the importance of the sEV in the communication between cells in OVCA, focusing on the ferroptosis process. These findings could be the beginning form to study the molecular mechanism ferroptosis transmission through sEV.

Fluorescence Imaging of Intracellular Glutathione Levels in the Endoplasmic Reticulum to Reveal the Inhibition Effect of Rutin on Ferroptosis.

Ferroptosis is an emerging form of nonapoptotic cell death, and the search for novel ferroptosis inhibitors is of great importance to explore unique cytoprotective strategies against ferroptosis-relevant diseases. In this work, we present an endoplasmic reticulum-targeting fluorescent probe (ER-G) for the imaging of intracellular glutathione (GSH) levels and revealed the inhibition effect of rutin on ferroptosis. Structurally, ER-G utilized a cyclohexyl sulfonylurea as the endoplasmic reticulum-targeting unit, and single-crystal X-ray diffraction analysis confirmed that ER-G possessed a N-oxide pyridine sulfinyl group instead of sulfone. After the response of ER-G to GSH, the fluorescence intensity at 523 nm displayed a significant increase by 3900-fold. ER-G showed extreme sensitivity and selectivity to GSH. The fluorescence imaging results demonstrated that ER-G exhibited excellent endoplasmic reticulum-targeting properties and could be applied to monitor GSH levels in the endoplasmic reticulum during the erastin-induced ferroptosis process. By the fluorescence imaging of GSH levels in the endoplasmic reticulum, it was demonstrated that rutin could efficiently block the depletion of GSH during erastin-induced ferroptosis and potentially act as a novel ferroptosis inhibitor. Moreover, unlike traditional ferroptosis inhibitors, it was speculated that the inhibition mechanism of rutin to ferroptosis was the integration of the chelate effect on Fe(II) ions and antioxidant effect. We expect that fluorescence imaging of GSH levels in the endoplasmic reticulum could provide a convenient and feasible method to evaluate the inhibition effect of small molecules on ferroptosis.

In Situ and Quantitatively Monitoring the Dynamic Process of Ferroptosis in Single Cancer Cells by Scanning Electrochemical Microscopy.

Ferroptosis, as a promising therapeutic strategy for cancers, has aroused great interest. Quantifying the quick dynamic changes in key parameters during the early course of ferroptosis can provide insights for understanding the underlying mechanisms of ferroptosis and help the development of therapies targeting ferroptosis. However, in situ and quantitatively monitoring the quick responses of living cancer cells to ferroptosis at the single-cell level remains technically challenging. In this work, we selected HuH7 cells (hepatocellular carcinoma (HCC) cells) as a cell model and Erastin as a typical ferroptosis inducer. We utilized scanning electrochemical microscopy (SECM) to quantitatively and in situ monitor the early course of ferroptosis in HuH7 cells by characterizing the three key parameters of cell ferroptosis (i.e., cell membrane permeability, respiratory activity, and the redox state). The SECM results show that the membrane permeability of ferroptotic HuH7 cells continuously increased from 0 to 8.1 × 10-5 m s-1, the cellular oxygen consumption was continuously reduced by half, and H2O2 released from the cells exhibited periodic bursts during the early course of ferroptosis, indicating the gradually destroyed cell membrane structure and intensified oxidative stress. Our work realizes, for the first time, the in situ and quantitative monitoring of the cell membrane permeability, respiratory activity, and H2O2 level of the early ferroptosis process of a single living cancer cell with SECM, which can contribute to the understanding of the physiological process and underlying mechanisms of ferroptosis.

Construction of a ferroptosis scoring system and identification of LINC01572 as a novel ferroptosis suppressor in lung adenocarcinoma.

Background: Ferroptosis is a novel process of programmed cell death driven by excessive lipid peroxidation that is associated with the development of lung adenocarcinoma. N6-methyladenosine (m6a) modification of multiple genes is involved in regulating the ferroptosis process, while the predictive value of N6-methyladenosine- and ferroptosis-associated lncRNA (FMRlncRNA) in the prognosis of patients remains with LUAD remains unknown. Methods: Unsupervised cluster algorithm was applied to generate subcluster in LUAD according to ferroptosis-associated lncRNA. Stepwise Cox analysis and LASSO algorithm were applied to develop a prognostic model. Cellular location was detected by single-cell analysis. Also, we conducted Gene set enrichment analysis (GSEA) enrichment, immune microenvironment and drug sensitivity analysis. In addition, the expression and function of the LINC01572 were investigated by several in vitro experiments including qRT-PCR, cell viability assays and ferroptosis assays. Results: A novel ferroptosis-associated lncRNAs-based molecular subtype containing two subclusters were determined in LUAD. Then, we successfully created a risk model according to five ferroptosis-associated lncRNAs (LINC00472, MBNL1-AS1, LINC01572, ZFPM2-AS1, and TMPO-AS1). Our nominated model had good stability and predictive function. The expression patterns of five ferroptosis-associated lncRNAs were confirmed by polymerase chain reaction (PCR) in LUAD cell lines. Knockdown of LINC01572 significantly inhibited cell viability and induced ferroptosis in LUAD cell lines. Conclusion: Our data provided a risk score system based on ferroptosis-associated lncRNAs with prognostic value in LUAD. Moreover, LINC01572 may serve as a novel ferroptosis suppressor in LUAD.

Tracking endoplasmic reticulum viscosity during ferroptosis and autophagy using a molecular rotor probe.

Ferroptosis is a unique non-apoptotic cell death process associated with endoplasmic reticulum (ER) stress-related diseases. We have designed and synthesized a far-red emitting and ER targetable viscosity-sensitive fluorophore to track ER-phagy. Furthermore, the ER viscosity alteration during the ferroptosis process was investigated via intensity and lifetime-based spectroscopy and microscopy.

A chemiluminescent sensor for imaging endogenous hydrogen polysulfides in a living system.

By constructing 2-(benzoylthio)benzoate and a 2-fluoro-4-nitrobenzoate structure in an adamantylidene-dioxetane system, we designed and synthesized two novel chemiluminescent probes for the detection of H2Sn from other RSS. Under the same conditions, the maximum luminescence emission intensity of the probe CL-HP2 could reach 150 times that of the probe CL-HP1, and the chemiluminescence signal still existed at low concentrations. Therefore, CL-HP2 was more suitable for H2Sn detection as a chemiluminescent probe. The probe CL-HP2 exhibited a good linear relationship with Na2S4 in a wide range (0.025-10 mM). Interestingly, a good linear relationship (R2 = 0.997) was also observed at low concentrations (0-100 μM) with a LOD as low as 0.23 μM. CL-HP2 has been effectively employed to visualize endogenous H2Sn within living cells. Moreover, it has been applied for live imaging of bacterially infected murine models and the ferroptosis process in tumor-bearing mouse models.

PAR-CLIP Assay in Ferroptosis.

Ferroptosis is a regulatory cell death process that is accompanied by large amounts of iron ion accumulation and lipid peroxidation. Photoactivated ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) is a method used to identify the binding sites of RNA-binding proteins (RBPs) on target RNAs with high resolution at the nucleotide level. By inserting photosensitive ribonucleoside analogs into new RNA transcripts of living cells, characteristic mutations can be generated during reverse transcription and be used to accurately locate the crosslinking position of RNAs and RBPs. The use of PAR-CLIP to detect interactions and determine precise crosslinking sites between RNAs and RBPs, or to search for RNAs upstream or downstream of ferroptosis pathways genes through known proteins, can help to clarify and verify the occurrence and regulation mechanisms of the various signaling pathways of ferroptosis. Furthermore, it may reveal new targets for ferroptosis detection and improve the treatment efficiency of ferroptosis-related diseases such as cancer and neurodegenerative diseases. Here, we introduce a specific PAR-CLIP protocol for monitoring the ferroptosis process.

In-Cell Western Assay in Ferroptosis.

Ferroptosis is a regulated form of cell death caused by the excessive accumulation of iron-dependent lipid peroxidation. It has been implicated in various pathological processes and diseases, and its modulation involves multiple proteins associated with iron and lipid metabolism. To better understand these mechanisms and monitor the ferroptosis process, there is a need for reliable and high-throughput methods to evaluate variations in protein expression levels. In-Cell Western assays provide a simple and rapid assay method for detecting biomarkers and signaling proteins in whole cells using antibodies. This assay involves seeding cells in microtiter plates, followed by fixation/permeabilization and subsequent labeling with primary antibodies and infrared-conjugated secondary antibodies. In this chapter, we introduce the protocol for the In-Cell Western assay for detecting intracellular proteins during ferroptosis.

Detection of Ferroptosis by Immunohistochemistry and Immunofluorescence.

Ferroptosis is a type of regulated cell death driven by oxidative damage, characterized by iron overload and lipid peroxidation, and regulated by a network of distinct molecules and organelles. Impaired ferroptotic response is implicated in multiple physiological and pathological processes, including tumorigenesis, neurodegeneration, and ischemia-reperfusion damage. Classical techniques of immunohistochemistry (IHC) and immunofluorescence (IF) can be employed to exhibit antigen expression and location in tissues observed with microscopy, making them powerful tools in studying the ferroptosis process. In this chapter, we introduce commonly used protocols and summarize typical markers used in IHC and IF to monitor ferroptosis.

Carbonic anhydrase IX-targeted nanovesicles potentiated ferroptosis by remodeling the intracellular environment for synergetic cancer therapy.

Ferroptosis is one critical kind of regulated cell death for tumor suppression, yet it still presents challenges of low efficiency due to the intracellular alkaline pH and aberrant redox status. Herein, we reported a carbonic anhydrase IX (CA IX)-targeted nanovesicle (PAHC NV) to potentiate ferroptosis by remodeling the intracellular environment. CA IX inhibitor 4-(2-aminoethyl) benzene sulfonamide (AEBS) was anchored onto nanovesicles loaded with hemoglobin (Hb) and chlorin e6 (Ce6). Upon reaching tumor regions, PAHC could be internalized by cancer cells specifically by means of CA IX targeting and intervention. Afterwards, the binding of AEBS could elicit intracellular acidification and alter redox homeostasis to boost the lipid peroxidation (LPO) level, thus aggravating the ferroptosis process. Meanwhile, Hb served as an iron reservoir that could efficiently evoke ferroptosis and release O2 to ameliorate tumor hypoxia. With the help of self-supplied O2, Ce6 produced a plethora of 1O2 for enhanced photodynamic therapy, which in turn favored LPO accumulation to synergize ferroptosis. This study presents a promising paradigm for designing nanomedicines to heighten ferroptosis-based synergetic therapeutics through remodeling the intracellular environment.