Progress in the Mechanism of the Effect of Fe3O4 Nanomaterials on Ferroptosis in Tumor Cells

Ferroptosis is a new form of iron-dependent programmed cell death discovered in recent years, which is caused by the accumulation of lipid peroxidation (LPO) and reactive oxygen species (ROS). Recent studies have shown that cellular ferroptosis is closely related to tumor progression, and the induction of ferroptosis is a new means to inhibit tumor growth. Biocompatible Fe3O4 nanoparticles (Fe3O4-NPs), rich in Fe2+ and Fe3+, act as a supplier of iron ions, which not only promote ROS production but also participate in iron metabolism, thus affecting cellular ferroptosis. In addition, Fe3O4-NPs combine with other techniques such as photodynamic therapy (PDT); heat stress and sonodynamic therapy (SDT) can further induce cellular ferroptosis effects, which then enhance the antitumor effects. In this paper, we present the research progress and the mechanism of Fe3O4-NPs to induce ferroptosis in tumor cells from the perspective of related genes and chemotherapeutic drugs, as well as PDT, heat stress, and SDT techniques.


Introduction
Global Statistics of 2020 [1] showed that there were an estimated 19.3 million new cancer cases and nearly 10 million cancer deaths, of which female breast cancer surpassed lung cancer as the most commonly diagnosed cancer with an estimated 2.3 million new cases (11.7%), followed closely by lung cancer (11.4%), colorectal cancer (10.0%), prostate cancer (7.3%), and stomach cancer (5.6%). Lung cancer remained the leading cause of cancer deaths with an estimated 1.8 million deaths (18%), followed by colorectal cancer (9.4%), liver cancer (8.3%), stomach cancer (7.7%), and female breast cancer (6.9%). The proliferation and apoptosis of corresponding tumor cells are closely related to tumor formation and progression. Thus, in the past few decades in oncology, scientific researchers have been devoted to the development of various apoptosis-related drugs to reverse the fate of non-senescent and over-proliferating tumor cells [2][3][4].
However, the therapeutic outcome is far from satisfactory due to the inherent or acquired apoptosis resistance of tumor cells [5]. Lung cancer, with the highest agestandardized incidence rate (ASIR) and age-standardized mortality rate (ASMR) worldwide, and with 2.1 million new cases and 1.8 million deaths every year, has a very high mortality rate [6][7][8][9]. Small cell lung cancer (SCLC) is one of the most malignant types of lung cancer. It has a short multiplication time, patients are prone to rapid drug resistance during treatment, and the disease deteriorates rapidly after recurrence. Currently, there is no effective second-line single-agent chemotherapy regimen other than topotecan [10]. In addition, the incidence and mortality of hepatocellular carcinoma (HCC) has rapidly

Ferroptosis
Ferroptosis, a new form of cell death discovered in recent years, is an iron-dependent non-apoptotic form of cell death characterized by the accumulation of intracellular ROS [18]. This modality disrupts the redox homeostasis of cells and has a great potential to kill cancer cells [49,50]. Studies on the mechanisms of ferroptosis have focused on lipid peroxidation (LPO) and iron loading. Under normal circumstances, ROS is mainly produced by normal metabolism in mitochondria and is essential for cell signaling and tissue homeostasis [51]. Polyunsaturated fatty acid (PUFA) peroxidation induced by ROS is the main cause of ferroptosis. Furthermore, ferroptosis is regulated by a complex interaction between cellular redox homeostasis, lipid metabolism, and iron metabolism [52]. In terms of cell morphology, cells undergoing ferroptosis show some specific changes, such as the appearance of smaller mitochondria than normal, wrinkled mitochondrial membranes, reduced or absent mitochondrial cristae, and fragmented extracellular membrane [53]. The biochemical features of ferroptosis are cystine deficiency, GSH depletion, and glutathione peroxidase 4 (GPX4) inactivation [54]. However, the underlying mechanisms have not been fully elucidated.
The main cause of ferroptosis is the change of cell metabolism, which leads to the accumulation of LPO and ROS in the cells. According to existing research reports, the changes in cell metabolic pathways caused by ferroptosis mainly include the following aspects ( Figure 1): (1) GSH synthesis pathway is inhibited and LPO accumulates, thus inducing ferroptosis in cells [55]. (2) Iron metabolism is altered. Iron is a redox-active metal involved in ROS formation and LPO diffusion, and a rising iron level could increase cellular susceptibility to irondependent cell death [56]. The accepted explanation today is that Fe 2+ can transfer electrons to intracellular oxygen, and then react with intracellular lipids to form LPO, which further induces ferroptosis [57,58]. Iron metabolism genes and iron metabolism regulation genes play a key role in intracellular system iron homeostasis. For example, the gene of Iron Responsive Element Binding Protein 2 (IREB2) is a key player in the Erastin-induced ferroptosis of HT-1080 fibrosarcoma cells and Calu-1 lung cancer cells [59]. Thus, intracellular iron overload is critical for ferroptosis [60]. (3) ROS metabolic pathway effects. This pathway also plays an important role in ferroptosis. Cytosolic cystine/glutamate transport receptor (System Xc-) and voltagedependent anion channels (VDACs) [18] in the outer mitochondrial membrane, GPX4 and ferroptosis suppressor protein 1 (FSP1) ferroptosis-related proteins [61,62], and p62/keap1/Nrf2 [63], p53-related pathway [64,65], and ACSL4/LPCTA3/LOX [66] ferroptosis-related pathways play their roles in regulating ferroptosis by affecting ROS metabolism pathways [67].
(2) Iron metabolism is altered. Iron is a redox-active metal involved in ROS formation and LPO diffusion, and a rising iron level could increase cellular susceptibility to iron-dependent cell death [56]. The accepted explanation today is that Fe 2+ can transfer electrons to intracellular oxygen, and then react with intracellular lipids to form LPO, which further induces ferroptosis [57,58]. Iron metabolism genes and iron metabolism regulation genes play a key role in intracellular system iron homeostasis. For example, the gene of Iron Responsive Element Binding Protein 2 (IREB2) is a key player in the Erastin-induced ferroptosis of HT-1080 fibrosarcoma cells and Calu-1 lung cancer cells [59]. Thus, intracellular iron overload is critical for ferroptosis [60]. (3) ROS metabolic pathway effects. This pathway also plays an important role in ferroptosis. Cytosolic cystine/glutamate transport receptor (System Xc-) and voltagedependent anion channels (VDACs) [18] in the outer mitochondrial membrane, GPX4 and ferroptosis suppressor protein 1 (FSP1) ferroptosis-related proteins [61,62], and p62/keap1/Nrf2 [63], p53-related pathway [64,65], and ACSL4/LPCTA3/LOX [66] ferroptosis-related pathways play their roles in regulating ferroptosis by affecting ROS metabolism pathways [67].

Mechanism of the Effect of Fe3O4-NPs on Ferroptosis in Tumor Cells
Research on tumor detection, diagnosis, and treatment methods has been a popular topic, and the development of nanotechnology has provided new strategies in this field [68][69][70][71][72][73][74][75]. Fe 2+ and Fe 3+ , rich in Fe3O4-NPs, contain abundant lone-pair electrons and can form coordination bonding with organic molecules. Thus, Fe3O4-NPs can easily realize surface modification and surface drug loading. After surface modification, Fe3O4-NPs are more difficultly encapsulated by plasma proteins and can avoid the phagocytosis of the reticuloendothelial system, reduce their clearance rate, and prolong the circulation time in blood [76][77][78][79].
Moreover, both Fe 2+ and Fe 3+ can react with hydrogen peroxide to generate ROS, catalyze the occurrence of the Fenton reaction, and then induce ferroptosis in cells [57]. Furthermore, it can enhance immunotherapy, which has attracted much more attention. Overall, iron-based nanoparticles as an emerging ferroptosis inducer can enhance iron ion and ROS levels. For example, Fe3O4-based nanoparticles prepared by Song et al. can

Mechanism of the Effect of Fe 3 O 4 -NPs on Ferroptosis in Tumor Cells
Research on tumor detection, diagnosis, and treatment methods has been a popular topic, and the development of nanotechnology has provided new strategies in this field [68][69][70][71][72][73][74][75]. Fe 2+ and Fe 3+ , rich in Fe 3 O 4 -NPs, contain abundant lone-pair electrons and can form coordination bonding with organic molecules. Thus, Fe 3 O 4 -NPs can easily realize surface modification and surface drug loading. After surface modification, Fe 3 O 4 -NPs are more difficultly encapsulated by plasma proteins and can avoid the phagocytosis of the reticuloendothelial system, reduce their clearance rate, and prolong the circulation time in blood [76][77][78][79].
Moreover, both Fe 2+ and Fe 3+ can react with hydrogen peroxide to generate ROS, catalyze the occurrence of the Fenton reaction, and then induce ferroptosis in cells [57]. Furthermore, it can enhance immunotherapy, which has attracted much more attention. Overall, iron-based nanoparticles as an emerging ferroptosis inducer can enhance iron ion and ROS levels. Tian et al. [81] synthesized Fe 3 O 4 -NPs with different sizes (with an average size of 2, 4, 10, and 100 nm) and evaluated their antitumor effects ( Figure 3). The experimental results showed that, compared to larger Fe 3 O 4 -NPs, the ultra-small (<5 nm) Fe 3 O 4 -NPs could accumulate in the nucleus and had a higher efficiency of •OH and ROS generation and a higher ability of cytokines secretion. Additionally, the ultra-small Fe 3 O 4 -NPs were more efficient in enhancing the toxicity of H 2 O 2 due to the rapid release of Fe 2+ . Nevertheless,  Tian et al. [81] synthesized Fe3O4-NPs with different sizes (with an average size of 2, 4, 10, and 100 nm) and evaluated their antitumor effects ( Figure 3). The experimental results showed that, compared to larger Fe3O4-NPs, the ultra-small (<5 nm) Fe3O4-NPs could accumulate in the nucleus and had a higher efficiency of •OH and ROS generation and a higher ability of cytokines secretion. Additionally, the ultra-small Fe3O4-NPs were more efficient in enhancing the toxicity of H2O2 due to the rapid release of Fe 2+ . Nevertheless, 10 nm Fe3O4-NPs displayed the best antitumor effect in vivo. It shows that selecting the right size of Fe3O4-NPs is more effective in treating tumors.  Tian et al. [81] synthesized Fe3O4-NPs with different sizes (with an average size of 2, 4, 10, and 100 nm) and evaluated their antitumor effects ( Figure 3). The experimental results showed that, compared to larger Fe3O4-NPs, the ultra-small (<5 nm) Fe3O4-NPs could accumulate in the nucleus and had a higher efficiency of •OH and ROS generation and a higher ability of cytokines secretion. Additionally, the ultra-small Fe3O4-NPs were more efficient in enhancing the toxicity of H2O2 due to the rapid release of Fe 2+ . Nevertheless, 10 nm Fe3O4-NPs displayed the best antitumor effect in vivo. It shows that selecting the right size of Fe3O4-NPs is more effective in treating tumors. Ferulic acid glycerol, a nano-Fe supplementation, was found to have antileukemic effects under low levels of iron transport protein (FPN) in leukemic cells by inducing ferroptosis in vitro and in vivo [82]. Leukemic cells are unable to export intracellular Fe 2+ produced by methyl ferulate, which can cause an increase in ROS levels through the Fenton reaction and then affect leukemic cells [83]. In addition, Zanganeh et al. showed that triglyceride ferulate has an intrinsic therapeutic effect on the growth of liver and lung metastases in early breast cancer and lung cancer [84].

Effect of Fe 3 O 4 -NPs on the Expression of Ferroptosis-Related Genes
In addition to lipid peroxidation [85][86][87], the role of genes involved in ferroptosis should also not be ignored. It has been shown that p53 activation is required for ferroptosis in cancer cells [88]. p53 can mediate ferroptosis in fibroblasts, osteosarcoma cells, and breast cancer cells through the transrepression of SLC7A11 [64,88]. It is the first piece of evidence of ferroptosis-induced p53 gene expression. p53 plays a dual role in ferroptosis. On the one hand, p53, as a tumor suppressor, down-regulates SLC7A11, thereby inhibiting the cellular uptake of cysteine, increasing cellular LPO and ultimately leading to ferroptosis. On the other hand, p53 inhibited Erastin-induced ferroptosis by blocking dipeptidyl peptidase 4 (DPP4) activity in human colorectal cancer (CRC) [89]. Fe 3 O 4 -NPs could promote the expression of p53 and acetylated p53 protein and accelerate their translocation from cytoplasm to nucleus in human umbilical vein endothelial cells; silencing p53 could up-regulate SLC7A11 and GPX4 mRNA expression levels ( Figure 4) [90]. For macrophages, RNA sequencing results showed that Fe 3 O 4 -NPs induced ferroptosis through up-regulating p53 gene expression and down-regulating SLC7A11 gene expression [91]. In ovarian cancer cells incubated with superparamagnetic iron oxide, iron repletion could promote p53 expression [92,93] and up-regulated p53 promoted ferroptosis. These results suggest that Fe 3 O 4 -NPs could promote the expression of p53, which is involved in the expression of SLC7A11 and GPX4 to regulate ferroptosis. ferroptosis in vitro and in vivo [82]. Leukemic cells are unable to export intracellular Fe 2+ produced by methyl ferulate, which can cause an increase in ROS levels through the Fenton reaction and then affect leukemic cells [83]. In addition, Zanganeh et al. showed that triglyceride ferulate has an intrinsic therapeutic effect on the growth of liver and lung metastases in early breast cancer and lung cancer [84].

Effect of Fe3O4-NPs on the Expression of Ferroptosis-Related Genes
In addition to lipid peroxidation [85][86][87], the role of genes involved in ferroptosis should also not be ignored. It has been shown that p53 activation is required for ferroptosis in cancer cells [88]. p53 can mediate ferroptosis in fibroblasts, osteosarcoma cells, and breast cancer cells through the transrepression of SLC7A11 [64,88]. It is the first piece of evidence of ferroptosis-induced p53 gene expression. p53 plays a dual role in ferroptosis. On the one hand, p53, as a tumor suppressor, down-regulates SLC7A11, thereby inhibiting the cellular uptake of cysteine, increasing cellular LPO and ultimately leading to ferroptosis. On the other hand, p53 inhibited Erastin-induced ferroptosis by blocking dipeptidyl peptidase 4 (DPP4) activity in human colorectal cancer (CRC) [89]. Fe3O4-NPs could promote the expression of p53 and acetylated p53 protein and accelerate their translocation from cytoplasm to nucleus in human umbilical vein endothelial cells; silencing p53 could up-regulate SLC7A11 and GPX4 mRNA expression levels ( Figure 4) [90]. For macrophages, RNA sequencing results showed that Fe3O4-NPs induced ferroptosis through up-regulating p53 gene expression and down-regulating SLC7A11 gene expression [91]. In ovarian cancer cells incubated with superparamagnetic iron oxide, iron repletion could promote p53 expression [92,93] and up-regulated p53 promoted ferroptosis. These results suggest that Fe3O4-NPs could promote the expression of p53, which is involved in the expression of SLC7A11 and GPX4 to regulate ferroptosis. In addition, there are other genes that play an equally important role in ferroptosis such as BRCA1-Associated Protein 1 (BAP1). BAP1, as a tumor suppressor, promotes ferroptosis and inhibits cancer cell growth by inhibiting SLC7A11 [94]. The mechanism may be that BAP1 suppresses SLC7A11 transcription by reducing histone 2A In addition, there are other genes that play an equally important role in ferroptosis such as BRCA1-Associated Protein 1 (BAP1). BAP1, as a tumor suppressor, promotes ferroptosis and inhibits cancer cell growth by inhibiting SLC7A11 [94]. The mechanism may be that BAP1 suppresses SLC7A11 transcription by reducing histone 2A ubiquitination (H2Aub) on the SLC7A11 gene. Gao et al. [83] developed a cancer treatment named gene interference ferroptosis therapy (GIFT) by combining a DMP-controlled gene interference tool with DMSA-coated Fe 3 O 4 -NPs. GIFT consists of a gene interference vector (GIV) and Fe 3 O 4 -NPs, and DMP, an NF-κB-specific promoter, consisting of an NF-κB decoy and a minimal promoter. In a variety of cancer cells treated with Fe 3 O 4 -NPs, the expression of FPN and LCN2 (iron metabolism genes) was selectively down-regulated by Cas13a or microRNA controlled by the NF-κB-specific promoter and ferroptosis was significantly induced, but the same treatment had little effect on normal cells.
The Beclin1/ATG5-dependent autophagy pathway is also closely related to ferroptosis induced by Fe 3 O 4 -NPs. Wen et al. [95] demonstrated that down-regulated Beclin1/ATG5 could significantly inhibit the ferroptosis induced by ultra-small iron oxide nanoparticles (USIONPs), while overexpressed Beclin1/ATG5 could significantly enhance ferroptosis. It suggests that USIONP-induced ferroptosis is regulated through the Beclin1/ATG5-dependent autophagy pathway, which could lead to the degradation of USIONPs, the release of iron ions, and the accumulation of ROS and LPO, and ultimately induce cellular ferroptosis.

Fe 3 O 4 -NPs Enhance the Sensitivity of Tumor Cells to Anticancer Drugs
There is growing evidence that the induction of ferroptosis enhances susceptibility to anticancer drugs [96,97]. Inducing ferroptosis with Erastin and RSL-3 has been reported to enhance the anticancer effects of cisplatin and reduce the resistance of anticancer drugs by inhibiting System Xc-in lung cancer, CRC, ovarian cancer, and pancreatic ductal adenocarcinoma [98]. Drug-loaded microspheres, as a commonly used clinical chemoembolic agent, suffer from inhomogeneous particle size and unstable efficacy. Chen et al. [106] introduced Fe 3 O 4 -NPs into the chemoembolic agent system to prepare gelatin microspheres co-loaded with doxorubicin (ADM/Fe 3 O 4 -MS) to improve its anti-liver cancer effect. Ferroptosis was also involved in the process of tumor cell death, in which GPX4, a marker of ferroptosis, was significantly reduced and ACSL4 was significantly increased. Meanwhile, ferroptosis inhibitors reversed the killing effect induced by ADM/Fe 3 O 4 -MS plus microwave irradiation. This study demonstrates that Fe 3 O 4 -NPs can significantly improve the overall efficacy of drug-loaded microspheres, which has promising applications especially in tumor chemoembolization [107].
Ferroptosis plays an important role in the treatment of triple-negative breast cancer (TNBC) cells. Yao et al. [108] prepared a novel ferroptosis nanodrug by loading simvastatin (SIM) into amphoteric polymer-coated magnetic nanoparticles (Fe 3 O 4 @PCBMA) to improve the therapeutic effect of TNBC. SIM could inhibit the expression of HMG-CoA reductase (HMGCR) and down-regulated the mevalonate (MVA) and GPX4 pathways, and consequently induced ferroptosis in cancer cells. Fe 3 O 4 -NPs serve as a good carrier to deliver antitumor drugs to tumor tissues through passive targeting. Based on this study, it is known that an Fe 3 O 4 -NP nanosystem can overcome the drug resistance caused by apoptotic drugs in tumor cells in clinical practice.
Wu et al. [109] developed a nanomedicine with both nanocatalytic and glutaminase inhibitor properties, which was made by integrating ultrasmall Fe 3 O 4 -NPs and CB-839 into dendritic mesoporous silica nanoparticles and then etched with Mn (DFMC) (Figure 6). The Fe 2+ and Mn 2+ in DFMC exerted Fenton/Fenton-like activity and could effectively catalyze the decomposition of H 2 O 2 to be highly toxic ·OH under acidic conditions for tumor therapy. The GSH depletion ability of DFMC not only consumes existing GSH, but also prevents the synthesis of GSH, which weakens the GSH-related antioxidant defense system (ADS), thereby enhancing the sensitivity of ROS-mediated tumor catalytic therapy and oxaliplatin chemotherapy, and finally achieving higher tumor clearance rate. Drug-loaded microspheres, as a commonly used clinical chemoembolic agent, suffer from inhomogeneous particle size and unstable efficacy. Chen et al. [106] introduced Fe3O4-NPs into the chemoembolic agent system to prepare gelatin microspheres co-loaded with doxorubicin (ADM/Fe3O4-MS) to improve its anti-liver cancer effect. Ferroptosis was also involved in the process of tumor cell death, in which GPX4, a marker of ferroptosis, was significantly reduced and ACSL4 was significantly increased. Meanwhile, ferroptosis inhibitors reversed the killing effect induced by ADM/Fe3O4-MS plus microwave irradiation. This study demonstrates that Fe3O4-NPs can significantly improve the overall efficacy of drug-loaded microspheres, which has promising applications especially in tumor chemoembolization [107].
Ferroptosis plays an important role in the treatment of triple-negative breast cancer (TNBC) cells. Yao et al. [108] prepared a novel ferroptosis nanodrug by loading simvastatin (SIM) into amphoteric polymer-coated magnetic nanoparticles (Fe3O4@PCBMA) to improve the therapeutic effect of TNBC. SIM could inhibit the expression of HMG-CoA reductase (HMGCR) and down-regulated the mevalonate (MVA) and GPX4 pathways, and consequently induced ferroptosis in cancer cells. Fe3O4-NPs serve as a good carrier to deliver antitumor drugs to tumor tissues through passive targeting. Based on this study, it is known that an Fe3O4-NP nanosystem can overcome the drug resistance caused by apoptotic drugs in tumor cells in clinical practice.
Wu et al. [109] developed a nanomedicine with both nanocatalytic and glutaminase inhibitor properties, which was made by integrating ultrasmall Fe3O4-NPs and CB-839 into dendritic mesoporous silica nanoparticles and then etched with Mn (DFMC) ( Figure  6). The Fe 2+ and Mn 2+ in DFMC exerted Fenton/Fenton-like activity and could effectively catalyze the decomposition of H2O2 to be highly toxic ·OH under acidic conditions for tumor therapy. The GSH depletion ability of DFMC not only consumes existing GSH, but also prevents the synthesis of GSH, which weakens the GSH-related antioxidant defense system (ADS), thereby enhancing the sensitivity of ROS-mediated tumor catalytic therapy and oxaliplatin chemotherapy, and finally achieving higher tumor clearance rate.

Fe3O4-NPs Can Enhance the Efficacy of Drugs or Synergize with Them to Promote Ferroptosis
In one study, magnetic nanoparticles (MNP) were coated with the zwitter polymer Poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) and then loaded sorafenib (SRF) to obtain the drug-loaded nanoparticles composite MNP@PMPC

Fe 3 O 4 -NPs Can Enhance the Efficacy of Drugs or Synergize with Them to Promote Ferroptosis
In one study, magnetic nanoparticles (MNP) were coated with the zwitterionic polymer Poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) and then loaded with sorafenib (SRF) to obtain the drug-loaded nanoparticles composite MNP@PMPC-SRF [110].
Fe 3 O 4 -NPs can also improve the anticancer efficiency of artemisinin (ART). The PFH/ART@PLGA/Fe 3 O 4 -EFA constructed by Wang et al. [110] was exposed to Low Intensity Focused Ultrasound (LIFU) irradiation to induce perfluorohexane (PFH) phase transition and nanoparticle collapse, which promoted the release of ART and Fe 3 O 4 in vitro. The efficiency of this approach is attributed to the synergistic effect of intracellular Fe 2+ and ART, which play a key role in the induction of ferroptosis in tumor cells by promoting ROS production in vitro.

Fe 3 O 4 -NPs in Combination with PDT, Heat Stress, and SDT Further Induced Ferroptosis in Tumor Cells
The combination of Fe 3 O 4 -NPs with other technologies further exploits their advantages and expands the scope of application [111][112][113][114][115][116]. The results of established studies have shown that Fe 3 O 4 -NPs are of great interest as a good cellular ferroptosis inducer, especially in the field of tumor therapy. However, current iron-based nanoparticles, due to their low ROS generation efficiency, require synergistic use with other therapeutic approaches or high dose application to achieve effective treatment [117].

Synergy of Photodynamic Therapy (PDT)
PDT is a novel tumor treatment method. However, the efficacy of PDT is greatly affected by oxygen concentration [118], which applied alone is not satisfactory. Fe 3 O 4 -NPs are highly biocompatible in vivo. When they enter cancer cells by endocytosis, they can be effectively released into Fe 2+ and Fe 3+ in the acidic environment of cancer cells, thus enhancing the Fenton reaction [119,120]. The Fenton reaction can produce ·OH and O 2 , increase the oxygen concentration in tumor cells, enhance the PDT effect, and then realize the treatment of cancers.
Chen et al. [121] designed a nanosystem coated with the FDA-approved poly (lactic-coglycolic acid) (PLGA) containing Fe 3 O 4 -NPs and chlorin E6 (Ce6) for synergistic ferroptosisphotodynamic anticancer therapy (Figure 7). The nanosystem can dissociate in acidic TME to release Fe 2+ /Fe 3+ and Ce6. Fe 2+ /Fe 3+ may react with the excess intracellular H 2 O 2 to trigger the Fenton reaction, which allows intracellular ROS accumulation and LPO generation, providing the necessary conditions for the onset of ferroptosis. Meanwhile, Ce6 could also increase the generation and accumulation of ROS under laser irradiation, and then provide PDT to further promote cellular ferroptosis. It demonstrates that the synergistic effect of PDT and ferroptosis induced by Fe 3 O 4 -NPs has an efficient effect on tumor suppression.
Ultrafine Fe 3 O 4 @PGL complex nanoparticles, designed by Liang et al. [122], were formed by amphiphilic porphyrin-grafted lipid (PGL) self-assembly on the hydrophobic surface of ultrasmall Fe 3 O 4 -NPs. This complex could not only trigger PDT under laser action, but also induce a large amount of ROS through the activity of tumor-associated macrophages (TAMs). Experimental results confirmed that most cells followed a PDT-based cell death pathway, and Fe 3 O 4 -mediated ferroptosis can further improve the therapeutic efficiency, showing a strong synergistic effect. Under the synergistic effect of ferroptosis and PDT, tumor cells regressed [123].
In summary, Fe 3 O 4 -NPs in an acidic environment (such as TME and lysosomes) can dissociate and then release Fe 2+ /Fe 3+ diffusing into the cytoplasm, which accelerates the Fenton reaction, promotes ferroptosis, and leads to intracellular ROS accumulation and LPO generation [124]. PDT can also promote ROS production and accumulation, which in turn promotes the occurrence of ferroptosis. Therefore, the combination of Fe 3 O 4 -NPs and PDT can effectively promote ferroptosis.
in acidic TME to release Fe 2+ /Fe 3+ and Ce6. Fe 2+ /Fe 3+ may react with the excess intracellular H2O2 to trigger the Fenton reaction, which allows intracellular ROS accumulation and LPO generation, providing the necessary conditions for the onset of ferroptosis. Meanwhile, Ce6 could also increase the generation and accumulation of ROS under laser irradiation, and then provide PDT to further promote cellular ferroptosis. It demonstrates that the synergistic effect of PDT and ferroptosis induced by Fe3O4-NPs has an efficient effect on tumor suppression. Ultrafine Fe3O4@PGL complex nanoparticles, designed by Liang et al. [122], were formed by amphiphilic porphyrin-grafted lipid (PGL) self-assembly on the hydrophobic surface of ultrasmall Fe3O4-NPs. This complex could not only trigger PDT under laser action, but also induce a large amount of ROS through the activity of tumor-associated macrophages (TAMs). Experimental results confirmed that most cells followed a PDTbased cell death pathway, and Fe3O4-mediated ferroptosis can further improve the therapeutic efficiency, showing a strong synergistic effect. Under the synergistic effect of ferroptosis and PDT, tumor cells regressed [123].
In summary, Fe3O4-NPs in an acidic environment (such as TME and lysosomes) can dissociate and then release Fe 2+ /Fe 3+ diffusing into the cytoplasm, which accelerates the Fenton reaction, promotes ferroptosis, and leads to intracellular ROS accumulation and LPO generation [124]. PDT can also promote ROS production and accumulation, which in turn promotes the occurrence of ferroptosis. Therefore, the combination of Fe3O4-NPs and PDT can effectively promote ferroptosis.

Metabolism Modulation by Heat Stress
Peptide-modified and 1H-perfluoropentane (1H-PFP)-coated Fe 3 O 4 -NPs (GBP@Fe 3 O 4 ) have been rationally designed by researchers to propose a hypothesis of heat-triggered tumor-specific ferroptosis (Figure 8) [125]. When a GBP@Fe 3 O 4 complex is irradiated by an 808 nm laser, the phase transition of 1H-PFP could be triggered by localized heat (45 • C), which leads to an abrupt release of Fe 3 O 4 -NPs in situ, and then generates intense ROS through the Fenton reaction in the TME. Oxidative damage, antioxidant inhibitory response, and the unique reprogramming of lipid metabolism during heat stress are amplified to induce tumor ferroptosis and achieve sufficient antitumor effects. Biocompatible Fe 3 O 4 -NPs act as a supplier of iron ions and can not only promote ROS production, but also participate in iron metabolism associated with ferroptosis [126]. Moderate heat stress has no significant effect on cellular ROS production, but it can significantly amplify Fe 3 O 4 -NPinduced oxidative stress and induce apoptosis in cancer cells [125]. Therefore, moderate heat stress acts synergistically with Fe 3 O 4 -NPs to weaken the self-defense response of tumors and affect their metabolism, thus regulating their mode of death.
A plate-like Bi 2 Se 3 -Fe 3 O 4 /Au (BFA) nanoplatform was designed for clinical diagnosis and therapy [127], which can increase the rate of the Fenton reaction to enhance ferroptosis therapy with active-passive targeting. This platform benefits from the internal synergistic effects of Fe 3 O 4 -NPs and Au-NPs, as well as external near-infrared light (NIR)-mediated hyperthermia, and could promote hydroxyl radical (·OH) production to enhance intracellular oxidative stress and further induce ferroptosis by inactivating GPX4. At the same time, BFA-NPs can be used as an effective diagnostic agent for photoacoustic (PA), magnetic resonance (MR), and X-ray imaging to guide the synergistic treatment of photothermal-ferroptosis. CdSe/Fe 3 O 4 nanoplatforms coated with multi-mode polymers had both optical and magnetic functions [128,129]. They co-wrapped with CdSe quantum dots and Fe 3 O 4 -NPs, and it was demonstrated for the first time that they may have biomedical applications based on two-photon and magnetothermal therapies such as bioimaging and anticancer therapy. However, co-wrapping limits CdSe/Fe 3 O 4 nanoplatforms inducing iron-dependent and LPO-mediated ferroptosis. Chen et al. [106] confirmed the synergistic effect of hyperther-mia and Fe 3 O 4 -NPs from another aspect. They designed an ADM/Fe 3 O 4 -MS complex to enhance their antitumor effects by activating ferroptosis under microwave heating. antioxidant inhibitory response, and the unique reprogramming of lipid metabolism during heat stress are amplified to induce tumor ferroptosis and achieve sufficient antitumor effects. Biocompatible Fe3O4-NPs act as a supplier of iron ions and can not only promote ROS production, but also participate in iron metabolism associated with ferroptosis [126]. Moderate heat stress has no significant effect on cellular ROS production, but it can significantly amplify Fe3O4-NP-induced oxidative stress and induce apoptosis in cancer cells [125]. Therefore, moderate heat stress acts synergistically with Fe3O4-NPs to weaken the self-defense response of tumors and affect their metabolism, thus regulating their mode of death. A plate-like Bi2Se3-Fe3O4/Au (BFA) nanoplatform was designed for clinical diagnosis and therapy [127], which can increase the rate of the Fenton reaction to enhance ferroptosis therapy with active-passive targeting. This platform benefits from the internal synergistic effects of Fe3O4-NPs and Au-NPs, as well as external near-infrared light (NIR)mediated hyperthermia, and could promote hydroxyl radical (·OH) production to enhance intracellular oxidative stress and further induce ferroptosis by inactivating GPX4. At the same time, BFA-NPs can be used as an effective diagnostic agent for photoacoustic (PA), magnetic resonance (MR), and X-ray imaging to guide the synergistic treatment of photothermal-ferroptosis. CdSe/Fe3O4 nanoplatforms coated with multi-mode polymers had both optical and magnetic functions [128,129]. They co-wrapped with CdSe quantum dots and Fe3O4-NPs, and it was demonstrated for the first time that they may have biomedical applications based on two-photon and magnetothermal therapies such as Extensive studies have shown that thermotherapy is an effective sensitizer of tumor chemoresistancy, which can greatly improve the effectiveness of chemotherapy [130] and enhance the antitumor effect of chemotherapeutic agents [131]. Therefore, the introduction of hyperthermia into the chemoembolization system could significantly improve the efficacy of transcatheter arterial chemoembolization (TACE). In conclusion, moderate heat, rather than a heat source, is essential for the induction of ferroptosis in combination therapy [125].

Promotion of Sonodynamic Therapy (SDT)
Recently, researchers have proposed a combination therapy strategy for glioma based on a non-invasive blood-brain barrier (BBB) opening, biomimetic sound-therapy-systemmediated SDT, and ferroptosis [132]. In this study, multifunctional homologous tumortargeting biomimetic nanoparticles (PIOC@CM-NPs) coated with Fe 3 O 4 -NPs and Ce6 were constructed as a nanoultrasound sensitizer, which combined the biomimetic nanoacousticsensitizer-mediated SDT with ferroptosis to achieve the effect of synergistic treatment of glioma. It demonstrated that the glioma C6 cell membrane on the surface of nanoparticles allowed selective accumulation of nanosensitized agents in tumors by homologous targeting in vitro. After effective internalization in C6 cells, PIOC@CM-NPs can significantly improve ROS levels and deplete GSH upon ultrasound irradiation, resulting in a loss of GPX4 activity, thereby promoting SDT and ferroptosis to kill glioma C6 cells.

Conclusions and Perspectives
Ferroptosis, a new iron-dependent programmed death discovered in recent years, has received increasing attention as a unique mechanism of occurrence and resistance, unlike apoptosis, cell necrosis, and cell autophagy. The activation of ferroptosis is widely recognized as a new target for drug discovery, and an increasing number of small molecule compounds have been identified and characterized to induce ferroptosis directly or indi-rectly by targeting iron metabolism and LPO. However, there are relatively few reports on the relationship between nanomaterials and cell ferroptosis, especially Fe 3 O 4 -NPs, which are rich in Fe 2+ and Fe 3+ and have unique advantages in inducing ferroptosis; however, the specific role and influencing mechanism still requires further research. Although there are many in vitro assays available to detect ferroptosis, such as by measuring cell viability, iron content, and ROS levels, confirming the presence of ferroptosis in vivo is more difficult. Ferroptosis is a double-edged sword, and we need to fully investigate the potential toxicities of inducers or inhibitors of key proteins and pathways of ferroptosis to ensure tumor-specific triggering of the Fenton response and avoid off-target toxicity to normal tissues causing carcinogenesis or other diseases [133,134].