Non-Enzymatic lipid peroxidation is a chain reaction driven by free radicals. It is essentially the ROS-induced lipid peroxidation of PUFAs on cell membranes (Hassannia et al., 2019). Intracellular ROS are formed mainly through three modalities: intracellular mitochondrial oxidative phosphorylation; the transmembrane transfer of electrons by nicotinamide adenine dinucleotide phosphate oxidase (NOX); and the Fenton reaction (Kuang et al., 2020). Hydroxyl radicals are one of the most dominant ROS and are mainly derived from the Fenton reaction between iron and hydrogen peroxide.

Recent studies have found that hydrogen peroxide, which undergoes the Fenton reaction, may be associated with intracellular enzymes. Nicotinamide adenine dinucleotide phosphate (NADPH)–cytochrome P450 oxidoreductase (POR) is an oxidoreductase located on the endoplasmic reticulum that transfers electrons from NADPH to microsomal cytochrome P450, cytochrome b5 and haeme oxygenase (HO), thus maintaining the intracellular redox homeostasis (Riddick et al., 2013). NADH–cytochrome b5 reductase 1 (CYB5R1) is another oxidoreductase whose deletion inhibits lipid peroxidation and ferroptosis. It was found that POR bound to CYB5R1 can induce lipid peroxidation in cells. The specific mechanism involves the binding of POR to CYB5R1 to form hydrogen peroxide, followed by the Fenton reaction of hydrogen peroxide with Fe2⁺ to generate hydroxyl radicals (Koppula et al., 2021; Yan et al., 2021).

The first step in non-enzymatic lipid peroxidation is the extraction of hydrogen by hydroxyl radicals from PUFAs in the cell membrane, thereby generating carb-centric lipid radicals (L•). Subsequently, oxygen molecules (O₂) react with carbon-centred lipid radicals to form lipid peroxide radicals (LOO), which can take up hydrogen from adjacent PUFAs to form lipid hydroperoxide (LOOH) and new lipid radicals to complete the lipid radical chain reaction. In addition, lipid hydroperoxides (LOOH) are converted to alkoxides (LO•) via Fe²⁺, which then react with adjacent PUFAs to induce another lipid radical chain reaction. These processes occur when there is an imbalance between lipid oxidation and the antioxidant system, leading to cell rupture and death and eventually ferroptosis (Hassannia et al., 2019).

Enzymatic lipid peroxidation is mainly mediated by the lipoxygenase (LOX) family, which is a group of haeme-type iron-containing enzymes that catalyse PUFAs, primarily adrenergic acid (Ada) and arachidonic acid (AA). The first step in enzymatic lipid peroxidation involves that Acetyl-CoA carboxylase (ACAC)-mediated fatty acid synthesis or lipophagy-mediated fatty acid release induces the accumulation of intracellular free fatty acids. The next step in enzymatic lipid peroxidation involves the formation of the corresponding derivatives AA-CoA and Ada-CoA catalysed by ACSL4 (Hassannia et al., 2019). Subsequently, lysophosphatidylcholine group transferase 3 (LPCAT3) catalyses the reaction of AA-CoA and Ada-CoA with phosphatidylethanolamine (PE) to form AA/PE and Ada/PE, respectively (Dixon et al., 2015; Doll et al., 2017). Finally, LOX promotes the peroxidation of AA/PE and Ada/PE to form AA/Ada-PE-OOH, leading to oxidative damage to the cell membrane. Therefore, the ACSL4–LPCAT3–LOX signalling axis is an intracellular pathway that promotes lipid peroxidation in ferroptosis, and inhibition of this pathway may inhibit ferroptosis.

The glutathione (GSH) peroxidase (GPX) family is a group of essential enzymes that intracellularly inhibit antioxidants. GPX4 is considered the most important antioxidant enzyme. It uses GSH as a substrate to reduce intracellular peroxides to non-toxic lipid alcohols. Therefore, once GPX activity is inhibited by inhibiting GSH synthesis or by directly inhibiting GPX, inhibition of peroxides can be directly reduced, thus achieving increased intracellular lipid peroxide accumulation and cellular ferroptosis.

GSH is synthesised by cysteine in the presence of r-glutamylcysteine synthetase, and GSH synthetase (GSS). Cysteine synthesis depends on the cystine/glutamate reverse transporter (system XC^–), which is a membrane Na⁺-dependent amino acid reverse transporter widely distributed in the phospholipid bilayer of biological cells. The transporter system XC⁻ is also a heterodimer composed of a light chain solute carrier family 7 member 11 (SLC7A11) and a heavy solute carrier family three members 2 (SLC3A2). This transporter system excretes a molecule of cystine whenever a molecule of cystine is taken up into the cell (Pitman et al., 2019). Intracellular cystine is reduced to cysteine mediated by thioredoxin reductase 1 (TXNRD1) (Mandal et al., 2010). Followed by this, glutathione (GSH) was synthesized by using cysteine as the substrate. Cysteine–GSH–GPX4 signalling axis is considered the most classical pathway in the regulation of ferroptosis. Inhibiting the system XC^– transporter reduces intracellular GSH synthesis and further inhibit GPX4 activity directly, which affects the capacity of the intracellular antioxidant system and thereby leading to the development of ferroptosis. Extracellular glutamate and glutamine levels can affect the occurrence of ferroptosis by affecting system XC^–. When extracellular glutamate levels are elevated, system XC^– can be prevented from performing glutamate/cystine exchange, thereby reducing GSH synthesis and subsequently triggering ferroptosis (Hirschhorn and Stockwell, 2019; Battaglia et al., 2020).

Two recent studies have identified a regulatory protein similar to GPX4 that is involved in the onset of cellular ferroptosis, namely ferroptosis suppressor protein 1 (FSP1) (Bersuker et al., 2019; Doll et al., 2019). FSP1 was previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2) and has significant homology to apoptosis-inducing factor (AIF) (Wu et al., 2002), a flavoprotein described initially as a P53-responsive gene (Horikoshi et al., 1999) that induces apoptosis.

FSP1 is recruited to the cell membrane via myristoylation and can subsequently reduce ubiquinone (i.e. coenzyme Q [CoQ10]) to ubiquinol in the form of an oxidoreductase. By such reaction, FSP1, acts as a lipophilic radical trap and inhibits cell membrane lipid peroxidation (Bersuker et al., 2019; Doll et al., 2019). In addition, FSP1, as an AIF family protein, can catalyse the regeneration of CoQ10 in reaction with NAD(P)H [31]. In some cases, FSP1 can also inhibit ferroptosis in a pathway parallel to that of CoQ10, such as through the membrane repair mechanism of ESCRT-III (a protein complex). However, the exact mechanism remains unknown (Dai et al., 2020). The FSP1 inhibitor iFSP1, which induced ferroptosis in GPX4-knockdown cells and FSP1-overexpressing cells, was found to exert the intracellular antioxidant effect of FSP1 (Doll et al., 2019).

In addition, a recent study revealed that an antioxidant system might exist in cellular mitochondria (Mao et al., 2021). Dihydroorotate dehydrogenase (DHODH) is located in the inner mitochondrial membrane and is responsible for catalysing the fourth step of pyrimidine nucleotide synthesis, that is, the oxidation of dihydroorotate (DHO) to orotate (OA) and the reduction of CoQ in the inner membrane to ubiquinol (CoH₂) by receiving electrons. CoH₂ can act as a trapping antioxidant to prevent intracellular lipid peroxidation (Vasan et al., 2020).

The DHODH inhibitor brequinar was found to induce ferroptosis in GPX4-low cells and enhance the susceptibility of GPX4-high cells to ferroptosis. The results were further validated in an in vivo assay via tumour formation in a nude mouse. The assay identified an alternative oxidative pathway to GPX4 in the cytosol and mitochondria and FSP1 in the cell membrane. DHODH in the inner mitochondrial membrane inhibits lipid peroxidation in the mitochondria by reducing CoQ₁₀ to CoQ₁₀H₂, indicating that DHODH can be a novel therapeutic target in cancer therapy by inducing ferroptosis.

In addition, a study demonstrated that a CRISPR/dCas9 overexpression screening using a genome-wide activation library identified the genes signature that inhibit ferroptosis, among which GTP cyclic hydrolase 1 (GCH1) and its metabolic derivative tetrahydrobiopterin/dihydrobiopterin (BH₄/BH₂) are shown to be the most prominent (Kraft et al., 2020). In cells, GTP can be hydrolysed by GCH1 to generate BH₄ and mediates its role in cellular lipid remodelling, thus acting as an antioxidant. It was further found that, unlike the antioxidant mechanism of GPX4, the antioxidant effect of GCH1–BH₄/BH2 did not protect all PUFA-containing phosphates (PUFA-PLs) from oxidation; instead, it can selectively protect specific phosphates containing two PUFA acyl chains from oxidative degradation, thereby inhibiting ferroptosis. In addition, under oxidative stress following ferroptosis induction, BH₄ inhibited ferroptosis by increasing CoQ10 levels. Therefore, the implication of GCH1–BH₄/BH₂ axis in inhibiting ferroptosis can be either by directly playing the intracellular antioxidant role in protecting cell membrane phospholipids, or by promoting CoQ10 synthesis and further regulating oxidative stress levels. The combination of these two regulating approaches can together form an antioxidant system independent of Cysteine/GSH/GPX4 axis (Kraft et al., 2020).

Genes are essential in life processes and can encode proteins involved in various cellular activities. Some genes have recently been found to play a crucial role in the occurrence of ferroptosis. Therefore, gene technology targeting ferroptosis can be used to treat cancer. The primary gene technologies include gene transfection and gene knockout.The P53 pathway, a classical tumour suppression pathway, has recently been found to sensitise cells to ferroptosis by inhibiting SLC7A11 (Berkers et al., 2013), a positive regulatory gene of ferroptosis. However, ACSL4 is an essential pro-ferroptosis gene (Shen Z. et al., 2018), and ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid (5-HETE) contributes to ferroptosis and is significantly downregulated in ferroptosis-resistant cells, thus positively regulating ferroptosis (Shen Z. et al., 2018). Therefore, by transfecting target cells with ferroptosis-positive regulatory genes such as P53 and ACSL4, ferroptosis can be induced for cancer treatment (Shen Z. et al., 2018). HSPB1 is a negative regulator of ferroptosis. HSPB1 knockdown in cells enhances erastin-induced ferroptosis, whereas HSPB1 overexpression inhibits erastin-induced death (Sun et al., 2015). Ferroptosis-negative regulatory genes can be knocked down in tumour cells using RNA interference technology, which induces cellular ferroptosis and increases anticancer activity. Therefore, gene technology (e.g. gene transfection or gene knockout) can be used in cancer therapy to introduce a relevant regulatory gene into a cell or to silence a gene, thereby regulating cellular ferroptosis (Shen Z. et al., 2018).

The traditional treatment modalities often fail to achieve good results in clinical practice owing to various drawbacks. Immunotherapy has gradually become an essential strategy for tumour treatment, in which immune-blocking agents exert their antitumour effects mainly by promoting the clearance function of cytotoxic T cells and preventing the escape of tumour cells (Khalil et al., 2016). Cancer immunotherapy is effective in treating some solid tumours including breast cancer, lung cancer, gastric carcinoma. However, compared with other conventional treatment modalities, immunotherapy is less effective in treating some tumours (Pham et al., 2018). Therefore, it is necessary to combine immunotherapy with other treatment modalities to improve its therapeutic effects (Adams et al., 2019). Recent studies have revealed a close relationship between activated CD8⁺ T cells in tumour immunotherapy and ferroptosis. The underlying mechanism involves the downregulation of SLC3A2 and SLC7A11, the two subunits system XC^–, by interferon-gamma released from immunotherapy-activated CD8⁺ T cells, thus reducing intracellular cysteine synthesis, decreasing antioxidant capacity and promoting lipid peroxidation and ferroptosis in tumour cells (Wang et al., 2019). In addition, it was found in an in vivo study in mice that when an immunosuppressant was combined with cysteine proteases (which degrade intracellular cysteine and cystine), they induced ferroptosis in tumour cells and simultaneously enhanced the efficacy of the immunosuppressant, achieving a synergistic effect (Wang et al., 2019) (Figure 5).

Ferroptosis interacts with the immune system through various mechanisms. In addition to the activation of CD8⁺ T cells by immunotherapy to promote ferroptosis, it has been demonstrated that ferroptosis is a form of immunogenic cell death (ICD) (Efimova et al., 2020). Therefore, ferroptotic cells can release a range of DAMPS, including high-mobility group protein B1 (HMGB1) and calreticulin (CRT), which may interact with pattern recognition receptors (PRRs) and phagocytic receptors on immune cells to activate the immune response and adaptive immune system (Shi et al., 2021). Therefore, it is of great clinical significance to study the immunogenicity of ferroptotic cancer cells and investigate their therapeutic effects.

Furthermore, the excellent biological properties of nanomaterials can be used to improve therapeutic effects by combining ferroptosis with tumour immunotherapy. In a study, a bionic magnetic cyst was constructed, in which Fe₃O₄ magnetic nanoclusters (NCs) as the core and leukocyte membrane as the shell were combined with transforming growth factor-β inhibitor (Ti) and PD-1 antibody (Pa) to promote the synergistic effect of ferroptosis and immunomodulation. Under magnetic resonance imaging (MRI)-guided drug delivery, Pa and Ti in bionic magnetic vesicles created an immunogenic environment after entering the tumour interior, which increased the concentration of hydrogen peroxide in polarised M1 macrophages, promoting a Fenton reaction with iron ions released from NCs. Hydroxyl radicals generated from the Fenton reaction subsequently induced ferroptosis in tumour cells, and the tumour antigens released by the dead cells subsequently promoted ferroptosis. The tumour antigens released by the dead cells, in turn, enhanced the immunogenicity of the microenvironment. Therefore, immunotherapy and ferroptosis exert a powerful synergistic effect (Zhang et al., 2019).

Although the relationship between ferroptosis and immunotherapy is not well understood, preliminary studies have been conducted on immunotherapy and ferroptosis in animal models. A study developed an iron oxide-loaded nanovaccine (IONV) for combined ferroptosis–immunotherapy treatment (Ruiz-de-Angulo et al., 2020). The principle of this nanovaccine was based on chemically integrated iron catalysts and drug delivery systems by exploiting the properties of the tumour microenvironment. IONV activation in the tumour microenvironment increased oxidative stress in tumour cells, which in turn promoted ferritin deposition, activated M1 macrophages, improved tumour antigen cross-presentation and enhanced the immunotherapeutic efficacy. The findings from the above-mentioned studies suggest that the combination of ferroptosis and immunotherapy is a promising cancer treatment strategy, which requires in-depth research.

Chemotherapy is a common clinical approach to cancer treatment, and the commonly used chemical agents include methotrexate, cyclophosphamide and vincristine. Although chemotherapy has made significant progress in clinical cancer treatment, the epithelial-to-mesenchymal transition, in which tumour cells can metastasise and become resistant to drugs, has limited the effectiveness of anticancer therapies (van Staalduinen et al., 2018; Yang et al., 2020). Epithelial-to-mesenchymal transition is a process wherein epithelial cells lose polarity and intercellular adhesion and gradually transform into invasive and metastatic mesenchymal cells (Yang et al., 2020). However, it has been found that mesenchymal cells formed after epithelial-to-mesenchymal transition have a higher susceptibility to ferroptosis (Hangauer et al., 2017; Viswanathan et al., 2017). Therefore, the use of ferroptosis appears to be one of the strategies to address tumour drug resistance and metastasis (Xu et al., 2021).

Inducing ferroptosis in drug-resistant tumour cells can overcome drug resistance, and varied underlying mechanisms have been proposed. Ferroptosis inducers can reverse tumour drug resistance by promoting intracellular ROS accumulation, thereby inducing ferroptosis in drug-resistant tumour cells (Friedmann Angeli et al., 2019). Furthermore, NRF2 can reduce intracellular ROS accumulation via the P62–Keap1–NRF2 pathway, which in turn is involved in the resistance of cancer cells to sorafenib; however, NRF2 inhibitors can reverse the resistance of cancer cells by increasing intracellular ROS accumulation (Roh et al., 2017).

Furthermore, ferroptosis inducers can induce ferroptosis in drug-resistant tumour cells by decreasing cellular TF levels and increasing intracellular iron transfer. For example, artesunate can induce ferroptosis in Adriamycin-resistant leukaemia cells by decreasing TF levels and promoting the therapeutic effect of Adriamycin on leukaemia (Wang et al., 2017; Park et al., 2018).

Ferroptosis agonists that act by inhibiting the system XC^– transport function can also lead to lipid peroxidation in resistant cells, thereby reversing drug resistance in tumour cells (Lim et al., 2005). For example, erastin can reverse cisplatin resistance in ovarian cancer cells by inhibiting the transport function of system XC^– (Sato et al., 2018).

In addition to reversing tumour cell resistance, the use of ferroptosis agonists in combination with chemotherapeutic agents for non-drug-resistant cancer cells can also achieve more desirable results. Ferroptosis can adopt the mechanism of action of traditional chemotherapeutic drugs, such as cisplatin, which function by reducing intracellular GSH by inhibiting GPX4 activity to induce cellular ferroptosis (Guo et al., 2018). Therefore, a combination of the chemotherapeutic drug cisplatin and ferroptosis inducers may enhance therapeutic efficacy. For example, erastin combined with cisplatin provides better results than when cisplatin is used alone (Guo et al., 2018). Another ferroptosis inducer, RSL3, exerts its induction effect by inhibiting GPX4 when combined with cisplatin, which enhances the therapeutic influence of cisplatin both in vitro and in vivo (Zhang et al., 2020). In addition to cisplatin, other classical chemotherapeutic agents can also exert better effects when used along with ferroptosis inducers. For example, the classical chemotherapeutic drug paclitaxel can further regulate glutamate catabolism (one of the pathways through which ferroptosis occurs) in colorectal cancer cells by reducing the expression of intracellular glutamate-catabolism-related genes such as SLC7A11 and SLC1A5 and inducing an increased expression of P53 and P21, which altogether inhibit tumour growth (Lv et al., 2017). In mutant-P53 pharyngeal squamous carcinoma, when low doses of paclitaxel were combined with the ferroptosis inducer RSL3, paclitaxel upregulated mutant P53 and promoted RLS3-induced ferroptosis, providing a synergistic therapeutic effect (Ye et al., 2019).

Therefore, the emergence of ferroptosis combined with chemotherapy as a new treatment modality offers a new strategy for tumour treatment.

Radiotherapy is one of the therapeutic modalities for the clinical treatment of tumours, which involves direct and indirect mechanisms of action. Regarding its direct mechanism, high-energy ionising radiation disrupts the double-stranded structure of cellular DNA and leads to cell cycle arrest. Regarding its indirect mechanism, radiation leads to cellular alterations including radiolysis of intracellular water and stimulation of oxidative enzymes. These alterations can generate ROS such as hydroxyl radicals and hydrogen peroxide. ROS overproduction can further damage nucleic acids, proteins and lipids, thereby inducing cell death (Lei et al., 2020). A recent study demonstrated that radiotherapy can exert therapeutic effects by inducing ferroptosis in cells, and the combination of radiotherapy and ferroptosis inducers results in enhanced antitumour effects when compared with the use of either treatment alone. The results of the study indicated that the combination of ferroptosis and radiotherapy might provide a promising strategy for treating tumours (Lang et al., 2019).

In addition, recent studies have reported that radiotherapy can induce ferroptosis in tumour cells. The four primary underlying mechanisms can be summarised as follows (Figure 6: First, radiotherapy can upregulate the expression ofataxia–telangiectasia mutated (ATM) gene and further inhibit the transport function of system XC–through inducing DNA double strand breaks (DBSs), which leads to the inhibition of antioxidant activity of GSH and GPX4, therebyresulting in lipid peroxidation (Lang et al., 2019). Second, radiotherapy can either increase the production of ROS or upregulate the gene expression of ACSL4. The conversion of PUFA to PUFA-CoA is catalysed by ACSL4. Subsequently, the PUFA-CoA enzyme is converted to PUFA-PL, which is catalysed by LPCAT3. The PUFA-PL product can induce lipid peroxidation owing to the catalysis of arachidonate lipoxygenases (ALOXs). Therefore, radiotherapy can promote intracellular lipid peroxidation and induce ferroptosis (Lei et al., 2020; Lei et al., 2021). Third, tumour cells treated with radiation can release tumour cell-released microparticles (RT-MPs) into the extracellular environment, which can promote the effect of radiotherapy by inducing ferroptosis in other tumour cells (Wan et al., 2020). Fourth, radiotherapy induces intracellular DNA damage, thereby activating the CGAS–cGAMP signalling pathway and subsequently inducing autophagy-dependent ferroptosis (Li et al., 2021).

Regarding the application of ferroptosis in radiotherapy, the combination of radiotherapy and the ferroptosis inducer (FIN) was found to act synergistically in tumour treatment by increasing lipid peroxidation and upregulating PTGS2 expression, with FINS exerting significant radio-sensitising effects in vitro and in vivo (Lei et al., 2020). Another study reported similar results by demonstrating that the ferroptosis inducer sulfasalazine acted as a radiosensitiser and increased the sensitivity of gliomas to Gamma Knife radiosurgery. The underlying mechanism involves blocking the uptake of cystine by system XC^–, leading to GSH depletion (Sleire et al., 2015). Another study investigating lung adenocarcinoma and glioma established a murine xenograft model and human patient-derived models to analyse the beneficial effects of combining radiotherapy with the ferroptosis inducer sorafenib in vivo, resulting in the improved efficacy of radiotherapy and inhibition of tumour growth (Ye et al., 2020).

Furthermore, a study reported that ferroptosis was also involved in radiotherapy-induced cellular damage in normal cells instead of only affecting tumour cells (Li et al., 2019). Based on this study, the combined application of ferroptosis inducers and radiotherapy in cancer treatment needs to be considered with caution. Whether the combination causes less harm to normal tissues than to tumour tissues needs to be investigated. Further studies to address this issue are warranted, and nanomaterials may be a future research direction. Altogether, these studies demonstrate that the combination of radiotherapy and ferroptosis may be considered a novel therapeutic strategy and also provides directions for future cancer research.

Photodynamic therapy (PDT) is a non-invasive cancer treatment modality. The action mechanism of PDT involves absorbing energy from ionising radiation and generating cytotoxic singlet oxygen (Dolmans et al., 2003). However, the efficacy of PDT in tumour treatment is limited owing to the inherent hypoxic nature of the tumour microenvironment. Therefore, oxygen supplementation in the tumour microenvironment is an essential measure to improve the efficacy of PDT (Cui et al., 2018). Because ferroptosis can generate lipid ROS, it is an excellent strategy to synergise with PDT.

Some studies have reported the applications of ferroptosis-related drugs in PDT therapy. A previous study designed a co-assembled nanosystem using the combination of erastin (ferroptosis inducer) and chlorin e6 (photosensitiser Ce6). This nanosystem was formed via intermolecular interactions of hydrogen bonding and π–π stacking. Erastin increased the concentration of intracellular oxygen and significantly improved the therapeutic efficiency of PDT by generating ROS (Zhu et al., 2019). Another example of the combined use of oxygen-enhanced PDT and ferroptosis is a 2-in-1 nanoplatform (SRF@HB-Ce6), which was designed by piggybacking the photosensitiser Ce6 on sorafenib (ferroptosis inducer) attached to the haemoglobin (Hb) (Xu et al., 2020). As for this nanoplatform, the capacity of Hb in providing endogenous iron and carrying oxygen provided the conditions for photodynamic-type therapy by enhancing ferroptosis (Xu et al., 2020). In addition, PDT can enhance ferroptosis and sensitise tumour cells to ferroptosis by recruiting immune cells to secrete interferon-gamma. Such 2-in-1 nanosystems combine the therapeutic advantage of both ferroptosis-related drugs and PDT and play a significant role in promoting antitumour effects (Xu et al., 2020). In addition to these nanosystems established by incorporating specialised photodynamic sensitisers (e.g. ferroptosis inducers), another design method has recently emerged based on the ability of some nanomaterials to generate ROS under certain conditions. For example, graphene oxide was recently found to release ROS under near-infrared (NIR) irradiation (He et al., 2017). Based on this theory, a research group prepared a new graphene oxide-based NIR-absorbing nanoparticle agent that was decorated with iron hydroxide/oxide (GO-FeO xH). In vitro experiments on this nanomaterial demonstrated that GO-FeO x H released oxygen upon exposure to NIR, which improved the treatment outcome of PDT (He et al., 2017).

Owing to the unique physicochemical properties of nanomaterials, bio-nanotechnology has become a focused research topic in the field of cancer treatment. Drug treatment can be improved by delivering anticancer drugs via nano-drug delivery systems (nano-DDS), which can significantly reduce the toxic side effects of drugs.

Recent studies have reported the use of nanomaterials to efficiently deliver ferroptosis targeting drugs (Shen Z. et al., 2018). Because ferroptosis is cell death caused by intracellular iron ion-dependent lipid peroxidation and lipid ROS accumulation, nanomedicines developed for ferroptosis are mainly used for intracellular ROS assembly. Therefore, based on the action mechanism of ferroptosis, ferroptosis-related nanodrugs can be classified into the following three categories: first, ferroptosis-related nanodrugs that can promote intracellular Fenton reaction; second, nanodrugs that can inhibit intracellular GPX4 activity, thus reducing intracellular antioxidant capacity; third, nanodrugs that can be used for exogenous regulation of lipid peroxidation in tumour cells (Shan et al., 2020). The regulatory role and application of these three categories of ferroptosis-related nano-drugs will be discussed in this section.

The first category of ferroptosis-related nanodrugs (for promoting intracellular Fenton reaction): The Fenton reaction is defined as the reaction between intracellular iron ions and hydrogen peroxide, leading to the production of the harmful hydroxyl radicals. Ferroptosis can induce cell death by promoting the intracellular Fenton reaction (Qian et al., 2019). Compared with normal cells, tumour cells possess more hydrogen peroxide and hence served as a substrate for the Fenton reaction, further facilitating the induction of ferroptosis. In addition, the tumour microenvironment is slightly acidic compared with the normal cell environment, and such Fenton reactions generally do not occur in the relatively neutral normal cells/tissues. Therefore, based on the Fenton reaction, ferroptosis-related drugs can accurately target tumour cells instead of destroying normal cells, which is a major advantage, especially involving less toxic side effects.

The Fenton reaction can be induced in tumour cells by enhancing the substrate of intracellular Fenton reaction (e.g. Fe⁺ and H₂O₂) via sustained release by a nano-delivery system. A typical example is nanocatalysts, which are prepared by adding ultrafine Fe₃O₄ nanoparticles and natural glucose oxidase (GOD) to silica nanoparticles with large pore size. GOD can effectively consume glucose in tumour cells, thus generating a large amount of hydrogen peroxide. Furthermore, Fe₃O₄ reacts with hydrogen peroxide, leading to a Fenton reaction, followed by the overproduction of hydroxyl radicals, which increases ROS levels and further induces ferroptosis in cells (Huo et al., 2017).

The second category of ferroptosis-related nanodrugs (for inhibiting intracellular GPX4 activity): In addition to promoting the intracellular Fenton reaction, these nanodrugs can inhibit GPX4 activity, thus reducing intracellular GSH levels. Currently, there are two primary modes of combining inhibition of GPX4 activity with nanomaterials: mode 1, intracellular administration of GPX4 inhibitors via nano-delivery systems; mode 2, direct GPX4 inhibition using nanomaterials or nanodrugs (Shan et al., 2020).

Mode 1 (direct administration of GPX4 inhibitors via nano-delivery systems): In a study, a nanomaterial scaffold was incorporated with three components (small molecule GPX4 inhibitor sorafenib, Fe³⁺ and tannic acid). Such nanodrugs can not only inhibit GPX4 activity but also convert Fe³⁺ to Fe²⁺ via tannic acid. These alterations eventually induce ferroptosis in cancer cells (Liu et al., 2018).

Mode 2 (nanosystems with GPX4 inhibition): This mode involves designing nanomaterials or drugs with GPX4 inhibition. A typical example is low-density lipoprotein (LDL) nanoparticles reconstituted with the natural omega-3 fatty acid docosahexaenoic acid (LDL-DHA), which were reported to induce ferroptosis in hepatocellular carcinoma cells by inhibiting intracellular GPX4 expression and reducing GSH levels (Ou et al., 2017). Another example is arginine-rich manganese silicate nanobubbles (AMSNs), which were found to induce iron sagging by inactivating GPX4, leading to an efficient GSH depletion capacity. The surface coating of arginine and the nanobubble structure improved the GSH depletion efficiency of manganese silicate nanobubbles when compared with the conventional nanoparticles (Wang et al., 2018).

The third category of ferroptosis-related nanodrugs (for regulating exogenous lipid peroxidation): Ferroptosis is mainly caused by lipid peroxidation, and the primary substrates of lipid peroxidation are PUFAs present in the phospholipid membranes. Therefore, increasing PUFA concentration exogenously may be a reasonable strategy for regulating lipid peroxidation and effectively inducing ferroptosis. This drug design strategy can efficiently improve the antitumour activity of ferroptosis inducers. To verify this hypothesis, some studies have been recently conducted using nano-delivery systems to exogenously modulate lipid peroxidation. A typical example is the nanosystem design based on Fenton-like reactions wherein linoleic acid hydroperoxide (LAHP) was incorporated into iron oxide nanoparticles (IONPs) using sub-stable FeO and Fe₃O₄ as iron sources to achieve on-demand Fe²⁺ release from the nanosystem under acidic conditions in tumours. The released Fe²⁺ generated tumor-specific ¹O₂ using LAHP, ultimately inducing ferroptosis in tumour cells (Zhou et al., 2017).