Nanoscale View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM REVIEW Cite this: Nanoscale, 2021, 13, 2266 View Journal | View Issue Nanoparticle-induced ferroptosis: detection methods, mechanisms and applications Huizhen Zheng, a Jun Jiang,a Shujuan Xu,a Wei Liu,b Qianqian Xie,a Xiaoming Cai,c Jie Zhang,d Sijin Liu b and Ruibin Li *a Although ferroptosis is an iron-dependent cell death mechanism involved in the development of some severe diseases (e.g., Parkinsonian syndrome, stroke and tumours), the combination of nanotechnology with ferroptosis for the treatment of these diseases has attracted substantial research interest However, it is challenging to differentiate nanoparticle-induced ferroptosis from other types of cell deaths (e.g., apoptosis, pyroptosis, and necrosis), elucidate the detailed mechanisms and identify the key property of nanoparticles responsible for ferroptotic cell deaths Therefore, a summary of these aspects from current research on nano-ferroptosis is important and timely In this review, we endeavour to summarize some convincing techniques that can be employed to specifically examine ferroptotic cell deaths Then, we discuss the molecular initiating events of Received 29th November 2020, Accepted 7th January 2021 DOI: 10.1039/d0nr08478f rsc.li/nanoscale nanosized ferroptosis inducers and the cascade signals in cells, and therefore elaborate the ferroptosis mechanisms Besides, the key physicochemical properties of nano-inducers are also discussed to acquire a fundamental understanding of nano-structure–activity relationships (nano-SARs) involved in ferroptosis, which may facilitate the design of nanomaterials to deliberately tune ferroptosis Finally, future perspectives on the fundamental understanding of nanoparticle-induced ferroptosis and its applications are provided a State Key Laboratory of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, 215123 Jiangsu, China E-mail: liruibin@suda.edu.cn b State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085, China c School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, 215123 Jiangsu, China d Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062, Shandong, China Introduction Iron is a crucial and essential nutrient for all organisms because it participates in various biological processes, such as oxygen transport, mitochondrial respiration, DNA/RNA synthesis, and lipid metabolism.1 Iron deficiency often leads to nutritional disorder and growth issues, whereas excess iron may elicit genetic disorders (e.g., hemochromatosis) and organic lesions (e.g., Alzheimer’s disease and stroke) most likely via the newly reported ferroptosis mechanism Huizhen Zheng received her Ph.D Degree in 2017 from Dalian Institute of Chemical Physics, Chinese Academy of Sciences She is currently an Associate Professor in the School of Radiation Medicine and Protection, Soochow University Her current research interests include three-dimensional cell culture and nano–bio interactions Huizhen Zheng 2266 | Nanoscale, 2021, 13, 2266–2285 Jun Jiang is a Graduate Student under the supervision of Prof Li in Soochow University He is focused on increasing therapeutic effects via the combination of radiotherapy and ferroptosis Jun Jiang This journal is © The Royal Society of Chemistry 2021 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Nanoscale Review Therefore, living systems have evolved with sophisticated feedback loops to maintain iron homeostasis However, some exogenous stimuli may disrupt iron homeostasis, resulting in severe pathological changes in mammalian tissues Liu and co-workers found that polychlorinated biphenyls led to disordered iron metabolism and lipid accumulation in the liver by dysregulation of hepcidin.2 Auranofin as an anti-rheumatoid arthritis drug was found to reduce transferrin saturation and mitigate systemic iron by activating the hepatic IL-6/hepcidin signalling pathway.3 Besides these small chemicals, some nanoparticles (NPs) have been reported to disrupt iron metabolism4 and induce ferroptosis signals in mammalian cells Consequently, this has caused considerable concerns on nanosafety since nanomaterials are increasingly synthesized and applied in catalysis, electronics, energy storage, mechanics, textiles, cosmetics etc., which are employed in the production of more than 8900 commercial products.5,6 A significant amount of nanoparticles have been released into the environment and exposed to mammals during their lifecycle via the synthesis and transportation of materials, and the fabrication, consumption and recycling of nanoproducts.6 In terms of the biomedical applications of nanoparticles, they are expected to activate ferroptosis to overcome some issues, especially tumour resistance in cancer therapy.7,8 Considering that iron deficiency has been well elucidated in the past decades,9 it is not discussed in this review In contrast, ferroptosis induced by the overloading of iron in specific biological organisms or subcellular compartments is emphasized, especially from the perspective of nanosized inducers 1.1 Iron metabolism in cells Cellular iron homeostasis can be maintained by a feedback loop, i.e., the iron regulatory protein (IRP)–iron responsive element (IRE) signalling pathway.10 This pathway enables fine tuning of the iron levels in cells via the regulation of iron uptake proteins (transferrin receptor (TfR1) and divalent metal transporter (DMT1)), iron transport protein (transferrin), iron storage protein (ferritins) and iron efflux protein (ferroportin (FPN)) Generally, IRP1 and IRP2, as two cytoplasmic proteins, bind to IRE in RNA stem-loops in the untranslated region (UTR), and subsequently regulate the translation of target mRNA encoding proteins involved in iron metabolism For instance, high iron levels alter the IRP-IRE binding activity to regulate the translation of target transcripts, such as promotion of efficient translation of ferritin and FPN mRNA and destabilization of TfR1 and DMT1 mRNA, which can reduce cellular iron levels by accelerating iron efflux and reducing iron uptake.11 On the contrary, once cellular iron is deficient, IRPs tightly bind with IREs of TfR1 or DMT1 mRNA to stabilize the transcripts and increase their expression for iron uptake.12 A conventional metabolic process of iron in cells involves four steps, including collection of Fe3+, valence transition, intracellular transportation and elimination Firstly, extracellular Fe3+ ions bound to transferrin can be recognized by TfR1 and internalized into endosomes by endocytosis via clathrin-coated pits, which eventually mature into lysosomes Besides, aged ferritin may be transported into lysosomes by autophagosomes for the recycle of Fe3+ The enzymatic and acidic lysosomal compartment can facilitate the dissociation of Fe3+ from transferrin The reduction of Fe3+ is a critical step in iron metabolism as labile iron is more biologically active During the fusion of endosomes with lysosomes, Fe3+ dissociated from the receptor– ligand under an acid environment may be reduced into Fe2+ by ferrireductase, a six-transmembrane epithelial antigen of prostate (STEAP3).13 Fe2+ can be released into the cytoplasm through DMT1 and enters a pool termed the “labile iron pool” (LIP) These entrapped Fe2+ ions in the LIP have three possible destinations as follows: (i) imported into mitochondria14 for Fe– S cluster biogenesis, energy generation, and heme synthesis;15 (ii) sequestration and storage by ferritin to avoid toxic effects;16 and (iii) export by FPN that is a multi-transmembrane iron export protein regulated by the IRP-IRE system and liable to transport ferrous ions out of cells with the assistance of ferrous oxidase ceruloplasmin or hephaestin.17 1.2 Ferroptosis Overloading of labile iron in cells may lead to ferroptosis, a new type of programmed cell death, which was described for Shujuan Xu is a Graduate Student under the supervision of Prof Li in Soochow University She is conducting research on the mechanism of pulmonary toxicity induced by 2D nanomaterials Shujuan Xu This journal is © The Royal Society of Chemistry 2021 Wei Liu is a Graduate Student under the supervision of Prof Liu at the Research Center for Eco-Environmental Sciences (RCEES) at CAS His research interests focus on iron metabolism disorders Wei Liu Nanoscale, 2021, 13, 2266–2285 | 2267 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Review Nanoscale the first time in 2012.18 Different from apoptosis, necrosis and pyroptosis, ferroptosis as an iron-dependent mode of cell death is prevailingly featured by the excessive accumulation of lipid hydroperoxides Besides, an increase in the release of labile iron into the cytoplastic compartment initiates ferroptosis as this ion may catalyze the generation of oxygen radicals by Fenton reactions for lipid peroxidation In contrast, glutathione peroxidase (GPX4), as another prominent biomarker of ferroptosis, can catalyse the degradation of lipid peroxides Insufficient expression of GPX4 is often accompanied in ferroptotic cell death Similar to GPX4, ferroptosis suppressor protein (FSP1) was discovered by Doll et al and Bersuker et al in 2019 Interestingly, this enzyme can convert ubiquinone into ubiquinol in cell membranes to suppress lipid peroxidation and protect cells from ferroptosis.19,20 Different to the dramatic cell morphology changes in apoptosis, necrosis and pyroptosis, ferroptotic cells display intact cell membranes, normal-sized nuclei and dispersive chromatins.18,21 However, ferroptotic cells have been found to show dramatic changes in their mitochondria, such as obvious shrinkage with an increased membrane density and the reduction or absence of mitochondria cristae 1.3 Ferroptosis and disease Increasing evidence has revealed that ferroptosis is closely linked to several diseases, such as neurodegenerative diseases (Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease),22 cardiovascular and cerebrovascular diseases (stroke, ischemia reperfusion injury (IRI), and myocardial injury)23,24 and cancer (liver, pancreatic, kidney and breast cancer).25 Thus, numerous studies have explored the correlation between iron and AD Iron accumulation has been observed in the brain regions in patients diagnosed with AD, especially in amyloid plaques as a mineralized magnetite species.26,27 Iron dyshomeostasis was found to be involved in AD progression including β-amyloid (Aβ) deposition and abnormal phosphorylation of Tau proteins Telling et al demonstrated that elevated iron levels can promote the aggregation and oligomerization of Aβ peptides.28 In addition, iron overload was revealed to regulate Tau hyperphosphorylation and aggregation, which is another hallmark of AD.29 Thus, ferroptosis in AD tissue can be considered a diagnostic index Similarly, iron accumulation was also identified in the substantia nigra pars compacta, the main regions of dopaminergic neurodegeneration and cell loss, which are the typical features of Parkinson’s disease.30 Febbraro et al reported that α-synuclein, as a protein biomarker in neurodegeneration, was regulated by iron at the translational level via IRE in the 5′-UTR.31 Also, hydroxyl radicals induced by iron via Fenton reactions can elevate oxidative stress and lipid peroxidation to trigger the loss of nigral dopaminergic neurons in Parkinson’s patients.32 Furthermore, Rhodes et al suggested that TfR2 mutations may play a protective role in PD owing to the reduction in iron uptake.33 In ischemic stroke, free iron and ferritin may invade the brain parenchyma via defects in the blood brain barrier (BBB), eliciting Fenton reactions, lipid peroxidation and ferroptotic cell death.34,35 During cardiac IRI, the hypoxia-inducible factor may upregulate the expression of TfR1 and enhance iron acquisition, leading to iron overload, ROS generation, lipid oxidative damage and cell death.36,37 Nowadays, emerging evidence has revealed that cancer cells evading other forms of cell death maintain or acquire sensitivity to ferroptosis.38 Mammalian cells with oncogenic mutations were described to show greater sensitivity to erastin or cystine deprivation-induced ferroptosis with oxidative stress generation.39,40 Therefore, ferroptosis has been extensively explored for tumour therapy since it displays different signalling pathways to apoptosis, and via its activation, conventional chemotherapy and radiotherapy can eliminate cancer cells Especially, ferroptotic cell death provides opportunities to overcome drug resistance in cancer therapy Substantial reports have demonstrated that ferroptosis inducers, such as RSL3, FIN56, and ML162, enable the aggravation of tumour cell death or sensitization of radioresistant cancer cells to ionizing radiation.41,42 Besides, nanomaterials, such as low-density lipoprotein–docosahexaenoic acid NPs, have been demonstrated to selectively trigger ferroptosis in human hepatocellular carcinoma cells by lipid peroxidation, GSH depletion, and GPX4 inactivation.43 Qianqian Xie is a Graduate Student under the supervision of Prof Li in Soochow University Her current research focuses on the bio-function of lipid peroxides Qianqian Xie 2268 | Nanoscale, 2021, 13, 2266–2285 Xiaoming Cai is an Associate Professor in the School of Public Health, Soochow University Her current research interests include the discovery of biomarkers for nanotoxicity assessment, structure–activity relationships of nanomaterials and interactions of biomolecules with ENMs Xiaoming Cai This journal is © The Royal Society of Chemistry 2021 View Article Online Nanoscale Table Biomarkers of ferroptosis Metabolites Proteins Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Genes 1.4 Review Biomarkers Mechanisms Iron accumulation, lipid peroxidation, MDA, 4-HNE, GSH, NADPH and selenium contents SLC7A11, GPX4, ATG5-ATG7-NCOA4, IREB2, p62-Keap1NRF2, p53-SAT1-ALOX15, ACSL4 and LPCAT3 GPX4, TFR1, SLC7A11, NRF2, NCOA4, p53, HSPB1, ACSL4, FSP1 and KOD Iron-metabolized dysfunction, ROS generation, Fenton reaction acceleration, lipid peroxidation, and GPX4 inhibition Regulating GSH/GPX4, iron metabolism and lipid metabolism pathways Encoding proteins associated with ferroptosis pathways Nano-ferroptosis studies Detection methods of ferroptosis The development of nanotechnology has led to a high risk of exposure to NPs in the environment Reactive oxygen species (ROS) are considered to be the major cause of apoptotic cell death by most toxic NPs Although endocytosis has been considered to play a major role in the internalization of NPs in cells, lysosomal dysfunction by some NPs may elicit NLRP3 inflammasome activation, cathepsin B release and iron metabolism disruption.44–46 Consequently, efforts have been made to deliver cargo molecules by nano-carriers for the induction of ferroptotic cell death For instance, Zhao et al designed a hypoxia-responsive polymer micelle to encapsulate RSL3, which enabled the release of RSL3 in the hypoxia tumour microenvironment to inhibit GPX4 activity and induce ferroptosis.47 Recently, a few reviews have discussed the potential utilities of ferroptosis in nanomedicine.27,28 Besides, ferroptosis has been observed in epithelial, immune, neurone and cancer cells merely exposed to iron-based and iron-free nanomaterials.7,48,49 These papers developed some new techniques to characterize nanoparticle-induced ferroptosis, proposed some unique cellular mechanisms and discussed the key physicochemical properties of NPs responsible for ferroptosis However, there is lack of a review on these findings to summarize the detection methods, ferroptosis mechanisms, and structure–activity relationships (SARs) between the physicochemical properties of NPs (e.g., surface vacancy, size, surface group and ion release) and ferroptosis Jie Zhang Jie Zhang received her Ph.D Degree from the Research Center for Eco-Environmental Sciences, Chinese Academy of Science in 2019 She is currently an Assistant Professor at the Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Shandong First Medical University & Shandong Academy of Medical Sciences Her research interests focus on the health risks of environmental pollutants, including nanoparticles and fine air particles This journal is © The Royal Society of Chemistry 2021 2.1 Biomarkers involved in ferroptosis Although morphological identification is always considered as the most direct and intuitive evidence for cell death, there are almost no morphological changes in cell membranes, nuclei and chromatin in ferroptosis compared to other types of cell death Subcellular morphology examination by transition electron microscopy (TEM) in ferroptotic cells revealed some alterations in the membrane and crista of mitochondria.50 For instance, Fadeel and co-worker performed TEM to observe the morphologies of Jurkat cells in apoptosis, necrosis, and ferroptosis, and successfully differentiated ferroptotic cells by comparison of both their mitochondrial and nuclear changes.51 Since mitochondria play a critical role in ferroptosis, Gao et al demonstrated that mitochondria-depleted cells (Parkinmediated mitophagy) were less sensitive to ferroptosis induced by cysteine starvation or erastin.52 Besides, the molecular biomarker of ferroptosis can be determined in three categories, including biochemical metabolism, proteins and regulatory genes (Table 1) Firstly, considering that ferroptosis is closely dependent on the iron metabolic process, the cell death can be indexed via some metabolic reactions, such as iron overload, lipid peroxidation and the reduction of GSH.53 An increase in intracellular labile iron may result in oxidative radical generation by Fenton reactions, further leading to lipid peroxidation Although lipid Sijin Liu Sijin Liu is a Professor at the State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (CAS) His research interests include: (i) environmental behaviors and biological effects of engineered nanomaterials and fine air particles and (ii) the molecular mechanisms responsible for environmental pollutant-induced oncogenic effects Nanoscale, 2021, 13, 2266–2285 | 2269 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Review Nanoscale Fig Regulatory pathways of ferroptosis Three metabolism pathways include: (a) amino acid metabolism involving cystine and glutamate exchange, and GSH synthesis and consumption; (b) lipid metabolism involving PL-PUFA-OOH accumulation and p53-SAT1-ALOX15-regulated pathway; (c) iron metabolism mediated by TRF1 and lysosomal metabolism, ferritin storage pathway and p62-Keap1-NRF2-regulated pathway peroxides are the major cause of ferroptotic cell death, it is difficult to directly detect these molecules because of their super activity and short lifetime Lipid peroxides may react with some reductive biomolecules, including proteins, lipids and DNA to inactivate enzymes, degenerate proteins, and impair DNA and phospholipid structures.54,55 Alternatively, the secondary products of lipid peroxidation, such as MDA and 4-HNE, are stable markers to assess lipid peroxidation.196 NADPH as a reductant responsible for the eradication of lipid hydroperoxides can also be considered a ferroptosis biomarker.57 Currently, more research has been focused on protein markers in ferroptosis as these markers are extremely useful for the design of ferroptosis inducers or antagonists in pharmacy Based on the amino acid metabolism, iron metabolism Ruibin Li Ruibin Li is a Professor in the State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Soochow University His research interests focus on: (i) understanding nano–bio interactions; (ii) developing predictive toxicology methods for nanosafety assessment; and (iii) using nanotechnologies for immunotherapy, anti-resistant bacterial coating, radiotherapy and radio imaging 2270 | Nanoscale, 2021, 13, 2266–2285 and lipid metabolism pathways relating to ferroptosis (Fig 1), the protein markers can be assigned into these three networks, including reductase GPX4, transmembrane transporter protein SLC7A11 (commonly known as xCT), p53, ACSL4, and LPCAT3.50 Among them, the amino acid metabolism pathway, namely the GSH/GPX4 pathway, involves system xc−, glutamine metabolism, sulfur transfer and the p53 regulatory axis.58 Notably, GPX4 is a core protein enzyme that controls cellular lipid peroxide levels by catalyzing the reduction of fatty acid hydroperoxide System xc−, consisting of the transporter subunit SLC7A11 and regulatory subunit SLC3A2, is responsible for the modulation of intracellular cysteine and GSH Since SLC3A2 with pleiotropic functions is involved in the transportation of other amino acids and glucose, SLC7A11 in system xc− has been identified as a typical biomarker for ferroptosis.59 The downregulations of GPX4 and SLC7A11 were popularly examined to validate ferroptotic cell death For instance, erastin was found to inhibit SLC7A11 for suppressing cysteine uptake and GSH synthesis, which subsequently elicited lipid peroxidation and ferroptosis.60 Friedmann Angeli et al demonstrated that the knockout of GPX4 may cause ferroptotic cell death in GPX4−/− mice, and this effect could be rescued by Liproxstatin-1 treatment.61 The increment in GPX4 biosynthesis by the supplementation of selenium was reported to be an effective method to enhance tolerance against ferroptotic cell death.62 Besides, transport, reductive and storage proteins in iron metabolism, including TfR1, DMT1, STEAP3 and ferritin are another type of protein biomarker in ferroptosis These proteins can maintain iron homeostasis by regulation of the IREB2, ATG5-ATG7-NCOA4, p62-Keap1-NRF2 and HSPB1 pathways.50 Song et al demonstrated that DMT1 was highly This journal is © The Royal Society of Chemistry 2021 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Nanoscale Review expressed in ferroptotic cells in the infraction myocardium.63 Li et al found that NCOA4 could increase the level of intracellular free iron to induce ferroptosis in LPS-treated cardiomyocytes by promoting the degradation of ferritin.64 Since lipid peroxidation is the major hazardous signal in ferroptosis, the proteins in the lipid metabolism pathways, such as p53SAT1-ALOX15, ACSL4 and LPCAT3 are all relevant to ferroptosis Doll et al disclosed that ACSL4 was an essential component for ferroptosis execution, which was preferentially expressed in a panel of basal-like breast cancer cell lines.65 Accordingly, the genes encoding critical protein markers (GPX4, TfR1, SLC7A11, NRF2, NCOA4, TP53, HSPB1, ACSL4, and FSP1) of ferroptosis have been demonstrated to dictate ferroptotic cell death.19,50 For instance, Cheng et al demonstrated that ACSL4 could exert anti-proliferative effects in glioma cells by the induction of a ferroptosis pathway Overexpression of ACSL4 led to a reduction in GPX4, accumulation of ferroptotic markers, increment in lactate dehydrogenase release and inhibition of glioma cell proliferation In contrast, ACSL4 suppression by siRNA could ameliorate sorafenibinduced cell death.66 Besides, substantial efforts have been made to use shRNA-mediated silencing and genomics for the exploration of the pivotal genes controlling ferroptosis Jiang et al disclosed a novel mechanism of intercellular interaction mediated by E-cadherin to suppress cancer cell ferroptosis by activating the cadherin–NF2–HippoYAP signaling pathway.67 Recently, Distefano et al demonstrated that the kiss of death (KOD) gene could encode a short peptide to regulate GSH depletion, ROS accumulation and Ca2+ metabolism in heat stress-induced ferroptosis in plants.68 2.2 Ferroptosis inducers and inhibitors Based on the ferroptosis biomarker and regulatory pathways, several small molecular reagents have been developed to Table Inducer Inducers and inhibitors of ferroptosis Mechanism Small molecules Nanoparticles Inhibition system xc− NA Suppression GPX4 Erastin, PE, IKE, other erastin analogs; sulfasalazine; glutamate; sorafenib (1S,3R)-RSL3; ML162, FIN56; DPI family members Iron dyshomostasis ferric ammonium citrate GSH depletion Cystine/cysteine deprivation, BSO, DPI2, cisplatin; cysteinase, acetaminophen FINO2 Statins Lipid peroxidation CoQ10 biosynthesis inhibition NRF2 inhibition Inhibitor inhibit or induce this effect (Table 2) More than seven categories of inducers have been developed to induce ferroptosis, underlying different mechanisms, including inhibition of system xc− and GPX4, disruption of iron homeostasis, depletion of GSH, induction of lipid peroxidation and blocking CoQ10 biosynthesis and NRF2 The induction of ferroptosis by system xc− inhibitors (e.g., erastin, sulfasalazine, glutamate, and sorafenib) can trigger cell death by preventing cystine import, resulting in GSH depletion and lipid peroxide accumulation.69–71 GPX4 inhibitors (e.g., RSL3, ML162, FIN56, and DPI compounds) are commonly used to covalently target or degrade GPX4, which also lead to the accumulation of lipid peroxides and ferroptosis.42,48 Buthioninesulfoximine (BSO), cisplatin, cysteinase and Acetaminophen can disrupt amino acid metabolism to induce ferroptosis by the restriction of GSH synthesis or increase of GSH depletion.72 Ferroptosis can also be elevated by the exposure of cells to ferric ammonium citrate (FAC) for direct overloading of intracellular iron.73 FINO2 is reported to induce ferroptosis through a combination of oxidation of ferroptosis-relevant substrates and inhibition of GPX4 activation.48 In addition, other small molecule reagents, including statins, cysteinase and trigonelline, can be exploited as ferroptosis inducers by blocking CoQ10 biosynthesis and inhibiting NRF2 activity for the disruption of amino acid metabolism and iron metabolism.74,75 Beside of these small chemicals, NPs are another important category of ferroptosis inducers These materials can be divided into two classes, iron-based and iron-free NPs Zheng et al demonstrated that superparamagnetic iron oxide nanoparticles (SPION) released free divalent iron to accelerate Fenton reactions and lipid peroxide accumulation for the induction of ferroptosis in ischemic cardiomyocytes.76 Encapsulation of H2O2 in the hydrophilic core of an Fe3O4-poly (lactic-co-glycolic acid) (PLGA) could also induce ferroptosis by Iron chelators Reduction of lipid peroxidation System xc− activation GPX4 upgradation Selenoprotein increment Iron-free nanomaterials, e.g., WS2, MoS2, Co NP Iron-based nanomaterials, e.g., Iron oxide (IO), Iron-organic NP, FePt ZnO NP WS2, MoS2, Iron-organic NP, FePt NA Trigonelline, brusatol NA DFO, CPX, DFP, DFX Vitamin E, trolox, tocotrienols, BHT, BHA, Fer-1, Lip-1; CoQ10, idebenone; XJB-5-131; deferoxamine, cyclipirox, deferiprone; CDC, baicalein, PD-146176, AA-861, zileuton; vildagliptin, alogliptin, and linagliptin Cycloheximide, beta-mercaptoethanol Dopamine Selenium NA CPS This journal is © The Royal Society of Chemistry 2021 NA NA NA Nanoscale, 2021, 13, 2266–2285 | 2271 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Review Fenton reactions to provide excessive oxidized lipids in cancer cells.77 Chen et al designed ultrasmall polydopamine NPs to load Fe2+/Fe3+ ions and release them in cells, resulting in the generation of ROS and lipid peroxidation.44 Metal–organic frameworks (MOFs) and metal–organic networks (MONs) were also reported to induce ferroptosis by increasing the iron levels in cells for the acceleration of Fenton reactions.44,78,79 Besides these iron-based nano-inducers, the endocytic internalization of iron-free NPs may initiate the key signals (Fe2+ or ROS) of ferroptosis in cells by interactions with lysosomal enzymes or acidic substances Xu et al found that the lysosomal internalization of WS2 and MoS2 could induce the release of labile iron in the cytoplastic compartments.87 Co NPs were discovered to release Co2+ from vesicles/endosomes to regulate the reduction of GPX4 expression and depletion of cellular GSH levels.49 Zhang et al discovered that zinc ions from ZnO NPs disrupted intracellular ROS and iron homeostasis.45 Additionally, Wu et al observed an increase in iron levels, ROS production and oxidative lipids in mitochondria followed by the endocytosis of metal-free graphene quantum dots (GQD) in microglia.80 These hazard signals may eventually cumulate in ferroptotic cell death Since ferroptosis is considered to be a relevant cause in neurodegenerative, cardiovascular and cerebrovascular diseases, ferroptosis inhibitors may have therapeutic effects on these diseases Although diverse chemicals have displayed inhibitory effects in ferroptotic cell death, few of them specifically work on ferroptosis For instance, iron chelators including deferoxamine (DFO), ciclopirox (CPX), deferiprone (DFP), and deferasirox (DFX)81 can prevent ferroptotic cell death by reducing the free labile iron in cells Potent antioxidants, e.g., vitamin E, trolox, tocotrienols, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ferrostatin (Fer-1) and liproxstatin-1 (Lip-1), have been found to attenuate lipid peroxidation.82 Cycloheximide and β-mercaptoethanol mainly target system xc− to enhance GSH levels for the effective elimination of lipid peroxides Dopamine and selenium were reported to elevate GPX4 expression in cells by blocking its degradation or increasing its biosynthesis Interestingly, Li et al found that carboxyl-modified polystyrene nanoparticles (CPS) showed potent anti-ferroptosis effects in RAW264.7 cells by nucleus translocation of transcription factor EB (TFEB) to enhance the expression of lysosomal protein and SOD, even stronger than DFO.83 Considering the colocalization of NPs and Fe3+/Fe2+ in lysosomes, this finding offers a new approach to specifically inhibit ferroptosis by accurate alteration of lysosomal iron metabolism rather than iron levels in whole cells 2.3 Techniques used for ferroptosis assessment Currently, apoptosis, necrosis, ferroptosis and pyroptosis are the major types of cell death in response to NP exposure Two or more death types are often involved in cells exposed to specific NPs, but cell death assay kits provide poor examinations of different death mechanisms Since ferroptosis displays limited biological features compared to the other three types of cell death, it is urgent to find appropriate techniques for the 2272 | Nanoscale, 2021, 13, 2266–2285 Nanoscale convincing characterization of ferroptosis Although there are a cascade cellular events of small molecules, proteins, genes and subcellular organelles involved in ferroptosis, tiered assessments are highly recommended to accurately identify ferroptosis from other types of cell death since the molecular mechanisms involved in ferroptosis are still unknown and there is no distinct morphology change in ferroptotic cells Therefore, multiple complementary techniques are required for the successful identification of ferroptotic cell death A summary of the ferroptosis detection methods may greatly assist researchers to rapidly undertake nano-ferroptosis studies Microscopy imaging Microscopy, as one of the most widely used techniques, can be conducted to visualize the fine structures of subcellular organelle and metabolite/protein/gene changes at the molecular level, such as TEM and confocal microscopy Unlike other nanoparticle-induced cell death, ferroptotic cells merely display some changes in the morphology of mitochondria Since this organelle is closely related to ferroptotic cell death, high-resolution visualization of mitochondria may provide some desirable evidence For instance, Zhang et al observed the shrinkage of mitochondria with fused mitochondrial cristae in ZnO-treated HUVEC cells under TEM.45 Similarly, these cellular mitochondria with a great number of fragments and short tubes were observed by super-resolution confocal microscopy after MitoTracker® Deep Red FM staining.45,80 Li et al visualized substantial abnormal mitochondria with shrinkage and reduction of cristae in lung tissues by TEM for the validation of ferroptosis in radiationinduced lung injury.84 Besides subcellular organellar imaging, labile iron accumulation and lipid peroxidation as two characteristic biochemical reactions in ferroptosis can be examined by fluorescence microscopy Considering the transient features of both Fe2+ and lipid peroxides, fluorescent substrates rapidly reacting with these two targets have been exploited for microscopy imaging, such as FeRhoNox-1 and BODIPY® 581/591 C11 reagent Specifically, FeRhoNox-1 is a non-fluorescent cell-permeable compound that can selectively react with labile Fe2+ in living cells for the emission of intense fluorescence at 575 nm.85 This staining agent has been recently exploited to detect the intracellular ferrous ions in bone marrow mononuclear cells from C57BL/6 mice exposed to iron dextran86 and Fe2+ release in the cytoplasm of epithelial and macrophage cells incubated with WS2 and MoS2 nanosheets (Fig 2).87 BODIPY® 581/591 C11 is a fluorescent phenylbutadiene segment that can shift its fluorescence from red to green upon reaction with lipid peroxides in live cells.88 Xu et al employed this fluorophore to visualize the cellular lipid peroxidation in BEAS-2B and alveolar macrophage cells by fluorescence microscopy.87 Beside of mammalian cells, this substrate could be extended to examine lipid peroxidation in bacteria Li and co-workers observed significant lipid peroxidation in E coli cells exposed to graphene oxides and their nanocomposites.89,90 Mass spectrometry Recently, mass spectrometry (MS) has been exploited as a potential tool for the detection of lipid peroxidation products Basically, MS measures the mass-to-charge This journal is © The Royal Society of Chemistry 2021 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Nanoscale Review Fig Visualization of the Fe2+ distribution in cells by FeRhoNox-1 staining (a) Representative images of Fe2+ in BEAS-2B cells by confocal microscopy after treatment with Fe(NH4)2(SO4)2 (a positive control), WS2 and MoS2 Scale bar: 10 μm (b) Representative images of Fe2+ in alveolar macrophages from lung tissue after exposure to WS2 with or without Fer-1 pre-treatment Scale bar: 10 μm Reproduced from ref 87, with permission from Springer Nature ratio of lipid molecules and produces a mass spectrum, which can offer information on the molecular mass, elemental composition and even chemical structure of lipids Compared to the ferric thiocyanate test, MS is more sensitive and can directly identify the products of lipid hydroperoxides in ferroptosis Various MS-based techniques have been developed for the identification of the structure–activity relationship between lipid hydroperoxides and ferroptosis.56,91 For instance, Kagan et al detected the structure of lipid hydroperoxides in ferroptotic cells using liquid chromatography coupled with MS (LC-MS).56 They found that only the oxidized phosphatidylethanolamines with two or three oxygens were ferroptosis biomarkers Isabel Weigand et al utilized matrix-assisted laser desorption/ionization (MALDI)-based MS to investigate the role of oxidized polyunsaturated fatty acids in the process of ferroptosis.92 Western blotting Western blotting is the gold-standard technique in molecular biology for the qualitative and quantitative identification of specific proteins It is achieved via electrophoresis separation in gels based on protein sizes and identification based on antigen–antibody interactions Based on the protein markers in ferroptosis, western blotting experiments were conducted in nearly all studies to verify the ferroptotic effect, especially for SLC7A11 reduction, GPX4 suppression and TfR1 expression.93 Eleftheriadis et al assessed the expression of these biomarkers by western blotting in proximal renal tubular epithelial cells and discovered the critical role of SLC7A11 and ferritin in the resistance to warm anoxia-reoxygenation.94 Wang et al employed this technique to explore the mechanism of glycyrrhizin on ferroptosis in acute liver failure The high-expression of GPX4, NRF2 and HO-1 in glycyrrhizintreated hepatocytes and mouse liver showed alleviated effects This journal is © The Royal Society of Chemistry 2021 in acute liver failure by inhibiting ferroptosis.95 Other protein markers involved in the p62-Keap1-NRF2, p53-SLC7A11, p53SAT1-ALOX15, HSPB1-TfR1, and FSP1-COQ10-NAD(P)H regulation pathways were examined by western blotting in ferroptosis studies.96,97 Besides, lipid peroxidation markers, such as SOD2 and 4-HNE, were examined via western blotting in the study by Zhou et al They found that the GPX4 and SOD2 proteins progressively decreased in ferroptosis, but 4-HNE increased during 12 h in a rat IRI model.98 Genetic analysis There are mainly two type of genetic techniques in ferroptosis investigations, including genetic analysis and gene mutagenesis Genetic analysis, including RNA interference (RNAi) screening and genomic screening, can be used to identify the key relevant genes For instance, to systemically study the mechanisms of ferroptosis, Gao et al performed RNAi screening for the large-scale functional analysis of ferroptotic cells Consequently, previously known ferroptosis genes (e.g., TFRC, IREB2, and SLC38A1) and some uncorroborated genes (e.g., ULK1, ATG3, ATG13, BECN1, and NCOA4) were found to be associated with ferroptosis.99 In general, although advances in gene analysis may be significantly beneficial for the identification of ferroptosis-related genes, gene mutagenesis is the most powerful technique to validate the findings Cao et al used genome-wide human haploid cell genetic screening technology to directly identify genes that regulate intracellular GSH abundance and confirm their role in ferroptosis regulation.100 Based on the screening results, the ABCC1/MRP1 gene as a negative regulator of intracellular GSH levels was identified from five candidate genes (KEAP1, ABCC1/MRP1, GSTO1, SETD5 and NAA38) by CRISPR-Cas9 technology This technique was also exploited by Doll and Bersuker et al to examine the protective role of FSP1 in Nanoscale, 2021, 13, 2266–2285 | 2273 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Review ferroptosis.19,20 Yang et al used siRNA to knockdown GPX4 in HT-1080 cells, which exhibited hypersensitivity to (1S,3R)-RSL3 lethality and rendered resistance to ferroptosis after GPX4 over-expression.101 Recently, more ferroptosis-related genes (e.g., NRF2, HSPB1, and ACSL4) have been examined by gene mutagenesis.102 Besides gene mutations in cells, knockout/knockin (KO/KI) mice have been widely studied to verify the effect of ferroptosis in many diseases To verify the effect of forebrain neurons ferroptosis in AD patients, Hambright et al established a novel mouse model (namely GPX4BIKO mouse) with conditional ablation of GPX4 in forebrain neurons They revealed that the cognitively impaired GPX4BIKO mice susceptible to ferroptosis exhibited hippocampal neurodegeneration, which could be attenuated by vitamin E with anti-ferroptosis activity.103 Notably, full deletion of GPX4 is lethal in adult mice by eliciting renal failure,104 implying that ferroptosis may take part in more basal biological processes Wang and workers produced various KO/KI mouse models, e.g., global SLC39A14-knockout mice and hepatocyte-specific SLC39A14-knockout mice,105 SLA7A11 conditional KO/KI mouse model106 and FPN KO mouse model.107 Other techniques Since ferroptosis is featured by its dependence on iron, total iron content or the ratio of Fe2+/Fe3+ is very relevant to ferroptotic cell death Inductively coupled plasma mass spectrometry (ICP-MS) is one of the most accurate techniques to quantify the contents of iron in biological systems Pepper et al developed a novel solvent extraction method to specifically extract Fe3+ in the organic phase with bis(2-ethylhexyl) hydrogen phosphate for distinguishing ferrous and ferric iron in biological solutions by ICP-MS.108 Moreover, fluorescence spectrophotometry is commonly used to identify Fe2+ and Fe3+ in biological solutions using fluorescent probes, which can selectively chelate Fe2+ (bathophenanthroline and ferrozine) and Fe3+ (Rhodamine B hydrazonespirolactam) with alterations in their spectra.109 Absorption near edge spectroscopy (XANES)110 and Mössbauer spectroscopy111,112 are two suitable techniques to distinguish ferric and ferrous phases by the positions of the Fe K-edge or center shifts derived from spectral fitting of resonant absorption However, these two techniques are rarely exploited for the assessment of iron metabolism in biological samples Mechanisms of nanoparticleinduced ferroptosis Understanding the mechanisms of nanoparticle-induced ferroptosis will have significant implications in nanomedicines and nanosafety Although ferroptosis mediated by nanomaterials displays the same classical characteristics as small molecule inducers, e.g., GPX4 inhibition, iron overloading and lipid peroxidation, the initial molecular events in the ferroptosis pathway of nanosized inducers are totally different Based on the reported ferroptosis signals induced by NPs, we propose three ferroptosis pathways, including membrane 2274 | Nanoscale, 2021, 13, 2266–2285 Nanoscale impairment, damage 3.1 lysosomal dysfunction and mitochondrial Membrane impairment The cellular plasma membrane is the first defence barrier to deny the free access of exogenous NPs In contrast to the lysosomal internalization of most nanomaterials, a few NPs, such as fumed silica113 and graphene oxide88 were found to display strong association with plasma membranes but rarely identified in lysosomes Considering that system xc− and TfR1 are important upstream proteins in iron metabolism, NP binding on membranes may affect the biological functions of these proteins Coincidently, GO and fumed silica have been extensively reported to elicit cell death in THP-1, BEAS-2B, A549 and HCT116 cells.88,113 Thus, it will be interesting to examine whether ferroptosis plays an important role in their hazardous effects Besides, other metal-based NPs may also impact the activities of DMT1 and TfR1 by dissolved metal ions Herbison et al reported that Co(II)Tf and Mn(II)Tf upregulated TFR1 and reduced ferritin, which affect iron homeostasis.114 Mn2+ can share the same importer (DMT1) with Fe2+, which may competitively affect the uptake of ferrous ions.115 E-cadherin as the intercellular interaction protein was identified to suppress ferroptosis by activation of the NF2 and Hippo signaling pathways.67 Recently, AuNPs were reported to increase E-cadherin expression,116 whereas rGO exposure promoted a decrease in the expression of E-cadherin.117 These NPs may impact ferroptosis via the cadherin–NF2–Hippo–YAP pathway Besides, membrane phospholipids containing polyunsaturated fatty acids (PUFAs) are susceptible to oxidative radicals Hydroxyl, hydroperoxyl and superoxide anion radicals may lead to lipid peroxidation.118 These molecules may serve as fuses to induce substantial lipid peroxides Recently, we found that the carbon radicals on graphene oxide nanosheets could elicit intense lipid peroxidation in THP-1 cells and alveolar macrophages of mouse lungs.88 Furthermore, the secondary products of lipid peroxidation of PUFAs, such as lipid hydroperoxides (LOOHs), MDA and 4-HNE, also play critical roles to initiate ferroptosis.70 Notably, lipid peroxidation occurs not only in the cytoplasmic membrane, but also in the mitochondrial and lysosomal membrane, which can substantially alter the properties of the lipid bilayer, including the area per lipid, membrane width, curvature, and lipid diffusion.119 These alterations may synergistically contribute to the tolerance of membrane to NPs For instance, Rossi et al demonstrated that the membrane width (hydrophobic regions) and lipid diffusion could determine the partial embedding of gold NPs across the membrane.120 Thus, according to the above discussion, it can be speculated that NPs may induce ferroptosis by dysfunction of ferroptosis-related proteins in membranes or initiation of PUFA oxidation for cascaded lipid peroxidation (Fig 3a) 3.2 Lysosomal dysfunction Ferroptosis has a close relation with lysosome dysfunction.121 This is not surprising as lysosome is an important depository This journal is © The Royal Society of Chemistry 2021 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Nanoscale Review Fig Mechanisms of nanoparticle-induced ferroptosis (a) Membrane impairment induced by nanoparticles involving lipid peroxidation and inactivation of system xc−; (b) lysosome dysfunction induced by nanoparticles including disruption of lysosomal membrane, alteration of acidic environment, modification of STEAP3 and DMT1 activities; and (c) mitochondrial damage induced by nanoparticles including destruction of mitochondrial morphology and dysregulation of the mitochondrial antioxidant defense as well as iron dyshomeostasis for large amounts of redox-active iron.122 This organelle may suffer undesirable interactions with endocytic NPs Consequently, some NPs may transform in this acidic and enzymatic compartment to elicit lysosomal impairment by redox reactions,123 denature lysosomal biomolecules,124 and physical interactions.125,126 Our recent study showed that lysosomal dysfunction by MoS2 and WS2 led to the release of ferrous ions in the cytoplasm The labile Fe2+ prompted the generation of oxidative radicals by Fenton reactions and induction of lipid peroxidation, finally eliciting ferroptosis.87 This iron-dependent cell death induced by WS2 and MoS2 nanosheets was further validated by the down expression of GPX4, amelioration effects of iron-chelators (DFP and DFX) and TfR knockdown Notably, modification of the WS2 and MoS2 nanosheets by Na2S or methanol ameliorated lysosomal impairment and diminished the release of Fe2+ in the cytoplasm, which ultimately contributed to the improvement of cell viability.87 Besides, cathepsins and ATPase in lysosomes were also found to regulate ferroptosis Wang et al reported that aminemodified polystyrene NPs incubated with cells for 12 h could induce lysosomal swelling, leading to the release of lysosomal enzymes (such as cathepsins B, D and L) and iron to activate ferroptosis.127 Additionally, lysosome-associated membrane protein-2 (LAMP2) deficiency was demonstrated to increase the risk of ROS-induced ferroptosis.128 However, severe lysosomal damage may trigger pyroptosis, which is dependent on the lysosomal release of cathepsin B and NLRP3 inflammation activation.129 Thus far, the impact of NPs on lysosomal iron metabolism still unclear The biotransformation of NPs may affect the release of iron from ferritin, reduction of Fe3+ and This journal is © The Royal Society of Chemistry 2021 export of Fe2+ (Fig 3b) Thus, substantial efforts are required to elucidate the detailed mechanisms 3.3 Mitochondrial damage Mitochondria as the central subcellular organelles to regulate substance and energy metabolism are involved in many types of programmed cell death, such as apoptosis, autophagy and ferroptosis.130 Here, we mainly focus on the mitochondrial metabolic regulations and morphological architecture alterations involved in nanoparticle-induced ferroptosis Plenty of evidence revealed that diverse cellular metabolic pathways, including iron, lipid and amino acid metabolism can trigger ferroptosis Zhang et al developed an efficient ferroptosis agent, FePt@MoS2 nanocomposites, which could release more than 30% Fe2+ within 72 h in the tumour microenvironment for the induction of ferroptosis by speeding up Fenton reactions.46 Huang et al disclosed that zero-valent iron nanoparticles (ZVI NPs) governed ferroptosis by the oxidative conversion of ZVI to Fe2+ to assist Fenton reactions for the induction of mitochondrial lipid peroxidation and MDA production.131 Besides iron-based nanomaterials, iron-free NPs with ion-leaking properties can also disturb mitochondrial iron homeostasis and metabolism for ferroptosis activation Zhang et al demonstrated that Zn2+ dissolved from ZnO NPs upregulated the mitochondrial voltage-dependent anion channel (VDAC) proteins, which are responsible for the transport of metabolites and irons from outer membrane of mitochondria.45 Mitochondrial ultrastructure changes are the hallmark of ferroptosis, including volume reduction, bilayer membrane density increment, outer mitochondrial membrane disruption Nanoscale, 2021, 13, 2266–2285 | 2275 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Review and the disappearance of mitochondrial cristae.132 SPION internalized in cardiomyocytes could be degraded into free Fe2+ in lysosomes The labile iron subsequently accumulated in the mitochondria, resulting in mitochondrial lipid peroxidation accumulation and mitochondrial membrane potential dissipation to mediate ferroptosis.76 Also, a positively charged lipophilic nanocarrier (Fe-CO@Mito-PNBE) was found to target the negatively charged mitochondrial membrane, leading to mitochondrial collapse and eventually cell ferroptosis.133 Mitochondrial injuries derived from the exposure of iron-free NPs could also modulate ferroptotic cell death SH-SY5Y cells treated by Co NPs were found to display swollen and degenerated mitochondria without any morphological changes in their nuclei.65 In addition to the alteration of the mitochondrial morphology, Ca2+ dyshomeostasis and GSH depletion were detected to induce ROS production and lipid peroxidation to mediate ferroptosis.65 Primarily, the metal ions (e.g., Fe2+/Fe3+, Zn2+, and Co2+) released from NPs may destroy the mitochondrial morphology and dysregulate the mitochondrial antioxidant defense system, resulting in ferroptosis Interestingly, NPs without ion release, such as salinomycin conjugated with gold nanoparticles (Sal-AuNPs) were also reported to induce ferroptosis by targeting breast cancer stem cells with a reduction in the mitochondrial membrane potential.134 These reports indicated that NPs can impair mitochondria by released metal ions or intact NPs to burst ROS generation, disrupt iron metabolism and elicit lipid peroxidation (Fig 3c) Structure–activity relationships Understanding the SARs of nanoparticles and ferroptosis requires the identification of key physicochemical properties responsible for ferroptotic cell death SAR knowledge can further assist the rational design of nanomaterials and nanoproducts.135,136 On one hand, enhancement of the key properties will be an effective strategy to improve the therapeutic effects of ferroptosis nano-inducers On the other hand, passivation of ferroptosis-related properties may offer insight for the precise design of safe nanoproducts 4.1 Surface vacancies Vacancies are defined as a type of defect in a crystal, where an atom is missing from one of the lattice sites.137 Surface vacancies on nanomaterials are capable of tuning various physical and chemical surface properties, such as conductivity, hydrophilicity, oxidizability, and catalytic activity.138,139 The impacts of vacancies were well documented in the study by Xu et al., where WS2 and MoS2 were found to induce ferroptosis by their active surfaces They prepared a comprehensive library of 2D transition metal dichalcogenides (TMDs) consisting of 20 nanosheets with changed gradients in diameter, thickness, surface area, surface charge, hydrodynamic size and vacancy density The in silico analysis between property intensities and cell viabilities revealed the critical role of vacancies in 2276 | Nanoscale, 2021, 13, 2266–2285 Nanoscale Fig Surface vacancy-induced ferroptosis (a) Representative HR-TEM images of vacancies on pristine and modified TMD surfaces The scale bar is nm (b) Cell viability of pristine and modified TMD in THP-1 and BEAS-2B cells Cell viability data is presented as mean ± SD Reproduced from ref 87, with permission from Springer Nature ferroptosis The identified SAR was further validated by visualization of vacancies under high-resolution TEM and vacancy healing experiments (Fig 4) These vacancies led to lysosomal dysfunction by oxidative reactions or phospholipid extraction.87 In addition, surface vacancies may modulate the catalytic activity of NPs (Fig 5a).140,142 Esmailpour et al demonstrated that the enhancement of oxygen vacancy levels on ceria could intensify its performance in catalytic ozonation.141 Gao et al reported that the dehydrogenase activity of SnSe was partially determined by Sn vacancies.138 Given that Fenton reactions may occur in the catalytic sites of NPs, modulation of the density of vacancies can impact the generation of oxidative radicals in cells Zhang and co-workers designed a novel nanocluster with plenty of oxygen vacancies on Gd-doped Fe3O4, which captured oxygen molecules to enhance the catalytic activity of Fenton reactions The intracellular ROS accumulation could dramatically down-regulate the NADPH and GSH levels, resulting in the inhibition of GPX4 expression and activity.143 Since some nanozymes have been identified to display oxidase/peroxidase-like activity by Fenton reactions,144,145 we speculate that these NPs may also elicit ferroptosis in cells 4.2 Surface groups Chemical modification of the surface of NPs (–COOH, –NH2, –OH, etc.) plays a decisive role in ferroptosis by modulating the surface properties of NPs, such as surface charge, hydrophilicity and corona formation (Fig 5b).146,147 These surface properties have been well documented to account for the hazar- This journal is © The Royal Society of Chemistry 2021 View Article Online Published on 07 January 2021 Downloaded by RMIT University Library on 3/27/2021 1:56:05 PM Nanoscale Review Fig Structure–activity relationships of nanoparticle properties and ferroptosis (a) Surface vacancy induces ferroptosis by modulation of oxidation capability or surface catalysis activity to trigger lysosome dysfunction or membrane impairment; (b) surface chemical groups affect surface charge, hydrophilicity and corona formation to determine nano–bio interactions; (c) different particle sizes decide their cellular uptake to regulate ferroptosis; and (d) ions released from nanoparticles trigger lysosome dysfunction or mitochondrial damage by Fenton-like reactions dous effects of NPs Although positively charged and hydrophobic NPs display more hazardous effects,148,149 corona formation can greatly attenuate their cytotoxicity For instance, protein corona-coated gold clusters were rejected by HCT116 cells and displayed excellent stability (fluorescence and morphology) in the gastrointestinal tract, whereas the cells had good affinity for the positively charged clusters.150 Thus, the surface functionalities may dramatically impact the cellular uptake of NPs Recently, Wu et al prepared a series of graphene quantum dots (GQDs) with similar sizes (