Nanoparticle induced ferroptosis detection methods, mechanisms and applications

suggested that TfR2 mutations may play a protective role in PD owing to the reduction in iron uptake.33In ischemic stroke, free iron and ferritin may invade the brain parenchyma via defe

Cite this: Nanoscale, 2021, 13, 2266

Received 29th November 2020,

Accepted 7th January 2021

DOI: 10.1039/d0nr08478f

rsc.li/nanoscale

Nanoparticle-induced ferroptosis: detection methods, mechanisms and applications

Huizhen Zheng, aJun Jiang,aShujuan Xu,aWei Liu,bQianqian Xie,aXiaoming Cai,c Jie Zhang,dSijin Liu band 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 ferrop-tosis for the treatment of these diseases has attracted substantial research interest However, it is challenging

to di fferentiate 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 speci fically examine ferroptotic cell deaths Then, we discuss the molecular initiating events of 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.

1 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 syn-thesis, 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

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 inter-actions

Jun Jiang

Jun Jiang is a Graduate Student under the supervision of Prof Li

in Soochow University He is focused on increasing thera-peutic effects via the combi-nation of radiotherapy and ferroptosis

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

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Therefore, living systems have evolved with sophisticated

feed-back loops to maintain iron homeostasis

However, some exogenous stimuli may disrupt iron

homeo-stasis, resulting in severe pathological changes in mammalian

tissues Liu and co-workers found that polychlorinated

biphe-nyls 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.3Besides these small

chemi-cals, some nanoparticles (NPs) have been reported to disrupt

iron metabolism4 and induce ferroptosis signals in

mamma-lian cells Consequently, this has caused considerable

con-cerns on nanosafety since nanomaterials are increasingly

syn-thesized 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

sig-nificant 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

fab-rication, 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.7,8Considering

that iron deficiency has been well elucidated in the past

decades,9it is not discussed in this review In contrast,

ferrop-tosis 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.10This pathway enables fine

tuning of the iron levels in cells via the regulation of iron

uptake proteins (transferrin receptor (TfR1) and divalent metal

transporter 1 (DMT1)), iron transport protein (transferrin),

iron storage protein (ferritins) and iron efflux protein

(ferro-portin (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 pro-motion 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.11On 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, intra-cellular transportation and elimination Firstly, extraintra-cellular

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 fer-ritin may be transported into lysosomes by autophagosomes for the recycle of Fe3+ The enzymatic and acidic lysosomal com-partment 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 pros-tate 3 (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 mitochondria14for 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

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 nano-materials

Wei Liu

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 metab-olism disorders

the first time in 2012.18Different 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

peroxi-dase 4 (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 1 (FSP1)

was discovered by Doll et al and Bersuker et al in 2019

Interestingly, this enzyme can convert ubiquinone into

ubiqui-nol in cell membranes to suppress lipid peroxidation and

protect cells from ferroptosis.19,20Different to the dramatic cell

morphology changes in apoptosis, necrosis and pyroptosis,

fer-roptotic cells display intact cell membranes, normal-sized

nuclei and dispersive chromatins.18,21However, ferroptotic cells

have been found to show dramatic changes in their

mitochon-dria, 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

myo-cardial 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.26,27Iron 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

aggre-gation and oligomerization of Aβ peptides.28In addition, iron

overload was revealed to regulate Tau hyperphosphorylation

and aggregation, which is another hallmark of AD.29Thus, fer-roptosis in AD tissue can be considered a diagnostic index Similarly, iron accumulation was also identified in the sub-stantia 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.31Also, 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.33In ischemic stroke, free iron and ferritin may invade the brain parenchyma via defects in the blood brain barrier (BBB), eliciting Fenton reactions, lipid per-oxidation 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 sensi-tivity 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 signal-ling pathways to apoptosis, and via its activation, conventional chemotherapy and radiotherapy can eliminate cancer cells Especially, ferroptotic cell death provides opportunities to over-come 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 demon-strated to selectively trigger ferroptosis in human hepatocellu-lar carcinoma cells by lipid peroxidation, GSH depletion, and GPX4 inactivation.43

Qianqian Xie

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 per-oxides

Xiaoming Cai

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, struc-ture–activity relationships of nanomaterials and interactions

of biomolecules with ENMs

1.4 Nano-ferroptosis studies

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

con-sidered 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

metab-olism disruption.44–46Consequently, 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

ferrop-tosis.47Recently, a few reviews have discussed the potential

uti-lities of ferroptosis in nanomedicine.27,28Besides, 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

tech-niques to characterize nanoparticle-induced ferroptosis,

pro-posed some unique cellular mechanisms and discussed the

key physicochemical properties of NPs responsible for

ferrop-tosis 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

2 Detection methods of ferroptosis

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 elec-tron microscopy (TEM) in ferroptotic cells revealed some alterations in the membrane and crista of mitochondria.50For instance, Fadeel and co-worker performed TEM to observe the morphologies of Jurkat cells in apoptosis, necrosis, and ferrop-tosis, and successfully differentiated ferroptotic cells by com-parison of both their mitochondrial and nuclear changes.51 Since mitochondria play a critical role in ferroptosis, Gao et al demonstrated that mitochondria-depleted cells (Parkin-mediated mitophagy) were less sensitive to ferroptosis induced

by cysteine starvation or erastin.52Besides, the molecular bio-marker 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 peroxi-dation and the reduction of GSH.53An increase in intracellular labile iron may result in oxidative radical generation by Fenton reactions, further leading to lipid peroxidation Although lipid

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

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 beha-viors and biological effects of engineered nanomaterials and fine air particles and (ii) the molecular mechanisms respon-sible for environmental pollu-tant-induced oncogenic effects

Table 1 Biomarkers of ferroptosis

Metabolites Iron accumulation, lipid peroxidation, MDA, 4-HNE, GSH,

NADPH and selenium contents

Iron-metabolized dysfunction, ROS generation, Fenton reaction acceleration, lipid peroxidation, and GPX4 inhibition

Proteins SLC7A11, GPX4, ATG5-ATG7-NCOA4, IREB2,

p62-Keap1-NRF2, p53-SAT1-ALOX15, ACSL4 and LPCAT3

Regulating GSH/GPX4, iron metabolism and lipid metabolism pathways

Genes GPX4, TFR1, SLC7A11, NRF2, NCOA4, p53, HSPB1, ACSL4,

FSP1 and KOD

Encoding proteins associated with ferroptosis pathways

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.54,55Alternatively, the

second-ary 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

hydroperox-ides 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

phar-macy Based on the amino acid metabolism, iron metabolism

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 LPCAT3.50Among 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 respon-sible 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 fer-roptosis.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 eli-cited lipid peroxidation and ferroptosis.60 Friedmann Angeli

et al demonstrated that the knockout of GPX4 may cause fer-roptotic cell death in GPX4−/−mice, and this effect could be rescued by Liproxstatin-1 treatment.61The increment in GPX4 biosynthesis by the supplementation of selenium was reported

to be an effective method to enhance tolerance against ferrop-totic cell death.62Besides, transport, reductive and storage pro-teins 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.50Song et al demonstrated that DMT1 was highly 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) under-standing nano–bio interactions;

(ii) developing predictive toxi-cology methods for nanosafety assessment; and (iii) using nano-technologies for immunotherapy, anti-resistant bacterial coating, radiotherapy and radio imaging

Fig 1 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.

expressed in ferroptotic cells in the infraction myocardium.63

Li et al found that NCOA4 could increase the level of

intra-cellular free iron to induce ferroptosis in LPS-treated

cardio-myocytes 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

p53-SAT1-ALOX15, ACSL4 and LPCAT3 are all relevant to

ferropto-sis Doll et al disclosed that ACSL4 was an essential

com-ponent 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

fer-roptotic cell death.19,50 For instance, Cheng et al

demon-strated that ACSL4 could exert anti-proliferative effects in

glioma cells by the induction of a ferroptosis pathway

Over-expression 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

sorafenib-induced 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

inhibit or induce this effect (Table 2) More than seven cat-egories 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.69–71GPX4 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.72Ferroptosis can also be elevated by the exposure of cells to ferric ammonium citrate (FAC) for direct overloading of intracellular iron.73 FINO2is 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 biosyn-thesis 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 nano-particles (SPION) released free divalent iron to accelerate Fenton reactions and lipid peroxide accumulation for the induction of ferroptosis in ischemic cardiomyocytes.76 Encapsulation of H2O2in the hydrophilic core of an Fe3O4-poly (lactic-co-glycolic acid) (PLGA) could also induce ferroptosis by

Table 2 Inducers and inhibitors of ferroptosis

Inducer Inhibition system xc− Erastin, PE, IKE, other erastin analogs; sulfasalazine; glutamate;

sorafenib

NA Suppression GPX4 (1S,3R)-RSL3; ML162, FIN56; DPI family members Iron-free nanomaterials, e.g., WS2,

MoS2, Co NP Iron dyshomostasis ferric ammonium citrate Iron-based nanomaterials, e.g., Iron

oxide (IO), Iron-organic NP, FePt GSH depletion Cystine/cysteine deprivation, BSO, DPI2, cisplatin; cysteinase,

acetaminophen

ZnO NP Lipid peroxidation FINO2 WS2, MoS2, Iron-organic NP, FePt CoQ10 biosynthesis

inhibition

Reduction of lipid peroxidation

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

CPS

System xc−activation Cycloheximide, beta-mercaptoethanol NA

Selenoprotein increment

Fenton reactions to provide excessive oxidized lipids in cancer

cells.77Chen 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

intern-alization 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

lysoso-mal internalization of WS2and MoS2could induce the release

of labile iron in the cytoplastic compartments.87Co NPs were

discovered to release Co2+from vesicles/endosomes to regulate

the reduction of GPX4 expression and depletion of cellular

GSH levels.49Zhang 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.80These hazard signals may eventually cumulate

in ferroptotic cell death

Since ferroptosis is considered to be a relevant cause in

neurodegenerative, cardiovascular and cerebrovascular

dis-eases, ferroptosis inhibitors may have therapeutic effects on

these diseases Although diverse chemicals have displayed

inhibitory effects in ferroptotic cell death, few of them

specifi-cally work on ferroptosis For instance, iron chelators

includ-ing 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

liprox-statin-1 (Lip-1), have been found to attenuate lipid

peroxi-dation.82Cycloheximide andβ-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.83Considering 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

convincing characterization of ferroptosis Although there are a cascade cellular events of small molecules, proteins, genes and subcellular organelles involved in ferroptosis, tiered assess-ments are highly recommended to accurately identify ferropto-sis from other types of cell death since the molecular mecha-nisms involved in ferroptosis are still unknown and there is no distinct morphology change in ferroptotic cells Therefore, mul-tiple complementary techniques are required for the successful identification of ferroptotic cell death A summary of the ferrop-tosis 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 struc-tures of subcellular organelle and metabolite/protein/gene changes at the molecular level, such as TEM and confocal microscopy Unlike other nanoparticle-induced cell death, fer-roptotic cells merely display some changes in the morphology

of mitochondria Since this organelle is closely related to fer-roptotic cell death, high-resolution visualization of mitochon-dria may provide some desirable evidence For instance, Zhang

et al observed the shrinkage of mitochondria with fused mito-chondrial 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 mito-chondria with shrinkage and reduction of cristae in lung tissues by TEM for the validation of ferroptosis in radiation-induced lung injury.84

Besides subcellular organellar imaging, labile iron accumu-lation 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.85This staining agent has been recently exploited to detect the intra-cellular ferrous ions in bone marrow mononuclear cells from C57BL/6 mice exposed to iron dextran86and Fe2+release in the cytoplasm of epithelial and macrophage cells incubated with

WS2and MoS2nanosheets (Fig 2).87BODIPY® 581/591 C11 is a fluorescent phenylbutadiene segment that can shift its fluo-rescence from red to green upon reaction with lipid peroxides

in live cells.88Xu et al employed this fluorophore to visualize the cellular lipid peroxidation in BEAS-2B and alveolar macro-phage cells by fluorescence microscopy.87Beside of mammalian cells, this substrate could be extended to examine lipid peroxi-dation 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 per-oxidation products Basically, MS measures the mass-to-charge

ratio of lipid molecules and produces a mass spectrum, which

can offer information on the molecular mass, elemental

com-position 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

ferrop-tosis Various MS-based techniques have been developed for

the identification of the structure–activity relationship between

lipid hydroperoxides and ferroptosis.56,91For instance, Kagan

et al detected the structure of lipid hydroperoxides in

ferropto-tic cells using liquid chromatography coupled with MS

(LC-MS).56 They found that only the oxidized

phosphatidy-lethanolamines with two or three oxygens were ferroptosis

bio-markers Isabel Weigand et al utilized matrix-assisted laser

de-sorption/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

tech-nique in molecular biology for the qualitative and quantitative

identification of specific proteins It is achieved via

electro-phoresis separation in gels based on protein sizes and

identifi-cation 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-reoxy-genation.94Wang 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

glycyrrhizin-treated hepatocytes and mouse liver showed alleviated effects

in acute liver failure by inhibiting ferroptosis.95Other protein markers involved in the p62-Keap1-NRF2, SLC7A11, p53-SAT1-ALOX15, HSPB1-TfR1, and FSP1-COQ10-NAD(P)H regu-lation pathways were examined by western blotting in ferropto-sis 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 pro-teins 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 tech-niques in ferroptosis investigations, including genetic analysis and gene mutagenesis Genetic analysis, including RNA inter-ference (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 fer-roptotic cells Consequently, previously known ferroptosis genes (e.g., TFRC, IREB2, and SLC38A1) and some uncorrobo-rated 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 ben-eficial 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

Fig 2 Visualization of the Fe 2+ distribution in cells by FeRhoNox-1 staining (a) Representative images of Fe 2+ in BEAS-2B cells by confocal microscopy after treatment with Fe(NH4)2(SO4)2(a positive control), WS2and MoS2 Scale bar: 10 μm (b) Representative images of Fe 2+ in alveolar macrophages from lung tissue after exposure to WS2with or without Fer-1 pre-treatment Scale bar: 10 μm Reproduced from ref 87, with per-mission from Springer Nature.

ferroptosis.19,20Yang 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

fer-roptosis 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

elicit-ing renal failure,104implying 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

depen-dence 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

accu-rate 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

fluo-rescent probes, which can selectively chelate Fe2+

(bathophe-nanthroline and ferrozine) and Fe3+(Rhodamine B

hydrazone-spirolactam) 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

absorp-tion However, these two techniques are rarely exploited for the

assessment of iron metabolism in biological samples

3 Mechanisms of

nanoparticle-induced ferroptosis

Understanding the mechanisms of nanoparticle-induced

fer-roptosis will have significant implications in nanomedicines

and nanosafety Although ferroptosis mediated by

nano-materials displays the same classical characteristics as small

molecule inducers, e.g., GPX4 inhibition, iron overloading and

lipid peroxidation, the initial molecular events in the

ferropto-sis pathway of nanosized inducers are totally different Based

on the reported ferroptosis signals induced by NPs, we

propose three ferroptosis pathways, including membrane

impairment, lysosomal dysfunction and mitochondrial damage

3.1 Membrane impairment The cellular plasma membrane is the first defence barrier to deny the free access of exogenous NPs In contrast to the lyso-somal internalization of most nanomaterials, a few NPs, such

as fumed silica113and graphene oxide88were found to display strong association with plasma membranes but rarely identi-fied 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 exten-sively 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 compe-titively affect the uptake of ferrous ions.115E-cadherin as the intercellular interaction protein was identified to suppress fer-roptosis by activation of the NF2 and Hippo signaling path-ways.67Recently, AuNPs were reported to increase E-cadherin expression,116 whereas rGO exposure promoted a decrease in the expression of E-cadherin.117These NPs may impact ferrop-tosis via the cadherin–NF2–Hippo–YAP pathway

Besides, membrane phospholipids containing polyun-saturated fatty acids (PUFAs) are susceptible to oxidative rad-icals Hydroxyl, hydroperoxyl and superoxide anion radicals may lead to lipid peroxidation.118These 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 alveo-lar macrophages of mouse lungs.88Furthermore, the second-ary 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 substan-tially 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

for large amounts of redox-active iron.122This 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.125,126Our recent study showed that

lyso-somal 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

induc-tion of lipid peroxidainduc-tion, 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

MoS2nanosheets by Na2S or methanol ameliorated lysosomal

impairment and diminished the release of Fe2+in the

cyto-plasm, 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

amine-modified 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.128However, 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

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 altera-tions 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@MoS2nanocomposites, which could release more than 30% Fe2+within 72 h in the tumour microenvironment for the induction of ferroptosis by speeding up Fenton reac-tions.46 Huang et al disclosed that zero-valent iron nano-particles (ZVI NPs) governed ferroptosis by the oxidative con-version of ZVI to Fe2+to assist Fenton reactions for the induc-tion of mitochondrial lipid peroxidainduc-tion and MDA pro-duction.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 trans-port 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

Fig 3 Mechanisms of nanoparticle-induced ferroptosis (a) Membrane impairment induced by nanoparticles involving lipid peroxidation and inacti-vation of system xc−; (b) lysosome dysfunction induced by nanoparticles including disruption of lysosomal membrane, alteration of acidic environ-ment, modi fication 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.