Fenton reaction-independent ferroptosis therapy via glutathione and iron redox couple sequentially triggered lipid peroxide generator

Journal Pre-proof Fenton reaction-independent ferroptosis therapy via Glutathione and iron redox couple sequentially triggered lipid peroxide generator Yu-Jing He, Xiao-Ying Liu, Lei Xing, Xing Wan, Xin Chang, Hu-Lin Jiang PII:

S0142-9612(20)30157-5

DOI:

https://doi.org/10.1016/j.biomaterials.2020.119911

Reference:

JBMT 119911

To appear in:

Biomaterials

Received Date: 25 October 2019 Revised Date:

11 February 2020

Accepted Date: 23 February 2020

Please cite this article as: He Y-J, Liu X-Y, Xing L, Wan X, Chang X, Jiang H-L, Fenton reactionindependent ferroptosis therapy via Glutathione and iron redox couple sequentially triggered lipid peroxide generator, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2020.119911. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Credit Author Statement Hu-Lin Jiang and Yu-Jing He conceived this project. Hu-Lin Jiang, Yu-Jing He, Xiao-Ying Liu, and Lei Xing designed and supervised the project and commented on the project. Yu-Jing He, Xiao-Ying Liu, and Xing Wan prepared and characterised the LPOgener, performed in vitro and in vivo experiments and analysed the data. Hu-Lin Jiang, Yu-Jing He, Xiao-Ying Liu, and Lei Xing wrote the paper. All the authors contributed to the discussion during the whole project.

Fenton Reaction-Independent Ferroptosis Therapy via Glutathione and Iron Redox Couple Sequentially Triggered Lipid Peroxide Generator Yu-Jing He1,2,†, Xiao-Ying Liu1,†, Lei Xing1,3,4,5,†, Xing Wan1, Xin Chang1, Hu-Lin Jiang1,3,4,5,* 1

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China

Pharmaceutical University, Nanjing 210009, China 2

School of Pharmaceutical Sciences, Shandong First Medical University & Shandong

Academy of Medical Sciences, Taian 271016, China 3

Jiangsu Key Laboratory of Druggability of Biopharmaceuticals,

China

Pharmaceutical University, Nanjing 210009, China 4

Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University,

Nanjing 210009, China 5

Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China

Pharmaceutical University, Nanjing 210009, China † These authors contributed equally to this work. * Corresponding author. E-mail: [email protected] Abstract: Ferroptosis, a newfound non-apoptotic cell death pathway, results from the accumulation of iron-dependent lipid peroxide (LPO). Recently, emerging iron-based nanomaterials have been extensively developed to induce Fenton reaction-dependent ferroptosis for cancer therapy. However, insufficient amount of H2O2 and limited acidity of tumor could not satisfy the optimal conditions for Fenton reaction, which extremely limited the efficacy of ferroptosis therapy. Herein, we report a novel glutathione (GSH) and iron redox couple sequentially triggered LPO generator (LPOgener) which can directly supply the Fenton reaction-independent downstream executioner of ferroptosis for cancer therapy. By harnessing GSH-mediated Fe3+ reduction and the well-established iron redox couple-mediated lipid peroxidation, LPOgener was constructed by complete ferric ammonium citrate (FAC) and unsaturated lipids-rich phosphatidylcholine, and formed as FAC loaded liposome. The Fe3+ encapsulated in LPOgener could be efficiently reduced to Fe2+ under high GSH level in tumor cells. Subsequently, the formed iron redox couple could trigger overwhelming lipid peroxidation for Fenton reaction-independent ferroptosis. Superior anticancer therapeutic effect with little systemic toxicity demonstrated that

LPOgener was a potent ferroptosis-inducing agent for cancer therapy. Therefore, to directly supply the druglike, easily prepared, GSH and iron redox couple sequentially triggered LPOgener would provide a new direction in designing strategies for ferroptosis therapy. Keywords: ferroptosis; iron redox couple; ferric ammonium citrate; liposome; lipid peroxide

1. Introduction As a newly annotated mode of regulated cell death, ferroptosis is biochemically, genetically, and morphologically distinct from apoptosis, necrosis and autophage[1-3]. The classical feature of ferroptosis is the overwhelming, iron-dependent accumulation of lethal lipid peroxide (LPO)[4-6], which will perturb cellular redox homeostasis and has great potential in killing cancer cells[7-10]. Recently, various iron-based nanomaterials have been widely investigated to induce ferroptosis for the proposed reason that Fenton reaction between irons and tumor cellular H2O2 could generate reactive oxygen species (ROS), which further causes the accumulation of LPO[11-17]. However, insufficient amount of endogenous H2O2 in tumor region severely restricts iron-mediated Fenton reaction[18,19]. Meanwhile, the tumor microenvironment is difficult to fulfill the optimal condition (pH = 3-4) for Fenton reaction[20,21]. Therefore, the ferroptosis induced by current iron-based nanomaterials is far from satisfactory, which generally requires a very high Fe dose, wrapping exogenous H2O2 or stimulating the production of endogenous H2O2 to enhance Fenton reaction induced lipid peroxidation[22-24]. Since the accumulation of iron-dependent LPO is considered as the downstream executioner in the process of ferroptotic cell death[25], exploiting and applying a Fenton reaction-independent LPO generator (LPOgener) could effectively promote the efficacy of ferroptosis therapy. Fortunately, it is well established that iron redox couple (Fe2+/Fe3+) could initiate lipid peroxidation in the absence of H2O2[26-30]. The coexist of Fe2+ and Fe3+ mediates electron transfer for the redox reaction between oxygen and unsaturated lipids, while complete Fe2+ or complete Fe3+ results in the condition that do not promote lipid peroxidation. Therefore, the critical factors of iron redox couple-triggered ferroptosis-inducing agents are proposed as the simultaneous participation of: (1) iron redox couple, and (2) unsaturated lipids. Moreover, Fe3+ could be efficiently reduced

into Fe2+ by various reductive agents, especially high level of GSH in tumor cells[31,32], and then formed iron redox couple for lipid peroxidation. This is a favorable indication that designing a ferroptosis-inducing LPOgener by co-delivery of complete Fe3+ and unsaturated lipids can cause iron redox couple-triggered lipid peroxidation especially in tumor sites, but avoid damage to normal tissues. Herein, we report a ferroptosis-inducing agent with GSH and iron redox couple sequentially triggered LPOgener for Fenton reaction-independent ferroptosis therapy (Scheme). Taking advantages of unsaturated lipids-rich phosphatidylcholine and biocompatible ferric ammonium citrate (FAC)[33,34], the LPOgener was prepared through extrusion, and formed as FAC loaded liposome. Upon LPOgener internalization into tumor cells via endocytosis, overwhelming lipid peroxidation was triggered by GSH-mediated Fe3+ reduction and the subsequently formed iron redox couple. The efficiencies of LPOgener for LPO generation and cancer therapy were evaluated both in vitro and in vivo. As a consequence, the GSH and iron redox couple sequentially triggered LPOgener would overcome the limitations of Fenton reaction induced ferroptosis and provide a new approach for cancer therapy.

Scheme. GSH and iron redox couple sequentially triggered LPOgener for Fenton reaction-independent ferroptosis therapy. The lipid peroxidation process of (A) LPOgener and (B) Fenton reaction-dependent LPO. (C) Schematic illustration of GSH and iron redox couple sequentially triggered LPOgener mediated ferroptosis. LPOgener was internalized into tumor cells via endocytosis. Fe3+ could be efficiently reduced into Fe2+ under high GSH level in tumor cells. Subsequently, the formed iron redox couple initiated lipid peroxidation, which further caused ferroptotic cancer cell death.

2. Materials and Methods 2.1. Materials Ferric ammonium citrate (FAC), Deferoxamine (DFO), Sodium azide (NaN3), Ferrostatin-1 (Fer-1) and 3-methyladenine (Aut) were purchased from Sigma-Aldrich (St.

Louis,

MO,

USA).

Phosphatidylcholine

1,2-Dimyristoyl-sn-glycero-3-phosphocholine Southeast

pharmaceuticals

Co.,

(DMPC) Ltd

(PC, were

98%)

and

purchased

from

(Soochow,

China).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Vitamin C (VC), Vitamin E (VE), Necrostatin-1 (Nec-1), trypan blue dye and amiloride hydrochloride were purchased from J&K (Beijing, China). Glutathione (GSH) was purchased from Biosharp (Hefei, China). β-Cyclodextrin (β-CD) was purchased from Sinopharm

Chemical

Reagent

Co.,

Ltd

(Shanghai,

China).

1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine Iodide (DiR) was purchased from Beijing Fanbo Biochemicals Co., Ltd (Beijing, China). Coumarin 6 (C6) was purchased from Tokyo chemical industry Co., Ltd (Tokyo, Japan). Z-VAD-FMK (Apo) was purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China). Lysotracker Green, Mitotracker Green, DIO, Hoechst 33342, MDA Assay Kit, Calcein-AM/PI Double Stain Kit, RPMI 1640 and DMEM were purchased from KeyGEN Biotech (Nanjing, China). Trypsin with EDTA solution (0.25%) was obtained from Gibco (Burlington, ON, Canada). Fetal bovine serum (FBS) and C11-BODIPY581/591 were purchased from Thermo Fisher (Waltham, MA, USA). 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and GSH Assay Kit were purchased from Beyondtime (Nanjing, China). All other chemicals and reagents were analytical grade.

2.2. Preparation of FAC loaded liposome (LPOgener), empty liposome (Lip), coumarin 6 loaded liposome (Lip@C6) and DiR loaded liposome (Lip@DiR) Briefly, PC was dissolved in ethanol and dropwise added into saline solution of FAC under continuous stirring. The mixed solution was dialyzed against saline by using dialysis bag with molecular weight cut-off (MWCO) of 14 KDa to remove residual ethanol and the unloaded FAC. Ultimately, LPOgener was obtained by extrusion with a polycarbonate membrane of 50 nm. Lip was prepared according to the method described above without adding FAC. Coumarin 6 (C6) was dissolved in DMF, and PC was dissolved in ethanol. The mixed solution of C6 and PC was dropwise added into saline under continuous stirring. After dialysis against saline, Lip@C6 was obtained by extrusion with a membrance of 50 nm. DiR was dissolved in DMSO, and PC was dissolved in ethanol. The mixed solution of DiR and PC was dropwise added into saline under continuous stirring. After dialysis against saline, Lip@DiR was obtained by extrusion with a membrance of 50 nm. 2.3. Lipid peroxidation of LPOgener under GSH and FeSO4 Briefly, Lip and LPOgener were mixed with (1) GSH, (2) FeSO4, respectively. After incubating overnight, the samples were detected by mass spectrometer (Waters, Q-TOF micro). 2.4. Characterization of LPOgener, Lip, Lip@C6 and Lip@DiR The size distribution of LPOgener, Lip, Lip@C6 and Lip@DiR were measured by ZetaPlus particle size analyzer (Brookhaven Instruments, USA). For transmission electron microscope (TEM) analysis, LPOgener was directly dropped onto a copper grid followed by phosphotungstic acid staining and vacuum drying, and then observed by TEM (JEM-200CX, JEOL, Japan) of 100 kV accelerating voltage. 2.5. Stability of LPOgener LPOgener was dispersed in saline, sequentially diluted in different times, and the particle size was determined to obtain the dilution stability curve. Meanwhile,

appropriate amount of LPOgener was dispersed in equal volume of saline, 10 mM PBS, and 10% FBS for a week to get the solvent stability curve[35]. 2.6. Release profiles of LPOgener The amount of FAC ultraviolet-visible

(UV-Vis)

encapsulated in spectrophotometer

LPOgener was measured by with

maximum

absorbance

wavelength at 260 nm which was not interfered by PC[36]. Briefly, LPOgener was dissolved in ethanol for demulsification, and then FAC could be detected using a pre-established calibration curve. To evaluate the release behavior of LPOgener, dialysis method was applied using dialysis bag (MWCO, 14 KDa) in 10 mM PBS at different pH values (pH 4.5, 5.5, 6.8, 7.4)[37]. Essentially, 4 mL of LPOgener was added into dialysis bag and transferred into 50 mL PBS solution and gently shaken at 37 oC with 120 rpm. At predetermined time intervals, 3 mL of media was withdrawn and replaced with equivalent fresh buffer to retain a constant volume. The amount of released FAC was determined by UV-Vis absorption method. Each sample was performed in triplicate. 2.7. Cell culture Multidrug-resistant human breast cancer MCF-7/ADR cells, human breast cancer MCF-7 cells, mouse breast cancer 4T1 cells and human hepatic L02 cells were purchased from American type culture collection (ATCC). MCF-7 cells and MCF-7/ADR cells were cultured in RPMI 1640 medium, 4T1 cells and L02 cells were cultured in DMEM medium, which supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 U/mL of penicillin and 100 U/mL of streptomycin) at 37 oC and 5% CO2. In addition, 1 µM DOX was added for the culture of MCF-7/ADR cells to maintain the resistant phenotype. 2.8. Cell morphology, trypan blue staining and live/dead staining 4T1 cells were seeded in 24-well plates at a density of 1.0×105 per well in 1 mL of complete DMEM and incubated for 24 h. FAC, Lip and LPOgener were added and cultured for 24 h. After removing the medium and washing with PBS for 3 times, the cell morphology was photographed under an inverted fluorescent microscope (Nikon

ti-s, Japan). Furthermore, trypan blue staining and live/dead staining were used to assess the cytotoxicity of LPOgener on 4T1 cells. Briefly, 4T1 cells were seeded in 24-well plates at a density of 1.0×105 per well in 1 mL of complete DMEM and incubated for 24 h. FAC, Lip and LPOgener were added and cultured for 24 h. After removing the medium and washing with PBS for 3 times, trypan blue (0.05%) was added into each well for 5 min staining and then washed with PBS for 3 times. The cells were photographed under an inverted fluorescent microscope (Nikon ti-s, Japan). For live/dead staining, the treated cells were stained according to the protocol of Calcein-AM/PI Double Stain Kit. The cells were also photographed under an inverted fluorescent microscope (Nikon ti-s, Japan). 2.9. In vitro cytotoxicity for optimizing the formulation In order to optimize the formulation of LPOgener, we evaluated the cell cytotoxicity on 4T1 cells with different mole ratio of FAC and Lip. Briefly, 4T1 cells were seeded in 96-well plates at a density of 1.0×104 per well in 200 µL of complete DMEM and incubated for 24 h. Subsequently, we set the concentration of FAC at 10, 20, 30, 40, 50, 60 µM. Under every concentration of FAC, Lip was added with different concentrations varing from 10 to 60 µM. After 48 h treatment, MTT solution (20 µL, 5 mg/mL) was added into each well and incubated for an additional 4 h in 37 o

C incubator. The medium was then removed, and the formazan crystals were

dissolved with 150 µL DMSO. The absorbance at 490 nm was measured by a microplate reader (Thermo Scientific Multiskan Go, USA) to determine the relative cell viability. 2.10. Cell cytotoxicity of LPOgener on different cell lines Cell cytotoxicity of LPOgener (mole ration of FAC and Lip: 1:2) was performed on MCF-7/ADR, MCF-7, 4T1, L02 cell lines. These cells were seeded in 96-well plates, respectively. When the density reached 70-80%, a series of concentrations of LPOgener at an equivalent FAC dose of 10, 15, 20, 25, 30, 35 µM were added and incubated for 48 h. After adding MTT solution (20 µL, 5 mg/mL) and incubating for an additional 4 h, the formazan crystals were dissolved with 150 µL DMSO. The

absorbance at 490 nm was measured by a microplate reader (Thermo Scientific Multiskan Go, USA) to determine the relative cell viability. 2.11. In vitro cytotoxicity of LPOgener treated 4T1 cells with various inhibitors 4T1 cells were seeded in 96-well plates, when the density reached 70-80%, LPOgener was added into every well, and then the ferroptosis inhibitors including Fer-1 (1 µM), DFO (100 µM) and VE (200 µM), the ROS eliminator VC (200 µM), a necroptosis inhibitor (Nec-1, 1 µM), an apoptosis inhibitor (Apo, 160 µM), and an autophagy inhibitor (Aut, 120 µM) were added to LPOgener treated cells simultaneously. In addition, the complexes of DMPC with FAC and Lip with Zn2+ were set as control treatments. After incubation for additional 48 h, the medium was removed and the cells were washed with PBS for 3 times. MTT solution (20 µL, 5 mg/mL) was added into each well and incubated for an additional 4 h in 37 oC incubator. The medium was then removed, and the formazan crystals were dissolved with 150 µL DMSO. The absorbance at 490 nm was measured by a microplate reader (Thermo Scientific Multiskan Go, USA) to determine the relative cell viability. 2.12. Cellular uptake assay The cellular uptake behavior was studied in 4T1 cells by using flow cytometry. Cells were seeded in 24-well plates at a density of 1.0×105 per well in 1.0 mL of complete DMEM and cultured for 24 h. The cells were treated with free C6 and Lip@C6 (0.1 µM, 0.2 µM of C6) for 1, 2, 4 and 6 h, thereafter, culture medium was removed and cells were washed with cold PBS for three times and treated with trypsin. Data for 1.0×104 gated events were collected and analysis was performed by means of a flow cytometer (BD Accuri C6, USA). 2.13. The cellular uptake mechanism of LPOgener In order to verify the cellular internalization mechanism of LPOgener, inhibitors of specific endocytosis pathways were used with no-treatment as a control. 4T1 cells were seeded in 24-well plates and cultured for 24 h. Cells were first treated with Lip@C6 at C6 concentration of 0.2 µM for 1 h. Then, the cells were washed with cold PBS thrice, followed by incubation at 37 oC for 30 min with various kinds of

endocytosis inhibitors: inhibitor of caveolin-mediated endocytosis, β-CD (5 mM); cell energy metabolism inhibitor, sodium azide (NaN3, 0.1%, w/v); inhibitor of clathrin-mediated endocytosis, chlorpromazine (CMZ, 10 µg/mL); inhibitor of macropinocytosis, amiloride (133.3 µg/mL). In addition, a whole cells were incubated at 4 oC as energy suppression condition. Finally, the cells were washed with cold PBS, and then collected and analyzed by flow cytometry (BD Accuri C6, USA). 2.14. In vitro cytoplasmic ROS The generated cytoplasmic ROS of the cells was determined by ROS sensitive probe, DCFH-DA. 4T1 cells were seeded in 24-well plates and incubated for 24 h. Lip, FAC, DMPC, the complexes of FAC and DMPC, and LPOgener were added and cultured for 24 h. The cells were stained with DCFH-DA (10 µM) for 30 min, thereafter, the cells were washed with PBS thrice and collected for flow cytometry analysis (BD FACS Calibur, USA). 2.15. Intracellular LPO measurement C11-BODIPY581/591, a lipophilic fluorescent dye that can insert into lipid membranes and been oxidized by intracellular LPO, was used to investigate the intracellular LPO generation. Upon oxidation, its fluorescence shifts from red (λex: 581 nm, λem: 591 nm) to green (λex: 488 nm, λem: 510 nm). In this study, 4T1 cells were seeded in 24-well plates and incubated for 24 h. FAC, Lip and LPOgener were added and cultured for 24 h. The cells were stained with C11-BODIPY581/591 (5 µM) for 30 min, thereafter, the cells were washed with PBS thrice and collected for flow cytometry analysis (BD Accuri C6, USA). For the observation of lipid ROS under Confocal Laser Scanning Microscope (CLSM, Zeiss, Germany), 4T1 cells were seeded in 3.5 cm-confocal dishes and incubated for 24 h. FAC, Lip and LPOgener were added and cultured for 24 h. The cells were stained with C11-BODIPY581/591 (5 µM) for 30 min, and then washed with PBS thrice and stained with Hoechst 33342 for 15 min. Finally, CLSM was used to get the fluorescent photograph of treated cells. Furthermore, the intracellular LPO content was measured using MDA assay kit according to the manufacturer’s instructions.

2.16. The mitochrondria morphology of LPOgener treated 4T1 cells 4T1 cells were seeded in 6-well plates and incubated for 24 h. After treating with LPOgener for 24 h, the cells were collected and washed with electron microscopic fixative (2.5% glutaraldehyde). The cells were further fixed at 4 oC overnight. After dehydration, cells were embedded and sliced with a thickness of 50 nm. Finally, the cell sections were stained with 5% uranyl acetate for 15 min and 2% lead citrate for 15 min before TEM imaging. 2.17. Intracellular GSH measurement 4T1 cells were seeded in 6-well plates and incubated for 24 h. FAC, Lip and LPOgener were added and cultured for 12 h. Intraceullar GSH content was detected using a GSH Assay Kit according to the manufacturer’s instructions. 2.18. Intracellular GSH-dependent peroxidases 4 (GPX4) measurement 4T1 cells were seeded in 6-well plates and incubated for 24 h. FAC, Lip and LPOgener were added and cultured for 12 h. Proteins collected from the cell lysate were analyzed by a 12% SDS-PAGE and transferred from the gel to nitrocellulose membrane by electroblotting. Then, 5% skim milk in Tris Buffered Saline Tween was used to block. The next step was using primary antibodies against GPX4 (Abcam, UK) to detect GPX4 on the membrane and the secondary antibody (Beyotime, Shanghai, China) was conjugated with the enzyme horseradish peroxidase, which could catalyze a reaction with enhanced chemiluminescence (ECL) detection reagents (Tanon, Shanghai, China) to produce light and imaged by a CCD imaging system (Tanon 4200, China). 2.19. Study of the localization of lipid ROS in organelles 4T1 cells were seeded in 3.5 cm-confocal dishes and incubated for 24 h. LPOgener was added and cultured for 24 h. The cells were stained with Lysotracker Green, Mitotracker Green, DIO, respectively, and then washed with PBS thrice and stained with Hoechst 33342 for 15 min. Finally, CLSM was used to get the fluorescent photograph of treated cells. 2.20. Plasma pharmacokinetics, in vivo imaging and biodistribution

Female BALB/c mice (6-8 weeks old and weighing 18-22 g) were supplied by SIPPR/BK (shanghai, China). Animal care and handing procedures were according to the guidelines evaluated and approved by the regional ethics committee of China Pharmaceutical University. Female BALB/c mice (n = 5) were treated with FAC and LPOgener at a FAC dose of 2.65 mg/kg via tail vein injection. Blood samples was withdrawn from orbital sinus of the mice at different time points (0 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 11 h, 24 h and 48 h) after administration. Then collected blood samples in heparinized tubes were centrifugated at 3500 rpm for 10 min, and the concentration of Fe in the supernatants was determined by inductively coupled plasma mass spectrometry (ICP-MS). The in vivo circulating half-life of FAC and LPOgener were calculated based on a double-compartment pharmacokinetic model. 4T1 tumor-bearing mice were selected as the animal model to assess the in vivo experiments. 4T1 cells (1×106 cells in 100 µL serum-free DMEM medium) were subcutaneously injected into the region near the mammary of mice. To determine in vivo biodistribution, 4T1 tumor-bearing mice were injected with 100 µL of free DiR and Lip@DiR at a dose corresponding to 10 mg/kg of DiR via tail vein. The real-time distribution and tumor accumulation of free DiR and Lip@DiR were recorded at 24 h post-injection using a Kodak In-Vivo FX Pro imaging system equipped with DiR filter sets (excitation/emission, 720/790 nm). In vivo biodistribution experiments were also performed on 4T1 tumor-bearing mice. When the tumor volume reached about 150 mm3, 10 mice were divided into two groups (n = 5) and treated with FAC and LPOgener at a FAC dose of 2.65 mg/kg via tail vein injection, respectively. At 24 h post injection, mice were sacrificed and the major organs (heart, liver, spleen, lungs, kidneys, tumor) were removed. To analyze the concentration of Fe, the major organs were dissolved in digesting chloroazotic acid (HNO3/HCl = 3:1) and determined by ICP-MS. 2.21. In vivo efficacy When the tumor volumes reached ~50 mm3, 4T1 tumor-bearing mice were

intravenously injected with saline, FAC, Lip and LPOgener at a FAC dose of 1.325 mg/kg respectively twice per week for three weeks (n = 6 per group). Tumor volume was determined by a caliper, and the tumor volume was calculated as (tumor length)×(tumor width)2/2. In the end, all of the organs and tumor tissues were taken out, and the tissue samples were embedded in paraffin blocks, sectioned into 5 mm slices and mounted onto the glass slides. After hematoxylin-eosin (H&E) staining, C11-BODIPY581/591 staining and Sirius staining, the sections were examined by a digital microscope (Olympus, DP 73, Japan). 2.22. Statistical analysis All results were presented as mean ± standard deviation. Statistical analysis of differences between the two groups was determined by an unpaired t-test, and the differences among multigroup were determined by ANOVA. ***P<0.001, **P<0.01, *P<0.05.

3. Results and Discussion 3.1. Identity of GSH and iron redox couple sequentially triggered lipid peroxidation Fe3+ can be reduced to Fe2+ due to GSH-catalyzed reductive reaction (Fig. 1A), which will provide iron redox couple to initiate lipid peroxidation. To investigate the reduction efficiency of FAC into Fe2+ by GSH, o-phenanthroline was used to detect the generation of Fe2+[16]. The complexes of o-phenanthroline and Fe2+ have an obvious absorption at 510 nm, while complexes with FAC do not. As shown in Fig. 1B, Fe2+ was emerging while GSH was added into FAC solution, and the generation of Fe2+ increased along with increasing level of GSH and reaction time (Fig. S1). In order to verify iron redox couple-triggered lipid peroxidation (Fig. 1C), Fe2+ and GSH were added into empty liposome (Lip) and LPOgener, respectively. The appearance of Lip showed no significant change after mixing with individual Fe2+, or GSH, while obvious lipid peroxidation induced yellow precipitation occurred in LPOgener groups in the presence of Fe2+, or GSH (Fig. 1D). Thereafter, all above complexed samples were detected by mass spectrometry. As shown in Fig. 1E, all complexes had the molecular ion peak of unsaturated lipids (m/z 804), while the molecular ion peak of oxidation products (m/z 836) only appeared in the complexes

of LPOgener with Fe2+, or GSH[38]. These results indicated that the coexist of Fe2+/Fe3+ could initiate the redox reaction of oxygen and unsaturated lipids into LPO, while complete Fe2+ or complete Fe3+ could not. In addition, the unsaturated lipids also could be oxidized into LPO in the coexist of FAC/GSH due to GSH-catalyzed iron redox couple. The proposed formulation of iron redox couple-triggered lipid peroxidation was shown in Fig. 1F, and LPOgener was a potential GSH and iron redox couple sequentially triggered LPOgener for ferroptosis therapy.

Fig. 1. Identity of GSH and iron redox couple sequentially triggered lipid peroxidation. (A) The reduction mechanism of Fe3+ to Fe2+ via GSH-catalyzed

reductive reaction. (B) The detection of GSH-mediated reduction of FAC to Fe2+. Fe2+ was detected using o-phenanthroline. (C) Iron redox couple oxidized unsaturated lipids of Lip into LPO. (D) The appearance of Lip and LPOgener in the presence of Fe2+, or GSH. (E) Mass spectrometry of Lip and LPOgener in the presence of Fe2+, or GSH. (F) The formulation of iron redox couple-triggered lipid peroxidation of unsaturated lipids into LPO. 3.2. Preparation and characterization of LPOgener As shown in Fig. 2A, LPOgener was prepared by simple modified ethanol injection method[36] and further extrusion with a polycarbonate membrane of 50 nm. To optimize the proportion of Lip and FAC for the preparation of LPOgener, cytotoxicity test was performed on 4T1 cells. Exhilaratingly, both Lip and FAC had no toxicity on 4T1 cells (Fig. 2B), while the complexes in different proportion of Lip and FAC caused cytotoxicity in various degree (Fig. 2C). Briefly, the cytotoxicity was dose-dependent with Lip and FAC, and the amount of Lip played a key role in killing 4T1 cells. Therefore, 2:1 was chosen as the final molar ratio of Lip and FAC to fabricate the LPOgener. Favorably, we obtained desired liposome with light yellow appearance and obvious Tyndall effect under laser irradiation (Fig. 2D). The encapsulation efficiency of FAC in LPOgener detected by Ultraviolete-Visible (UV-Vis) spectrophotometry was about 98.5 wt%, indicating a good encapsulation capability of liposome. Transmission electron microscopy (TEM) image showed that LPOgener was nanometer-sized particles with spherical morphology (Fig. 2E). Further size distribution analysis indicated that the average diameter and polydispersity index of LPOgener were 94.6 ± 0.8 nm and 0.122, respectively (Fig. 2F). Similarly, the particle sizes of Lip and substituted liposome (coumarin 6 loaded liposome and DiR loaded liposome) were also around 90-100 nm (Fig. S2, A to C). By observing the variation of particle size, it was found LPOgener could keep stable at different dilution times (Fig. 2G) and media (including saline, 10 mM PBS and 10% FBS) for a week (Fig. 2H), ensuring long-term storage and in vivo application. Dialysis method was used to investigate the release behavior of LPOgener in media with different pH values. As shown in Fig. 2I, a pH-responsive release was exhibited, and low-pH environment accelerated the FAC release. The release rates (49.05%, 31.17%) of pH 4.5 and 5.5 (mimicking microenvironment in lysosome and endosome, respectively) were

significantly higher than those (22.43%, 18.33%) at pH 6.5 and 7.4 (extracelluar environment in tumor tissue and blood plasma, respectively). This phenomenon may be due to the pH-responsive coordination bond between FAC and phosphate group of liposome[39]. Acidic environment accelerated the release rate of FAC for effective iron redox couple-triggered lipid peroxidation.

Fig. 2. Preparation and characterization of LPOgener. (A) Preparation process of LPOgener. (B) Cytotoxicity of Lip and FAC on 4T1 cells. (C) Cytotoxicity of different proportion complexes of Lip and FAC on 4T1 cells. (D) The appearance of LPOgener solution and the Tyndall effect of LPOgener under laser irradiation, exhibiting a stable, transparent solution. (E) TEM image of LPOgener. (F) Size distribution analysis of LPOgener. (G) Dilution stability of LPOgener. (H) The stability of LPOgener at three media (including saline, 10 mM PBS and 10% FBS). (I) Release profiles of LPOgener in PBS with different pH values. 3.3. Cellular internalization and in vitro cytotoxicity of LPOgener To understand the internalization behavior of LPOgener based on the enhanced anti-proliferation activity, we prepared a substituted liposome loading coumarin 6 (Lip@C6) and investigated their uptake on 4T1 cells using flow cytometry[40]. Obviously, Lip@C6 had enhanced internalization and accumulation in a dose- and time-dependent manner compared with free C6 (Fig. 3A). Furthermore, the cellular uptake mechanism was studied with various endocytic inhibitors by flow cytometry on 4T1 cells (Fig. 3B)[37]. Compared with Lip@C6 without inhibitors, the cellular

uptake was significantly inhibited by β-CD (inhibitor of caveolae-mediated endocytosis), which proved their caveolae-mediated cellular uptake pathway. In addition, the endocytosis was mildly inhibited when incubated at 4 oC, or in the presence of sodium azide (NaN3, energy inhibitor), which indicated their energy dependent endocytosis. Moreover, the cellular endocytosis was inhibited by chlorpromazine (CMZ, inhibitor of clathrin-mediated endocytosis), but not affected by amiloride (inhibitor of macropinocytosis-dependent endocytosis). These results suggested

that

the

cellular

uptake

mechanism

of

Lip@C6

was

mainly

caveolin-mediated and multipathway including clathrin-mediated endocytosis. Based on above uptake mechanism, LPOgener could be successfully taken into lysosome, which contributed to further rapid and massive release of FAC for effective lipid peroxidation. After treated with LPOgener, the morphology of 4T1 cells had obvious changes including rounding up and detachment[41,42], while Lip and FAC groups had no variations (Fig. 3C). In addition, trypan blue staining (Fig. 3D), live/dead staining (Fig. 3E) and cell viability tests also showed that LPOgener had good killing capacity on 4T1 cells (Fig. 3I), while Lip and FAC had no effect on cell survival. To further validate the therapeutic performance of LPOgener, two additional kinds of tumor cells (MCF-7/ADR cells and MCF-7 cells) and one kind of normal cells (L02 cells) were treated with various preparations. Favourably, Lip and FAC also showed nontoxicity on these cells (Fig. S3). The possible reason for FAC group was that most of the internalized FAC was stored as a component of ferritin in the Fe3+ form[43,44], and the remained low level of free FAC was reduced to Fe2+. In addition, the limited iron redox couple-induced lipid peroxidation can be inhibited by cellular antioxidant agents such as vitamin E (VE), etc[45]. Moreover, liposome is a promising carrier with good safety and biocompatibility, which also had no damage to the cells. Meanwhile, LPOgener exhibited powerful cytotoxicity on MCF-7/ADR cells (Fig. 3G) and MCF-7 cells (Fig. 3H) but slight cytotoxicity on L02 cells (Fig. 3J). Especially, it was found that low concentration of LPOgener showed strong killing capability for resistant cancer cells. The half maximal inhibitory concentration (IC50) of LPOgener against MCF-7/ADR cells was about 7-fold lower than MCF-7 cells, and about 9-fold lower than L02 cells (Table S1). These results were probably in that high GSH level in resistant cancer cells[46] could effectively reduce FAC to Fe2+, and then increasing iron redox couple enhanced lipid peroxodation for ferroptosis therapy. Oppositely, the

concentration of GSH is low to restrict redox reaction in normal cells[47], which further inhibited the lipid peroxidation and provided enough protection for cell survival (Fig. 3F). All above demonstrated that LPOgener was an ideal GSH and iron redox couple sequentially triggered LPOgener, which would cause lipid peroxidation in tumor environment but keep silence in normal cells.

Fig. 3. Cellular internalization and in vitro cytotoxicity of LPOgener. (A) Cellular uptake of free C6 and Lip@C6 at 1 h, 2 h, 4 h, 6 h on 4T1 cells. (B) Cellular uptake efficiency of Lip@C6 on 4T1 cells in the presence of various endocytosis inhibitors. **P < 0.01, ***P < 0.001 vs control. (C) Visualization, (D) Typan blue staining and (E) Calcein-AM/PI staining of 4T1 cells treated with FAC, Lip, and LPOgener, scale bar: 100 µm. (F) The scheme of GSH-dependent cytotoxicity. Cytotoxicity of LPOgener on different cells: (G) MCF-7/ADR cells, (H) MCF-7 cells, (I) 4T1 cells, (J) L02 cells. 3.4. Mechanism of LPOgener induced cell death via ferroptosis Upon the effective in vitro GSH and iron redox couple sequentially triggered lipid peroxidation and high tumor killing capability of LPOgener, we hypothesized that iron redox couple-triggered lipid peroxidation could cause the overwhelming accumulation of LPO for ferroptosis therapy. In order to confirm the cell death pathway of LPOgener, specific inhibitors associated with cell death pathways were used to regulate cell viability (Fig. 4A)[48]. Briefly, Z-VAD-FMK (APO, apoptosis inhibitor), necrostatin-1 (Nec-1, necroptosis inhibitor) and 3-methyladenine (Aut,

autophagy inhibitor) could hardly rescue 4T1 cells from death, while ferrostatin-1 (Fer-1, ferroptosis inhibitor) significantly prevented the LPOgener induced cell death. Furthermore, the addition of deferoxamine (DFO, iron chelating agent) could dramatically inhibit the cytotoxicity in response to LPOgener. In addition, vitamin E (VE, the antioxidant of LPO) could obviously reduce the toxicity of LPOgener, whereas the water-soluble ROS antioxidant vitamin C (VC) exhibited poor ability of rescuing LPOgener treated cells. To confirm the importance of two critical events (iron redox couple and unsaturated lipids) for ferroptosis, we performed various preparations

including

the

complexes

1,2-Dimyristoyl-sn-glycero-3-phosphocholine

(DMPC,

of a

FAC kind

of

and saturated

phospholipid), and the complexes of Lip and Zn2+ on 4T1 cells. All above complexes were nontoxic to 4T1 cells. These results demonstrated that LPOgener treated cells suffered from ferroptosis, which needed the coexistence of unsaturated lipids and FAC. Biological electron microscope images showed LPOgener treated 4T1 cells had smaller mitochondria with condensed mitochondrial membrane densities (Fig. 4B), which was consistent with the feature of ferroptosis[49]. The remarkably rising intracellular

oxidative

stress

induced

by

LPOgener

was

determined

by

dichlorofluorescein diacetate (DCFH-DA) using flow cytometry (Fig. 4C). While the complexes of FAC and DMPC had no impact on intracellular redox balance, the significantly elevated cytoplasmic ROS level of LPOgener group indicated that iron redox couple and unsaturated lipid-induced lipid peroxidation could perturb redox homeostasis by improving oxidative stress. Especially, the accumulation of LPO, a gold

standard

of ferroptosis,

was

detected

by lipid

peroxidation

probe

C11-BODIPY581/591. As shown in Fig. 4D and Fig. 4E, LPOgener treated cells showed stronger green fluorescence intensity (representing more LPO) than Lip and FAC treatment groups. Higher LPO content of LPOgener treated cells was also detected by MDA assay kit (Fig. 4F). These results also proved that LPOgener could efficiently produce LPO in tumor cells. Meanwhile, as a key role in ferroptosis repair systems, GPX4 can be inactivated by GSH depletion[48]. Reduced intracellular GSH content (Fig. 4G) and GPX4 expression level (Fig. 4H) suggested that LPOgener could also inhibit ferroptosis repair pathway for enhanced LPO accumulation. In addition, LPO mainly located in lysosomes, mitochondria and cytomembrane (Fig. 4, I to K), which was consistent with previous reports[50].

Fig. 4. Mechanism of LPOgener induced cell death via ferroptosis. (A) Cell viability of LPOgener treated 4T1 cells after the addition of Fer-1, DFO, APO, Nec-1, Aut, VC, VE, the complexes of FAC and DMPC, and the complexes of Lip and Zn2+. (B) TEM images of LPOgener treated 4T1 cells, scale bar: 2 µm. (C) Cytoplasmic ROS of 4T1 cells treated with Lip, FAC, DMPC, the complexes of FAC and DMPC, and LPOgener. (D) Confocal Laser Scanning Microscope (CLSM) images of LPO stained with fluorescent C11-BODIPY581/591, scale bar: 20 µm. C11-BODIPY581/591: green signal. Nucleus: blue signal. (E) Fluorescence intensity of LPO stained with fluorescent C11-BODIPY581/591. (F) LPO content of 4T1 cells treated with FAC, Lip and LPOgener. (G) Relative GSH content of 4T1 cells treated with FAC, Lip and LPOgener. (H) Western blot results for GPX4 expression level in 4T1 cells after treatment with FAC, Lip and LPOgener. Confocal microscopic images of lysosome (I), mitochondria (J) and cytomembrance (K) of LPOgener treated 4T1 cells. C11-BODIPY581/591: green signal. Nucleus: blue signal. Cellular organs: red signal. Scale bar: 10 µM. 3.5. In vivo anticancer effect on 4T1 tumor-bearing mice Due to the positive results of in vitro experiments, 4T1 tumor-bearing mice were

chosen to conduct in vivo tests. According to the experimental scheme shown in Fig. 5A, the samples were intravenously administrated when the tumor volume reached about 50 mm3. As showed in Fig. 5B, FAC or Lip failed to inhibit tumor growth compared with that in saline group, suggesting the tumor growth was not affected by individual events of ferroptosis. But in contrast with above three groups, tumors treated with LPOgener exhibited the slowest growth speed and smallest volumes (***P < 0.001) during the treatment, which was further supported by C11-BODIPY581/591 staining and hematoxylin and eosin (H&E) staining of tumor tissues. As shown in Fig. 5C, C11-BODIPY581/591 staining confirmed that LPOgener could efficiently produce LPO, which further led to ferroptotic cancer cell death. In addition, LPOgener administrated group had significant tumor cell death and many cavities, whereas large amount of live cancer cells were found in the other three groups (Fig. 5D). All these results indicated that LPOgener could effectively induce tumor tissue damage through ferroptosis therapy. The significantly improved anticancer efficacy of LPOgener could be ascribed to the smart formulation. Indeed, DiR (a near-infrared fluorescent dye) loaded liposome (Lip@DiR) was more effectively accumulated and retained in the tumor after 24 h post injection via EPR effect (Fig. S4A), while most fluorescence of free DiR was observed in lung with 4T1 tumor-bearing mice, which was proved by biodistribution experiments. Furthermore, in order to reveal the in vivo behaviors of LPOgener, we studied the pharmacokinetics of FAC and LPOgener by intravenously injection and measured the levels of Fe in major organs by ICP-MS. According to the blood circulation of Fe (Fig. S4B), the pharmacokinetic parameters of FAC and LPOgener were obtained (Table S2). The first and second phase blood circulating half-lives of LPOgener were determined to be 0.0498 h and 4.0166 h, respectively, which were longer than FAC (0.0170 h and 3.2847 h, respectively). In addition, LPOgener presented a higher tumor accumulation than FAC at 24 h post injection (Fig. S4C), which is consistent with the in vivo imaging results. Therefore, the longer circulation time and higher tumor accumulation of LPOgener attributed to the enhanced anti-tumor effect. According to the reports showing that particles with small size have strong tumor permeation ability[51], which allows them to reach the depth region of the tumor for thorough treatment, we visualized the tumor penetration of Lip@C6 in vitro and in vivo. Briefly, 4T1 tumor spheroids were incubated with free C6 or Lip@C6 for 24 h.

The fluorescence intensity of Lip@C6 treated group was higher in the interior of the tumor spheroid than free C6 (Fig. S4D). In addition, 4T1 tumor-bearing mice were intravenously injected with free C6 and Lip@C6, and sacrificed 24 h later. As shown in Fig. S4E, the fluorescence intensity in the tumor from Lip@C6 treated mice was higher than that from free C6 treated mice, indicating that Lip@C6 exhibited strong penetration both in vitro and in vivo. Above all, the enhanced accumulation and tumor penetration ability of LPOgener led to a wide distribution in tumor tissues, which improved the subsequent iron redox couple-triggered LPO and ultimately exhibited better anti-tumor activity for cancer therapy. During the entire treatment period, there was no significant difference in body weight among groups administrated with saline, FAC, Lip and LPOgener (Fig. 5E). At the end of the experiment, liver tissues were excised from the mice. As shown in Fig. 5F, the liver of LPOgener had no obvious morphology damage. In addition, H&E staining showed no significant pathological changes (Fig. 5G), and Sirus red staining did not indicate obvious hepatic fibrosis phenomenon (Fig. 5H). LPOgener was also determined to be innocuous through blood biochemical markers with particular attention of liver and kidney (Fig. 5, I to L). Furthermore, the other major organs, including heart, spleen, lung and kidney, displayed no remarkable pathological changes in any groups (Fig. S5). Apparently, the favorable biosafety renders LPOgener a promising ferroptosis-inducing agent for cancer treatment.

Fig. 5. In vivo anticancer effect on 4T1 tumor-bearing mice. (A) A scheme showing the experiment design. (B) Tumor growth curves after intravenous injection with various samples (Saline, Lip, FAC and LPOgener) (n = 6, ***P < 0.001). (C) C11-BODIPY581/591 staining of tumor sections. (Magnification: 200×) (D) H&E staining of tumor sections. (Magnification: 200×) (E) Body weight changes during the treatment. (F) Appearance, (G) H&E staining and (H) Sirius red staining of liver tissues of mice. (Magnification: 200×) Blood biochemistry analysis of liver function markers: AST, aspartate aminotransferase (I); ALT, alanine aminotransferase (J); kidney function markers: BUN, blood urea nitrogen (K); CR, creatinine (L) (n = 6).

4. Conclusions In summary, we have developed a GSH and iron redox couple sequentially triggered LPOgener for Fenton reaction-independent ferroptosis therapy. LPOgener with small particle size was prepared by facile extrusion and showed enhanced tumor accumulation and tumor permeability. The detailed mechanism study revealed that

Fe3+ could be efficiently reduced into Fe2+ under high GSH level in tumors, and the subsequently formed iron redox couple catalyzed unsaturated lipids into LPO which further induced ferroptosis. Importantly, in vitro and in vivo anticancer experiments indicated that LPOgener treatment not only suppressed the tumor growth but also had no significant side effects. Under this exploration, designing liposomal iron nanocarriers as iron redox couple-triggered LPOgener would open a new approach for the development of Fenton reaction-independent ferroptosis-inducing agents with high biocompatibility, multifunctionality and efficacy.

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Acknowledgments This work was financially supported by the National Science and Technology Major Project (2017YFA0205400) and the National Natural Science Foundation of China (81773667, 81573369), the Project Program of State Key Laboratory of Natural Medicines of China Pharmaceutical University (SKLNMZZJQ201601) and the Outstanding Youth Fund of Jiangsu Province of China (BK20160031). This work was also supported by the “111” Project from the Ministry of Education of China and the State Administration of Foreign Experts Affairs of China (B16046). We thank the Cellular and Molecular Biology Center of China Pharmaceutical University for assistance with confocal microscopy work.

Author contributions Hu-Lin Jiang and Yu-Jing He conceived this project. Hu-Lin Jiang, Yu-Jing He, Xiao-Ying Liu, and Lei Xing designed and supervised the project and commented on the project. Yu-Jing He, Xiao-Ying Liu, and Xing Wan prepared and characterised the LPOgener, performed in vitro and in vivo experiments and analysed the data. Hu-Lin Jiang, Yu-Jing He, Xiao-Ying Liu, and Lei Xing wrote the paper. All the authors contributed to the discussion during the whole project. Competing financial interests The authors declare no competing financial interest.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: