Boosting the Ferroptotic Antitumor Efficacy via Site-Specific

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Biological and Medical Applications of Materials and Interfaces

Boosting the Ferroptotic Antitumor Efficacy via Sitespecific Amplification of Tailored Lipid Peroxidation Yang An, Jundong ZHU, Fang Liu, Jian Deng, Xuan Meng, Guangqin Liu, Huiyuan Wu, Aiping Fan, Zheng Wang, and Yanjun Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10954 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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ACS Applied Materials & Interfaces

Boosting the Ferroptotic Antitumor Efficacy via Sitespecific Amplification of Tailored Lipid Peroxidation Yang An#, Jundong Zhu#, Fang Liu, Jian Deng, Xuan Meng, Guangqin Liu, Huiyuan Wu, Aiping Fan, Zheng Wang, and Yanjun Zhao*, School of Pharmaceutical Science & Technology, Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China

 To whom correspondence should be addressed. Prof. Yanjun Zhao School of Pharmaceutical Science & Technology, Tianjin University 92 Weijin Road, Nankai District, Tianjin 300072, China Tel: +86-22-2740 7882, Fax: +86-22-2740 4018 Email: [email protected]

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ABSTRACT: Ferroptosis is an iron-dependent cell death pathway that can eradicate certain apoptosisinsensitive cancer cells. The ferroptosis-inducing molecules are tailored lipid peroxides whose efficacy is compromised in hypoxic solid tumor, and is lack of tumor selectivity. It has been demonstrated that ascorbate (Asc) in pharmacological concentrations can selectively kill cancer cells via accumulating hydrogen peroxide (H2O2) only in tumor extracellular fluids. It was hypothesized that Asc-induced, selective enrichment of H2O2 in tumor coupled with Fe3+ co-delivery could simultaneously address the above two problems via boosting the levels of hydroxyl radicals and oxygen in tumor site to ease peroxidation initiation and propagation, respectively. The aim of this work was to synergize the action of Asc with lipid-coated calcium phosphate (CaP) hybrid nanocarrier that can concurrently load polar Fe3+ and nonpolar RSL3, a ferroptosis inducer with a mechanism of inhibiting the lipid peroxide repair enzyme (GPX4). The hybrid nanocarriers showed accelerated cargo release at acidic conditions (pH 5.0). The combinational approach (Asc plus nanocarrier) produced significantly elevated levels of hydroxyl radicals, lipid peroxides, and depleted glutathione under hypoxia, which was accompanied with a strong cytotoxicity (IC50 = 1.2 ± 0.2 µM) in the model 4T1 cells. In the 4T1 tumor-bearing xenograft mice model, the intravenous nanocarrier delivery plus intraperitoneal Asc administration resulted in a superior antitumor performance in terms of tumor suppression, which did not produce supplementary adverse effects to the healthy organs. This work provides a novel approach to enhance the potency of ferroptotic nanomedicine against solid tumors without inducing additional side-effects.

KEYWORDS: Ferroptosis, drug delivery, nanocarrier, lipid peroxidation, ascorbate.


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INTRODUCTION Ferroptosis is an iron-dependent, programmed cell death pathway that is different to traditional apoptosis, necrosis and autophagy in many aspects.1,2 The three key elements of ferroptosis are redoxactive iron (Fe2+), defective repair of lipid peroxides, and two phosphoethanolamines (PEs) containing arachidonic acid (AA) or adrenic acid (AdA), namely 18:0-20:4 PE (PE-AA) and 18:0-22:4 PE (PE-AdA) (Figure S1, Supporting Information).3-5 The two tailored lipids, PE-AA or PE-AdA are oxidized to the corresponding peroxides (PE-AA-OOH and PE-AdA-OOH) via either the Fenton reaction and/or lipoxygenase-dependent route which both involve iron.6 Then these “custom-made” lipid peroxides act as the “death signals” to induce ferroptosis that causes the imbalance of intracellular redox homeostasis. Ferroptosis can also be induced by direct inhibition of the activity or expression of glutathione peroxidase 4 (GPX4), the lipid peroxide repairing enzyme, and/or indirect depletion of intracellular glutathione (GSH) that acts as the cofactor of GPX4.7 The unique machinery of ferroptosis has drawn increasing attention in the field of anti-tumor therapy due to a number of benefits.8-12 The solid tumor is characterized with a complex microenvironment that is featured with hypoxia, acidity, inflammation, carcinoma-associated fibroblasts, and intratumoural heterogeneity.13 The tumor microenvironment together with the presence of cancer stem cells (CSCs) contributes to the activation of epithelial-to-mesenchymal transition (EMT) program.14 Such switch elicits several changes of cellular physiology, including the formation of spindle-like cell morphology, dissolution of tight junctions, apical-basal polarity loss, and mobility acquisition.15 All these lead to the invasiveness, metastasis, multidrug resistance, and hence therapy failure. However, the cells at the mesenchymal state show sensitivity to ferroptosis because of its exceptional ability to target lipid metabolism and induce oxidative stress.16-21



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Although the peculiar ability of ferroptosis to regulate redox homeostasis opens new avenues of nonapoptotic antitumor medicine discovery, the hypoxic solid tumor as well as the extremely low level of labile iron pool (ca. 1 µM Fe2+) limits the therapeutic potency of ferroptosis.22 The lack of Fe2+ hinders the initiation of peroxidation of ferroptotic lipids by hydroxyl radical that is usually produced by Fenton reaction.23 Previous work has developed a series of approaches for iron delivery to induce or enhance ferroptosis in tumor site.10,11,24 However, the extent of Fenton chemistry is also dependent on the level of intracellular hydrogen peroxide that is relatively limited (at µM scale). In addition, the low level of oxygen further inhibits the propagation of peroxidation reaction,25 resulting in a limited amount of ferroptotic “death signals”, i.e. PE-AA-OOH and PE-AdA-OOH. Another potential problem of ferroptosis is the tumor targeting. Targeted killing tumor cells via ferroptosis has been challenging due to the widespread distribution of GPX4 in most organs and the difficulty in selective increasing Fe2+ level and PE-AA/PEAdA concentrations in disease site.26 Such dilemma of ferroptosis would cause the adverse effects to the healthy organs. Since both hydroxyl radical and oxygen play critical roles in lipid peroxidation, it was hypothesized that their site-specific enrichment only in the hypoxic tumor tissue would increase the levels of ferroptosisinitiation molecules (i.e. PE-AA-OOH and PE-AdA-OOH), and hence enhance the ferroptotic antitumor potency without the problem of side-effects. The Fenton reaction involving Fe2+ and hydrogen peroxide (H2O2) is a robust means to produce hydroxyl radicals.23 It has been well-known that ascorbate (Asc) in pharmacological millimolar concentrations can exclusively accumulate H2O2 in tumor extracellular fluids.27-29 This is a consequence of the reduction of protein-centered metal by the Asc-donated electron, followed by superoxide formation with subsequent dismutation to H2O2 at the acidic tumor microenvironment (ca. 6.8).27 In contrast to the Fe2+ that shows high oxidation potential, the Fe3+ is easy to deliver to the cells. The Asc-produced H2O2 can be decomposed to oxygen with the help of Fe3+ as the


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catalyst.30,31 Moreover, Fe3+ can also be reduced to Fe2+ by the intracellular iron reductase (e.g. STEAP3) and superoxide; then hydroxyl radical can be produced by Fe2+ together with H2O2.32,33 Therefore, the aim of this work was to enhance ferroptotic antitumor efficacy in vivo using a Fe3+-loaded nanocarrier coupled with concurrent intraperitoneal high dosing of Asc to address the targeting and potency issues related to ferroptotic therapy (Scheme 1). The calcium phosphate (CaP) core-lipid shell hybrid nanocarrier was selected;34 ferric ammonium citrate (FAC) and hydrophobic GPX4 inhibitor (RSL3) was physically encapsulated in the nanocarrier core and shell, respectively (Figure S2, Supporting Information).

Scheme 1. Schematic illustration of selective boosting ferroptosis potency in tumor. Ferric ammonium citrate (FAC) and RSL3 was loaded in the lipid-coated calcium phosphate nanocarrier that was intravenously (i.v.) administrated. Concurrently, high dose of ascorbate (Asc) was intraperitoneally (i.p.) delivered. Selective accumulation of H2O2 in tumor site can generate both hydroxyl radial and oxygen with the interplay with Fe3+ to boost lipid peroxidation without additional toxic burden to the healthy organs. STEAP3 is an iron reductase. RSL3 is an inhibitor to the lipid peroxide repair enzyme (GPX4). PE-AA and PE-AdA are precursor of ferroptotic “death signals” (i.e. the corresponding peroxides). RESULTS AND DISCUSSION



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Hybrid nanocarriers: cargo loading and controlled release. The lipid-coated CaP nanoplatform is a robust vehicle for concurrent loading of both polar and nonpolar payloads via the electrostatic and hydrophobic interactions, respectively.35 In the current work, both the control and co-delivery nanocarriers were within nanometer range in terms of hydrodynamic size (< 200 nm). The incorporation of cargos (CaP-RSL3 and CaP-Fe/RSL3) slightly increased the particle size compared to the drug-free

Figure 1. Physicochemical characterization of lipid-coated calcium phosphate (CaP) hybrid nanocarrier (n = 3). (A) Hydrodynamic size and (B) Zeta potential of placebo nanocarrier (CaP), RSL3-loaded nanocarrier (CaP-RSL3), and co-delivery nanocarrier (CaP-Fe/RSL3); (C) Elemental (Ca, P and Fe) mapping of CaP-Fe/RSL3 nanocarriers (Scale bar: 200 nm); (D) TEM images of CaP-Fe/RSL3 nanocarriers at pH 7.4, 6.8 and 5.0 (Scale bar: 200 nm); Cumulative release of (E) RSL3 and (F) iron from CaP-Fe/RSL3 at three different pH conditions (7.4, 6.8 and 5.0); (G) Stability of CaP-Fe/RSL3 nanocarriers in PBS (pH 7.4) solution containing with 10% fetal bovine serum; (H) pH-dependent


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nanocarrier stability at pH 7.4 and 5.0 with the hydrodynamic size as the indicator; (I) Illustration of pHdependent nanocarrier disassembly and drug release. control particles (CaP) (Figure 1A), which was in good agreement with previous investigations.36 All three nanocarriers displayed a negative surface charge due to the presence of anionic phospholipids (Figure 1B), which would be beneficial for the systemic circulation of nanoparticles.37 The elemental mapping showed that charged Fe3+ was successfully encapsulated in the inorganic CaP core (Figure 1C). The hydrophobic RSL3 cargo was simultaneously accommodated in the asymmetric lipid bilayer shell that was deposited onto the surface of pre-formed CaP core. The average drug loading was determined at ca. 6.0 ± 0.5% (w/w, Fe) and 1.1 ± 0.1% (w/w, RSL3), correspondingly. The hybrid nanocarriers showed a pH-dependent cargo release profile in vitro (Figure 1E and 1F). At the mimicked lysosome pH (5.0), the cargo release rate was much faster than that at pH 6.8 and pH 7.4, which was accompanied with nanocarrier disassembly and expansion (Figure 1D). The stability of hybrid nanocarriers at both neutral and acidic conditions were tested using the hydrodynamic diameter as the indicator, which proved the particle dis-integration at pH 5.0 (Figure 1H). In a biologically relevant medium (pH 7.4), the nanocarrier stability was also well maintained (Figure 1G). This was because the nanocarrier lost its integrity at acidic conditions as a result of the reduction of electrostatic interaction between Ca2+ and phosphate group (pKa1 = 2.1, pKa2 = 7.2, and pKa3 = 12.7).36 Such behavior can be explained by the fact that hydrogen phosphate (HPO42-) and dihydrogen phosphate (H2PO4-) are the dominant species at pH 7.4 and pH 5.0, respectively (Figure 1I). Fenton reaction-induced lipid peroxidation. The murine breast cancer cells (4T1) were used as a model to examine the level of total reactive oxygen species (ROS) and lipid peroxides produced by the co-delivery nanocarriers under hypoxia. The fluorescent 2′,7′-dichlorofluorescein (DCF) precursor probe (DCFH-DA) was employed to assess the degree of oxidative stress produced by different formulations (Figure 2A).38 The placebo cells (control) showed negligible green fluorescence signal. The Asc-treated



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cells displayed strong fluorescence due to the extracellular production of H2O2 that can diffuse into the cytoplasm. Both free RSL3 and RSL3-loaded nanocarriers (CaP-RSL3) could produce lipid peroxides due to their capability in inactivating GPX4 that was essential for the reduction of lipid

Figure 2. Fluorescent imaging of (A) total reactive oxygen species (ROS) and (B) lipid peroxides in 4T1 cells under hypoxia. The cells were treated by different samples including free ascorbate (Asc), free RSL3, CaP-RSL3, CaP-Fe/RSL3, and CaP-Fe/RSL3+Asc. The dose of RSL3 and Asc was fixed at 5 µM and 4 mM, respectively. ROS and peroxides were imaged by DCFH-DA probe and Liperfluo probe, respectively. Scale bar: 20 µm.


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hydroperoxides to lipid alcohols.39 Compared to iron-free nanocarrier (CaP-RSL3), the co-delivery nanocarrier (CaP-Fe/RSL3) was able to produce more ROS (p < 0.001). This was presumed due to the presence of Fenton reaction-produced hydroxyl radicals and the corresponding elevation of lipid radicals levels. Fe2+ is critical for Fenton reactions; the nanocarrier-enriched Fe3+ could be converted to Fe2+ by the intracellular ferric reductases and/or superoxide.32,33 The CaP-Fe/RSL3 nanocarrier plus Asc produced the highest degree of oxidative stress among all samples (p < 0.001); this was because of the RSL3mediated GPX4 activity inhibition and the amplified Fenton reaction as a consequence of parallel increase of Fe2+ and H2O2 (by Asc) (Figure S3, Supporting Information). The concentration of intracellular lipid peroxides was quantified by Liperfluo® that is a lipid peroxidespecific fluorescence probe (Figure S4, Supporting Information).40 Unsurprisingly, the control cells and Asc-treated cells showed poor red fluorescence due to the low level of lipid peroxides (Figure 2B). In contrast, free RSL3 and CaP-RSL3 nanocarrier generated dramatically higher fluorescence as RSL3 was a potent GPX4 inhibitor to silence the defense system against lipid oxidation. However, there was no significant difference in terms of intracellular fluorescence intensity in cells incubated with both samples (p > 0.05) (Figure S5, Supporting Information); this was presumed due to the rapid endosomal escape of hybrid CaP nanocarriers (Figure S6, Supporting Information).35 Analogous to the total ROS level, the intracellular concentration of lipid peroxides in 4T1 cells under hypoxia ranked as follows: CaPFe/RSL3 (+ Asc) > CaP-Fe/RSL3 > CaP-RSL3 (p < 0.01). Similarly, this was attributed by the augmented Fenton reaction that enriched the hydroxyl radicals to facilitate the initiation of lipid oxidation, as well as the H2O2-decompsed oxygen to aid the propagation of peroxidation process.23, 30, 31 GPX4 inhibition and glutathione depletion. The ferroptosis induction by GPX4 enzyme activity inhibition also requires GSH as a cofactor (Figure 3A).15 Therefore, coincident inhibition of GPX4 activity and depletion of GSH would have a synergistic effect to increase ferroptosis. GSH is one of the



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major antioxidant components in the cell. The production of H2O2 by Asc at the pharmacological concentrations (e.g. 4 mM in the current work) could diminish GSH under hypoxia, which was observed in the cells treated by either free Asc (p < 0.01) or co-delivery nanocarrier plus Asc (p < 0.001) (Figure 3B). The CaP-RSL3 nanocarrier did not significantly deplete GSH (p > 0.05) compared to the control

Figure 3. Analysis of intracellular factors involved in RSL3 and ascorbate-mediated cell death under hypoxia (n = 3). (A) The interplay of RSL3, GPX4, and GSH during lipid peroxidation; (B) Intracellular GSH level in 4T1 cells upon treatment with CaP-RSL3, CaP-Fe/RSL3, free ascorbate (Asc), and CaPFe/RSL3+Asc; * p < 0.05, ** p < 0.01, *** p < 0.001, and n.s. indicates no significant difference; (C) The GPX4 activity in 4T1 cells incubated with four samples; (D) Western blots of GPX4 in 4T1 cells upon different formulation treatment for 12 h (RSL3 dose: 5 μM; Asc dose: 4 mM); (E) Intracellular imaging of hydroxyl radicals in 4T1 cells treated by seven formulations with the aid of hydroxyphenyl fluorescein (HPF) probe. The dose of Fe, RSL3 and Asc was fixed at 210 µM, 5 µM and 4 mM, respectively. Scale bar: 20 μm. because RSL3 only acted on GPX4, which was consistent with previous observations. 41,42 However, the iron doping in CaP-Fe/RSL3 induced the reduction of GSH concentration, possibly via the Fenton reaction-mediated ROS surge to counteract the antioxidant system (p < 0.05). The GPX4 activity was


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analyzed by a commercial assay kit and all three RSL3-loaded nanocarriers, i.e. CaP-RSL3, CaP-Fe/RSL3, and CaP-Fe/RSL3 plus Asc, induced the loss of GPX4 activity (Figure 3C), which was further verified by the western blotting analysis (Figure 3D). Among all samples, the CaP-Fe/RSL3 plus Asc produced the highest level of hydroxyl radical under hypoxia as a consequence of the amplified Fenton reaction (Figure 3E and Figure S7, Supporting Information). These data also agreed well with the highest extent of GSH depletion by the same sample (Figure 3B). Hybrid nanocarrier recovers hypoxia-induced efficacy loss of RSL3. At low oxygen levels, the ability of RSL3 in inducing ferroptotic cell death was compromised; there was an almost 5 times difference in terms of corresponding half maximal inhibitory concentrations (IC50) at 1.1 ± 0.1 µM (normoxia) and 4.7 ± 0.5 µM (hypoxia) (Figure 4A). Actually we presumed that this phenomenon was not caused by the reduction of RSL3 potency in inhibiting GPX4. Instead, it should be a result of the reduced peroxidation of tailored lipids (PE-AA/PE-AdA) under hypoxia, which arose from the decreased propagation reaction at the low concentration of oxygen. The potency of Asc under hypoxia was also determined with a corresponding IC50 at 6.3 ± 0.6 mM, which was three orders higher than that of RSL3 (Figure 4B). The presence of iron in the nanocarrier (CaP-Fe/RSL3) considerably increased the cytotoxicity of RSL3, which was evidenced by a lower IC50 at 2.8 ± 0.2 µM (Figure 4C). The supplement of Asc (4 mM), i.e. CaPFe/RSL3 + Asc, further reduced the IC50 down to 1.2 ± 0.2 µM (Figure 4C). These data were also supported by the vivid live-dead cell staining results (Figure 4D). The cargo-free placebo CaP nanocarrier showed no cytotoxicity, which excluded the vehicle effect (Figure S8, Supporting Information). Nevertheless, the combination of CaP-RSL3 and Asc only slightly enhanced the cytotoxicity compared to that without Asc, indicating the key role both Fe2+ and H2O2 in the Fenton reaction (Figure S9, Supporting Information). The morphological analysis of mitochondria in 4T1 cells corroborated that all RSL3-containing formulations induced the decrease of mitochondria size and concurrent increase of



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mitochondria membrane density compared to the control; these features are specific markers for ferroptosis (Figure 4E). Although free Asc could produce H2O2, no morphological change of mitochondria was observed in 4T1 upon Asc treatment because H2O2 could not directly induce ferroptotic cell death (Figure 4E).

Figure 4. The viability of 4T1 cells in response to treatment by different formulations under hypoxia or normoxia (n = 3). (A) Free RSL3 under both hypoxia and normoxia; (B) Asc under hypoxia; (C) CaPRSL3, CaP-Fe/RSL3 and CaP-Fe/RSL3+Asc under hypoxia; (D) Live-dead cell imaging by confocal laser scanning microscopy upon formulation treatment (RSL3: 5 μM; Asc: 4 mM); the cells were stained with Calcein AM (green, live cells) and PI (red, dead cells) (Scale bar: 20 μM); (E) Transmission electron microscope images of mitochondria in 4T1 cells treated with the same five formulations with RSL3 and Asc dose being fixed at 5 µM and 4 mM, respectively (white arrows: nuclei; black arrows: mitochondria).


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When Fe3+ was delivered to the 4T1 cells via hybrid nanocarriers, the intracellular ferric reductase together with superoxide could convert it to the reduced form (Fe2+).32,33 When no external H2O2 was supplemented, the obtained Fe2+ would react with cytoplasmic H2O2 to produce hydroxyl radicals that were believed to enhance lipid peroxidation, increase the levels of PE-AA-OOH/PE-AdA-OOH, and hence boost ferroptosis efficacy.15, 23 This was the case for CaP-Fe/RSL3 hybrid nanocarrier. However, the addition of Asc provided dual beneficial effects. First, it could dramatically increase intracellular H2O2 concentration, and hence the hydroxyl radical level by Fenton reaction. Second, the extent of hypoxia could be comparatively relieved due to the decomposition of H2O2 to oxygen and water with Fe3+ as the catalyst, which was demonstrated in vitro (Figure S10, Supporting Information).30 The presence of intracellular catalase should also facilitate oxygen release from H2O2. Then the newly created oxygen would facilitate the oxidation of PE-AA/PE-AdA because of the improved propagation of peroxidation reaction. As a result, the levels of ferroptotic “death signals” (PE-AA-OOH/PE-AdA-OOH) were elevated and the problem of ferroptosis efficacy reduction under hypoxia could be addressed accordingly. Furthermore, the Fenton reaction-induced ROS upsurge would also breed the apoptotic cell death (Figure S11, Supporting Information). This was further proved by the enhanced cytotoxicity of CaP-Fe with the Asc supplement (Figure S12, Supporting Information). Passive tumor targeting via hybrid nanocarriers. The shell of hybrid nanocarrier contained lipids that were conjugated with poly(ethylene glycol) (PEG). The surface PEGylation would extend the blood circulation of nanocarriers and the nanoscale size would enable passive particle targeting to the solid tumor via the enhanced permeability and retention (EPR) effect.43,44 As the nanocarrier was not intrinsically fluorescent, we loaded a typical near-infrared fluorescence-emitting dye (Cy5) in hybrid nanocarrier, i.e. CaP-Fe/Cy5 to monitor the kinetic deposition of nanocarrier in tumor site (Figure 5).45 The loading of Cy5 in the fluorescent nanocarrier was 3.4 ± 0.4% (w/w). The free water-soluble Cy5 was used as the



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control, compared to which CaP-Fe/Cy5 nanocarrier delivered more payloads to the tumor (Figure 5A). This behavior reached the peak at 6 h post intravenous dose administration, which was consistent with the characteristic tumor targeting by EPR effect (Figure 5C).37 The ex vivo fluorescence of tumor tissue 24 h post dosing proved the accumulation of more Cy5 in the tumor with respect to the nanoparticle formulation (Figure 5B, 5D). The strong fluorescence in both liver and kidney was because both are key organs for nanocarrier elimination. However, the employment of nanocarrier cannot exclude the drug deposition in the healthy organs as a result of biodistribution, which would cause adverse effects to a certain extent.46,47 This has been one of the major challenges in the field of antitumor nanomedicine.48

Figure 5. Biodistribution and tumor targeting of hybrid nanocarrier in 4T1 tumor-bearing xenograft mice. (A) Kinetic distribution of free Cy5 and CaP-Fe/Cy5 nanocarrier upon intravenous administration; (B) Kinetic fluorescence intensity of Cy5 in tumors (n = 3); (C) Cy5 fluorescence in tumor and major healthy organs ex vivo 24 h post dosing; (D) Comparison of Cy5 fluorescence in tumor (Tu) and major organs including heart (He), liver (Li), spleen (Sp), lung (Lu), and kidney (Ki) 24 h post dose administration (n = 3). Nanoencapsulation plus Asc co-delivery boosted the ferroptosis efficacy in vivo. Due to the presence of biocompatible lipids on the nanocarrier surface, all types of nanocarriers are not hemolytic (Figure S13 and Table S1, Supporting Information). The in vivo efficacy of RSL3-loaded hybrid nanocarriers was evaluated using the 4T1 tumor-bearing xenograft mice model (Figure 6). The negative control, phosphate


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buffered saline (PBS), was not capable of suppressing tumor growth. The intraperitoneal dosing of Asc demonstrated visible tumor growth inhibition, which was because the Asc-produced ROS could induce both genotoxic stress (DNA damage) and metabolic stress (ATP depletion), followed by tumor cell death.29 Intravenous delivery of free RSL3 also resulted in evident suppression of tumor cell proliferation, which should be attributable to the ferroptosis machinery as a consequence of GPX4 inhibition. Nanoencapsulation (CaP-RSL3) marginally improved the antitumor efficacy of RSL3 compared to the free drug. This result can be simply explained by the enhanced tumor deposition of RSL3 by virtue of EPR effect of nanocarriers.43,44 The iron doping in hybrid nanocarrier (CaP-Fe/RSL3) could produce additional hydroxyl radical as well as oxygen that would enhance lipid peroxidation and ferroptotic cell death, which was observed both in vitro and in vivo. The co-supplement of intraperitoneal Asc with intravenous CaP-Fe/RSL3 ended up with the best antitumor effect among all investigated formulations (Figure 6A). Four weeks post the dose initiation, the mice treated by co-delivery hybrid nanocarrier plus Asc displayed the lowest tumor mass (Figure 6B, Figure S14, Supporting Information). The superiority of CaP-Fe/RSL3 plus Asc over CaP-Fe/RSL3 alone in terms of tumor suppression was due to the Ascinduced H2O2 enrichment that amplified the Fenton reaction, relieved hypoxia, and eventually boosted lipid peroxidation. The in vivo GPX4 level in tumors concurred well with the tumor growth inhibition curves (Figure S15, Supporting Information). In addition, the heightened ROS could also directly provoke cell death via apoptosis mechanism that was caused by impaired glycolysis, DNA and mitochondria damage, and ultimately ATP depletion to induce an energetic crisis.27,49 Therefore, the antitumor efficacy of CaP-Fe/RSL3 plus Asc was practically a combinational effect of both ferroptosis and apoptosis (Figure 6D). The PBS-treated mice showed perceivable body weight loss compared to those treated by other formulations, which was thought due to the reduced food uptake caused by the uncontrolled tumor growth



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(Figure 6C). The histological hematoxylin & eosin (H&E) staining and apoptotic TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) imaging results proved the

Figure 6. In vivo efficacy of hybrid nanocarriers plus ascorbate (Asc) in 4T1 tumor-bearing xenograft mice. (A) Tumor growth inhibitory curve upon treatment by 6 formulations; (B) Quantification of tumor mass 4 weeks post the onset of treatment; (C) Constant monitoring of mice body weight during the treatment course; (D) H&E and TUNEL staining of tumor tissues when the efficacy study was over. The data were presented as the mean ± standard deviation (n = 6). * p < 0.05, ** p < 0.01. Scale bar: 100 µm (H&E); 20 µm (TUNEL). exclusive antitumor potency of CaP-Fe/RSL3 plus Asc in a hypoxic tumor microenvironment, which was conceived due to Asc-induced H2O2 production, passive tumor deposition of iron by nanocarrier, and subsequent boosting of ferroptosis through Fenton reaction and oxygen release (Figure 6D). The hypoxia relief was also indirectly evidenced by the decreased expression of hypoxia-inducible factor 1 alpha (HIF1α) (Figure S16, Supporting Information). The co-delivery hybrid nanocarrier coupled with concurrent Asc delivery improved in vivo antitumor efficacy, but the boosted potency could be primarily constrained to the tumor region without supplementary injury to the healthy organs (Figure S17, Supporting


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Information). The traditional antitumor nanomedicine has been suffering from a dilemma of potency enhancement and side-effects uplifting.50,51 The current work could circumvent this problem via employing millimolar Asc for selective H2O2 accumulation within the tumor extracellular fluids, which was united with nanoparticulate iron/RSL3 delivery for amplifying Fenton reaction and relieve hypoxia to selectively enhance lipid peroxidation in tumor sites. CONCLUSION To fully emancipate the power of ferroptotic cell death as therapies of solid tumor, we employed the approach of hybrid nanocarrier for concurrent delivery of Fe3+, a GPX4 inhibitor (RSL3), and a H2O2 presenter (Asc) to address the difficulty of targeted ferroptosis enhancement to tumor site and the problem of reduced lipid peroxidation under hypoxia. The trick of this strategy lay in the selective H2O2 deposition only in the tumor site by pharmaceutically acceptable, high dose of Asc. Subsequently, the interplay of H2O2 and Fe3+ inside the cells produced both hydroxyl radicals and oxygens that both were key players in lipid peroxidation to generate the “death signals” of ferroptosis. Because the amplified lipid oxidation mainly occurred in the tumor tissue, the ferroptotic tumor targeting was achieved in a 4T1 tumor-bearing xenograft mice model without added side-effects to the non-tumor organs. The current work opened the avenues of enhancing ferroptotic antitumor efficacy in hypoxic tumors via the use of Asc as the unique pro-oxidant agent. Such proof-of-concept could be extended to the combinational integration of ferroptosis with other antitumor approaches for managing various solid tumors. MATERIALS AND METHOD Materials. Dioleoylphosphatidic acid (DOPA) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt (DSPE-PEG2000) was provided by Ponsure Biotechnolog (Shanghai, China). L-αPhosphatidylcholine (PC) and cholesterol were obtained from A.V.T. Pharmaceutical Co., Ltd. (Shanghai,



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China). Branched polyoxyethylene nonylphenylether (Igepal®CO-520), calcium chloride (CaCl2), and sodium phosphate dibasic dodecahydrate (Na2HPO4) were purchased from Sigma-Aldrich (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Baibei Biotechnology Co., Ltd. (Tianjin, China). Ammonium ferric citrate (FAC) was sourced from Heowns Biochem Co., Ltd. (Tianjin, China). (1S, 3R)-RSL3 was purchased from D&C Chemicals (Shanghai, China). L-ascorbic acid (Asc), ethanol and cyclohexane were purchased from J&K Scientific Ltd. (Beijing, China). Methanol and acetonitrile were obtained from Concord Technology Co., Ltd. (Tianjin, China). 3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). Cy5 dye was purchased from InnoChem Science & Technology Co., Ltd. (Beijing, China). All other chemicals were purchased from Jiangtian Fine Chemical Technology Co. Ltd. (Tianjin, China). Hybrid nanocarrier preparation. The CaP cores were prepared by a water-in-oil microemulsiontemplated method.34,36 Briefly, two microemulsions were prepared and both contained cyclohexane as the oil phase. One colloidal system contained 150 μL of 500 mM CaCl2 (pH 7.0) that was dispersed in a mixture of cyclohexane and Igepal®CO-520 (5 mL, 71/29, v/v). The other disperse system comprised of 150 μL of 100 mM Na2HPO4 (pH 9.0) and 250 mM ammonium ferric citrate (FAC) that was dispersed in 5 mL of cyclohexane doped with 58 μL dioleoylphosphatydicacid (DOPA, 25 mg/mL) chloroform solution. Then the two colloidal microemulsions were mixed and stirred at room temperature for 2 hours to form the CaP inorganic core coated by DOPA. After 2 hours, 10 mL of absolute ethanol was added to precipitate DOPA-protected CaP, followed by particle collection by centrifugation (10,000 g, 15 min). This washing step was repeated in triplicate, followed by vacuum-drying the sediment and dispersion in 500 μL chloroform. To prepare the final CaP-Fe/RSL3 nanoparticles, the above obtained DOPA-coated CaP colloid dispersion was mixed with 150 μL of 20 mM cholesterol, 75 μL of 20 mM DSPE-PEG2000,


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150 μL of 15 mg/mL PC and 60 μL of 5 mg/mL RSL3 (all in chloroform) to realize external lipid deposition and RSL3 loading. After solvent evaporation, the obtained hybrid nanocarriers were suspended in deionized water, followed by dialysis against distilled water (MWCO: 5,000 Da) using a regenerated cellulose tube. Afterwards, the dispersion was centrifuged at 8,000 g for 10 min and the supernatant was lyophilized to obtain the final CaP-Fe/RSL3 nanocarrier. The preparation procedure of CaP-RSL3 and CaP-Fe was similar to that of CaP-Fe/RSL3 nanocarrier without the supplement of FAC or RSL3, respectively. The drug-free CaP nanocarriers were also prepared without adding any payloads (e.g. FAC and RSL3). Hybrid nanocarrier characterization. The particle size and surface charge of hybrid nanocarriers were tested by a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK).52 Transmission electron microscopy (TEM) images of nanocarriers at different pH (5.0, 6.8 and 7.4) were taken using a HITACHI HT7700 TEM microscope. The elemental mapping of P, Ca and Fe of CaP-Fe/RSL3 nanocarrier was carried out using a FEI Tecnai G2-F20 TEM. The RSL3 loading in nanocarriers was determined by high performance liquid chromatography (HPLC, Waters e2695) coupled with an ultraviolet detector (230 nm). A Phenomenex Gemini C18 column (250 mm × 4.6 mm, 5 μm) was used for separation. The temperature was set at 25oC and the mobile phase was a mixture of acetic acid aqueous solution (1%, v/v) and acetonitrile (40:60, v/v); the flow rate was constant at 1 mL/min with an injection volume of 20 μL. The iron level was quantified by Atomic Absorption Spectroscopy (Hitachi High-Technologies Co. Ltd., Shanghai, China). The stability of CaP-Fe/RSL3 nanocarrier (1 mg/mL) at 37oC was monitored by monitoring their hydrodynamic size in PBS (pH 7.4 and 5.0) at 0 h, 2 h, 8 h, and 24 h post sample dispersing. The serum stability of CaP-Fe/RSL3 nanocarrier was tested by monitoring their size in PBS (pH 7.4) solution containing with 10% fetal bovine serum (FBS). Briefly, CaP-Fe/RSL3 nanocarrier (1



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mg/mL) was incubated in the above medium at 37°C for up to 24 h, and the hydrodynamic size and poly dispersity index (PDI) was recorded at 0 h, 2 h, 4 h, 8 h, and 24 h post sample dispersing. In vitro cargo release. The release profile of RSL3 and Fe from the CaP-Fe/RSL3 nanocarriers utilized one of our previously published methods with minor modification.53 The static Franz-type diffusion cells were employed and the temperature was maintained at 37oC to mimic the physiological conditions (n = 3). The receiver chamber was filled with PBS (pH 7.4/0.18 M, or pH5.0/0.15 M) with the presence of 5% (w/v) sodium dodecyl sulfate. The buffer was stirred by a magnetic flea. The donor chamber contained 10 mg nanocarriers that were dispersed in the same buffer as the receiver fluid. The diffusion membrane was the regenerated cellulose membrane (MWCO: 1,000 Da) that separated the donor and receptor chambers. At pre-designed time points, 0.5 mL of receiver medium was withdrawn for drug concentration analysis by HPLC. The mobile phase was a mixture containing aqueous solution of acetic acid (1%, v/v) and acetonitrile (40:60, v/v). The flow rate was set at 1 mL/min with an injection volume of 20 µL. An ultraviolet detector was used and the detection wavelength was 230 nm. The iron concentration was spectrophotometrically quantified with the aid of orthophenanthroline. The cargo release curve was created by plotting the accumulatively released RSL3/Fe against time. Oxygen release in vitro. The ferric iron-catalyzed oxygen release from H2O2 was carried out in a biologically relevant medium containing 90% deionized water and 10% DMEM culture medium containing 1% FBS. The CaP-Fe/RSL3 nanocarrier was first damaged by HCl (pH 4.0) to release iron prior to being transferred to the above medium (20 mL), followed by H2O2 supplement. The final dose was set at 0.5 mM for both H2O2 and Fe. The oxygen level was continuously monitored using a JPB-607A dissolved oxygen meter (Shanghai Instrument Electric Science Instrument Ltd.) The system containing FAC (0.5 mM) and H2O2 (0.5 mM) was used as the control.


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Endosomal escape analysis. To explore the endosomal escape ability of the hybrid nanocarriers, a fluorescent probe (Rhodamine/Rho) was loaded in the nanocarriers, i.e. CaP-Fe/Rho. 4T1 cells were plated in 20 mm confocal plates (8 × 104 cells per well). After 24 h, the cells were further cultured under hypoxia for 12 h. Then the cells were incubated with CaP-Fe/Rho (3 mg/mL) for 2 h prior to PBS washing. Subsequently, the cells were incubated with fresh medium for another pre-designed period (0 h, 2 h and 4 h), followed by incubation with DMEM containing Lyso Traker Green DND99 (40 nM) at 37oC for 30 min, and then Hoechest 33342 for another 10 min. The UltraView Vox CLSM (PerkingElmer, USA) was used to record the fluorescence images of cells. The excitation wavelength of Hoechest 33342, LysoTraker Green DND99, and Rhodamine were 405 nm, 488 nm, and 561 nm, respectively. Cell and animal model. Murine breast cancer cells (4T1) were provided by the State Key Laboratory of Medicinal Chemical Biology (Nankai University). DMEM medium was supplemented together with 10% fetal bovine serum and 1% penicillin/streptomycin. For experiments under normoxia, all cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. In contrast, the hypoxic conditions were obtained by incubating the cells in a Billups-Rothenberg modular incubator chamber that was supplemented with a humidified mixture of 1% O2, 5% CO2 and balanced N2.54 Female BALB/c mice (6 weeks, 16−19 g) were sourced from Huafu Kang Bioscience Co. Inc. (Beijing, China). The 4T1 tumor-bearing BALB/c mice model was established by subcutaneously inoculating 1 × 106 4T1 cells into the right flank of mouse. The tumor volume was calculated according to the following formula: (L×W2)/2, where L and W stands for the longest and shortest diameters of the tumor, respectively. The tumor volume was allowed to grow to 200 mm3 ready for further use. All procedures related to animal experiments were implemented in accordance with the guidelines for care and use of laboratory animals of Tianjin University and approved by the animal ethics committee of Tianjin University.



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Detection of ROS, hydroxyl radicals and lipid peroxides. The level of intracellular total ROS was fluorescently determined by the standard probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA). In detail, 4T1 cells were plated in 20 mm confocal plate (8 × 104 cells per well). After 24 h, the cells were further cultured for 12 h under hypoxic condition. Subsequently, the cells were incubated with different samples, including free medium (control), free RSL3, free Asc, CaP-RSL3 and CaP-Fe/RSL3 for 2 h, respectively. The concentration of RSL3 and Asc was fixed at 5 μM and 4 mM for all formulations. The Asc sample was adjusted to neutral (pH 7.0) by sodium hydroxide. Regarding the combination group (CaP-Fe/RSL3 + Asc), the cells were incubated with 4 mM Asc under hypoxia for additional 2 h post nanocarrier treatment. Afterwards, the cells were washed with PBS and incubated with DMEM containing DCFH-DA (20 µM) at 37oC for 30 min. The Leica TCS SP8 confocal laser scanning microscope was used to record the fluorescence images of cells (Ex = 488 nm, Em = 500-600 nm). Similarly, fluorescent detection of hydroxyl radicals employed a different probe, hydroxyphenyl fluorescein (HPF), based on the product protocol. HPF concentration was fixed at 20 µM (Ex = 488 nm, Em = 490-590 nm). The detection of intracellular lipid hydroperoxides levels relied on the use of a selective probe Liperfluo whose concentration was set at 10 µM (Ex = 532 nm, Em = 535-650 nm).40 GSH detection and GPX4 activity assay. The 4T1 cells (1 × 107) maintained under hypoxia were treated with different formulations including CaP-RSL3, CaP-Fe/RSL3, free Asc and CaP-Fe/RSL3+Asc, wherein the RSL3 and Asc dose was kept at 5 μM and 4 mM, respectively. The cells without formulation treatment were used as the control. After 4 h, the cells were harvested and re-suspended in 600 µL PBS, followed by sonication for 10 min on ice. Then the cell lysate was analyzed based on the product protocol of the commercial GSH assay kit. The results were presented as GSH content with reference to unit protein mass (n = 3). Similarly, GPX4 activity in these treated cells was also examined using a commercial assay (Beyotime Biotechnology, China), the mechanism of which was based on the GPX4-catalyzed oxidation


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of GSH with the consumption of NADPH (reduced nicotinamide adenine dinucleotide phosphate). The GPX4 activity was indirectly represented by the NADPH level that was kinetically reflected by the absorbance value at 340 nm at a 60 s interval over the 12 min time course. Western blotting. The 4T1 cells (1 × 107) were seeded in 10 cm dishes under hypoxia and treated with different formulations, including free RSL3, CaP-RSL3, CaP-Fe/RSL3 and CaP-Fe/RSL3 plus Asc, wherein the RSL3 and Asc dose was set at 5 μM and 4 mM, respectively. The cells without formulation treatment were used as the control. After 12 h, the cells were harvested and washed three times with PBS. Cells were lysed in 400 μL radioimmunoprecipitation assay (RIPA) lysis buffer containing 10 μM phenylmethylsulfonyl fluoride (PMSF). The protein content was quantified by the commercial bicinchoninic acid (BCA) assay kit (Solarbio, China). Then each sample containing 40 μg protein was resolved on SDS-polyacrylamide gels. The electrophoresed proteins were transferred onto a nitrocellulose membrane, blocked with 5% skim milk, and incubated with primary antibodies (GPX4-specific antibody at 1 : 2500 or rabbit anti-β-actin polyclonal antibody at 1 : 5000) at 4oC overnight, followed by incubation with secondary antibody (horseradish peroxidase (HRP)-conjugated anti-rabbit IgG H&L at 1 : 2500) for 2 h at room temperature. After washing off the unbound antibodies, the bands were recorded using Fusion SoloS imaging system (Vilber, France) upon incubating with ECLTM western blotting detection reagents (Adcansta, USA). Cytotoxicity analysis. The cytotoxicity of hybrid nanocarriers and corresponding controls in 4T1 cells was detected by the MTT cell viability assay. In details, 4T1 cells were seeded to 96-well plates (4 × 103 per well) containing 100 μL culture medium per well. After 24 h’s standard culture, the cells were maintained under normoxia or hypoxia for 12 h, followed by the supplement of different formulations including free RSL3, free Asc, CaP-RSL3 and CaP-Fe/RSL3 and cell culture at the same condition. The Asc sample was adjusted to neutral (pH 7.0) by sodium hydroxide. After 2 h, the cells were washed by



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PBS, and maintained in fresh medium under normoxia or hypoxia for additional 24 h. Regarding the combination formulation (CaP-Fe/RSL3+Asc), the cells were first treated with CaP-Fe/RSL3 for 2 h under hypoxia, followed by PBS washing. Then the cells were further incubated with Asc (4 mM) for 2 h, followed by additional culturing for 24 h in fresh medium under hypoxia. The IC50 of different samples was calculated accordingly. The cytotoxicity assay was also accompanied with the live/dead cell staining.55 The 4T1 cells were treated by the same formulations as described above with the corresponding dose at 5 μM (RSL3) and 4 mM (Asc) under the same conditions as cytotoxicity assay. Afterwards, the cells were stained by Calcein-AM (2 μM) and PI (2 μM) for 30 min at 37°C. The viable cells (green color) can be stained by Calcein AM (Ex/Em = 488 nm/490-530 nm), while dead cells (red color) can be stained by PI (Ex/Em = 532 nm/540-650 nm). Mitochondria morphology analysis. The 4T1 cells (1 × 107) were seeded in 10 cm dishes under hypoxia and treated with different formulations including free Asc, free RSL3, CaP-RSL3, CaP-Fe/RSL3 and CaPFe/RSL3+Asc, wherein the RSL3 and Asc dose was kept at 5 μM and 4 mM, respectively. The cells with no formulation treatment were used as the control. After 4 h post treatment, the cells were harvested and fixed in 2.5% electron microscopy grade glutaraldehyde at 4oC overnight. Then, the samples were washed by 0.1 M phosphate buffer (pH 7.0) for 15 min in triplicate, followed by fixing with 1% aqueous osmium tetroxide for 1-2 h, and PBS (pH 7.0, 0.1 M) washing three times (15 min each time). Afterwards, the samples were gradually dehydrated with ethanol (30%, 50%, 70%, 80%, 90% and 95%); the treatment time was 15 min at each concentration. Eventually the samples were successively treated with 100% ethanol for 20 min, acetone for 20 min, a mixture of embedding agent and acetone (1:1, v/v) for 1 h, a mixture of embedding agent and acetone (3:1, v/v) for 3 h, and then the embedding agent overnight. After osmotic treatment, the samples were embedded and maintained at 70oC overnight. Thin sample sections


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were produced using an ultramicrotome (LEICA EM UC7), and stained with 1% uranyl acetate and 0.4% lead citrate prior to TEM (HITACHI H-7650) imaging Hemocompatibility analysis. The hemocompatibility of hybrid nanocarrier was examined by hemolysis assay. In detail, 2 mL blood was collected from of healthy BALB/c mice and stored in heparinized tubes. The fibrinogen was removed by stirring with a bamboo stick to make defibrillated blood. Then the blood was mixed with 10 times of saline (0.9% NaCl, w/v) and centrifuged for 15 min (352 g). The supernatant was removed, and the collected red blood cells were washed with saline for 3 times. Then the obtained red blood cells were diluted with saline (1:10 v/v) for further use. The red blood cells suspension (0.5 mL) was mixed with 0.5 mL of free Asc, free RSL3, CaP-RSL3 nanocarriers, and CaP-Fe/RSL3 nanocarriers saline solution (RSL3: 150 μM; Asc: 40 mM), respectively. Then, all the samples were maintained in 37oC for 3 h, followed by centrifugation at room temperature (352 g) for 15 min. The supernatant was placed in a 96-well plate, and the absorbance at the wavelength of 540 nm was measured. Saline and deionized water was used as the negative and positive control, respectively. Biodistribution of hybrid nanocarrier. To investigate the biodistribution of the hybrid nanocarriers, a NIR fluorescent probe (Cy5) was non-covalently loaded in the particles, i.e. CaP-Fe/Cy5 since RSL3 showed no fluorescence. The Cy5 content was quantified by a fluorescence spectrophotometer (Ex/Em = 635 nm/670-900 nm). The free Cy5 (hydrophilic version) was set as the control.45 Both CaP-Fe/Cy5 aqueous dispersion (150 µL) and free Cy5 aqueous solution (150 µL) were intravenously (i.v.) injected to the 4T1 tumor-bearing mice through the tail vein (n = 3). The dose of Cy5 was identical at 25 µg/mL. After the i.v. administration of two formulations to the 4T1 tumor-bearing xenograft mice, the fluorescent intensity of Cy5 in tumor site together with the whole mice images was constantly monitored at predesigned time points (2 h, 4 h, 6 h, and 24 h). The CRI Maestro in vivo imaging instrument was employed for the analysis (Cambridge Research & Instrumentation, Inc., MA, USA). Twenty four hours post dosing,



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the mice were sacrificed, and the tumors and the main organs (heart, liver, spleen, lungs and kidneys) were excised for ex vivo fluorescence imaging and intensity comparison (n = 3). In vivo antitumor efficacy of hybrid nanocarriers. For investigating the therapeutic effect of different ferroptotic hybrid nanocarriers, the 4T1 tumor-bearing BALB/c mice were treated by pre-designed formulations when the tumor volume reached approximately 200 mm3.56 In brief, the mice were randomly divided into six groups (n = 6) that corresponded to six formulations, including PBS (negative control), free Asc, free RSL3, CaP-RSL3 nanocarrier, CaP-Fe/RSL3 nanocarrier, and CaP-Fe/RSL3 nanocarrier plus Asc. All nanocarriers were dispersed in PBS. The vehicle for free RSL3 was a mixture of PEG 400 and PBS (30/70, v/v). The dosing strategy was detailed in Figure S18 (Supporting Information). The formulations were intravenously (i.v.) administrated three times at days 0, 3, and 6 (RSL3 dose at 3 mg/kg); in contrast, Asc (4 g/kg) was intraperitoneally (i.p.) injected each day from day 0 to day 8. There was 6 h difference between i.v. and i.p. dosing for the days (0, 3, and 6) when two injections were delivered in attempt to maximize the EPR effect. The i.v. administration was placed before Asc delivery. The tumor volume and body weight of each mouse was monitored every two days to plot the tumor inhibition curve. The mice body weight was also continuously recorded and plotted against time. The efficacy study was terminated at the 28th day post treatment initiation. In the end of efficacy study, the tumor tissues were harvested and weighted. The healthy organs were also collected including heart, liver, spleen, lung, and kidney for adverse effects evaluation by standard histological H&E staining. The tumor tissues were also analyzed via H&E, apoptotic TUNEL staining, and HIF-1α staining. The GPX4 content in tumor upon formulation treatment was also examined by western blot. The 4T1 tumor-bearing mice were randomly divided into four groups (n = 3) and subject to four formulation treatment including PBS (negative control), CaP-RSL3 nanocarrier, CaP-Fe/RSL3 nanocarrier, and CaP-Fe/RSL3 nanocarrier plus Asc. The dosing


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strategy and administration method were as same as that described above. At 24 h post the last injection, tumors were harvested for western blotting analysis following the above-mentioned procedures. Statistical analysis. The data were presented as mean ± standard deviation (SD). Sample difference was statistically compared via either Student’s t-test or analysis of variance integrated with Tukey’s post-hoc analysis. The threshold P value was set at 0.05. ASSOCIATED CONTENT Supporting Information. This supporting information is available free of charge via the Internet at http://pubs.acs.org. Chemical structure of ferroptotic lipids, and RSL3; relative fluorescence intensity of DCF, Liperfluoro, and HPF; mechanism of Liperfluoro probe; endosomal escape of rhodamine-loaded nanocarriers; cytotoxicity of placebo CaP, CaP-RSL3, CaP-Fe, CaP-RSL3 + Asc, and CaP-Fe + Asc nanocarriers; in vitro ironcatalyzed oxygen release; apoptosis analysis; hemolysis analysis; representative tumor image post treatment; western blot of GPX4 in tumor; HIF-1α staining of tumor; H&E staining of major healthy organs; in vivo dosing strategy. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions #

An Y and Zhu J made equal contribution to this work. The manuscript was written through contributions

of all authors. All authors have given approval to the final version of the manuscript.



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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We acknowledge the funding support from the National Basic Research Program of China (2015CB856500), and the Tianjin Research Program of Application Foundation and Advanced Technology (18JCZDJC35700). ABBREVIATIONS AA, arachidonic acid; AdA, adrenic acid; Asc, ascorbate; CaP, calcium phosphate; CLSM, confocal laser scanning microscope; CSCs, cancer stem cells; DCF, 2′,7′-dichlorofluorescein; DMEM, Dulbecco's modified Eagle's medium; DOPA, dioleoylphosphatidic acid; DSPE-PEG2000, 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt; EMT, epithelial-tomesenchymal transition; EPR, enhanced permeability and retention; FAC, ferric ammonium citrate; GPX4, glutathione peroxidase 4; GSH, glutathione; H&E, hematoxylin and eosin; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; IC50, half maximal inhibitory concentration; mPEG, methoxy poly(ethylene glycol); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; MWCO, molecular weight cut-off; PBS, phosphate buffer saline; PDI, poly dispersity index; Rho, rhodamine; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; TEM, transmission electron microscope; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. REFERENCES


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Boosting the Ferroptotic Antitumor Efficacy via Site-Specific

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