Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis

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Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis-Inducing Agent for Tumor-Targeted Theranostics Shuaifei Wang, Fangyuan Li, Ruirui Qiao, Xi Hu, Hongwei Liao, Lumin Chen, Jiahe Wu, wu haibin, Meng Zhao, Jianan Liu, Rui Chen, Xibo Ma, Dokyoon Kim, Jihong Sun, Thomas P. Davis, Chunying Chen, Jie Tian, Taeghwan Hyeon, and Daishun Ling ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06399 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis-Inducing Agent for Tumor-Targeted Theranostics Shuaifei Wang,†,# Fangyuan Li,†,# Ruirui Qiao,‡,# Xi Hu,† Hongwei Liao,† Lumin Chen, ┴ Jiahe Wu,† Haibin Wu,† Meng Zhao,† Jianan Liu,§ Rui Chen, ‖ Xibo Ma,◊ Dokyoon Kim,§ Jihong Sun, ┴ Thomas P Davis,‡,ψ Chunying Chen, ‖ Jie Tian,◊ Taeghwan Hyeon§,⁋ and Daishun Ling†,Δ,* †

Zhejiang Province Key Laboratory of Anti-cancer Drug Research, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China, Δ

Key Laboratory of Biomedical Engineering of the Ministry of Education, College of

Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou 310058, China, ‡ ARC

Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash

Institute of Pharmaceutical Sciences, Monash University, Australia, ψDepartment ┴ Sir

of Chemistry, University of Warwick, United Kingdom,

Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310058,

China, ‖ CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National

Center for Nanoscience & Technology of China, Beijing, 100190, China,

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◊Chinese

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Academy of Sciences Key Laboratory of Molecular Imaging, Institute of

Automation, Chinese Academy of Sciences, Beijing 100190, China, §Center

for Nanoparticle Research, Institute for Basic Science (IBS), Seoul, 08826, Korea,

⁋School

of Chemical and Biological Engineering, Seoul National University, Seoul, 08826,

Korea. #These

authors contributed equally to this work.

*E-mail: [email protected]

ABSTRACT: Ferroptosis, an iron-based cell death pathway, has recently attracted great attention owing to its effectiveness in killing cancer cells. Previous investigations focused on the development of iron-based nanomaterials to induce ferroptosis in cancer cells by the upregulation of reactive oxygen species (ROS) generated by the well-known Fenton reaction. Herein, we report a ferroptosis-inducing agent based on arginine-rich manganese silicate nanobubbles (AMSNs) that possess highly efficient glutathione (GSH) depletion ability and thereby induce ferroptosis by the inactivation of glutathione-dependent peroxidases 4 (GPX4). The AMSNs were synthesized via a one-pot reaction with arginine (Arg) as the surface ligand for tumor homing. Subsequently, a significant tumor suppression effect can be achieved by GSH depletion-induced ferroptosis. Moreover, the degradation of AMSNs during the GSH depletion contributed to T1-weighted magnetic resonance imaging (MRI) enhancement as well as on-demand chemotherapeutic drug release for synergistic cancer therapy. We anticipate that the GSH-depletion-induced ferroptosis


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strategy by using manganese-based nanomaterials would provide insights in designing nanomedicines for tumor-targeted theranostics.

KEYWORDS: nanobubbles, glutathione, GPX4, ferroptosis, theranostics


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Ferroptosis, a cell death pathway about iron-induced upregulation of reactive oxygen species (ROS), has been investigated extensively in the past few years.1, 2 Various iron-based nanomaterials such as ferumoxytol,3 amorphous iron nanoparticles,4 and iron-organic frameworks,5 have been adopted as ferroptosis-inducing agents for the reason that elevated levels of iron ions can trigger the Fenton reaction and then increase the ROS level to induce cell death.6, 7 However, the ferroptosis induced by current iron-based nanomaterials is far from satisfactory, which generally requires a very high Fe dose4 or an additional component for a combinational effect.5, 8, 9 In fact, ferroptosis can be also induced by another signaling pathway related to the inactivation of glutathione-dependent peroxidase 4 (GPX4), which leads to the irresistible lipid peroxidation to induce the onset of ferroptosis.10,

11

Consequently, other than the ROS-based ferroptosis, various GPX4-inactivating strategies including gene-transfection, gene-knockdown technologies, and small molecular agents, have been recently developed for ferroptosis associated tumor therapy.12, 13 For instance, erastin, a glutathione (GSH) scavenger, has been demonstrated as a classical initiator of ferroptosis to induce cell death via the inactivation of GPX4.10, 14 GSH levels are elevated in the tumor cells and are closely related to cancer progression and chemoresistance as a result of its antioxidant and detoxification capacity.15-18 Various GSH depletion strategies have been proposed to elevate the tumor therapeutic effects of chemotherapy, photodynamic therapy (PDT) and radiotherapy.15,

19-21

For instance,

manganese dioxide (MnO2) nanoparticles were used to reduce GSH levels during PDT and chemodynamic therapy via responsive biodegradation under tumor intracellular microenvironment, resulting in the enhanced therapeutic effect.22 However, the current 4 ACS Paragon Plus Environment

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nanoparticles designed for GSH scavenging only play a supplementary role for tumor therapy.23 Direct tumor killing induced by the cell death pathways using GSH scavenging nanoparticles remains challenging due to their relatively low GSH depletion efficiency. In this study, we report the one-pot synthesis of biocompatible arginine-rich manganese silicate nanobubbles (AMSNs) with high tumor killing activity via the GPX4-mediated ferroptosis pathway (Scheme 1). Arginine (Arg) was adopted as a surface capping ligand for the synthetic process to confer ideal water dispersibility, biocompatibility, and tumor homing capacity. The Arg based ultrathin surface capping layer and nanobubble structure contributed to a dramatically enhanced GSH depletion efficiency of AMSNs compared with conventional nanoparticles, thus allow highly efficient GPX4 inactivation of tumor cells. Systemic molecular biological studies were used to investigate the GPX4 inactivationinduced ferroptosis. Moreover, the degradation of AMSNs during the GSH depletion process led to the release of Mn ions and loaded drugs, resulting in enhanced T1-weighted magnetic resonance imaging (MRI) contrast and chemotherapeutic effects. Different from previously reported manganese-based nanomaterials which only provided an auxiliary function to consume GSH in tumor therapy, the rationally designed AMSNs directly induce ferroptosis both in vitro and in vivo for highly effective tumor-targeted theranostics.


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RESULTS AND DISCUSSION Synthesis and Characterization of AMSNs. L-Arg-capped silica nanoparticles (SiO2) of ~14.6 nm in size with a surface charge of −39.73 mV (Figures S1 and S2a,b) were first obtained by a modified Stöber method.24,

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The AMSNs were then synthesized by a one-pot

hydrothermal method as shown in Scheme 1. Briefly, L-Arg-capped SiO2 was hydrolyzed to form H4SiO4, and then active surface sites for the attachment of manganese ions were generated. During the process, ethanol was decomposed into CO2 under hydrothermal conditions, and the generated CO2 nanobubbles served as a soft template for AMSNs formation.26, 27 The single nanobubble of as-prepared AMSNs of 6.2 ± 1.0 nm in diameter were well-dispersed in the aqueous phase (Figures 1a,b and S3), with a hydrodynamic size of approximately 59 nm, and a zeta potential of −17.60 mV (Figure S2c,d). Owing to the simple synthetic process, this method can be easily scaled up to a gram-scale production of the AMSNs (Figure 1a, insert). The N2 absorption-desorption isotherm analysis (Figure 1c) reveals that the as-prepared AMSNs have a high surface area of 539.48 m2 g−1 and a pore volume of 123.95 cm3 g−1. The powder X-ray diffraction (XRD) pattern reveals that manganese silicate is the main component of the AMSNs (Figure S4a), which is further confirmed by X-ray photoelectron spectroscopy (XPS) (Figure 1d) and energy-dispersive X-ray spectroscopy (EDX) (Figure S4b). The 2p3/2 peak in the XPS data can be divided into three characteristic peaks at 640.8, 641.7, and 642.8 eV, each of which corresponds to the reported valence state values of MnO, Mn2O3, and MnO2 respectively, indicating the presence of Mn(II), Mn(III) and Mn(IV) (Figure 1d, insert).


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The successful capping of L-Arg on the surface of AMSNs, a key factor for good colloidal stability and biocompatibility, was validated by various methods, including ultraviolet (UV) spectrophotometry, thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). As shown in the UV-vis spectra (Figure 1e), the AMSNs exhibit a strong absorption peak at 200.5 nm, which is close to the maximum absorption peak of pure L-Arg at 201 nm. TGA result further confirms the presence of L-Arg on the AMSNs (Figure 1f). The first weight loss at 30–150°C can be attributed to the loss of free and bound water molecules, and the next weight loss at 150–410°C was ascribed to the loss of L-Arg. FTIR analysis revealed the absorption peaks at 1631, 1562, and 1427 cm−1, which can be attributed to the out-of-plane bending of –NH2, stretching of –CO–, and bending of –OH, respectively (Figure S5). These results demonstrated that the Arg remained structurally intact and successfully capped on the surface of AMSNs. The Mn–N coordinate bond may possibly formed between the guanidyl group of Arg and the surface Mn ions of AMSNs (Figure S6).28-30 In addition, as shown in Figure S2d, the AMSNs are negatively charged, indicating that the –COOH group of L-Arg is oriented toward the water phase and the guanidyl group is anchored to the surface of the AMSNs, which corroborates well with previous studies.31, 32 Colloidal Stability and Biodegradability of AMSNs. The colloidal stability of the AMSNs was evaluated in DI water, phosphate-buffered saline (PBS), and 10% fetal bovine serum (FBS) supplemented cell culture medium. As shown in Figure 2a, the Tyndall effect was observed for AMSNs, indicating their excellent colloidal stability,33 and no significant hydrodynamic size change was noticed within 2 days (Figure 2b). In addition, AMSNs 7 ACS Paragon Plus Environment

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retained excellent stability in blood serum without evident change in size or morphology as displayed in Figure S7. Furthermore, in vivo pharmacokinetic study demonstrated a relatively long blood half-life of 4.07 h (Figure 2c), which could be explained by their small size and stable surface capping ligands. Previous studies have demonstrated that –Mn–O– bonds can be decomposed by a mild acid or GSH in the tumor intracellular microenvironment,27, 34-36 and Mn with high valence would be reduced to Mn(II) by GSH as illustrated in Figure 2d. Therefore, we further investigated the biodegradation behavior of the AMSNs under various pH and GSH conditions. As shown in Figure 2e and S8, the solutions became transparent under acidic and GSH conditions, and the structural degradation of AMSNs was observed by the transmission electron microscope (TEM) images, demonstrating that the AMSNs were degraded at either pH 5.0 without any GSH or at a neutral pH with 10 mM GSH. The degradation is further enhanced at pH 5.0 with 10 mM GSH. In addition, the nanobubble structure collapsed and aggregated along with the degradation of AMSNs (Figure S9).27, 37 Mn ion release behavior was also monitored under different pH and GSH conditions. The accumulated Mn release amount increased at a low pH and high GSH concentrations, with a maximum release percentage of 85.84% at pH 5.0 in PBS with 10 mM GSH (Figures 2f and S10). The as-prepared AMSNs were characterized as antiferromagnetic at 300 K (Figure S11). Since the release of Mn ions subsequently increases the r1 relaxivity,37, 38 T1-weighted contrast enhancement was analyzed using a 3.0 T human clinical MRI scanner. As demonstrated in Figures 2g, S12 and S13, the r1 relaxivity is 0.16 mM−1 s−1 in neutral PBS, and a 4-fold increase was observed when the pH value decreased to 5.0. A greater increase from 0.16 to 1.46 mM−1 s−1 was further achieved 8 ACS Paragon Plus Environment

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following the incubation with 10 mM GSH at neutral pH value. Moreover, the combination of pH 5.0 and 10 mM GSH enabled the r1 value further increased to 4.59 mM−1 s−1. These results clearly demonstrate the potential of AMSNs as a stimuli-responsive T1 MRI contrast agent for targeted tumor imaging.

In vitro Tumor Cell Selective Uptake, in vivo Tumor Homing and MRI of AMSNs. The cellular uptake of AMSNs was monitored by T1 MRI and confocal laser scanning microscopy (CLSM). As the concentration and time increased, the signal intensity of T1weighted MRI increased, revealing positive correlations of cellular uptake with the AMSNs concentration and the incubation time (Figure S14). The AMSNs seem to internalize into cells via endo/lysosomes, as suggested by the CLSM investigation on Huh7 liver cancer cells that showed the co-localization of red (AMSNs/DOX) and green (endo/lysosome) fluorescence (Figure S15). Arg is known to feed auxotrophic tumor cells that cannot manufacture arginine themselves owing to argininosuccinate synthetase (ASS) deficiencies in various tumors, e.

g., breast cancer, renal cell carcinoma, melanoma and hepatocellular carcinoma.39-41 Consequently, considering that the consumption of Arg at tumor sites will lead to demand for Arg-modified nanomaterials, we hypothesized that Arg can be used as a targeting moiety for tumor diagnosis and therapy. We evaluated the tumor cell selectivity of AMSNs by CLSM and flow cytometry, and the Huh7 liver cancer cells exhibited a stronger fluorescence intensity than that of liver L02 normal cells after incubation with AMSNs/DOX (Figure 3a). The flow cytometry results showed the uptake rates for Huh7 9 ACS Paragon Plus Environment

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liver cancer cells were up to 92.4 and 99.3% at 4 and 8 h, respectively, whereas those of L02 liver cell groups only reached 10.6 and 57.6% (Figure S16). In addition, the quantitative cell uptake at different incubation time points were also evaluated by ICP-MS (Figure S17), which were consistent with the results of CLSM and flow cytometry. The cytotoxicity of AMSNs was also evaluated using both cell lines. As shown in Figure 3b, the cytotoxicity against Huh7 cells are generally greater than that for L02 cells at various concentrations, and this can be attributed to the Arg-mediated selective uptake of the AMSNs by Huh7 cells. The in vivo tumor homing capability of AMSNs was evaluated by MRI using two tumor xenograft mouse models of MDA-MB 231 and Huh7, respectively. The MRI signal intensity at the tumor site enhanced at 2 h post-injection and increased gradually until 5 h post-injection (Figures 3c, S18 and S19). The MRI signal could still be detected after 24 h, indicating the accumulation of the released Mn ions at the tumor site. The biodistribution of AMSNs in tumor was evaluated by inductively coupled plasma mass spectrum (ICP-MS) after intravenous administration of AMSNs. As shown in Figure 3d, the percentage of Mn ions in tumor increased from 0.23 % / ID/g to 2.20 % / ID/g at 2 h, and reached to a peak at 3.44% / ID/g at 5 h, which was in accordance with the MRI results. Advantages of the Nanobubble Structure of AMSNs in GSH Depletion. Owing to the nanobubble structure, the degradation of AMSNs is much faster than that of the solid counterparts (Figure 4a).37, 42, 43 Solid MnO nanoparticles were synthesized by a thermal decomposition method44,

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poly(ethylene glycol) (DSPE-PEG) to construct MnO-PEG nanoparticles (Figure S20),46-49 The DLS and zeta potential of MnO-PEG is about 13.83 nm and -1.97 mV, respectively (Figure S21). The GSH depletion rate of the AMSNs and the MnO-PEG nanoparticles were compared (Figure 4b,c), where the color of the AMSNs group was lighter than that of the MnO-PEG group along with time, and AMSNs showed a faster Mn ion release rate than that of MnO-PEG (Figure 4d), indicating that the nanobubble structure of the AMSNs was beneficial for the consumption of GSH. Moreover, the AMSNs showed elevated cytotoxicity than the MnO-PEG nanoparticles against Huh7 cells (Figure 4e,f), which can be attributed to the more efficient GSH depletion capacity of the AMSNs. These results demonstrated that Arg-grafted AMSNs with the nanobubble structure and ultrathin surface coating can be used as an effective agent for GSH consumption, leading to a ferroptosisinduced tumor killing effect. Mechanism Underlying AMSN-Induced Cell Death via Ferroptosis. Based on the effective Arg-mediated cellular uptake and high GSH depletion capability of the AMSNs, we hypothesized that the severe GSH depletion of AMSNs could cause the inactivation of GPX4 for ferroptosis mediated tumor cell death (Figure 5a). To verify the occurrence of ferroptosis, several inhibitors associated with cell death pathways were used. As shown in Figure 5b,c, ferrostatin-1 (Fer-1, an inhibitor of ferroposis) and deferoxamine mesylate (DFOM, iron chelating agent) could significantly alleviate the toxicity induced by AMSNs, whereas the necrostain-1 (inhibitor of necroptosis), Z-VAD-FMK (inhibitor of apoptosis) and 3-MA (inhibitor of autophagy) could scarcely rescue the Huh7 cells from death (Figure S22a-c). In addition, the addition of GSH or N-Acetyl-L-Cysteine (NAC; raw materials for 11 ACS Paragon Plus Environment

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GSH synthesis) could significantly suppress the cytotoxicity and improve the cell viability in response to AMSNs (Figures 5d and S22d). Vitamin E, a liposoluble antioxidant, could obviously relieve the toxicity of AMSNs, whereas the water-soluble antioxidant vitamin C could not rescue the cell from death induced by AMSNs (Figures 5e and S22e). These results demonstrated that the ferroptosis shoulder the crucial responsibility in AMSN-induced cell death. GPX4, which plays a key role in lipid repair systems; can be inactivated by GSH depletion, thereby inducing the ferroptosis.50-52 We have demonstrated that AMSNs could efficiently consume GSH; accordingly, we further analyzed the GSH depletion inside cells. As shown in Figure 5f, the GSH levels in Huh7 cells treated with AMSNs were significantly lower than that of the untreated group, possibly due to the oxidation of GSH into glutathione disulfide (GSSG). As the concentration and incubation time increased, the protein expression level decreased (Figure 5g), and the activity of GPX4 also decreased substantially (Figure 5h,i). Moreover, gene knockdown and over-expression of GPX4 were further applied to confirm the involvement of GPX4 in the AMSNs-induced ferroptosis (Figure S23). After the silencing of GPX4 mRNA using siRNAs that caused GPX4 knockdown, the Huh7 cells showed high sensitivity to AMSNs (Figure 5j). Conversely, when the GPX4 was overexpressed, strong resistance of cells to AMSNs was observed (Figure 5k). These results indicated that GPX4 plays a key role in the AMSNs-induced ferroptosis. Furthermore, due to the GSH depletion by AMSNs, the intracellular oxidative stress was also elevated, as determined by dichlorofluorescein diacetate (DCFH-DA) staining (Figures 5l and S24a). In addition, since the inactivation of GPX4 would suppress the lipid peroxides being converted 12 ACS Paragon Plus Environment

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into harmless lipid alcohols, the lipid peroxide level can be used as a significant indicator of the ferroptosis.53, 54 Thus, the lipid peroxidation probe C11-BODIPY581/591 was used to monitor the lipid peroxide level. As shown in Figures 5m and S24b, cells in AMSNs groups showed stronger fluorescence intensities than those of cells in the control group, and the intensity was proportional to the concentration and the incubation time. Above all, these results provide evidence that ferroptosis was mediated by the inactivation of the GPX4 pathway. Notably, AMSNs induced significant cytotoxicity even at a low Mn concentration (25 μg mL−1) (Figure 3b), in contrast with conventional iron-based ferroptosis-inducing agent.4

In vitro Drug Loading and Releasing Behaviors of AMSNs. The large specific surface area of the nanobubble structure, and the biodegradation capability make the AMSNs a highly efficient platform for drug loading and controlled release. The drug loading and release behaviors of the AMSNs were evaluated using DOX as a model drug. As DOX increased, the loading capacity of the AMSNs reached an optimal loading efficiency (58.08%) and embedding ratio (87.12%) at a weight ratio of 1:2 (AMSNs: DOX) (Figure S25). The DOX loaded AMSNs are well dispersed in aqueous solutions (Figure S26). The high drug loading efficiency and embedding ratio could be explained by the electrostatic interaction between the positively charged DOX and the negatively charged AMSNs, as well as the coordinate bonds between the N atoms in DOX and the Mn atoms in the AMSNs.35, 55 This optimal ratio used for subsequent experiments. AMSNs exhibited sustained DOX release, which was accelerated at high GSH concentrations and low pH values (Figures 6a–c). Importantly, as shown in Figure 6a, only 10.64% DOX released from the AMSNs/DOX after incubated at 13 ACS Paragon Plus Environment

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37 ℃ at pH 7.4 in PBS for 1 week, therefore, the AMSNs/DOX system was stable in physiological condition. These data demonstrated that the AMSNs had a high drug loading efficiency as well as tumor intracellular microenvironment-responsive drug release behavior. Moreover, the stronger cellular fluorescence intensity (Figure S27) and more severe cytotoxicity (Figure S28) of the AMSN/DOX group than those of the free DOX group indicated the successful cellular delivery of the AMSN/DOX. Interestingly, the AMSNs/DOX group shows a much lower cytotoxicity than that of free DOX in L02 cells, indicating the loading of DOX in AMSNs can potentially reduce the cytotoxicity of DOX in normal cells (Figure S29). In addition, the ferroptosis inhibitor Fer-1 showed little impact on the cytotoxicity of DOX. Therefore, the activation of ferroptosis was irrelevant with DOX (Figure S30).

In vivo Tumor Therapy Using AMSNs/DOX. The in vivo therapeutic efficacies of both AMSNs and AMSN/DOX were evaluated in Huh7 tumor xenograft nude mice. As shown in Figure 6d, compared with free DOX, the AMSN/DOX treated group exhibit negligible

in vivo systemic toxicity. After 14 days, the tumor growth of the AMSN/DOX group was significantly inhibited (Figure 6e,f). Interestingly, the AMSN group also exhibited greater suppression of tumor growth than those of the free DOX group owing to the Arg-mediated tumor homing and subsequent GSH depletion action in the tumor tissue via the AMSNinduced ferroptosis. However, the AMSN/DOX group showed the highest tumor suppression rate among all groups, indicating the combinational effect from the GSH depletion induced ferroptosis and the chemotherapy. Hematoxylin-eosin (H&E) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining 14 ACS Paragon Plus Environment

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showed significant tumor cell death and the formation of many cavities (Figure 6g), indicating that the AMSN/DOX can effectively induce tumor tissue damage. The AMSNs-induced ferroptosis was also verified in vivo by using tumor xenograft models. Compared with the PBS group and the liproxstatin-1+AMSNs group, the AMSNs-treated group showed obvious tumor growth suppression (Figure 7a-c). In addition, mRNA expression of Ptgs2 significantly elevated after the treatment with AMSNs, indicating the activation of ferroptosis.10 Moreover, liproxstatin-1 as a ferroptosis inhibitor decreased the high expression of Ptgs2 mRNA in AMSNs-treated tumors into the original level, implying the key role of ferroptosis in the mechanism of AMSNs-induced tumor killing (Figure 7d). Furthermore, the GPX4 inactivation effect induced by AMSNs can be relieved by the ferroptosis inhibitor liproxstatin-1 (Figure 7e). These results indicated that the ferroptosis induced by AMSNs played an important role in the tumor-growth inhibition. The H&E staining showed that the free DOX induced cardiotoxicity and liver toxicity, while AMSNs- and AMSNs/DOX-treated groups exhibited no notable toxicity, indicating the biocompatibility of AMSNs and AMSNs/DOX (Figure S31). Biosafety Evaluation of AMSNs. Biodegradation has a significant impact on the in vivo fate and biosafety profile of nanomaterials.56-58 The in vivo biodistribution profile of the AMSNs was evaluated by MRI and ICP-MS. The liver and kidney exhibited MRI signal enhancement, implying the accumulation of the AMSNs (Figure S32a–c), consistent with the ICP-MS results for the Mn ion bio-distribution (Figure S32d). As determined by ICPMS, the accumulated AMSNs in each major organ decreased markedly as time goes (Figure 15 ACS Paragon Plus Environment

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S33), indicating the AMSNs might be excreted out via the feces and urine (Figure S34). In addition, based on the long-term biodistribution analysis of AMSNs, the particles were mainly distributed in the liver, lung, and kidney after 24 h, and the Mn ion concentration decreased to normal levels after 30 d (Figure S35). Furthermore, the body weight curves (Figure S36), serum biochemical parameters (Figure S37a), hematological analysis (Figure S37b), and histopathological examination (Figure S38) supported the safety profile of AMSNs. Apparently, the favorable biosafety renders the AMSNs a promising platform for tumor theranostics. CONCLUSION In summary, we developed a ferroptosis-inducing agent based on biocompatible arginine-rich manganese silicate nanobubbles. The Arg-capped AMSNs were prepared by a facile one-pot hydrothermal reaction, which showed selective tumor cell uptake and in

vivo tumor homing due to the surface Arg. The AMSNs can be used as tumor intracellular pH/GSH sensitive T1 MRI contrast agent and drug delivery vehicle. Moreover, the nanobubble structure and ultrathin surface Arg layer of the AMSNs resulted in highly efficient GSH depletion inside tumor cells. Importantly, we demonstrated that the fast GSH depletion rate by AMSNs resulted in the inactivation of GPX4 and direct tumor cell death

via the ferroptosis pathway. Our study provides insight into the design of nanomedicine aimed a GPX4 inactivation-induced ferroptosis, which shall facilitate the development of next-generation ferroptosis inducing agents that are highly efficient, multifunctional and biodegradable. 16 ACS Paragon Plus Environment

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MATERIALS AND METHODS Materials: L-Arginine, tetraethyl orthosilicate (TEOS), 5,5 ′ -dithiobis (2-nitrobenzoic acid) (DNTB), cyclohexane, doxorubicin hydrochloride (DOX∙HCl) and 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Aladdin (Shanghai, China). KMnO4 and ethanol were purchased from Sinopharm (Beijing, China). Ferrostatin-1 (Fer-1), Z-VAD-FMK, 3-methladenine (3-MA), necrostatin-1, deferoxamine mesylate (DFOM), vitamine E and

L-(+)-ascorbic

acid were purchased from Target

Molecule Corp. (Shanghai, China). N-Acetyl-L-cysteine (NAC) and glutathione (GSH) were ordered from Beyotime (Jiangsu, China). Phosphate-buffered saline (PBS) power was bought from Solarbio (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), trypsin and penicillin-streptomycin solution were obtained from Gibco BRL (Gaithersburg, MD, USA). Paraformaldehyde (PFA) was purchased from Wuhan Boster Company (Wuhan, China). C11-BODIPY581/591 was purchased from Thermo Fisher (Waltham, MA, USA). 4 ′ ,6diamidino-2-phenylindole (DAPI) was purchased from Dawen Biotec (Hanghzou, China). All chemicals were used without further purification. Synthesis of Silica Nanoparticles:

L-Arginine

(50 mg) was dissolved in a solution

containing 40 mL of water and 3 mL of cyclohexane, and 3 mL of TEOS was added. The mixture was stirred (1000 rpm) at 50°C for 24 h, and then the small silica nanoparticles were separated using a separatory funnel. The upper cyclohexane layer was discarded and the lower aqueous phase was collected without further processing. 18 ACS Paragon Plus Environment

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Synthesis of Arginine-Rich Manganese Silicate Nanobubbles (AMSNs): A stock solution of the silica nanoparticles (1 mL, 11 mg mL−1) was dispersed in 2.0 mL of ethanol, add the solution of KMnO4 (0.025g, 3.5 mL) into the above solution, and then placed in a 20-mL Teflon-lined autoclave and heated at 190°C for 24 h. After cooling to room temperature, the final products were separated by centrifugation (9500 rpm) and washed several times with DI water. Characterization: TEM images were captured using a transmission electron microscope operated at 200 kV to observe the morphology of the AMSNs (Hitachi HT7700). The N2 adsorption/desorption isotherm was determined using a BEL Max (Quantachrome Instruments, Boynton Beach, FL, USA). XRD patterns were obtained using a PANalytical X'Pert PRO (PANalytical, Almelo, Netherlands). Size distribution and zeta potential were evaluated using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). XPS was performed using a Thermo Scientific ESCALAB 250 Xi XPS system. The Mn ion concentration was measured using inductively coupled plasma mass spectrum (ICP-MS) (NexION 300XX, PerknElmer, USA). For flow cytometry, an Accuri C6 (BD Bioscience, Franklin Lakes, NJ, USA) flow cytometer was used. For MRI, a 3T human clinical MR scanner (GE, Chicago, IL, USA) was used. Accumulated Release Profiles of Mn Elements: Using medium with different pH values (7.4, 6.5, and 5.0) and GSH concentrations (0, 5, and 10 mM), AMSNs were added and incubated at 37 ℃ in dialysis tube for different durations. The concentration of Mn at every time point was measured by ICP-MS. 19 ACS Paragon Plus Environment

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In vitro MRI of AMSNs: AMSNs were diluted to various concentrations (1, 0.5, 0.25, 0.125 and 0 mM) in PBS with different pH values (7.4, 6.5, and 5.0) and different GSH concentrations (0, 5 and 10 mM). Then, 200 μL of each solution was collected, and T1 MRI was performed using a 3.0 T human clinical scanner. MRI Observations of Cellular Uptake Behavior: Huh7 cells were seeded in cell-culture dishes. When the density reached 70–80%, AMSNs were added and incubated for 6 h with various concentrations (0, 1, 5, 10, and 20 μg mL−1). Other groups were incubated for different time periods using an AMSN concentration of 10 μg mL−1. The cells were harvested, washed with PBS three times and dispersed on a 1% agarose gel. Their MRI signals were measured using a 3.0 T human clinical scanner. CLSM Observations of the Tumor Homing Effect of AMSNs: Huh7 cells and L02 cells were seeded in a CLSM-specific dish for CLSM analysis. When the density reached 70– 80%, AMSNs/DOX were added (DOX, 5 μg mL−1), and the cells were cultured at 37°C in a humidified incubator for various time periods. Then, the cells were washed with PBS three times and nuclei were stained with DAPI. A CLSM unit (FV 1200, Olympus, Tokyo, Japan) was utilized to acquire fluorescence images of the cells. Flow Cytometry for the Quantification of the Tumor Homing Effect of AMSNs: Huh7 cells and L02 cells were seeded on a 6-well plate, respectively. When the density reached 70–80%, AMSNs/DOX was added to DMEM at an equivalent DOX dose of 5 μg mL−1. The cells were cultured at 37°C in a humidified incubator for various time periods. Then, the


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cells were washed with PBS three times and dispersed in 300 μL of PBS. A flow cytometry apparatus was used to record the fluorescence intensity of the cells.

In vitro Cell Viability Test of Huh7 Cells After Treatment with Various Inhibitors: Liver cancer Huh7 cells were seeded on a 96-well plate (approximately 3000 per well), and cultured at 5% CO2, 37°C overnight. AMSNs was added to every well, and then the ferroptosis inhibitors, including fer-1, DFOM, GSH, NAC, and VE, the ROS eliminator VC, a necroptosis inhibitor (Necrostain-1), an apoptosis inhibitor (Z-VAD-FMK), and an autophagy inhibitor (3-MA) were added to the AMSN treated cells after 6 h. After cells were cultured for another 24 h, the medium was removed and the cells were washed with PBS three times. MTT reagent was added and followed by the incubation at 37 °C for another 4 h. A microplate reader was used to measure the absorbance. Intracellular GSH Content: Liver cancer Huh7 cells were seeded on a cell-culture dish, and cultured at 5% CO2, 37°C overnight. AMSNs were added and cultured for 0, 0.25, 2, 4 and 8 h at 50 μg mL-1. The glutathione content was measured using a glutathione Assay kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. A microplate reader was used to measure absorbance. Intracellular GPX4 Activity Assay: Intracellular GPX4 activity was measured using a cellular glutathione peroxidase assay kit (Beyotime, Jiangsu, China). Liver cancer Huh7 cells were seeded on a 6-well plate and cultured at 5% CO2, 37°C overnight. AMSNs were added and cultured at various concentrations (0, 12.5, 25 and 50 μg mL-1) for 8 h or at 50 μg mL-1 cultured for 0, 2, 4 and 8 h. The cell lysates were collected and measured according 21 ACS Paragon Plus Environment

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to the manufacturer’s instructions. A microplate reader was used to measure the absorbance at 340 nm. Western Blot Analysis: Liver cancer Huh7 cells were seeded on a 6-well plate, and cultured at 5% CO2, 37°C overnight. AMSNs were added and cultured at various concentrations (0, 12.5, 25 and 50 μg mL-1) for 8 h or at 50 μg mL-1 cultured for 0, 2, 4 and 8 h. The cell lysates were collected and analyzed by electrophoresis run on 13% denaturing polyacrylamide gels. siRNA Transfection: The cells were transfected with three siRNAs targeting GPX4 (Genephama). Briefly, it was carried out by adding 0.5 mL of Opti-MEM (Genemute) containing 10 μL of lipo-RNAiMAX (Genemute) and the siRNA mixture to the Huh7 cells cultured in a 100 mm dish with 4.5 mL DMEM (supplemented with 10% serum). The cells were further cultured at 5% CO2, 37°C for 5 hours and then replaced with fresh medium. All the viability assays were conducted 48 hours after transfection. The sequences of the three siRNAs were: GPX4-homo-678, UGGUGAUAGAGAAGGACCUTT; GPX4-homo-445, CAGGGAGUAACGAAGAGAUTT; GPX4-homo-341, GACCGAAGUAAACUACACUTT. Generating Stable Huh7 Cell Lines Overexpressing GPX4: A cDNA of GPX4 (GeneBank ID: NM_002085.3) was cloned into a pLVX-puro vector. The plasmid (or the empty vector) was co-transfected into 293T cells seeded in a 100 mm petri-dish in company with pVSV-G and DR8.2 helper plasmids to generate lentivirus containing the expressed plasmid. Lentivirus was collected from the culture medium twice at 48 and 72 hours post transfection, respectively. The lentivirus-containing supernatant was filtered through 0.22 μm 22 ACS Paragon Plus Environment

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filter membrane and concentrated by centrifugation, and stored at -80 ℃ before use. To construct GPX4-overexpressing cells, Huh7 cells were seeded into a six-well plate at a confluence of 50% the night before infection. Cells were infected by incubating with 1 mL lentivirus-containing supernatant in the presence of 8 mg mL-1 polybrene. Following infection, cells were passaged several times in the culture media containing 2 μg mL-1 puromycin and grown in this media for all experiments performed. Intracellular Lipid Peroxide Measurement: The intracellular lipid peroxide content was determined by CLSM. Liver cancer Huh7 cells were seeded on a confocal dish, and cultured at 5% CO2, 37°C overnight. AMSNs were added and cultured at various concentrations (0, 25, 50 and 100 μg mL-1) for 8 h or at 50 μg mL-1 cultured for 0, 2, 4 and 8 h. The cells were stained with C11-BODIPY581/591 (2 μM) and incubated for 30 min. A CLSM unit (FV 1200, Olympus, Tokyo, Japan) was used to obtain the fluorescence images of cells.

In vitro DCFH-DA Assay: The ROS generated was measured by CLSM using an ROS sensitive probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Beyondtime). Liver cancer Huh7 cells were seeded on a confocal dish, and cultured at 5% CO2, 37°C overnight. AMSNs were added and cultured at various concentration (0, 25, 50 and 100 μg mL-1) for 8 h or at 50 μg mL-1 cultured for 0, 2, 4 and 8 h. The cells were stained by DCFH-DA (10 μM) and incubated for 20 min. The CLSM unit (FV 1200, Olympus, Tokyo, Japan) was used to obtain the fluorescence images of cells. Comparison of the GSH Depletion Rate between AMSNs and MnO: Ellman's reagent was used to measure GSH levels by the DTNB assay (Aladdin, Shanghai, China). Solutions with 23 ACS Paragon Plus Environment

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10 mM GSH were prepared and added with the same [Mn] amount of AMSNs or MnO nanoparticles. The solutions were incubated at 37°C for different durations (0 h, 1 h, 2 h, 4 h, 6 h, 8 h,10 h, and 12 h). The samples were centrifuged at 12000 rpm for 10 min, and each supernatant was collected to evaluate the GSH level. After 5 min of the co-incubation of the supernatant and DTNB solution (4 mM) at 25°C, absorbance was measured using a microplate reader.

In vitro Cytotoxicity: An MTT cell proliferation assay was used to evaluate cell viability. Liver cancer Huh7 cells were seeded in a 96-well plate (approximately 3000 per well), and cultured at 5% CO2, 37°C overnight. Free DOX and AMSNs/DOX were added at different concentrations (0, 1, 5, 10, and 25 μg mL−1), and cultured for 24 or 48 h. The medium was removed, the cells were washed with PBS three times, MTT solution was added to each well, and the plate was incubated at 37°C for 4 h. A microplate reader was used to measure the absorbance. CLSM Observations of Cellular Uptake Behavior: For CLSM observation, Huh7 cells were seeded on a CLSM-specific dish. When the density reached 70–80%, free DOX or AMSNs/DOX at an equivalent DOX dose of 5 μg mL−1 was added. The cells were cultured at 37°C in a humidified incubator for various time periods. Then, the cells were washed with PBS three times, and the nuclei were stained with DAPI. A CLSM unit (FV 1200, Olympus, Tokyo, Japan) was utilized to obtain the fluorescence images of the cells. Drug Loading and Release: Different amounts of doxorubicin hydrochloride (DOX·HCl) (2, 4, 8, 12, and 16 mg) were dispersed in 8 mL of DI water. AMSNs (4 mg) were dispersed 24 ACS Paragon Plus Environment

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in the DOX solution and stirred in the dark for 24 h. The DOX-loaded AMSNs were collected by centrifugation, washed three times, and dispersed in PBS. The supernatant was collected after centrifugation was also collected. The drug loading efficiency and embedding ratio were calculated based on a fluorescence intensity measured at 480 nm. The in vitro drug release behavior was evaluated in various release media. DOX-loaded AMSNs (2.5 mg) were put in a dialysis bag and placed in 20 mL of a PBS solution with different pH values (7.4, 6.5, and 5.0) and GSH concentrations (0, 5.0, and 10.0 mM). The solutions were incubated at 37°C with shaking at 150 rpm. At various time points, 2 mL of each solution was collected and replaced with 2 mL of fresh medium, and the drug release was estimated based on UV spectra. Animal Tumor Models: Four- or five-week-old BALB/c nude mice (about 20 g body weight) obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) were used for tumor xenograft establishment. All procedures with the approval of the animal experimental ethics committee of Zhejiang university. Huh7 or MDA-MB 231 cells with a density of 1×107 were used to establish tumor models. The cells were collected by centrifugation (1000 rpm, 3 min), washed with PBS three times, and then re-dispersed in a PBS buffer. The tumor model was established by the subcutaneous dorsal injection of 200 μL of Huh 7 or MDA-MB 231 cells in PBS per mouse.

In vivo MRI: Tumor-bearing nude mice were injected with an AMSNs solution at 10 mg kg−1, and MRI was performed using a 7.0 T animal MR imaging system. Images were captured at different time points (0, 0.5, 2, 5, 24, and 48 h). 25 ACS Paragon Plus Environment

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In vivo Antitumor Efficiency of AMSNs/DOX: To evaluate the efficiency of tumor-killing effect, tumor-bearing nude mice were divided into 5 groups and intravenously injected with PBS, free DOX, AMSNs, 2 × AMSNs and AMSNs/DOX. Body weights and tumor volumes were measured every 2 days after the treatment. After 2 weeks, the mice were sacrificed, and the primary organs were harvested for TUNEL and H&E staining. In vivo mechanism of ferroptosis induced by AMSNs: To study the in vivo mechanism of ferroptosis induced by AMSNs, tumor-bearing nude mice were divided into 3 groups and intravenously injected with PBS, AMSNs and AMSNs+liproxstatin-1. Each group was injected via the tail vein two times per week (5 mg kg-1 Mn), and the liproxstatin-1 was administrated via i.p. injection (10 mg kg-1). Body weights and tumor volumes were measured every 2 days during the treatment. The photos were obtained at 5 and 10 days post-injection. After 10 days, the mice were sacrificed, and tumors were harvested for the evaluation of Ptgs2 mRMA level and GPX4 activity. Ptgs2 level analysis: The tumor-bearing mice were sacrificed after different treatments as mentioned above. Tumors were harvested, cut into small pieces, and stored at -80 ℃ before the detection of Ptgs2 levels. After the total mRNA was extracted from the tumor tissue, it was reverse-transcribed into cDNA and followed quantitative on a Stepone real-time PCR system (Thermo) to detect Ptgs2 gene expression. Data for each sample were collected from 3 replicates. In vivo GPX4 activity assay: The in vivo tumor tissue of GPX4 activity was measured using a cellular glutathione peroxidase assay kit (Beyotime, Jiangsu, China). The tumor-bearing mice 26 ACS Paragon Plus Environment

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were sacrificed after different treatments as mentioned above, then the tumors were harvested, cut into smaller pieces, and stored at -80℃ before measurements of GPX4 activity. The tumor tissue lysates were collected and measured according to the manufacturer ’s instructions. A microplate reader was used to measure the absorbance at 340 nm.

In vivo Biocompatibility Assay: Healthy mice were injected with an AMSNs solution via intravenous tail vein administration at (180 μg [Mn] per mouse); PBS was injected as a control. Weight changes monitored and routine blood examinations were performed; blood biochemical indexes were tracked for 30 days. The mice were then sacrificed, and the primary organs were harvested for H&E staining. The amounts of Mn in various organs were quantitatively determined by ICP-MS.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Additional results for nanoparticle characterization, in vitro/ in vivo studies, and Figures (PDF) AUTHOR INFORMATION Corresponding Author 27 ACS Paragon Plus Environment

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*E-mail:

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[email protected]

Author Contributions #Shuaifei

Wang, Fangyuan Li, and Ruirui Qiao contributed equally to this work.

ACKNOWLEDGMENT We gratefully acknowledge the financial support providing by the National Key Research and Development Program of China (2016YFA0203600, 2016YFA0100900, and 2016YFA0201600), the National Natural Science Foundation of China (31822019, 51503180, 51611540345, 51703195, 81471739, and 81430040), “Thousand Talents Program” for Distinguished Young Scholars (588020*G81501/048), the Fundamental Research Funds for the Central Universities (520002*172210161, 2017XZZX001-04), the Research Center Program for the Institute of Basic Science (IBS-R006-D1), and the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036).

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Scheme 1. Schematic illustration of the designed synthesis of the arginine-rich manganese silicate nanobubbles (AMSNs) as well as the in vivo tumor homing after blood circulation, the AMSNs can be internalized into tumor cells by the Arg-driven intracellular delivery due to the ASS deficiency of tumor cells. The biodegradation of AMSNs accompanied by fast GSH depletion in tumor cells, inducing the inactivation of GPX4, failure of the conversion from lipid peroxides to non-toxic lipid alcohols, and eventually inducing the highly efficient tumor cell death via ferroptosis.


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Figure 1. Characterization of the AMSNs. a) Transmission electron microscope (TEM) image of AMSNs, and photograph of the gram-scale production of AMSNs (inset). b) Scanning TEM image of AMSNs. c) N2 absorption/desorption isotherm of AMSNs. d) XPS spectra of AMSNs and Mn 2p electronic energy (inset). e) UV-vis absorption spectra for AMSNs and pure L-Arg. f) Thermogravimetric analysis (TGA) curves forAMSNs and pure L-Arg.


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Figure 2. Colloidal stability and biodegradation of AMSNs. a) Tyndall effect of AMSNs in different solutions (water, PBS, 2× PBS, 5× PBS, and DMEM + 10% FBS). b) DLS profile of AMSNs in different aqueous solutions for 2 days. c) Pharmacokinetics profiles of AMSNs intravenous injected into the mice. d) Schematic illustration of the biodegradation of AMSNs in low pH and high GSH microenvironment. e) Photo and TEM images of AMSNs after incubation in buffers of different pH and GSH concentrations for 12 h. f) Accumulated release profiles of Mn ions in various pH (7.4, 5.0) and GSH concentrations (0 and 10 mM). g) Curves of Δ1/T1 versus the Mn concentration of AMSNs under different conditions, the slope indicates the specific relaxivity (r1).


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Figure 3. Tumor cell selective uptake and in vivo tumor homing behavior of AMSNs. a) Confocal laser scanning microscope (CLSM) images of the Huh7 liver cancer cells and L02 normal liver cells after incubation with AMSNs/DOX for various time periods. Scale bar: 20 µm. b) Cytotoxicity of AMSNs against to Huh7 cells and L02 cells after 24 h of incubation. c) In vivo T1-weighted MRI of tumor-bearing mice after the intravenous administration of AMSNs for different time periods. (B0=7.0T) d) In vivo distributions of AMSNs in the tumor site at different time points after i. v. injection as measured by ICPMS. (AMSNs/DOX: DOX-loaded AMSNs; AMSNs: without DOX loaded) (*P < 0.05, **P < 0.01, ***P < 0.001).


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Figure 4. Advantages of the nanobubble structure of AMSNs with respect to GSH depletion. a) Schematic illustration of ferroptosis pathways induced by GSH depletion from AMSNs. Both of the AMSNs and solid MnO-PEG could be internalized into tumor cells, however, the nanobubble structure and ultrathin surface modification of AMSNs make it much more preferable in GSH depletion than the solid MnO counterparts. Furthermore, the highly efficient GSH depletion could induce the tumor cell death directly. b) Photographs showing the degradation behavior of AMSNs and MnO-PEG nanoparticles in 10 mM GSH. c) The GSH levels of the solution after the treatment with AMSNs or solid MnO-PEG nanoparticles. d) Accumulated Mn ion release profiles for AMSNs and MnO-PEG nanoparticles in 10 mM GSH. e,f) Cytotoxicity of AMSNs and MnO-PEG nanoparticles against Huh7 cells after incubation for 24 (e) and 48 h (f). (*P < 0.05, **P < 0.01, ***P < 0.001).


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Figure 5. Mechanism evaluation of AMSN-induced cell death by ferroptosis. a) Schematic illustration of AMSN-induced ferroptosis via GSH depletion. With the fast GSH depletion by AMSNs, the activity of GPX4 can be suppressed, and the conversion of lipid peroxides to non-toxic lipid alcohols is inhibited, thereby induce the ferroptosis. b–e) The cell viability of Huh7 cells treated with AMSNs and Fer-1 (b), DFOM (c), GSH (d) and VE (e). f) GSH:GSSG level in Huh7 cells treated with AMSNs for different durations. g) Western blot results for GPX4 expression level in Huh7 cells after treatment with AMSNs. h,i) GPX4 43 ACS Paragon Plus Environment

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activity measured after treatment with AMSNs as the concentration and time increase. j) Knockdown of GPX4 with siRNA rendered Huh7 cells hypersensitive to AMSNs-induced cytotoxicity. k) Huh7 cells overexpressing GPX4 was highly resistant to AMSNs-induced cytotoxicity. Scr: Scramble siRNA; EV: Empty vector; GPX4-OE: GPX4 over-expression. l) DCFH-DA assay of Huh7 cells treated with AMSNs. (Scale bar: 100 μm) m) Lipid peroxides of Huh7 cells stained with C11-BODIPY581/591 after co-incubation with AMSNs. (Scale bar: 50 μm) (*P < 0.05, **P < 0.01, ***P < 0.001).


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Figure 6. Tumor therapy using AMSNs. a–c) Drug release profiles of AMSNs in buffers with different pH (a), with different GSH concentrations at pH 7.4 (b) and pH 5.0 (c). d) Body weight changes in mice 2 weeks after the treatment with AMSNs. e) Tumor-growth inhibitory effects of different treatments. f) Tumor photos after various treatments. g) H&E and TUNEL staining of tumor slices obtained from BALB/C mice after various treatments.

Figure 7. In vivo study of ferroptosis induced by AMSNs. a) Photos of tumor-bearing mice after various treatments. b) Tumor weight after different treatments. c) Changes in the tumor volume after different treatments (n=6). d) Relative Ptgs2 mRNA expression level after different treatments. e) GPX4 activity in tumor after different treatments.


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TOC GRAPHIC


Arginine-Rich Manganese Silicate Nanobubbles as a Ferroptosis

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