研选高/低氧细胞（动物）mini培养箱/三气培养箱/缺氧

Cyclic reactions-mediated self-supply of H2O2 and O2 for cooperative chemodynamic/starvation cancer therapy Xiaojuan Zhang a,1 , Chuanchuan He a,1 , Yan Chen a , Chen Chen a , Ruicong Yan a , Ting Fan a , Yongkang Gai b,c , Tan Yang a,***, Yao Lu a,**, Guangya Xiang a,* a School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China b Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China c Hubei Province Key Laboratory of Molecular Imaging, Wuhan, 430022, China ARTICLE INFO Keywords: Glucose oxidase Starvation therapy Chemodynamic therapy Hypoxia ABSTRACT Hydroxyl radical (⋅OH)-mediated chemodynamic therapy (CDT) and glucose oxidase (GOx)-based starvation therapy (ST) are two emerging antitumor strategies, limited by acid/H2O2 deficiency and tumor hypoxia, respectively. Herein, we developed a liposomal nanoplatform co-delivering Fe(OH)3-doped CaO2 nanocomposites and GOx molecules for synergistic CDT/ST with a complementary effect. Based on Fenton reactions initiated by iron ions, CaO2-supplied H2O2 could not only generate ⋅OH for H2O2-sufficient CDT, but also produce O2 to promote the catalytic efficiency of GOx under hypoxia. In return, the enhanced ST generated gluconic acid and H2O2, further amplifying CDT. Through in vitro and in vivo experiments, we demonstrated that such a mutually reinforced modality based on the cyclic Fenton/starvation reactions provided a novel and potent anticancer mechanism for the effective treatment of hypoxic cancers. 1. Introduction Owing to the potent cytotoxicity of hydroxyl radical (⋅OH) produced by Fenton reactions, chemodynamic therapy (CDT) that employs Fenton catalysts to eliminate cancer cells has attracted increasing research attention in recent years [1,2]. In contrast to photodynamic therapy (PDT), CDT needs no extra energy input, therefore avoiding the limitation posed by light penetration in tissues [3]. However, the efficiency of CDT largely relies on the concentrations of hydrogen ion (H+) and hydrogen peroxide (H2O2) in the tumor microenvironment (TME) [4]. On one hand, tumor acidosis is featured with acidic extracellular pH (pHe ≈ 6.5) and weak alkaline intracellular pH (pHi ≈ 7.2), severely hindering the effective implementation of CDT [5]. On the other hand, although cancer cells have a higher concentration of H2O2 than normal cells, the easily-consumable amount of H2O2 (10–50 μM) is variable between different types of cancer cells, which still limits its transformation for sufficient ⋅OH production and effective CDT [6]. As another new type of cancer treatment modality, starvation therapy (ST) has been proposed due to the unique catalytic properties of glucose oxidase (GOx) for glucose deprivation in cancer cells [7]. In this process, GOx can effectively catalyze the oxygen-dependent oxidation of glucose into gluconic acid and H2O2, resulting in an acidic and oxidative TME [8,9]. Inspired by the dependence of CDT on H+ and H2O2, considerable efforts have been made in the codelivery GOx and Fenton reagents [7,10–12]. Huo et al. constructed a sequential nanocatalyst by delivering GOx and Fe3O4 nanoparticles into dendritic mesoporous silica nanoparticles [13]. GOx in the nanocatalyst could effectively catalyze the intratumoral glucose into acidic H2O2, further catalyzed by Fe3O4 to liberate ⋅OH for CDT. However, due to the high dependency of GOx-catalytic reaction on O2, the intrinsic hypoxia of TME largely limited the catalytic performance of GOx, leading to the poor outcome of GOx-mediated ST and subsequent CDT [14]. Fortunately, exogenous H2O2 delivery is an applicable strategy to enhance CDT in an O2-independent manner [15]. Calcium peroxide (CaO2), a widely-explored O2-generating compound, has been constructed to bolster PDT and chemotherapy due to its biocompatible and * Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: yangtan0120@hust.edu.cn (T. Yang), yaolu11@hust.edu.cn (Y. Lu), gyxiang1968@hotmail.com (G. Xiang). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials https://doi.org/10.1016/j.biomaterials.2021.120987 Received 24 January 2021; Received in revised form 17 May 2021; Accepted 20 June 2021 Biomaterials 275 (2021) 120987 2 biodegradable properties [16,17]. As a matter of fact, CaO2 tends to produce H2O2 rather than O2 in acidic TME [18,19]. The codelivery of CaO2 and Fenton catalysts has already been investigated by several research groups to realize H2O2-sufficient CDT [20–22]. However, at the first step of Fenton reactions in CDT, the reduction of metal ions (e.g., Fe3+ to Fe2+, Cu2+ to Cu+, etc.) leads to the O2 generation and H2O2 consumption, which results in compromised CDT efficacy [23]. Inspired by the O2-consumption/dependence and H+/H2O2-production of GOx-based starvation reaction, it is rational to combine the H2O2-sufficient Fenton reagents with GOx to combat cancers: H2O2-sufficient Fenton reaction produces O2 to break the hypoxic barrier of ST, and in return, the enhanced ST supplies H+/H2O2 to improve CDT. Herein, we constructed a type of O2/⋅OH dual-generating Fenton’s reagents with CaO2/Fe(OH)3 nanocomposites, which were further coloaded with GOx molecules in a biocompatible liposome to formulate a combined nanomedicine. By simply adding aqueous FeCl3 into assynthesized CaO2 nanoparticles, Fe(OH)3 was formed in situ on the surface of CaO2 to obtain CaO2/Fe(OH)3 nanocomposites. Due to the surface coating by polyethylene glycol 200 (PEG200), the ultrasmall CaO2/Fe(OH)3 nanocomposites could be encapsulated into the lipid layer of liposomes [24]. As a hydrophilic biomacromolecule, GOx could be further loaded into the water-cavity of liposomes (Scheme 1A) [25]. After intravenous injection, the liposomes passively accumulated in tumor sites via the enhanced permeability and retention (EPR) effect (Scheme 1B) [26]. Firstly, the acidic TME enabled the decomposition of CaO2/Fe(OH)3 nanocomposites, leading to the generation of H2O2 and Fe3+ (Step I). Subsequently, H2O2 reacted with Fe3+ to generate O2 and ⋅OH, triggering Fenton reactions (Step II). The active ⋅OH possess the ability to disrupt cellular constitutes (e.g., lipids and proteins) for CDT-mediated cancer cell death. Meanwhile, the produced O2 promoted the catalytic efficiency of GOx, enhancing ST efficacy for further ablating tumors (Step III). Importantly, gluconic acid and H2O2 were generated in the starvation process, making an acidic and oxidative environment that contributed to elevating ⋅OH-production in CDT. Overall, the reactants of one reaction could be supplemented by the “byproducts” of another one, leading to a cycle-like catalytic process. Based on in vitro and in vivo results, we proved that such a mutually reinforced modality of Fenton reaction and starvation reaction provides a novel and potent anticancer mechanism for synergistic CDT/ST to effectively combat hypoxic cancers. 2. Experimental section 2.1. Materials Glucose oxidase (GOx), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1,10-dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine iodide (DIR) were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Dioleoylphosphatidylcholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (DSPE-PEG2000) were obtained from A.V.T. (shanghai) Pharmaceutical Co., Ltd. H2O2 assay kit was purchased from Solarbio (Beijing, China). Urea, Creatinine, etc colorimetric assay kits were bought from Elabscience Biotechnology Co., Ltd. All chemicals were used without further purifications. 2.2. Cell culture The human breast adenocarcinoma cell line MDA-MB-231 was obtained from KeyGEN Biotechnology Co. (Nanjing, China). The cells were cultured in a normoxic (21% O2) or hypoxic (1% O2) incubator with a complete DMEM medium (10% FBS, 1% penicillin-streptomycin). The anaerobic chamber was made by PUHE Biotechnology Company LTD (Wuxi. China). Scheme 1. (A) synthetic procedure of LipoCaO2/Fe(OH)3-GOx. (B) Illustration of cyclic Fenton/starvation reactions with Tai Ji diagram. X. Zhang et al. Biomaterials 275 (2021) 120987 3 2.3. Synthesis of CaO2/Fe(OH)3 nanocomposites 2 mL of CaCl2 aqueous solution (0.1 g/mL) in an opened grass flask was added 1 mL of ammonia solution (1 M) and 80 mL of PEG200. Then at quick stirring, 1 mL of 30% H2O2 was slowly added to the mixture at the rate of 1 drop per 10 s. The mixture was stirred for 6 h at room temperature, and then a clear and colorless solution was obtained. Further, NaOH solution (1 M) was slowly added to the mixture under ultrasound until the pH of the solution reached 11.5. The clear solution was thus changed to a white color or pale-yellow suspension. Then the mixture was centrifuged and a white precipitate was obtained, after washed with 0.1 M NaOH solution and ethanol, respectively, white CaO2 nanoparticles were obtained and stored in ethanol at 4 ◦C. For the synthesis of CaO2/Fe(OH)3 nanocomposites, FeCl3 aqueous solution was added into CaO2 ethanolic solution. And the feed ratio of FeCl3 and CaO2 was controlled at 1:20. After stirring for 5 min, the yellow product was obtained by centrifugation and washed with ethanol. Morphology of CaO2 and CaO2/Fe(OH)3 nanocomposites was observed by Field emission Transmission electron microscope (FTEM, Tecnai G2 20, FEI, The Netherlands). The successful synthesis of CaO2/Fe(OH)3 nanocomposites was confirmed by the high-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) analysis. 2.4. Preparation of GOx and CaO2/Fe(OH)3 encapsulated liposomes (LCFG) To prepare the combined nanoparticles, DOPC (7 mg), cholesterol (1 mg) and DSPE-PEG2000 (0.2 mg) were first dissolved in 2 mL chloroform/ethanol (v/v = 1:1) in a glass flask, and 0.2 mL of CaO2/Fe(OH)3 in ethanol (10 mg/mL) were added into the mixture. A thin lipid film was formed after rotary evaporation. 1 mL of GOx aqueous solution (0.1 mg/ mL) was added into the lipid film, after 30 s ultrasound treatment, a uniform lipid suspension was obtained. Then the suspension was centrifuged at 8000 rpm for 20 min, and the unloaded GOx supernatant solution was abandoned. A certain volume of ultrapure water was added into the precipitate to form liposomal LCFG. A similar procedure was used to prepare liposomal GOx (LipoGOx) but without the addition of CaO2/Fe(OH)3 in lipid film. 2.5. Characterization of LCFG Morphologies of LCFG and LipoGOx were observed by FTEM (JEM–2100 microscope). The hydrodynamic sizes of LCFG and LipoGOx in PBS solution (10 mM, pH 7.4) were measured by Zetasizer Nano ZS (Malvern Instruments) at 25 ◦C. 2.6. Quantitative analysis CaO2 was quantified by a commercial H2O2 assay kit based on its H2O2 production capability. Metal elements and GOx contents in nanoparticles were measured using inductively coupled plasma atomic absorption spectrometry (ICP-AAS) and bicinchoninic acid (BCA) protein assay kit, respectively. 2.7. Measure the changes of dissolved O2, pH, and H2O2 content in solution 1 mL of sample was added to 8 mL of PBS in pH 6.5 under stirring, and the dissolved O2 was determined by dissolved oxygen meter (DOG 3082) at 37 ◦C. The pH values were measured by the PHS-25 pH meter. And H2O2 contents were detected by the H2O2 assay kit as mentioned above. 2.8. Measure of ⋅OH levels in solution The ⋅OH was identified and detected by the ESR device of Bruker Biospin (Germany) with DMPO as a spin trap. In detail, 1 mM DMPO was added into LCFG solution (GOx concentration = 0.4 μg/mL) at pH 6.5 with or without 1 mg/mL glucose. After incubation for 1 h at 37 ◦C, ESR spectroscopy was then immediately performed on the mixture solution. The settings of ESR measurement parameters were as follows: 19.44 mW microwave power, 3445.2 center fields, 100 G sweep width, and 1.08 G field modulation. MB decolorization experiments were then recorded by a UV–vis spectrometer. 2.9. Intracellular O2 and reactive oxygen species (ROS) levels determination Intracellular O2 level was detected by [Ru(dpp)3]Cl2. First, MDA-MB231 cells were incubated in 12 well plates (NEST Biotechnology) under normoxic or hypoxic conditions overnight. And 10 μg/mL [Ru(dpp)3]Cl2 was added for a further 12 h incubation. Then the cells were treated with different nanoparticles. After 24 h, the cells were washed with PBS twice, and the fluorescence of [Ru(dpp)3]Cl2 was measured using a fluorescence microscope (λex = 450 nm, λem = 610 nm). The effects of nanoparticles on intracellular ROS generation were detected by DCFHDA assay (NJJCBIO). Briefly, cells were seeded (5 × 105 cells/well) in 12 well plates overnight, followed by refreshed with serum-free media. Thereafter, cells were further incubated with different nanoparticles for 6 h under normoxic or hypoxic conditions. Subsequently, 20 μM DCFHDA was added and the cells were incubated for a further 30 min. After 3 times of washing with PBS, the DCF fluorescence in the cells was observed on a fluorescence microscope. 2.10. Live/dead cells costaining MDA-MB-231 cells were seeded into 6-well plates (106 cells/well) and then cultured for 24 h in a humidified 21% or 1% O2 atmosphere. Then LipoGOx, LCF, or LCFG nanoparticles (GOx concentration: 0.4 μg/ mL) in DMEM media were added to each well, respectively. After another 24 h incubation, cells were stained with calcein-AM (live cells) and propidium iodide (PI, dead/late apoptotic cells), according to the manufacturer’s suggested protocol (Beijing Solarbio Science & Technology Co., Ltd.). Then, the cellular fluorescence was monitored on confocal microscopy. 2.11. Cell apoptosis analyses by FACS For apoptosis analysis, MDA-MB-231 cells were seeded on 6-well plates and cultured in a humidified 21% or 1% O2 atmosphere overnight. Then the cells were refreshed with a fresh medium. After culture with LipoGOx, LCF, or LCFG nanoparticles (GOx concentration: 0.4 μg/ mL) for 24 h, the cells were collected and stained with an Annexin VFITC/PI Apoptosis Detection Kit (KeyGen Biotech). A BD Accuri C6 Flow Cytometer was used for analysis. 2.12. Multicellular tumor spheroids (MCTS) inhibition 200 μL of MDA-MB-231 cell (103 cells/well) suspensions were seeded into ultra-low attachment round-bottom 96-well plates, and then cultured in a normoxic incubator. After 200–300 μm spheroids formation, MCTS were divided into 4 groups (n = 5). The selected MCTS were treated with LCF, LipoGOx, and LCFG. Untreated MCTS were employed as controls. The spheroids were incubated for 8 d. The diameter of the MCTS was imaged and recorded every two days using an optical microscope. X. Zhang et al. Biomaterials 275 (2021) 120987 4 2.13. Animals model BALB/c nude mice (5–6 weeks old, female) were purchased from Beijing Huafukang Bioscience Technology Co. (Beijing, China). The experimental protocol was approved by Committee on Ethical Animal Experiment at Huazhong University of Science and Technology. 2.14. In vivo imaging and biodistribution study The study was manipulated on MDA-MB-231 cells bearing mice by a noninvasive optical imaging system (Pearl Trilogy, LI-COR, USA). DIRloaded LCFG nanoparticles (LCFG@DIR) were prepared by coated hydrophobic fluorescein DIR into the lipid bilayers of LCFG with a film dispersion method. LCFG@DIR nanoparticles or free DIR were intravenously injected into tumor-bearing mice (5 μg DIR per mice). The fluorescence intensity images of each mouse were captured at different times. 2.15. In vivo antitumor studies When the tumor volume reached about 150 mm3 , the MDA-MB-231 bearing mice were randomly divided into 4 groups (n = 6) and injected intravenously with saline, LCF, LipoGOx, and LCFG at 0.5 mg/kg of GOx or its equivalent on days 1, 4, 7 and 10. Tumor sizes and animal body weights were recorded every three days after treatment. On day 16, the mice were sacrificed. Tumor tissues were acquired for comparison, weighing, and further immunohistochemistry assay. 2.16. Statistical analysis All data were taken from three independent experiments and then expressed as means ± standard deviation (SD). One-way ANOVA was performed to compare the statistical analysis by GraphPad Prism 5.0 (San Diego, CA, USA). 3. Results and discussion 3.1. The preparation and characterization of nanoparticles Firstly, PEG200-stabilized CaO2 nanoparticles were synthesized based on the reaction of CaCl2 with H2O2 in an alkaline environment [27]. Field emission transmission electron microscopy (FTEM) observations revealed the CaO2 nanoparticles dispersed well in ethanol with a size of about 20 nm in diameter (Fig. 1A). Under neutral conditions, CaO2 could react with water and produce hydroxyl ions, which could further coordinate with Fe3+ to form Fe(OH)3 precipitation [28]. Accordingly, by adding a few drops of FeCl3 aqueous solution into CaO2 nanoparticles in ethanol, we realized the in situ formation of Fe(OH)3 on the surface of CaO2 and obtained CaO2/Fe(OH)3 nanocomposites. As revealed in Fig. 1B, CaO2/Fe(OH)3 nanocomposites were similar to CaO2 nanoparticles and had rough surfaces, demonstrating that a bit of Fe-doping did not affect the size and morphology of CaO2 nanoparticles. Both nanoparticles exhibited good dispersion and uniform in ethanol by dynamic light scattering (DLS) analysis (Fig. 1C). The structure of CaO2/Fe(OH)3 nanocomposites was further confirmed by the high-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping (Fig. 1D), indicating that Ca (red) and O (yellow) were surrounded by Fe (green). Besides, Ca2+ representing peaks and Fe3+ signal were detected in the X-ray photoelectron spectroscopy (XPS) spectrum of CaO2/Fe(OH)3 nanoparticles (Fig. 1E), further confirming the formulation of CaO2 and Fe(OH)3 [29]. In the X-ray diffraction (XRD) result of CaO2 and CaO2/Fe(OH)3 nanoparticles, CaO2 representing specific peaks (2θ = 30.1◦, 35.6◦, 47.3◦) were displayed (Fig. 2A), proving the successful CaO2 preparation and the maintenance of crystal structure of CaO2 after the Fe(OH)3 introduction [30]. In addition, the surface Fe(OH)3 component is amorphous according to no XRD signal of the Fe(OH)3 phase [31]. Afterwards, the CaO2/Fe(OH)3 nanocomposites and GOx molecules were incorporated into a PEGylated liposome to form liposomal CaO2/ Fe(OH)3-GOx nanoparticles (denoted as LCFG). From FTEM images (Fig. 2B), LCFG nanoparticles with a diameter of about 100 nm were discovered. As control groups, GOx liposomes (LipoGOx) and liposomal CaO2/Fe(OH)3 (denoted as LCF) were prepared in similar methods. Zaverage hydrodynamic diameters of the obtained LipoGOx, LCF, and Fig. 1. FTEM images and high-resolution FTEM images (insert) of (A) CaO2 nanoparticles, (B) CaO2/Fe(OH)3 nanoparticles. (C) DLS of CaO2 and CaO2/Fe(OH)3 nanoparticles. (D) HAADF-STEM image and elemental mapping of CaO2/Fe(OH)3 nanoparticles. (E) XPS spectrum of CaO2/Fe(OH)3 nanoparticles. X. Zhang et al. Biomaterials 275 (2021) 120987 5 LCFG were measured by DLS, which turned out to be about 123.6 nm, 131.0 nm, and 145.6 nm, respectively (Fig. S1). The larger sizes determined by DLS than FTEM could be ascribed to the surface hydration of nanoparticles in DLS measurements. For the quantitative assay, the UV–vis spectrum method was carried out to determine the CaO2 contents in LCFG with the aid of an H2O2 kit, and the iron and calcium elements could be quantified by ICP-AAS. To measure the amount of GOx in nanoparticles, a BCA protein assay kit was utilized to amplify the GOx signal with a UV detector. The loading efficiency of GOx, CaO2, and Fe(OH)3 in LCFG was calculated to be 6.3%, 10.4%, and 0.7% (w/w), respectively. To confirm that CaO2 was able to reach the tumor site before decomposition, the stability study of CaO2 in nanoparticles was performed [32]. From the data shown in Fig. 2C, we found that less amount of Ca2+ was released in LCFG under neutral conditions, indicating that CaO2 nanoparticles were more stable in a liposomal formulation. Furthermore, in the weak acidic environment, the faster release of Ca2+ from CaO2 in LCFG was observed (Fig. 2D), which demonstrated the pH-responsive decomposition of CaO2. Hence, due to the protection of lipids and the pH-responsive property of CaO2, LCFG might be able to undergo blood circulation and then decompose in acidic TME without untimely disintegration. 3.2. In vitro catalytic profiles of LCFG nanoparticles It has been well-documented that GOx-based starvation reaction consumes O2 and produces H2O2/H+, which theoretically exhibit complementary effects with Fenton reactions. As shown in Fig. 3A and B, the GOx-catalytic glucose oxidation reaction increased H2O2 and consumed CaO2/Fe(OH)3-produced O2 in the LCFG system. Notably, the amount of H2O2 released from CaO2/Fe(OH)3 increased at the beginning and then decreased after several hours, which could be ascribed to the declining amount of CaO2 left and increasing iron ion concentrations for catalyzing H2O2 decomposition. Then we investigated the ⋅OH-generation capability of LCFG nanoparticles in solution by the electron spin resonance (ESR) technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as an ⋅OH trapper. Obviously, the introduction of glucose in the weak acidic environment (pH = 6.5) could enhance the ⋅OH-generation capability of LCFG nanoparticles, as manifested by the higher characteristic 1:2:2:1 ⋅OH radical signals in Fig. 3C. Moreover, methylene blue (MB) decolorization experiments were selected to investigate the mechanisms of the enhanced ⋅OH-generation. First, we proved that the ⋅OH generation is positively related to the H2O2 amount and H+ concentrations during Fenton reactions (Figs. S2 and S3). According to Fig. 3D, a significant decrease in MB absorbance was observed in LCFG-contained groups, showing the ⋅OH generation of nanoparticles. After the addition of glucose to initiate the catalytic activity of GOx, the ⋅OH-induced MB degradation increased due to the H2O2 and H+ production of GOx-mediated starvation reaction, which demonstrated the starvation-promoted Fenton reactions. Overall, the complementary effect between Fenton reactions and the starvation reaction was initially verified in solution, which is the basis of cooperative CDT/ST. 3.3. In vitro antitumor performance As described above, the catalytic reaction of GOx could consume O2. Conceivably, the hypoxic tumor environment might hinder the GOx efficiency and limit the starvation therapy outcomes. As expected, the IC50 value of LipoGOx against MDA-MB-231 cells under hypoxia was determined to be higher than that under normoxic conditions (Fig. 3E), showing that low O2 availability significantly reduced the antiproliferation efficacy of GOx. However, the LCFG group, in which CaO2/ Fe(OH)3 could provide sufficient O2, could obtain a recovered anticancer efficacy of GOx in hypoxic conditions (Fig. 3F). We also investigated the roles of individual components (CaO2 or Fe3+). As can be Fig. 2. (A) XRD spectrums of CaO2 and CaO2/Fe(OH)3 nanoparticles. (B) FTEM image and high-resolution FTEM image (insert) of LCFG nanoparticles. (C) Cumulative Ca2+ release of nanoparticles in conditions of pH 7.4. (D) Cumulative Ca2+ release of LCFG in conditions of pH 6.5 and pH 7.4. X. Zhang et al. Biomaterials 275 (2021) 120987 6 Fig. 3. (A) H2O2 amounts, (B) O2 concentrations of solutions containing nanoparticles and glucose at pH 6.5. (C) DMPO spin-trapping ESR spectra. (D) The absorbance of MB after incubation with nanoparticles at pH 6.5. (E) IC50 values of GOx in nanoparticles under normoxia and hypoxia. (F) Cell viability after incubation with different concentrations of nanoparticles. *p < 0.05, ***p < 0.001. Fig. 4. Confocal images of MDA-MB-231 cells stained with calcein-AM (green, live cells) and propidium iodide (red, dead cells) after different treatments. The concentrations of GOx were 0.4 μg/mL. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) X. Zhang et al. Biomaterials 275 (2021) 120987 7 seen in Fig. S4, the hypoxic cells were greatly eliminated only in the presence of both, and a single reagent (only CaO2 or Fe3+) failed to emulate the cytotoxicity of the combined group. These results collectively validated that CaO2/Fe(OH)3 nanocomposites enhanced the in vitro efficacy of GOx against hypoxic cancer cells. To visually assess the therapeutic effect of LCFG nanoparticles in vitro, calcein-AM and PI were used to identify live and dead/late apoptotic cells, respectively. As illustrated by Fig. 4, all the normoxic and hypoxic cells displayed green fluorescence in the control groups. In LipoGOx treated group, the dead cells under hypoxia were obviously less than that under normoxia according to the red fluorescence display, indicating the insufficient activity of GOx under hypoxia. However, in LCFG treated group, in which CaO2/Fe(OH)3 could provide sufficient O2, most of cells died and exhibited strong red fluorescence both in normoxia and hypoxia. In addition, we explored the capacity of LCFG to induce apoptosis by the annexin V-FITC/PI method. As revealed in Fig. 5A and B, the total of apoptotic cells (including early and late apoptotic cells) induced by LCFG reached 75.3% in normoxic MDA-MB231 cells, far exceeding the percents for LCF and LipoGOx. Under hypoxic conditions, the apoptotic cells treated with LCFG also reached 94.1%, breaking the hypoxic barrier of ST. Collectively, these results proved the improved antitumor effects of the combination treatment of CaO2/Fe(OH)3 and GOx at a cellular level. To further evaluate the combined treatment effect of LCFG, we established multicellular tumor spheroids (MCTS) by MDA-MB-231 cells as a mimic of in vivo breast tumor [33,34]. From the results in Fig. 5C and D, we found that compared with the compromised inhibition of MCTS with LCF and LipoGOx, the combined nanomedicine LCFG exhibited significant inhibition (even diminishment) of MCTS. This was another confirmation of the enhanced efficacy of synergistic CDT/ST against cancer. 3.4. Synergistic anticancer mechanism of combined CDT/ST The ROS generation of LCFG in cancer cells was studied using 2′ ,7′ - dichlorodihydrofluorescein diacetate (DCFH-DA) [35]. As illustrated in Fig. 6A and Fig. S5 obtained by flow cytometry, the LCFG-treated cells both in normoxic and hypoxic conditions showed stronger ROS signals compared with the LCF group, far exceeding the LipoGOx-treated group. By confocal microscopy, we observed apparent green fluorescence in cells treated with CaO2/Fe(OH)3-contained nanoparticle groups rather than other groups, indicating the ⋅OH generation of CaO2/Fe(OH)3 nanocomposites (Fig. 6B). Likewise, LCFG-treated cells showed brighter green fluorescence under normoxia and hypoxia, which could be attributed to such a mutually reinforced modality between CaO2/Fe (OH)3 and GOx. The H2O2/H+ generation of GOx catalysis enhanced Fenton reactions, and the O2-production of CaO2/Fe(OH)3 broke the hypoxic barrier of GOx catalysis. Next, we verified the O2 supply ability of CaO2/Fe(OH)3 in LCFG at the cellular levels. A commercial hypoxia probe, [Ru(dpp)3]Cl2, was Fig. 5. (A) Measurement of cell apoptosis by annexin V-FITC/PI double staining under normoxia and hypoxia. (B) Quantification of cell apoptosis, the concentrations of GOx were 0.4 μg/mL (C) MCTS inhibition by nanoparticles. (D) The relative volume changes of MCTS after treatment with LCF, LipoGOx, or LCFG for 8 d, the concentrations of GOx were 0.2 μg/mL ***p < 0.001. X. Zhang et al. Biomaterials 275 (2021) 120987 8 applied to detect the intracellular O2 level. In Fig. 7A, O2 quenched the red fluorescence of [Ru(dpp)3]Cl2 in normoxic cells. In GOx-treated cells, the mean fluorescence intensity increased along with the GOx concentrations (Fig. S6), which indicated the O2 consumption property of GOx activity. After introducing CaO2/Fe(OH)3 (LCF and LCFG groups), red fluorescence became weak, demonstrating the O2 supply capacity of CaO2/Fe(OH)3 could enhance the catalytic performance of GOx (Fig. 7B and Fig. S7). O2 could downregulate hypoxia-inducible factor-1α (HIF-1α) [36]. To further confirm the O2 generation ability of CaO2/Fe(OH)3, we determined the expression of HIF-1α by western blot. As depicted in Fig. 8A and B, LipoGOx treated groups showed higher levels of HIF-1α expression. However, the introduction of CaO2/Fe(OH)3 nanocomposites enabled the decrease of HIF-1α levels due to the O2-evolving by Fenton reactions. Thus, we concluded that the LCFG nanomedicine possessed the O2-supplied capacity without causing HIF-1 activation. 3.5. In vivo imaging and antitumor study A breast tumor (MDA-MB-231) xenograft model was employed to investigate the biodistribution and therapeutic efficacy of LCFG in vivo. As shown in Fig. 9A, only weak fluorescence was detected in the liver at Fig. 6. DCF fluorescence detection of MDA-MB-231 cells with (A) flow cytometer and (B) confocal microscopy after nanoparticles treatments under normoxia and hypoxia, the concentrations of GOx were 0.2 μg/mL. Fig. 7. O2 detection in cells using a commercial hypoxia probe, [Ru(dpp)3]Cl2 (A) after the treatment of different concentrations of LipoGOx under normoxia, (B) after the treatment of different nanoparticle groups under normoxia and hypoxia, the concentrations of GOx were 0.2 μg/mL. X. Zhang et al. Biomaterials 275 (2021) 120987 9 48 h post-injection because of fast metabolism and clearance of free DIR. However, with the same concentration of DIR, the tumorous fluorescence signal of the nanomedicine-treated mice lasted for more than 48 h. This result proved that the LCFG nanoparticles exhibited a long circulation and efficiently accumulated in the tumor to kill cancer cells with satisfactory selectiveness. To explore the therapeutic efficacy of LCFG in vivo, we started treatments when the average tumor volume reached about 150 mm3 . From the data shown in Fig. 9B–D, we found that the dual-therapies nanoparticle LCFG exhibited a stronger anticancer effect than any monotherapy group. Accordingly, CaO2/Fe(OH)3 and GOx co-loaded nanoformulation mediated the synergistic anticancer effect of CDT and ST was validated at an in vivo level. Furthermore, no significant weight loss was observed in mice, indicating the low toxicity of these lipid-based nanoformulations (Fig. 9E). Histological analysis also confirmed the biosafety of nanoparticles (Fig. S8). Moreover, as illustrated in Fig. S9, the LCFG nanoparticles did not induce nephrotoxicity and hepatotoxicity. The tumor section of LCFG-treatment showed obvious cell damage compared with other groups, further demonstrating the in vivo antitumor efficacy of combined nanomedicine (Fig. 10A). Finally, through TUNEL staining assay, we verified the enhanced apoptosis and cell death with the treatment of LCFG in tumor tissues (Fig. 10B). Fig. 8. (A) The expressions of HIF-1α protein and (B) the semi-quantitative results after different treatments: (1) Control; (2) LCF; (3) LipoGOx; (4) LCFG under normoxia and hypoxia. ***p < 0.001. Fig. 9. In vivo experiments. (A) Real-time fluorescence imaging of free DIR and LCFG@DIR nanoparticles treated mice. (B) Tumor photographs, (C) tumor volume, (D) average tumor weight, and (E) average mouse body weight of saline and other treated groups. ***p < 0.001. X. Zhang et al. Biomaterials 275 (2021) 120987 10 4. Conclusion In this work, we constructed biocompatible liposomes co-delivering CaO2/Fe(OH)3 nanocomposites and GOx to realize cooperative CDT/ST against hypoxic cancer. The synergistic mechanisms were also revealed: (1) CaO2/Fe(OH)3 initiated Fenton reactions and generated ⋅OH to induce CDT; (2) O2 as the “byproduct” of Fenton reactions reinforced the GOx-mediated glucose oxidation and thereby starving tumor cells without the limitation of hypoxia; (3) the augmented starvation reaction amplified CDT by the production of acidic H2O2. The improved anticancer efficacy of the combined CDT/ST was proved both in vitro and in vivo. This study offers a new combinational therapeutic paradigm by rationally integrating chemical reactions and making good use of the “byproducts” of individual reactions. Credit author statement Xiaojuan ZhangChuanchuan HeGuangya XiangXiaojuan ZhangYan ChenChen ChenRuicong YanTing FanTan YangYao LuYongkang GaiXiaojuan ZhangChuanchuan HeTan YangYao LuGuangya Xiangthe final Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments This work was supported by National Natural Science Foundation of China (Nos. 81973257, 81673368, 81703446) and the Fundamental Research Funds for the Central Universities (HUST: 2021yjsCXCY128). The authors thank the Analytical & Testing Center of Huazhong University of Science for FTEM, XRD, XPS. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.biomaterials.2021.120987. References [1] H. Ranji-burachaloo, P.A. Gurr, D.E. Dunstan, G.G. 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